Vous êtes sur la page 1sur 248

Phage Resource Guide

Science Education Alliance


Howard Hughes Medical Institute
Chevy Chase, Maryland

2011 by the Howard Hughes Medical Institute. All rights reserved.


Science Education Alliance
Tuajuanda C. Jordan, Ph.D.
Director
Lucia P. Barker, Ph.D.
Program Officer
Kevin W. Bradley
Research Technician
Razi Khaja
Bioinformatics Specialist
Melvina F. Lewis
Program Assistant
Credits
Design: VSA Partners, New York
Illustrations: Marjorie C. Leggitt
In Situ
Capture fig. 5, Capture fig. 6, Capture fig. 7,
Tame fig. 4, Tame fig. 6: Annie ONeill/Tom Little
Capture fig. 4: Adapted from an illustration at
www.sci.sdsu.edu/~ smaloy/MicrobialGenetics/topics/phage/lysis.html,
courtesy of Stanley Maloy, San Diego State University
Capture fig. 9: Adapted from fig. 6 in M. Feiss and W. Widner,
Bacteriophage l DNA packaging: scanning for the
terminal cohesive end site during packaging,
Proc Natl Acad Sci USA 79 (1982): 34983502,
courtesy of Michael Feiss. 1982 National Academy
of Sciences, USA.
Capture fig. 11: Adapted from fig. 9-15 in B. Alberts et al.,
Essential Cell Biology: An Introduction to the
Molecular Biology of the Cell (New York:
Garland Publishing, 1998), 288.
Dissect fig. 1: Lucia P. Barker
Appendix 1 photos: Annie ONeill/Tom Little;
DNA gels courtesy of FOTODYNE Incorporated
Appendix 5 photos: Courtesy of Fisher Scientific;
capillary tweezers courtesy of Ted Pella, Inc.;
DNA grids courtesy of Jim Burke, CIDDE/
University of Pittsburgh; Melvina Lewis
In Silico
Analyze figs. 24-27, 29, and 31: Lucia P. Barker

4000 Jones Bridge Road


Chevy Chase, Maryland 20815-6789
www.hhmi.org

Preface

The Science Education Alliance (SEA) and its first offering, the National Genomics
Research Initiative in Phage, are new ventures for HHMI. We believe that beginning
undergraduate students should experience real, inquiry-based science in person as
soon and as often as possible. We recognize that science is a collaborative and open
process. A very real and necessary part of science is sharing what does and does not
work and building new knowledge on a foundation of commonly held information.
Through the Alliance, we are creating a structure in which students and faculty share
ideas, problems, and findings in order to align the learning experience with the practice
of science.
Much of modern life science research is developing on the borders of biological and
physical science. Taking this into account, our course starts in vivo, in the field, with
the isolation of new viruses from nature. It then progresses to the laboratory bench for
an in vitro molecular analysis. Finally, it ends in silico, with a bioinformatics analysis
of the isolated genomes.
In short, answering questions by discovery and constructing new knowledge by sharing
carefully crafted information are all hallmarks of science. It is our hope that the Alliance,
the new course, and this guide should all help bring the excitement and fulfillment of
science to beginning students across the nation.
Peter J. Bruns, Ph.D.
Vice President, Emeritus
Howard Hughes Medical Institute

iii

Welcome

You are about to embark on a unique experience in which novice scientists across the
country will work together to address a single question of scientific interest. An
authentic research experience. An experience that will expose you, the new investigator,
to the process of doing science. This adventure will be about making discoveries
discoveries about mycobacteriophages, and discoveries about yourself. This means that
an observation has been made and a problem identified or a question posed, and scientific
tools will be available for you to use to address the problem or question. It means that
no one knows with absolute certainty whether those tools will work to address the
situation at hand. It means that you will have the opportunity to formulate testable
hypotheses, make predictions, design experiments to address the hypotheses, analyze
data, and decide what the next best step is. It means that there will be times of uncertainty. It means that there will be successes, and there will be some challenges.
You will learn that no one person ever has all the answers. You will not be alone in
that experience. Science is a collaborative and collegial endeavor. You are part of a
national network of experienced and novice scientists engaged in a single effort.
You have at your disposal a vast array of resources available from the Howard Hughes
Medical Institute, a national genome sequencing center, and colleges and universities
across the country. We are happy you are part of this alliance. We look forward to
learning of your discoveries.
The Staff of the Howard Hughes Medical Institutes
Science Education Alliance

Razi Khaja
Bioinformatics specialist

Tuajuanda C. Jordan
Director

ad le y
Ke vi n W. Br
hn ici an
Re se arch tec

Lu cia P. Ba rk
er
Pro gra m off ice
r

iv

M el vi na
F. Le w is
Pr og ra m as
si st an t

Overview

This guide is for college and university faculty to use for training novice scientists.
It provides a framework for exposing students to the process of doing science while
they explore mycobacteriophages, a focus of the National Genomics Research Initiative.
The NGRI has two major components: a wet lab and genomics. The guide breaks
these components down into steps that are correlated with the major steps of the
research experience: capture (the phage from the environment), tame (isolate and
purify a single phage), dissect (analyze aspects of the phages physical and molecular
structure), analyze (elucidate the genomic properties), discover (identify novel
genomic traits), and share (disseminate data). The first three sections take place in
the wet lab, and the last three involve the science of genomics.
The wet lab experience focuses on using techniques in microbiology, molecular biology,
and electron microscopy to characterize phages isolated from the environment. The
genomics portion focuses on using bioinformatics tools to facilitate genome finishing,
annotation, and comparison as ways to help us gain insight into the diversity of mycobacteriophages in the environment and the characteristics of mycobacteriophages
that render them unique and distinct from other phages.This type of information
could provide a framework for SEA scientists and other researchers to delve into the
possible utility of these organisms in a variety of biomedical, health, environmental,
and ecological applications.
This Resource Guide starts out with very detailed instructions for setting up work
areas and doing experiments. The level of detail drops off as the novice scientist gains
confidence and develops proficiency in designing experiments. The guide has four
main sections:
1. In Situ (Part 1)
This section acquaints young scientists with important aspects of an authentic
research experience and includes the protocols required to isolate, purify, and
characterize the mycobacteriophage and background information about phage
biology and nomenclature. It contains instructions for maintaining a laboratory
notebook and why that is important, the elements required to accurately label
reagents and data, and basic microbiological techniques and proficiencies.
Each procedure has the following sections: an overview of what is to be accomplished and a brief synopsis of the purpose of various components; key words
and concepts that are important for understanding the practical and theoretical
aspects of the procedure; required supplies and equipment; an implementation
timeline, to facilitate planning and setup; an objective, to provide the procedures
rationale; questions, to enhance the scientists critical thinking skills; and procedural notes, to provide specific information regarding reagent preparation or
experiment implementation.
v

In addition, the following are included throughout the experimental protocols:


The

Big Picture: Explains where the particular experiment or exercise fits in


with everything else

Alerts: Signal important information related to lab safety and special precautions

Notes

Food for Thought: Gets the scientist to think beyond the procedure

and Reminders: Suggest things that are important to consider


or remember

At key points throughout the procedure sections, Decision Trees guide the
scientist through the process of determining the next logical step.
2. In Silico (Part 2)

As in Part 1, each section contains an overview, key words and concepts, supplies,
an objective, questions, and procedural notes. A unique aspect of In Silico is that
the protocol listed in each section is divided into three sub-sections: input, which
describes how to access the bioinformatics tool and the type of data file required
to use it; procedure, which provides the step-by-step description of how to navigate each tool, uses multiple screenshots and, when appropriate, screencasts; and
output, which provides the type of data that will be obtained from the tool, contains a description of the file format, and explains how the file can be saved and/
or exported to another bioinformatics tool.
3. Glossaries and Appendices
Parts 1 and 2 have their own glossaries and appendices. The Glossaries are
available on the SEA wiki at http://www.hhmi.org/seawiki/display/EduRes/
Resource+Guide. Appendices for Part 1 provide additional resources and information, such as recipes, a troubleshooting guide, readings, and research paper
format. Those for Part 2 provide file formats for several of the bioinformatics
tools, a software-focused troubleshooting guide, and links and citations.
4.Video Resources
These videos are available on the SEA wiki at http://www.hhmi.org/seawiki/display/EduRes/Videos:

About safety. We take safety very seriously, and we expect each SEA member
institution to follow institutional safety guidelines as well as local, state, and
national rules and regulations. The video on safety issues, Practicing Safe
Science, can be downloaded from the SEA wiki and used as an ancillary
source.

vi

About

sequencing. The experiments during the first semester culminate with


DNA samples of the selected mycobacteriophage being sent to a genome sequencing center. The primary responsibilities of the sequencing center will be to sequence the phage genomes during the winter break and post sequence files for
subsequent analysis. Videos and links detailing several sequencing methods are
available on the SEA wiki. These resources describe what happens to the phage
DNA samples from the time they leave your institution until the data are posted
online.

 lthough the Resource Guide is for the use of faculty, a substantial portion of the
A
material is directed to the student. The students version, the Laboratory Manual, is
culled from the guides pages. The primary differences between the two texts are that
the manual does not contain procedural notes, implementation timelines, and recipes.
 pdated versions of the Resource Guide and course materials (e.g., PowerPoint slides
U
for selected topics) will be available on the web at www.hhmi.org/sea. We welcome
your comments on the guide. As you go through the protocols and other materials,
please note on our wiki (accessible via the SEA site) the places where the information
is unclear, incomplete, or too extensive and any information you would like us to add.

vii

Acknowledgments

This Resource Guide and the student Laboratory Manual are the result of hours
of discussions with a vast array of enthusiastically supportive individuals dedicated
to research and education. We would like to acknowledge and thank Elizabeth Fischer
and her staff at Rocky Mountain Laboratories/NIAID/NIH for their expert advice about
refining the protocols used for electron microscopy; Elizabeth Summer (Texas A&M
University) for hours of discussion concerning the joys and challenges associated
with phage biology and research-based laboratory courses and for providing us with
her refined DNA-isolation protocol, teaching resources, and editorial comments; the
Genomics Education Partnership (GEP) 2006 Cohort for lengthy discussions of appropriate ancillary materials to engage faculty and undergraduates in consortial research
projects; Erin Sanders-Lorenz (UCLA) for enthusiastic support of and editorial comments on the Resource Guide; Deborah Jacobs-Sera (University of Pittsburgh) for
mycobacteriophage protocols, materials, and enthusiastic support for the project;
Graham Hatfull (University of Pittsburgh) for his willingness to impart his expert
knowledge to the SEA community; the Department of Energys Joint Genome Institute
for their efforts to accommodate the Alliance and for providing the protocol to assess
genomic DNA quality; Annie ONeill and Tom Little for their expertise during the
Troubleshooting Guide photo shoot; Amy Vogelsberger (University of Pittsburgh) for
her solution recipes and tips; the students in the pilot course at the University of
Pittsburgh for their pioneer spirit and for their extreme patience in enduring the constant questions and comments from the SEA staff as we observed what was happening
in the course; Ed Lee (Lawrence Berkeley National Laboratory) for his enthusiasm for
all things bioinformatic and for building and refining the workflow; Steve Cresawn
for refining the Phamerator to meet the needs of the Alliance and for providing the
draft background and protocols; HHMI IT staff for working tirelessly to provide the
SEA with the tools and mechanisms necessary for creating, sustaining, and assessing
the Alliance; the HHMI Communications staff for providing the appropriate staff and
mechanisms to support these unique publications and the growing SEA community;
and finally, to the faculty and students of Cohort 2008 for their undeterred support,
insight, and constructive criticism of the materials and resources described herein.

ix

Contents

Part 1 In Situ Section


Before You Start

15 Decision Tree #1

F7 Procedural Notes

i Figure: In Situ Roadmap


ii Contents

17 Part C. FYI #2: Bacteriophage Lifestyles

Part A. Aseptic Technique


F1 Overview

Tame

F2 Timeline

iii Contents

1 Objective, Supplies, Equipment, and


Procedures

i Figure: Tame Roadmap


Part A. Purify the Phage: The Spot Test

F3 Procedural Notes

F1 Overview

Part B. Labels, Laboratory Notebooks and


Laundry Lists

F2 Timeline

F4 Overview

7 Objective, Supplies, Equipment, and


Procedures

F5 Procedural Notes

1 Objective, Supplies, Equipment, and


Procedures

F3 Procedural Notes

Part A. Purify the Phage: Plaque Streak

F4 Overview

Capture

F5 Timeline

i Figure: Capture Roadmap

ii Contents

1 P
 art A. FYI #1: Introduction to Bacteriophages

F1 A note from the SEA Staff: Enrichment vs.


Direct Plating

5 Objective, Supplies, Equipment, and


Procedures

F6 Procedural Notes

Part A. Purify the Phage: The Phage-Titer


Assay

F7 Overview

Part B. Isolate a Novel Phage from the Environment: Enrichment of Environmental


Samples

F8 Timeline

F2 Overview

F9 Procedural Notes

F3 Timeline

7 Objective, Supplies, Equipment, and


Procedures

9 Objective, Supplies, Equipment, and


Procedures
P
 art A. Purify the Phage: Final Plaque
Purification

F10 Overview

F4 Procedural Note

F11 Timeline

Part B. Isolate a Novel Phage from the Environment: Direct Plating of Environmental
Samples

15 Objective, Supplies, Equipment, and


Procedures

F5 Overview

F12 Procedural Notes

F6 Timeline

19 Decision Tree #2

11 O
 bjective, Supplies, Equipment, and
Procedures
continued
xi

Contents (continued)

P
 art B. Make Phage Stocks: Empirical Testing of Phage Lysates

F13 Overview

15 Decision Tree #3
F9 Procedural Notes

F14 Timeline

P
 art D. Evlaulate Genomic DNA Quality and
Send DNA to Sequencing Center

21 Objective, Supplies, Equipment, and


Procedures

F10 Overview

F15 Procedural Notes

16 Objective, Supplies, Equipment, Procedures,


and Questions

P
 art B. Make Phage Stocks: The 10-Plate
Phage Infection and Harvest

F16 Overview

F11 Timeline

19 Decision Tree #4
F12 Procedural Notes

F17 Timeline
25 Objective, Supplies, Equipment, and
Procedures

Appendices for Part 1


1 Appendix 1: The NGRI Troubleshooting


Guide

9 Appendix 2: Additional Resources for NGRI


Participants

F18 Procedural Notes


Dissect


i Figure: Dissect Roadmap


ii Contents
P
 art A. Analyze Phage Using Electron
Microscopy

F1 Overview

15 Appendix 3: Archiving and Reporting Your


Phage
21 Appendix 4: Format for Research Papers
23 Appendix 5: NGRI Equipment and Supplies

F2 Timeline

29 Appendix 6: Recipes for Stock Solutions,


Media, and Reagents

67 Appendix 7: Recipes for Bacterial Cultures

1 Objective, Supplies, Equipment, Procedures,


and Questions

F3 Procedural Notes

P
 art B. Isolate and Purify Phage Genomic
DNA

F4 Overview
F5 Timeline

4 Objective, Supplies, Equipment, Procedures,


and Questions

F6 Procedural Notes

P
 art C. Restrict and Analyze Phage Genomic
DNA

F7 Overview
F8 Timeline

8 Objective, Supplies, Equipment, Procedures,


and Questions

10 The Big Picture


xii

TAB
GOES
HERE

Situ
Overview
InInSitu
Overview
COLLECT ENVIRONMENTAL SAMPLE

ISOLATE PHAGE FROM


ENVIRONMENTAL SAMPLE

PURIFY THE PHAGE

CHARACTERIZE THE PHAGE

Rev. May 2011

Before You Start

Contents
Part A. Aseptic Technique, or Keeping It Clean
F1 Overview

Key Words and Concepts

Supplies and Equipment

F2 Implementation Timeline

1 Objective

Supplies (for students)

Equipment (for students)


Procedure: Getting Started with Aseptic
Technique
2 Procedure: Transferring Sterile Solutions
Using Aseptic Technique

4 The Big Picture

5 Notes

6 Questions

F3 Procedural Notes
Part B
 . Labels, Laboratory Notebooks, and
Laundry Lists
F4 Overview


7 The Principle of Autonomous Replication


Writing in a Laboratory Notebook
8 The Big Picture

9 Labeling Hints: Minimum Labeling


Requirements
Notes
10 Questions
F5 Procedural Notes

NOTE
We use live microorganisms in this course. These microorganisms are generally considered safe to use for those with a normal, noncompromised immune response.
If you have any health or other concerns associated with exposure, please
alert your instructor or the laboratory director immediately, or contact your
institutions safety office.
ii

Part A. Aseptic Technique, or Keeping It Clean

Overview
Aseptic technique is critical for the successful isolation of a
pure phage population. Students must become proficient at
it so that they can establish and maintain an aseptic bench
top and work area. The key components of the technique are
work-area preparation,
Bunsen burner use,
aseptic transfer of materials and reagents,
bench-top management, and
the storage and disposal of potentially contaminated
materials (potential biohazards).
These basic skills are required for all the microbiological
work the students will do. Avoiding contamination of
student samples with phages from other procedures or
samples is imperative because a pure phage population is
required for further steps, such as restriction analysis and
genome sequencing.
Many cases of contamination in the laboratory can be traced
to careless or nave work habits that are developed early in
a students scientific training and that remain undetected or
unchallenged over time. Conversely, even the most careful
of scientists will have to deal with contamination at one or
several points in their scientific careers. Our goal is to give
students an awareness of the likely causes of contamination
as well as ways they can decrease the likelihood of contamination while they are at their benches. Most importantly, if
students work slowly and deliberately until these principles
are second nature, they will be far less likely to deal with
contamination issues later on.

Supplies
For each student (these
materials do not need to be
sterilized; this is a practice
exercise1):
250-mL bottle or flask of water
(at least 50 mL water; 1 per 1 to
2 students)
Squeeze bottles containing
disinfectant2 (at least 1 per bench)
15-mL conical tube (1)
10-mL culture tube (1)
10-mL serological pipettes
(at least 2)
1-mL serological pipette (at least 1)
Microcentrifuge tubes (2)
Micropipettor tips (100-L)
(at least 3)

Equipment
Bunsen burners (1 per student)
Pipettors3 (e.g., Pipette-Aid; at least
1 per 2 students)
Micropipettor (100- or 200-L)
(1 per student)
Bottle of medium (water)

Key Words and Concepts


Aseptic technique, sterile field, disinfectant, autoclaving,
waste disposal, contamination.

Part1 Before You Start F1

Part A. Aseptic Technique, or Keeping It Clean

Implementation Timeline

LAB
Assemble all materials
at each bench.

t1D
Prep nonsterile bottles
of water.

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part1 Before You Start F2

Part A. Aseptic Technique, or Keeping It Clean

Objective
Each student will learn to minimize the chance of contamination.

Supplies







250-mL bottle or flask of water (at least 50 mL water;


1 per 1 to 2 students)
Squeeze bottles containing disinfectant
15-mL conical tube (1)
10-mL culture tube (1)
10-mL serological pipettes (at least 2)
1-mL serological pipette (at least 1)
Microcentrifuge tubes (2)
Micropipettor tips (100-L) (at least 3)

Equipment



Bunsen burners (1 per student)


Pipettors (e.g., Pipette-Aid; at least 1 per 2 students)
Micropipettor (100- or 200-L) (1 per student)
Bottle of medium (water)

Procedure: Getting Started with Aseptic Technique


A. Prepare your work area.
1. Tidy up your work bench by removing clutter, papers, bottles, etc.
2. Using a squeeze bottle containing disinfectant, such as 70% ethanol
(EtOH), dispense enough disinfectant to dampen the entire work
surface.
3. Using a paper towel or gauze pad, wipe the entire area, back
to front, to evenly spread the EtOH and moisten the entire
work surface.
4. If you need more EtOH, add more, and repeat the process from
back to front.
5. Allow the EtOH (or other disinfectant) to evaporatedo not
wipe dry.4
B. Ignite the Bunsen burner.

At a minimum, your
bench should be
wiped down with
70% ethanol at the
beginning and end of
every lab exercise.
The bench should
also be wiped down
whenever there is
any chance of
contamination or spill.

1. Light the Bunsen burner, but only after the disinfectant has
completely dried.
2. Adjust the flame so that a blue cone can be seen in the flame
(the tip of this cone is the hottest part of the flame).

Until one is familiar with using a Bunsen burner, it is important


to be constantly mindful of the location of the flame. The flame,
even with an apparent blue cone, is not always easy to see!

Part1 Before You Start 1

Part A. Aseptic Technique, or Keeping It Clean

Updraft
Before fig. 1
How a Bunsen burner works to
keep the work area aseptic.

Aseptic area around flame

Aseptic versus
sterile: These terms
are often used
interchangeably, but
they should not be.
Sterile means free
of all living microorganisms, including
bacteria, fungi, and
viruses. Aseptic
refers to an area
whose contamination
has been minimized.
In your procedures,
reference is often
made to supplies
that are sterile,
and the expectation
is that you will use
aseptic technique to
minimize contamination by undesired
microorganisms.

The flame is now providing an updraft (see Before fig. 1 and The Big
Picture, below). Because heat rises, microorganisms and dust particles
will be forced upward and away from the immediate work area. It is
important to work within this area, or zone, careful not to disturb the
updraft by rapid movements that dramatically change the air currents
around your bench. Work slowly, carefully, and deliberately.

Procedure: Transferring Sterile Solutions Using


Aseptic Technique
A. Arrange needed supplies on your bench so you have easy access.




A bottle of medium (i.e., water in this case)


A culture tube and a 15-mL conical tube in a rack
A 10-mL pipette
A pipettor
A Bunsen burner

B. Transfer 10 mL of medium into a 15-mL conical test tube.


1. Loosen (but do not remove) the tube and bottle caps.
2. Peel down the wrapper of the pipette (like a banana) from the top (opposite the tip). Hold the flaps against the pipette in your nondominant
hand (if you write with your right hand, thats your dominant hand).
3. Using your other (dominant) hand, place the pipette on the pipettor
and remove the wrapper. Do not touch the tip of the pipettewhich is
sterileto any surface!

Part1 Before You Start 2

Part A. Aseptic Technique, or Keeping It Clean

4. Using your nondominant hand, unscrew the top of the media bottle and
leave it between your forefinger and middle finger (see Before fig. 2).
5. Use your nondominant hand to pick up the bottle and pass the opening two to three times through the flame of the Bunsen burner. Do not
linger over the flame, and do not heat the top of the bottle or lip. This
technique is called flaming.
6. Immediately place the serological pipette into the bottle, and, using the
pipettor, draw up 10 mL.
7. Flame the top of the bottle again and replace the cover.

Do not turn the


cap upside-down
and do not set it
on the bench. Any
area inside the cap
of a sterile bottle
or sterile tube is
considered a sterile field, not to be
compromised!

Before fig. 2
Transferring liquid samples using
aseptic technique. Before beginning, dispense ethanol or other
disinfectant onto the surface of your
bench (A) and wipe from back to
front (B). Light your Bunsen burner
(C). Prepare all your materials, and
loosen all bottle and tube caps (D).
Peel down both sides of the pipette
package (E), and hold both flaps
while attaching the pipettor (F).
Using your first and second fingers,
remove the bottle top (G), and pick
up the bottle with the same hand
and flame (H). Remove your
sample (I), and reflame and recap
the bottle (J). Using the smallest
finger of your pipettor hand, remove the top of the tube (K and L),
and add your sample to the tube
(M). Recap the tube before discarding the pipette (N). When finished,
turn off your Bunsen burner, tidy
up, and wipe down your bench.

Part1 Before You Start 3

Part A. Aseptic Technique, or Keeping It Clean

8. Using the smallest finger on the hand holding the pipettor, remove the
top of the test tube.
9. Dispense the contents of the pipette into the tube.
The goal of
flaming is to gently
heat the air at
the bottle opening.
This creates a
mini-microbiological
force field because
the air is more likely
to come out of the
bottle than to fall
into the bottle when
the air at the opening is heated (the
updraft concept).

10. Cover the tube.


11. Discard the used pipette.
C. Repeat the entire process by transferring 5 mL of water into the 10-mL
culture tube.
1. Transfer 5 mL of water from the medium bottle into the glass culture
tube using aseptic technique, this time flaming the top of the glass tube.
2. Pour out the contents of the tube and practice this two to three times
until you are comfortable with the process.

The Big Picture


Bacteria and fungi (as well as pollen, spores, and
other particles) are found in surprisingly high
concentrations suspended in the air we breathe. One
estimate is that we each breathe in approximately
10,000 organisms per day. Creating an updraft with
the Bunsen burner minimizes the possibility of
organisms falling onto the bench or into open bottles,
tubes, plates, or flasks.

3. Choose a partner and critique each others technique.


Things to watch:
Remember to avoid
passing any of your
fingers over the top
of sterile bottles,
tubes, or flasks,
especially when
removing or replacing a cap or lid.

Hand and finger placements


Movement of pipette and pipette tips
Sterile field location(s)
Placement and handling of tube and bottle caps

D. Transfer 100 L of medium into a microcentrifuge tube.


1. Arrange on the bench so that you have easy access to each of these items:




A bottle of medium (water)


A 1.0-mL pipette
A 100- or 200-L micropipettor
A box of pipette tips
Microcentrifuge tubes (2) in a tube rack

2. Transfer 1.0 mL of medium from the bottle to a microcentrifuge tube using the aseptic technique described above, except do not attempt to flame
any plastic or microcentrifuge tubes!
Part1 Before You Start 4

Part A. Aseptic Technique, or Keeping It Clean

3. Wipe down the micropipettor with disinfectant (such as 70% ethanol),


and allow it to air-dry.5
4. Set the micropipettor to 100 L.
5. Using the micropipettor, remove a sterile tip from the box.
6. Open the tube containing the 1.0 mL of medium and remove 100 L
using the micropipettor.
7. Dispense the 100 L of liquid into the second microcentrifuge tube.
8. Pour out the contents of the tube, and practice this two to three times
until comfortable with the process.
9. Choose a partner and critique each others technique.

The micropipettor
has a two-stop
plunger system. The
first stop draws in
the desired volume
of liquid; the second
dispenses the liquid.

Things to watch:



Hand and finger placements


Movement of pipette and micropipettor, and pipette and
micropipettor tips
Sterile field location(s)
Placement and handling of tubes

E. Clean up your workbench.







Turn off the Bunsen burner.


Put away all supplies and solutions.
Put any potentially contaminated glassware into a
decontaminating solution.
Put any other potentially contaminated items into
infectious-waste containers.
Make sure the bench is clear and free from clutter.
Wipe down the bench with disinfectant (such as 70% EtOH),
and allow it to air-dry.

Notes
To keep an area aseptic, remember these important points:





Always know where your hands are.


Never pass your hands or fingers over the top of a sterile field
(such as open bottles or flasks, the inside of tube and bottle caps,
and agar plates).
Plastic culture tubes and microcentrifuge tubes cannot be flamed,
so it is doubly important to work with an open flame and to take
care not to pass fingers or hands over any tube openings.
The microcentrifuge tube should be kept closed until liquid is
transferred and should be closed immediately when done.
Never set a bottle or tube cap or Petri dish lid on a bench top.
Never go into a sterile solution with a used pipette or tip. In other
words, never reuse a pipette, even if great care has been taken
to keep it sterile.

Food
for Thought
Why is there an extra step?
In other words, why not transfer
100 L of media directly into
the microcentrifuge tube and
save some time and supplies?

Part1 Before You Start 5

Part A. Aseptic Technique, or Keeping It Clean

Always work with an open flame when opening sterile tubes or bottles.
Never have more than one tube, bottle, or flask open on the bench
at a time.
Even if someone else has recently used the bench and the bench
top was wiped down with disinfectant, always begin your laboratory
time by wiping down the laboratory bench. Always end laboratory
time by wiping down the bench. One bit of dust can contain hundreds
of organisms!

Questions
1. The food and beverage industries, though held to high standards, are not
required to perform this level of aseptic technique during the handling of
materials (except, of course, in the microbiological laboratories!). Why isnt
there more illness associated with our food supply?
2. Why do researchers concern themselves with contamination?
3. What do you expect to observe when something is contaminated? Why?

Part1 Before You Start 6

Part A. Aseptic Technique, or Keeping It Clean

Procedural Notes
1.
This exercise is designed to get the students comfortable with manipulating sterile bottles, tubes,
and solutions. The instructor is encouraged to modify it so that students are actually using all these
techniques as they pursue a question. For example, students can aliquot their own bacterial cultures
with or without sterile technique and plate the cultures to determine any contamination levels with
and without precautions.
2.
Any approved disinfectant (including those with 5% Amphyl or a quaternary ammonium compound)
will do. EtOH is a good general-purpose choice. Pure 100% EtOH is expensive and does not provide
the same desiccation effect as 70% EtOH. A solution of 95% EtOH can be used or diluted to 70%.
3.
Alternatives to electric or battery-operated pipettors include serological pipette bulbs or plastic pipettors (see www.sciencelab.com/page/S/PVAR/23779/60-379111002 for an example).
4.
This is an important point. The desiccation of organisms is one of the most effective ways to decontaminate a laboratory bench. It supplements the disinfectants bactericidal and fungicidal effects.
5.
The use of 70% EtOH on micropipettors and other laboratory equipment is generally innocuous. Other
disinfectants or a higher concentration of EtOH may, however, result in residue buildup or reactions
with certain plastics. If other disinfectants are used, be sure to test on a small area and be sure that
student exposure (e.g., handling disinfected micropipettors) is minimized.

Part1 Before You Start F3

Part B. Labels, Laboratory Notebooks,


and Laundry Lists

Overview
This section provides guidance for novice scientists. These
guidelines specify the minimum information required for
documenting lab results. In the likely event that the data
generated by students are published, there are ethical and
practical reasons to adhere to these guidelines.

Part1 Before You Start F4

Part B. Labels, Laboratory Notebooks,


and Laundry Lists

The Principle of Autonomous Replication


The goal of writing in a laboratory notebook is to create a record that anyone
can use to perform the same procedures and obtain the same resultsusing
only your laboratory notebook. This is known as the principle of autonomous
replication.
Indications that you have fulfilled the requirements for the principle of autonomous replication include:


The level of detail should be high enough that you can go back at any
time and troubleshoot your procedures if, for some reason, a procedure
does not work or yields questionable results.
Jargon, personal shorthand, and personal abbreviations should be kept
to a minimum (unless you have a list of abbreviations in the notebook).
Drawings, figures, and tables are encouraged. They must contain enough
information that another scientist can interpret them without extensive
reference to the text.

Writing in a Laboratory Notebook


This is an outline of the minimum information required in a laboratory notebook. Italicized portions are suggestions. You may, however, want to arrange
and organize your notebook in your own way.1

You must write in your lab notebook as you perform the procedure. Your
memory is not reliable. Experiments without sufficient documentation
must be discarded. The results cannot be disseminated!

A. Date. Date each page of the notebook. It is imperative that you record
everything in your notebook as you do it.
B. Aims and purpose. This is a thumbnail sketch of the reason you are performing the protocols and experiments for the day. It should stand alone as
your rationale. In other words, anyone should be able to open your notebook to any experiment and understand the why of the laboratory work
performed. State (or restate) any hypothesis or expected results here. This
acts as a framework from which you can build your analysis (below).

Part1 Before You Start 7

Part B. Labels, Laboratory Notebooks, and Laundry Lists

The Big Picture


When scientific data are published, the source of
the data (namely, the laboratory notebook) belongs
to, and must be retained by, the laboratory for at
least 7 years. The notebook should, therefore, be
a document that can be read and understood for
years to come even if, and especially when, science
moves on from the point of discovery outlined in
the notebook.

C. Materials. All materials should be listed. This helps you, and anyone reading the notebook, repeat procedures with the same supplies.
D. Procedures and protocols. This should be a comprehensive, accurate, and
detailed step-by-step accounting of your procedure. Even if you are repeating a protocol without any changes, write out the exact steps.
E. Results.


It is a good idea
to number and
initial each page as
you complete your
write-up.

The description of any results must be comprehensive and accurate.


Primary data (numerical values, photographs, printouts, and all
observations) should be listed first.
Anything taped into the notebook must be dated and initialed.
Primary data can later be sorted or organized into tables, graphs,
charts, or diagrams.
Any reader should be able to read the results of an experiment and
know exactly what happened.

F. Analysis and interpretation.



Part1 Before You Start 8

An objective and balanced analysis must be documented in the laboratory


notebook. Even if the results indicate a simple yes or no answer, it is
important to state that in this section.
Report any unexpected findings or problems during the course of the
experiments. This can act as a rationale for additional experiments, for
changing the protocol or materials, or for changing the path that your
research has been following.
Interpretation of data can sometimes be subjective, but your reasons for
a particular interpretation must be stated here.

Part B. Labels, Laboratory Notebooks, and Laundry Lists

G. Future plans.
On the basis of your interpretation of the data, you should end each
experiment or day of recording in your notebook with a short outline
of your next step or steps. The rationale for this should be clear in your
analysis section and does not necessarily have to be restated. This will
lead you and the reader into the next procedure with a clear idea of the
short- and long-term goals of your work.
This section can also include a to-do list for the next day of work
(Get more 5-mL pipettes, Dont forget to prewarm plates, etc.).2

Labeling Hints: Minimum Labeling Requirements


A. Each set of tubes, plates, and bottles should be labeled with


Your name or initials


The date
The designation (this can be numbers, letters, or more detailed
descriptors)

B. For prepared bottles of media or other solutions, tubes, cultures, or


stocks that are exclusively yours, each should be labeled with


Your name or initials


The contents
The date(s) received, prepared, and/or opened

C. For computer files or images



Use a consistent designator (e.g., your initials and the date, ML100208)
plus an image or file number.
Make sure that the label on or title of your file(s) matches the information in your laboratory notebook.

Notes
Every laboratory notebook belongs to the laboratory of the lead researcher
(faculty member) on your team. The original notebook cannot leave the laboratory, though laboratory personnel often make copies for their records.
Do not remove your notebook from the laboratory.
You may want to leave a few pages at the beginning of the notebook for a
table of contents (page numbers for each experiment and protocol) and a list
of abbreviations, both standard and personal.
At the beginning, you may want to limit notebook entries to the right side
of the open pages. The left side of the notebook can be used for notes, comments, and checklists.
Remember that everything that is labeled should be designated in your laboratory notebook with as much detail as possible. For example, if 10 samples

Part1 Before You Start 9

Part B. Labels, Laboratory Notebooks, and Laundry Lists

from the environment are labeled A to J on the bench (with, of course, the
date and the initials of the scientist), the laboratory notebook should list each
designation with the descriptors for each sample site.

Questions
1. In commercial and clinical laboratories, a supervisor is often required to sign
off daily on the entries of the laboratory scientist. In these settings, all notebooks are usually locked in a safe, or otherwise secured, until the next day of
work. Why do you suppose these precautions are taken?
2. What are the possible repercussions for a given laboratory or lead researcher
if, during a routine audit (i.e., an inspection of laboratory procedures and
practices, including documentation), the primary data used in a published
work cannot be found or cannot be interpreted? These audits do, in fact, occur,
especially if another laboratory obtains conflicting results!

Part1 Before You Start 10

Part B. Labels, Laboratory Notebooks, and Laundry Lists

Procedural Notes
1.
It is up to you to determine the best format for laboratory notebook notation and the amount of room
(if any) for personalization. You may want to encourage students to record any excitement, frustration,
doubts, or victories in their own words, but you may want to make clear that these personal entries
must not compromise the integrity of the data. Make sure students understand that the notebook can,
and will, be read by others.
2.
You may also want to emphasize that because of the nature of the SEA investigation, publication of
data generated by students is expected and very likely. Because of this, we encourage you to check
the laboratory notebooks of students regularly or hold conferences with individual students to give
advice and feedback regarding their documentation of procedures and experiments.

Part1 Before You Start F5

TAB
GOES
HERE

You
Are
Here
You
Are
Here
CAPTURE
COLLECT ENVIRONMENTAL
SAMPLE
Enrichment

or

Direct Plating

CULTURE SAMPLE
WITH BACTERIA

COLLECT UP TO 10
ENVIRONMENTAL SAMPLES

HARVEST LYSATE & FILTER

EXTRACT PHAGE

PREPARE PHAGE FILTRATE

INFECT BACTERIA

PLATE INFECTED BACTERIA;


CHECK FOR PLAQUES AFTER 24-48h

PURIFY THE PHAGE

CHARACTERIZE THE PHAGE

Rev. May 2011

Capture

Contents
Part A. FYI #1: Introduction to Bacteriophages

Direct Plating of Environmental Samples

1 What Are Bacteria?

F5 Overview

2 What Are Bacteriophages?

Key Words and Concepts

3 Who Discovered Phages?

Supplies and Equipment

4 What Happens When a Phage Infects


a Bacterium?

F6 Implementation Timeline

5 How Do You See (and Count) Bacteria


and Phages?

Objective

6 Why Study Phages Now?

The Big Picture

Part B. Isolate a Novel Phage from


the Environment

11 Overview

Supplies (for students)


Equipment (for students)


Procedure: Sample Collection and
Preparation

F1 A note from the SEA staff

13 Procedure: The First Round of Infection


Plaque Screening

Enrichment of Environmental Samples

15 Decision Tree #1

F2 Overview

16 Questions

Key Words and Concepts

F7 Procedural Notes

Supplies and Equipment

F3 Implementation Timeline

7 Overview

Objective

Supplies (for students)

Equipment (for students)

8 Procedure: Sample Collection and


Preparation

Procedure: Harvesting and Preparing the


Enriched Sample

9 Procedure: The First Round of Infection


Plaque Screening
10 Notes
F4 Procedural Note

ii

14 Notes

Part C. FYI #2: Bacteriophage Lifestyles


17 Lytic vs. Temperate Phages

Plaque Morphology and Phage Lifestyle

18 The Infection Process


19 The Lytic Cycle
20 The Temperate Phage Lifestyle
22 The Big Picture

Part A. FYI #1: Introduction to Bacteriophages

What Are Bacteria?


Bacteria are prokaryotes, the simplest free-living life-form.
They are quite small, usually in the range of 1 to 5 microns (m) (a human
red blood cell is 8 m across; a white cell is 12 to 20 m). See Cells Alive,
How big is a at

www.cellsalive.com/howbig.htm
Different bacteria can cause a variety of infections in humans, from food
poisoning to meningitis to boils.

LOG OF CELL NUMBER

Bacteria grow by binary fission (in other words, they split in half: 1 to 2 to 4
to 8 to 16, etc.). See Capture fig. 1.

C
D

Capture fig. 1
Bacterial growth over time.
When inoculated into fresh media,
bacteria will take some time to
recover without growth. This is
called the lag phase (A). Bacteria
will then enter a logarithmic (or
exponential) growth phase (B),
where the number of bacteria
doubles once every time period
(the period depends on the
bacterial species). Once nutrients
are used up, the bacterial number
will remain constant (C, the stationary phase) and eventually decline
(D, the decline, or death, phase).

TIME

Bacteria can grow very rapidly.


They are generally susceptible to antibiotics (unlike viruses, the other
germs).
Bacteria are ubiquitous.
The morphology (form and structure) of different bacteria varies (see Capture
fig. 2), but the basic components are these:
Outer structures. All bacteria have a plasma membrane made of lipids (or
fats) that surrounds the inner workings of the bacterial cell. The cell wall
is a collection of molecules that surround the plasma membrane and give
the bacteria structural strength. Bacteria can have an additional lipid membrane and/or a slimy capsule that protects them from harsh environments or
performs other functions. Some bacteria have appendages called pili and
flagella (singular is pilus and flagellum, respectively). These are hairlike
structures that protrude from the bacteria and perform specialized functions.

Part1 Capture 1

Part A. FYI #1: Introduction to Bacteriophages

Inner structures. All bacteria contain genetic material, or DNA. Since


bacteria do not have a nucleus, the free DNA, or chromosome, is called a
nucleoid. All bacteria also contain ribosomes, which translate the DNA
sequence into proteins.
For more information about bacterial cell components, see Cells Alive at

www.cellsalive.com/cells/bactcell.htm
flagellum
cell wall

Capture fig. 2
Bacterial structure.

cytoplasmic
membrane
cytoplasm
capsule

pili
ribosomes

bacterial
DNA

What Are Bacteriophages?


Bacteriophages (often shortened to phages) are a class of viruses (i.e., all
phages are viruses, but not all viruses are phages).
Phages are parasites specific to bacteria. The Ancient Greek word phagein
means to eat. A phage is an eater (or devourer) of bacteria.
Phage size ranges from 100 to 200 nm. See Cells Alive, How big is a, at

www.cellsalive.com/howbig.htm
Food
for Thought
How is it possible to isolate
individual phages if they can
only grow inside bacteria?

Part1 Capture 2

Phages cannot replicate or propagate outside their host bacteria.


Phages are not susceptible to antibiotics.
Phages are ubiquitous.
Phages are the most abundant life-form on earth (there are 1031 phages
worldwide).
Phages can survive in almost any environment, and they can be found both
inside and outside bacterial cells.

Part A. FYI #1: Introduction to Bacteriophages

The morphology of different phages varies (see Capture fig. 3), but there are
three basic components:

the capsid, also known as the head, which contains the genetic
material;

the genetic material, which in phages is mostly double-stranded DNA


(dsDNA); and

the tail, a hollow structure or tube. The tail serves two purposes. First, it
allows the phage to attach to bacteria. This is a very specific interaction.
The host range of the phage is defined by the kinds of bacteria to which
the phage can attach. Second, the DNA passes through the tail and into
the bacterium. These are the first two steps of infection of the bacterium
by the phage.

capsid

Capture fig. 3
Phage structure.

DNA

neck
tail sheath

tail fibers

end plate

pins

Phage can be singular or used in reference to a collection of the same type


of phage particle. For example:
My favorite phage is the Bronx Bomber.
I have 750 billion Bronx Bomber phage in this test tube.
Phages is the plural referring to a collection of different types of phage particles. For example:
We study Bronx Bomber and T3 phages in our laboratory.
Mycobacteriophages are phages that specifically infect species of bacteria in
the genus Mycobacterium.

Who Discovered Phages?


In 1910, Felix dHerelle saw clear spots on a lawn of bacteria. We call them
plaques. He filed this away in the back of his mind until, in 1915, he added
Part1 Capture 3

Part A. FYI #1: Introduction to Bacteriophages

some material from a plaque to a growing (and cloudy, or turbid) batch of


bacteria. This is how dHerelle described what happened:
The next morning, on opening the incubator, I experienced one of those moments
of intense emotion which reward the research worker for all his pains: at the
first glance I saw that the culture which the night before had been very turbid,
was perfectly clear: all the bacteria had vanished, they had dissolved away
like sugar in water. As for the agar spread, it was devoid of all growth and what
caused my emotion was that in a flash I had understood: what caused my clear
spots was in fact an invisible microbe, a filtrable virus, but a virus which is
parasitic on bacteria. (From The Bacteriophage, by Felix dHerelle, Science
News 14:4459 (1949). Translation by JL Crammer.)

Another scientist, Edward Twort, was credited for the independent


discovery of phages at the same time. dHerelle, however, coined the term
bacteriophages.

What Happens When a Phage Infects a Bacterium?


The phage attaches to the bacterium and injects its DNA into the bacterial
cell. The phage structure, now empty and attached to the bacterium, is no
longer infectious and is called a ghost. Using the bacteriums replication

Capture fig. 4
Phage life cycle, simple (lytic only).

Phage binds to receptor


on outer surface of
bacterial cell

Phage lysate

Phage DNA enters host cell


and circularizes by recombination
between the ends of the linear
phage DNA

Cell lysis
Progeny phage
accumulate; synthesis
of lysis proteins
Packaging of phage
DNA into phage heads;
attachment of tails
yielding intact phage

Part1 Capture 4

Replication of phage
genome

Expression of phage
proteins and assembly into phage heads
and tails

Part A. FYI #1: Introduction to Bacteriophages

machinery, the phage genomic DNA makes and assembles copies of itself
(including the capsid and tail) until the host bacterium lyses (Capture fig. 4).
See Cells Alive, Oh my goodness, my E. coli has a virus, at

www.cellsalive.com/phage.htm
How Do You See (and Count) Bacteria and Phages?
Use a microscope. You can see individual bacteria with a standard light
microscope, but to see phages, you generally need an electron microscope.
Grow a ton of them.
Bacterial colonies. These are visible collections of bacteria that arose from
one bacterium (see Capture fig. 5, left plate). When a bacterium is inoculated
onto an agar plate containing all the nutrients required for growth, it will
Capture fig. 5
Bacterial single colonies (left)
and lawn of bacteria with
clearing zones (right).

replicate and produce many bacteria. One bacterium becomes two; these two
become four, etc. Depending on the bacterial species and doubling time (the
time it takes a bacterial population to double), one can see a colonyliterally
a pile of bacteriaafter 16 to 24 hours. These bacteria are identical to the
original bacterium from which the colony was formed (i.e., they are clonal).
Phage plaques. If growing bacteria are inoculated into a solid (but soft)
medium (called top agar, or TA) and poured onto the top of an agar plate,
a cloudy suspension of bacteria will soon be visible throughout the medium.
If even one phage is present, it will infect one of the bacterial cells, replicate
within the cell, and lyse the bacterium (i.e., the bacterium will burst open).
This lysis will release many (up to 100) individual new phage. The number of
new phage released is called the burst size.
The new phage will travel via diffusion through the TA and infect other
bacteria. One infection-lysis cycle yields 100 100, or 10,000, new phage
particles. This cycle continues, and, because of the bacterial lysis, there is no
Part1 Capture 5

Part A. FYI #1: Introduction to Bacteriophages

cloudy suspension of bacteria radiating outward from the area that contained
the original phage. This is called a clearing zone, or plaque, indicating the
presence of an infectious phage particle (see Capture fig. 5, right plate). The
plaques contain billions of infectious phage particles, all identical to the
original phage (i.e., they are clonal).

Why Study Phages Now?


Genetics. Phages can be used as tools to move DNA around for cloning,
mutation, and other laboratory techniques. Genetic information about
different phages allows scientists to compare the phages (e.g., through
genome analysis), study biodiversity (e.g., through environmental patterns
of evolution), and identify new genes that may be useful for scientific or
therapeutic applications.

Food
for Thought
Name at least two challenges
that researchers might face
when trying to develop the use
of phages as treatments for
bacterial infections.

Epidemiology. Some phages can make their host bacterium more deadly to
humans. For example, a bacterium called Escherichia coli 0H157 causes foodborne illness that is sometimes fatal. The toxin that causes the symptoms was
delivered to the bacterium by a phage!
Therapeutics. Scientists would like to use phages to kill specific antibioticresistant bacteria that cause disease.

The Big Picture


You are now a part of the Science Education Alliance,
or SEA program. In this program, you will discover,
manipulate, and sequence a new phage. You will
label (or annotate) the entire genome and, in the
process, characterize a new life-form. This phage
will likely contain new genes and new gene arrangements. The data you and your classmates generate
will be used by other scientists to explore some of
the research avenues listed under Why Study
Phages Now? (above). Newly discovered phages
and new genes can lead to new strategies for molecular biology and comparative evolutionary biology
research, new therapeutics, and the discovery of
how different organisms and genes can cause
disease. You will be adding to the body of knowledge
in a discipline known as phage genomics.

Part1 Capture 6

Part B. Isolate a Novel Phage from the Environment

A note from the SEA Staff


In the first iteration of this resource guide, the direction was
given to use a direct plating method to isolate phage from the
environment. However, an enrichment protocol, developed by
a SEA associate institution, was available on the SEA wiki for
those inclined to use it. When all was said and done, it become
apparent that most of the phages that were characterized by the
inaugural Alliance members had been isolated using the enrichment protocol. The obvious question, then, is Which protocol
should be included in the revised resource guide? We took the
relatively easy approach and included both protocols. Thus,
the decision as to which to use is yours. To help you make an
informed decision, we have provided the pros associated with
each protocol, as identified by Alliance 2008, below. In addition
to tallying the pros for each method, you would also be wise to
take into consideration the nature of this course and the need to
balance gains in both science and education. You can, like many
Alliance members, decide to pursue both routes. You should be
aware, however, that the SEA staff has provided you with supplies and materials to pursue the enrichment route. Although
prudent pursuit of both lines of investigation will not significantly impact your supplies and materials, you should be aware
that any additional expenditure beyond what you have been
given is on your dime.Good luck!
Enrichment vs. Direct Plating
Direct Plating
Almost guaranteed success

Conceptually, easy for virtually all novices to understand from the outset

Greater chance of isolating relatively diverse phage populations

Allows capture of phage as it is (was) in the environment without the


in vitro evolution steps

Provides additional practice learning how to plate the cells

Relatively quick procedure to isolate a phage from the environment

Allows one to more accurately survey phage diversity in a given area

Allows one to more accurately count the number of phage in a given area

Forces students to think about bacterial ecology

Can screen many samples with fewer plates (just one per sample)

Facilitates discussion of the concept of selection versus enrichments

Enrichment

Part1 Capture F1

Part B. Isolate a Novel Phage from the Environment:


Enrichment of Environmental Samples

Overview
The enrichment culture technique creates conditions that
favor replication of specific bacterial phages. Since phages
are obligate intracellular parasites, large numbers of a
desired phage can be obtained by adding host bacteria and
bacterial media to an environmental sample. By seeding the
sample with host bacteria and using nutritional conditions
optimized for bacterial growth, the phages that are specific
to that bacterial species will infect the bacterial cells and
replicate to higher concentrations. These phages can then
be isolated at much higher frequency than with direct plating. This protocol is for the isolation of phages specific to
Mycobacterium smegmatis.
Key Words and Concepts
Discovery, enrichment, bacterial culture, replication, and
growth; aseptic technique, bacteriophage, infection.

Supplies
For each student (provided in kits)







A sample collection tube


A ruler
A laboratory notebook
A labeling pen
0.22-m sterilization filters (2)
1.0-mL tuberculin syringes (2)
A sample site marker
A 250-mL baffled Erlenmeyer flask
For each class

A GPS unit or Google EarthTM


Supplies and reagents for each
student (provided by SEA laboratories and prepared by instructors)
10 mL of phage buffer (PB) supplemented with 1 mM CaCl2
5 mL of an M. smegmatis culture
(48 hours)
0.5-mL aliquots of M. smegmatis culture
(7, in 10-mL glass culture tubes with lids
on a tube rack)
40 mL of top agar (TA) supplemented
with 1mM CaCl2
5 mL of 10X 7H9/glycerol broth
5 mL of AD supplement
0.5 mL of 100 mM CaCl2
Agar plates, 100-mm (7)
250-mL baffled Erlenmeyer flask
Sterile 15-mL conical tube with cap
Sterile 50-mL conical tube with cap
Sterile microcentrifuge tubes (7) and
tube rack
25-mL sterile disposable pipettes (2)
5-mL sterile disposable pipettes (14)
1-mL leur-lock syringe
Each student will also need
access to
A 1000-L and a 100-L or 200-L
micropipettor and tips
An automated pipettor for larger
volumes
A vortexer

Equipment
A 37C shaking incubator (or a shaker
in a large incubator)
A centrifuge with a rotor (or adaptors)
for spinning 50-mL conical tubes at
3000rpm
A microwave oven
A 37C incubator with room for 7
plates per student

Part1 Capture F2

Part B. Isolate a Novel Phage from the Environment

Implementation Timeline1

t+1D

LAB

t1D
Add CaCl2 to PB,
store at room
temperature.

t2D

t3D

 repare the
P
following:
P
 hage buffer (PB)
(Appendix 6).
Agar plates
(Appendix 6).

Prepare 5 mL
aliquots of saturated M. smegmatis cultures for
enrichment.
Prepare 1X
TA, add CaCl2,
dispense in 40-mL
aliquots, and
store at 55C.
Aliquot 10X 7H9/
glycerol, 5 mL per
student.
Aliquot AD
supplement, 5 mL
per student.

Aliquot 0.5 mL
of M. smegmatis into enough
10-mL culture
tubes for class (7/
student).
Prewarm agar
plates to RT (7/
student, >1hour
before class).
Dilute phage
stock for positive
control plate.

Aliquot 100 mM
CaCl2, 0.5 mL per
student.

Prep PB.
Prep 2X TA.

t4D
S
 aturated M.
smegmatis for
enrichment culture
and plaque screening (Appendix 7)
Part1 Capture F3

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part B. Isolate a Novel Phage from the Environment:


Enrichment of Environmental Samples

Overview
The enrichment culture technique creates conditions that favor replication of
specific bacterial phages. Since phages are obligate intracellular parasites,
large numbers of a desired phage can be obtained by adding host bacteria and bacterial media to an environmental sample. By seeding the sample
with host bacteria and using nutritional conditions optimized for bacterial
growth, the phages that are specific to that bacterial species will infect the
bacterial cells and replicate to higher concentrations. These phages can then
be isolated at much higher frequency than with direct plating. This protocol
is for the isolation of phages specific to Mycobacterium smegmatis.

Objective
To increase the likelihood of each student obtaining a novel phage from the
environment.

Supplies























Sterile 15-mL conical tube with cap


A ruler
A laboratory notebook
A labeling pen
0.22-m sterilization filters (2)
1.0-mL tuberculin syringes (2)
A sample site marker
A 250-mL baffled Erlenmeyer flask
A GPS unit or Google EarthTM
10 mL of phage buffer (PB) supplemented with 1 mM CaCl2
5 mL of an M. smegmatis culture (48 hours)
0.5-mL aliquots of M. smegmatis culture (7, in 10 mL glass culture tubes
with lids on a tube rack)
40 mL of top agar (TA) supplemented with 1 mM CaCl2
5 mL of 10X 7H9/glycerol broth
5 mL of AD supplement
0.5 mL of 100mM CaCl2
Agar plates, 100 mm (7)
Sterile 50-mL conical tube with cap
Microcentrifuge tubes (7) and tube rack
25-mL sterile disposable pipettes (2)
5-mL sterile disposable pipettes (14)
A 1000-L and a 100-L or 200-L micropipettor and tips
An automated pipettor for larger volumes
A vortexer

Equipment



A 37C shaking incubator (or a shaker in a large incubator)


A centrifuge with a rotor (or adaptors) for spinning 50-mL conical tubes
at 3000rpm
A microwave oven
37C incubator with room for 7 100-mm plates per student
Part1 Capture 7

Part B. Isolate a Novel Phage from the Environment: Enrichment of Environmental Samples

Procedure: Sample Collection and Preparation


A. Collect a soil sample
1. Label a 15-mL conical tube
2. Place soil into the tube
3. Mark your collection site and note site characteristics in your laboratory
notebook (see Capture, page B.6)
B. Back in the lab, prepare the enrichment culture.
1. Using a clean spatula, add approximately 1 gram (~1/2 teaspoon) of your
sample1 to a 250-mL baffled Erlenmeyer flask.
2. To this flask, aseptically add:
a. 40 mL of sterile H2O (using 2 x 25-mL pipettes).
b. 5 mL of sterile 10X 7H9/glycerol broth.
c. 5 mL of AD supplement.
d. 0.5 mL of 100 mM CaCl2.
3. Add 5 ml of late log/early stationary phase M. smegmatis culture (48
hour culture) to the flask.
C. Incubate the flask at 37C, shaking at 220rpm, for 24 hours.

Procedure: Harvesting and Preparing the Enriched Sample


A. Centrifuge the culture.
1. The next day, transfer the contents of the Erlenmeyer flask to a 50-mL
conical tube.
2. Balance the tube and spin at 3,000rpm (2,000xg) for 10 minutes to
pellet (i.e., force to the bottom of the tube) particulate matter, including
most of the bacterial cells.
B. Prepare a phage filtrate.
Assemble the following at your bench:




Microcentrifuge tubes (7)


A microcentrifuge tube rack
A permanent-ink marker
A 50 mL conical tube
A sterilization filter unit

1. Pour the supernatant (liquid) from the centrifuged sample into a fresh 50
mL conical tube.
2. Follow your instructors directions and filter-sterlize your enrichment
sample.
Part1 Capture 8

3. Immediately and aspetically cap the tube (be sure it is labeled properly).

Part B. Isolate a Novel Phage from the Environment: Enrichment of Environmental Samples

This is your 100 (undiluted) enrichment sample. This sample can be


stored at 40 C.
C. Dilute the enrichment sample 10010-4 by using serial 10-fold dilutions.
1. Arrange four microcentrifuge tubes in a tube rack and label them -1, -2,
-3, and -4.
2. Add 90 L of PB to each of these four tubes.
3. Label one microcentrifuge tube 0 and aseptically transfer 100 L of
your 100 (undiluted) enrichment sample into this tube.
4. Add 10 L of your 100 sample to the -1 tube and vortex well (see Capture fig. 6).

10 L

10 L

10 L

10 L

10 0

10-1

10-2

10-3

10-4

100 L

90 L

90 L

90 L

90 L

Capture fig. 6
The serial dilution of phage preparations for a titer. A volume of 10 L of
the undiluted phage sample (100) in
100 L of phage buffer is transferred into 90 L of PB in the 101
tube. After the tube is mixed, 10 L
of the 101 sample is transferred
into the 102 tube, and the tube is
mixed. This serial dilution continues until the desired dilutions
are obtained. The last tube will
contain a final volume of 100 L.

5. The -1 tube is your 10-1 (or 1:10) dilution.


6. Add 10 L of the -1 sample to the -2 tube and vortex well.
7. Continue each successive dilution until you get to the -4 tube.
D. For the positive control, obtain an aliquot of phage from your instructor.
E. For the negative control, filter-sterilize 1.0 mL of PB into an appropriately
labeled microcentrifuge tube.

Procedure: Plaque Screening

You may want to


label your diluted
enrichment samples
E0, E-1, E-2, etc.

A. Add 50 L of each sample (including controls) to 0.5 mL of M. smegmatis.


Assemble the following:




Culture tubes containing 0.5 mL of an M. smegmatis culture (7).


A 100-L or 200-L micropipettor.
Micropipettor tips.
Positive and negative control tubes.
Phage enrichment tubes (100-10-4).

For each sample, including the controls:


Part1 Capture 9

Part B. Isolate a Novel Phage from the Environment: Enrichment of Environmental Samples

1. Label the culture tubes containing 0.5 mL of M. smegmatis the same


way you labeled the microcentrifuge tubes.
2. Using a micropipettor:
a. Dispense 50 L of each sample into the appropriate culture tube.
b. For the negative control, add 50 L of filter-sterilized PB to the
phage-negative control tube.
c. For the positive control, add 50 L of the prepared phage solution to
the phage-positive control tube.
3. Mix each tube well by vortexing.
4. Allow the tubes to sit at room temperature for 15-30 minutes. This
allows the phage(s) to infect the bacteria.
B. Add 4.5 mL of TA and plate out each sample and each control.
1. Label each of 7 agar plates with sample or control information plus the
date and your initials.
2. Remove a bottle of TA from the 55C water bath.
3. For each sample, including the two control tubes:
a. Use a sterile 5-mL pipette to aseptically transfer 4.5 mL of TA to the
culture tube.
b. Immediately pull the mixture back up into the pipette.
c. Transfer the mixture to the appropriate plate and discard the pipette.
d. Swirl the plate gently to spread the TA evenly over the agar plate.
e. Let the plates sit undisturbed for at least 20 minutes until the TA has
completely solidified.
C. Incubate the plates at 37C.
1. After the TA has solidified, invert the plates rapidly but gently.
2. Put all inverted plates into the 37C incubator.
D. Check for plaques after 24 hours.

Notes
Google Earth can be downloaded at http://earth.google.com
If no plaques are apparent, check again at 48 hrs.
If you observe more than one plaque morphology on your enrichment plates,
be careful! Though you may well isolate more than one phage, the same
phage can produce different plaques depending upon the top agar, the density
of bacterial cells present, the age and thickness of the agar plate, etc. Careful
notes and tracking of samples is paramount.
Part1 Capture 10

Part B. Isolate a Novel Phage from the Environment: Enrichment of Environmental Samples

Procedural Note
1.

The students may want to enrich a liquid sample. They can add up to 40mL of their sample
to the flask. It is most important that they bring the final volume to 40mL with water before
the addition of the other materials.

Part1 Capture F4

Part B. Isolate a Novel Phage from the Environment:


Direct Plating of Environmental Samples

Overview
Students will go to different sites around their colleges and
universities, obtain soil samples, and record details about the
sites. The samples will be processed in the lab and used for
the infection of Mycobacterium smegmatis mc2155 for a plaque
assay and the eventual isolation of a new phage. Students
will use a screening process to discover a new phage that
they can name, characterize phenotypically and genotypically,
and make available to other scientists. Students will be able
to compare and contrast the locations and conditions of the
sites from which they obtained new phages and formulate a
hypothesis about the likelihood that different sites will yield
phages specific to M. smegmatis. They will also become familiar
with the basic skills required to perform a plaque assay.
Key Words and Concepts
Discovery, sample preparation, aseptic technique, the accurate
recording of sample-site characteristics and protocols in a
laboratory notebook, the accurate labeling of samples, plates,
and tubes.

Supplies
For each student (provided
in kits)
15-mL conical tubes (10)
A trowel, spatula, or spoon for
obtaining soil samples
A ruler
A laboratory notebook
A labeling pen (permanent-ink
marker)
0.22-m sterilization filters (10)
1.0-mL tuberculin syringes (10)
Microcentrifuge tubes (12) and a
tube rack
Site markers for sampling sites (10)
For each class
A GPS unit or Google EarthTM
Supplies and reagents for
each student (provided by SEA
laboratories and prepared by
instructors):
100 mL of phage buffer (supplemented with 1 mM (final) CaCl2)
0.5-mL aliquots of M. smegmatis
mc2155 culture (12, in 10-mL culture
tubes with lids on a tube rack)
60 mL of top agar (TA, supplemented with 1 mM (final) CaCl2)1
Agar plates, 100-mm (12)
10-mL sterile disposable pipettes
(at least 10)
5-mL sterile disposable pipettes
(at least 12)
An aliquot of a phage preparation
for use as a positive control
Each student will also need
access to
A 1000-L micropipettor and a
100- or 200-L micropipettor
and tips
An automated pipettor for larger
volumes (e.g., Pipette-Aid)
A vortexer

Equipment
37C incubator with room for
12 100-mm plates per student
A microwave oven
A shaking 37C incubator (or a
shaker in a large incubator)

Part1 Capture F5

Part B. Isolate a Novel Phage from the Environment: Direct Plating of Environmental Samples

Implementation Timeline

LAB
Melt 60-mL aliquots of TA.
Add CaCl2 to TA aliquots;
store in 55oC water bath.

t1D

t2D
Prep the following:
Phage buffer (PB)
(Appendix 6).

Add CaCl2 to PB
(Appendix 6); store
at room temperature
(RT).
Prepare top agar
(TA; 1X Middlebrook top
agar) and dispense in
60-mL aliquots
(Appendix 6).

Aliquot 0.5 mL of
saturated M. smegmatis
into enough 10-mL
culture tubes for class
(12 aliquots per student).
Prewarm agar plates
to RT (12/student;
> 1 h before class).
Dilute phage stock for
positive-control plate.

t4D
Saturated M. smegmatis
for plaque assays and
screening experiments
(Appendix 7).

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part1 Capture F6

Part B. Isolate a Novel Phage from the Environment:


Direct Plating of Environmental Samples
If you havent practiced aseptic technique yet, go back to the Before You
Start section.

Overview
You will go to different sites around your college or university, obtain soil
samples, and record details about the sites. The samples will be processed in
the lab and used for the infection of Mycobacterium smegmatis mc 2155 for a
plaque assay and the eventual isolation of a new phage. You will use a screening process to discover a new phage that you can name, characterize phenotypically and genotypically, and make available to other scientists. You will be
able to compare and contrast the locations and conditions of the sites from
which new phages have been obtained and formulate a hypothesis about the
likelihood that different sites will yield phages specific to M. smegmatis. You
will also become familiar with the basic skills required to perform a plaque
assay.

Objective
The objective of this procedure is for each student to discover a new phage.

Supplies



















15-mL conical tubes (10)


A trowel, spatula, or spoon for obtaining soil samples
A ruler
A laboratory notebook
A labeling pen (permanent-ink marker)
0.22-m sterilization filters (10)
1.0-mL tuberculin syringes (10)
Microcentrifuge tubes (12) and a tube rack
Site markers for sampling sites (10)
A GPS unit or Google EarthTM
100 mL of phage buffer (supplemented with 1 mM (final) CaCl2)
0.5-mL aliquots of M. smegmatis culture (12, in 10-mL culture tubes with
lids on a tube rack)
60 mL of top agar (TA, supplemented with 1 mM (final) CaCl2)
Agar plates, 100-mm (12)
10-mL sterile disposable pipettes (at least 10)
5-mL sterile disposable pipettes (at least 12)
An aliquot of a phage preparation for use as a positive control
A 1000-L micropipettor and a 100- or 200-L micropipettor and tips
An automated pipettor for larger volumes (e.g., Pipette-Aid)
A vortexer

Equipment


37C incubator with room for 12 100-mm plates per student


A microwave oven
A shaking 37C incubator (or a shaker in a large incubator)

Procedure: Sample Collection and Preparation2


A. Collect up to 10 soil samples in 15-mL screw-cap conical tubes from a
variety of locations around your campus, home, and/or city or town.3
Part1 Capture 11

Part B. Isolate a Novel Phage from the Environment: Direct Plating of Environmental Samples

1. Label each tube (with your name or initials, the date, and a designator).
2. Using a trowel, spatula, or spoon, transfer a sample to a clean tube. Soil
samples should fill the tube approximately one-third to one-half full.
3. For each sample, record the following in your laboratory notebook:

Date and time of sampling


Approximate air temperature, as indicated in a local weather report,
for example
Location (by GPS or Google EarthTM)4
Depth from which sample was obtained, to the nearest centimeter
Approximate moisture content (i.e., dry, moist, or saturated)
Defining features of the site, including

whether the area is urban or rural


proximity to buildings, foot traffic, major roads, cement, water,
and trees
if sloped, the sites compass direction (e.g., north-facing)
other: specify

B. Back in the lab, extract phage from the soil.


1. Using a 10-mL pipette and pipettor, add enough phage buffer (PB)3 to
flood each sample. The volume added should result (after settling) in a
liquid layer distinct from the solid layer.
2. Mix well using a vortexer.
3. Allow the sample to settle for at least 20 minutes.
C. Prepare a phage filtrate.
Assemble the following at your bench:




Microcentrifuge tubes (12)


A microcentrifuge tube rack
A permanent-ink marker
0.22-m filters (12)
1.0-mL syringes (12)

1. For each collected sample:


a. Use aseptic technique as you label the microcentrifuge tubes (10 sample
tubes plus 1 positive and 1 negative control5). Place each labeled tube
in the rack.
b. Open the packaging of the 0.22-m filter but do not remove the filter
from the packaging.

!
Part1 Capture 12

The bottom of the 0.22-m filter is a sterile field and should never be
touched. Use the plastic wrapper (not your bare fingers) to manipulate
the filter and attach it to the syringe.

Part B. Isolate a Novel Phage from the Environment: Direct Plating of Environmental Samples

c. Using the syringe, remove 1.0 mL of liquid from the top of the
sample tube.
d. Place the syringe into the top of the filter, and remove the filter
from the package.
e. Pushing the plunger, dispense the liquid through the filter into
the appropriately labeled sterile microcentrifuge tube. Immediately
cap the tube.

The filtrate and tube


are sterile and should
be manipulated using
aseptic technique.

f. Discard the syringe and filter.


2. For the positive control, obtain an aliquot (sample) of phage from
the instructor.
3. For the negative control, filter-sterilize 1.0 mL PB into an appropriately
labeled microcentrifuge tube.

Procedure: The First Round of InfectionPlaque Screening


A. Add 50 L of each sample (or control) to 0.5 mL M. smegmatis.
Assemble the following:




Culture tubes containing 0.5 mL of an M. smegmatis culture (12)


Micropipettor, 100-L or 200-L
Micropipettor tips
Positive control (i.e., phage-positive)
Phage buffer (PB)

For each sample, including the controls:


1. Label the 12 culture tubes containing 0.5 mL of an M. smegmatis culture
the same way you labeled the microcentrifuge tubes.
2. Using a micropipettor:
a. Dispense 50 L of each sample filtrate into the appropriate culture tube.
b. For the negative control, add 50 L of filter-sterilized PB to the phagenegative control tube.
c. For the positive control, add 50 L of the prepared phage solution to
the phage-positive control tube.
3. Mix each tube well by vortexing.
4. Allow the tubes to sit at room temperature for 15 to 30 minutes. This
allows the phage(s) to infect the bacteria.
B. Add 4.5 mL of top agar (TA, a solid but soft medium) and plate each
sample and the controls.
1. Label each of 12 agar plates with sample or control information plus the
date and your initials.

Agar plates should be


at room temperature.

2. Remove a 50-mL bottle of TA from the 55C water bath.


Part1 Capture 13

Part B. Isolate a Novel Phage from the Environment: Direct Plating of Environmental Samples

3. For each sample, including the two control tubes:


a. Use a sterile 5-mL pipette attached to a pipettor to aseptically transfer
4.5 mL of TA to the culture tube (which already contains the bacteria).
b. Immediately pull the mixture back up into the pipette. Try to avoid
bubbles; they can appear to be plaques on a plate.

The TA should not sit in the pipette more than a few moments, because
the agar will begin to solidify.

c. Transfer the entire mixture to the appropriately labeled plate, and


discard the pipette.
d. Swirl the plate gently to spread the TA evenly on top of the agar plate.
e. Let the plates sit, undisturbed, for at least 20 minutes.
C. Incubate plates at 37C.
1. After the TA has solidified, invert the plates rapidly (but gently). This
prevents condensation from dripping onto the TA.
2. Put all inverted plates in a 37C incubator.
D. Check plates for plaques (clear zones) after 24 hours.
Be sure to ascertain that

there are no plaques on the negative-control plate and


plaques are present on the positive-control plate.

If there are no plaques on your plates after 24 hours, do not discard!


Check them again at 48 hours.

Notes
If you see plaques at 24 or 48 hours, continue on to the Tame section.
Test anything that could possibly be a plaque by continuing with the plaque
assay or spot test (see Decision Tree #1).
Plaques may be difficult to see because they can be very small and the medium can be turbid, so look carefullyand remember, an air bubble in the agar
can appear to be a plaque.
If there are no possible plaques, dont be discouraged! Just go out and collect
new samples, and repeat the procedures until you discover your new phage.6
If you are not sure what a plaque might look like on the agar plate, consult
the Troubleshooting Guide (Appendix 1).
Part1 Capture 14

Decision Tree #1
From Soil Sample to Pure Phage

?
?

?
??
PROCESS SOIL SAMPLES

Are there plaques ?

NO

YES

SPOT ASSAY

Are there plaques ?


PHAGE TITRATION AND PURIFICATION

Do plaques all look the same ?

YES

REPEAT
at Least
times

YES

NO

NO

PURE PHAGE POPULATION

Part1 Capture 15

Part B. Isolate a Novel Phage from the Environment: Direct Plating of Environmental Samples

Questions
1. If students isolate and sequence the DNA of phages from different sites
nationwide, what kinds of research questions can be answered with the data
they collect, assuming they record all the requested sampling information?
2. Why are both controls needed?
3. Where are phages most likely to be found?
4. If there are thousands of phages in every square inch of soil, why are 10 sites
being sampled?
5. The filters used to process the supernatants are designated 0.22 m.
Whats the significance of 0.22 m relative to the things that might be
present in the supernatants?
6. Terry obtained the following results after 24 hours. Which plate in
Capture fig. 7 represents a negative result with respect to the presence
of phage? Explain.
Capture fig. 7
Two plates after 24 hours.

Part1 Capture 16

Part B. Isolate a Novel Phage from the Environment: Direct Plating of Environmental Samples

Procedural Notes
1.
Students will label aliquots of TA (stored in the 55C water bath) with their initials and the date they
opened their bottle. If less than 10 mL of TA remains in the bottle, the bottle should be rinsed out and
discarded (or washed, autoclaved, and reused). If, however, theres enough TA for additional procedures, the bottles can remain stored at 55C for 1 to 2 weeks. For example, if a student uses only 20
mL, the remaining TA in the bottle can be used the next time the student is in the lab. Do not store at
room temperature or 4C and remelt in the microwave, because the CaCl2 will precipitate. If a student
has a contaminated TA bottle, that will be apparent at the next meeting time. (For more about contamination, see Appendix 1. Troubleshooting Guide.) A contaminated bottle can be removed and used
as an example of a contaminated solution and, of course, discarded.
2.
Students should have obtained their samples by the second class meeting time. The first class can
be an introduction to the course and a review of aseptic technique and labeling methods. You should
also consider using part of the first class meeting time to discuss how samples are collected and to
demonstrate how the samples are processed (using, of course, aseptic technique). Have students
read Capture A. FYI #1 as homework for the second class meeting.
3.
Samples can be collected from virtually anywhere. Good starting places are sites rich in organic
material and decay. These include compost heaps, sewers, gardens, soil under bark, and mud or other
solids from stagnant ponds. Students who would like to collect something other than soil should be
encouraged to do so. For example, if they would like to obtain a water sample, they can draw it into
the sample tube and then add it directly to the filter when they return to the lab for filter sterilization. If
they want to try something else, the most important factor is that enough liquid is present (or phage
buffer added) to allow for the filter-sterilization step.
4.
If a GPS is not available, consult the SEA wiki for the procedure for determining location coordinates
of your site using Google Earth.
5.
The negative control is phage buffer (PB) only. Problems with contamination in either the bacterial
sample or the phage buffer will be immediately apparent, though not necessarily distinguishable,
using the negative control described. One additional control can be performed where nothing (no
phage buffer or sample) is added to the bacteria before plating in the top agar. Individual instructors
may want to set up one version of this control for the entire class.
6.
Some students will have difficulty obtaining plaques from their samples. This is more likely to be due
to bad luck than poor technique. Estimates are that only 1 in 20 to 1 in 30 random samples will actually
contain enough mycobacteriophage(s) to isolate by the direct plating method. Do not discard samples if
no plaques are apparent. Students who dont obtain phage after sampling the environment can use the
enrichment protocol.

Part1 Capture F7

Part C. FYI #2: Bacteriophage Lifestyles

Lytic vs. Temperate Phages


There are two categories of phages, defined by their life cycle: lytic and
temperate. Lytic phages have a straightforward life cycle, and they lyse all
the bacteria they infect. Temperate phages can use two different strategies
for replication and survival:
1. replicate and lyse the host bacteria they have infected, just like lytic
phages, or
2. enter a quiescent (dormant) state by incorporating their genetic
material, or DNA, into the DNA of the host bacterium.
The majority of bacteriophages are temperate.
Within a temperate phage population infecting a bacterial culture, it is likely
that some percentage of the phage will enter the lytic life cycle and some will
enter the quiescent state.

Plaque Morphology and Phage Lifestyle


Looking at Capture fig. 8, can you make a guess about which phage (left or
right plate) is likely to be lytic and which is likely to be temperate?
Capture fig. 8
Two phages, likely one temperate
and the other lytic.

You may notice a variety of plaque morphologies (appearances) in the plates


obtained from processing the soil samples. It is likely that clear plaques
(plaques in which very few or no bacteria remain intact) are the result of a
lytic phage. It is also likely that cloudy, or turbid, plaques are the result
infection with a temperate phage (some bacteria remain intact).
A lytic phage will likely produce clear plaques.
A temperate phage will likely produce turbid plaques.
A temperate phage can either enter the lytic cycle or incorporate its DNA into
the DNA of the host bacterium. When the latter happens,

To learn more
about phages
isolated by
other student
researchers,
see: http://
phagesdb.org/.

the phage genome incorporated into the bacterial DNA is called


a prophage,
the mechanism by which a nave bacterial cell becomes infected with a
prophage is called lysogeny, and
Part1 Capture 17

Part C. FYI #2: Bacteriophage Lifestyles

Capture fig. 9
Lytic and temperate phage life
cycle nomenclature.

the bacterium that contains the prophage is called a lysogen.


(See Capture fig. 9.)

LYTIC PHAGE

TEMPERATE PHAGE

receptor

receptor

bacterial DNA
phage
DNA
phage
replication

lysis

Bacterial cell

bacterial DNA
phage
DNA
lysogeny

prophage (phage DNA that is


incorporated into bacterial DNA)

Lysogen
(bacterial cell with prophage)

The Infection Process


Regardless of lifestyle, the first requirement for phage replication is infection
of a host bacterium. This happens in four discrete steps:
1. Adsorption of the phage to the surface of the bacterium. The tip of the
tail has specialized structures that adhere specifically to matching
receptors on the bacterial cell. This specificity defines the host range of
the phage. The phage tail will adhere to receptors on some bacteria but
not others. Interestingly, the receptors to which the phages adhere serve
a specific purpose for the bacterium, as structural proteins or pili, for
example. The phages have evolved to use the bacterial receptors to their
own advantage.
2. Irreversible attachment of the phage to the bacterial cell. Adsorption,
though very specific, is weak and reversible. The second phase of attachment is when the phage becomes tightly and irreversibly bound to the
bacterial cell.
3. Penetration of the bacterial cell wall. If a bacteriophage has a contractile
sheath (which resembles a vacuum cleaner hose), irreversible binding
of the phage to the bacterial cell leads to the contraction of the sheath.
The sheath contracts (the ribs on the hose move closer together),
and a hollow tail fiber pierces the bacterial cell envelope. Phages without
contractile sheaths have other mechanisms for penetrating the bacterial
cell wall. For example, some phages produce enzymes that eat away
at the wall.
Part1 Capture C.18

Part C. FYI #2: Bacteriophage Lifestyles

4. Nucleic acid injection from the phage head. When the phage has gotten
through the bacterial envelope, the DNA from the head passes through
the hollow tail and enters the bacterial cell. The capsid generally stays
attached to the outside of the bacterium as a ghost.
(Adapted from EP Mayer, Bacteriology: Bacteriophage [ch. 7], in Microbiology and
Immunology On-line, ed. RC Hunt [Columbia, SC: University of South Carolina School
of Medicine, 2007], http://pathmicro.med.sc.edu/mayer/phage.htm.)

The Lytic Cycle


The lytic cycle is very simple. It involves infection, viral assembly, and viral
release. See Capture fig. 4 for more details.
Injection of DNA. DNA is linear within the phage head, and it is injected as
a linear molecule. When the phage DNA gets inside the bacterium, it immediately circularizes.
Packaging of DNA. Newly synthesized DNA is loaded into the assembled
capsid of the phage in this way:
1. Head and tail components are synthesized and assembled.
2. The DNA is packaged (or loaded) into the capsid.
3. The tail and the capsid (containing the DNA) are attached to form
complete and infectious particles.
There are two mechanisms by which the DNA can be packaged
(see Capture fig. 10):

Cos-end packaging: Linear DNA packaged by this mechanism has


cos, or cohesive, ends. The DNA being packaged into the capsid is
cut at the cos site, loading specific and precisely cut DNA molecules
into the phage head. Later, when the new phage particle infects a
bacterium, the DNA is injected as before. The cos ends, which are
complementary, will stick together to form a circle of DNA.
Headfull packaging: Compared with cos-end packaging, this is much
less specific. Linear DNA, which is synthesized as a long chain of
repeat genomes, is packaged, or stuffed, into the phage head until
no more can fit.
Cos packaging

Headfull packaging

cos site

Capture fig. 10
Two kinds of packaging, cos and
headfull. During cos packaging, a
nuclease, or nucleic acid cutter
(represented by scissors in this
figure), recognizes the sequence at
the cos site and cuts the DNA into
discrete genomes. During headfull
packaging, a nuclease cuts the
DNA when the capsid can hold
no more DNA. Nuclease activity is
likely triggered by shear forces
acting on the DNA molecule.

Part1 Capture 19

Part C. FYI #2: Bacteriophage Lifestyles

The Temperate Phage Lifestyle


Temperate bacteriophages will not lyse their host bacterium until they switch
to the lytic life cycle.
Food
for Thought
What is the advantage (to the
phage) of integrating into the
host genome?

A genetic switch, called induction, can occur in a phage during lysogenic


growth that allows the phage to switch from lysogenic to lytic growth (see
Capture fig. 11).
BACTERIUM + PHAGE
(INFECTION)

Capture fig. 11
Temperate phage decisions.

LYSOGENIC GROWTH

?
induction

LYTIC GROWTH

Different genetic signals from the host bacterium will be produced depending
on many factors, including the health of the host bacterium, the availability
of nutrients, and the growth temperature. Certain signals will lead to phage
induction. The phage will then remove itself (or excise) from the host
genome and begin the lytic cycle.
The outcome of an infection of a bacterium by a temperate phage is entirely
dependent on genetic signals the phage receives from the bacterium:
1. the phage enters immediately into the lytic cycle or
Phages are not
lysogenic--they are
temperate!

2. the phage integrates (or inserts) into the bacterial genome:





The bacterium is now a lysogen.


The phage is now a prophage.
The whole process is called lysogeny.
The phage undergoes lysogenic growth.

A temperate phage does three key things during lysogeny:


1. Lets the host bacterium live. The temperate phage synthesizes a repressor that stops the expression of its lytic genes, thereby inhibiting lytic
growth (and thus the host is not lysed). Synthesis of this repressor protein also serves to stabilize the phage within the host bacterial genome.

Part1 Capture C.20

Part C. FYI #2: Bacteriophage Lifestyles

2. Ensures its own survival. The phage integrates its DNA into the host
genome, which keeps its own DNA safe. It does this via a mechanism
called site-specific recombination, in which the phage genomic DNA
inserts into a specific site in the bacterial genome. The site is selected
by the phage based on the DNA sequence that defines the site.
3. Ensures that its DNA gets passed along. Each time a bacterium replicates, the prophage is replicated. Each bacterial chromosome, therefore,
carries a copy of the prophage, so all bacterial progeny have a copy of
the prophage.
The viral DNA integrated in the host genome is called a prophage because
it is not truly a phage, but it has the capability of producing more phage.
Almost all bacteria that have been sequenced have at least one prophage in
their chromosome.
Induction occurs when genetic signals from the host bacterium decrease the
amount of phage repressor present. The prophage will excise during induction, circularize, and enter the lytic life cycle. When new phage particles are
assembled and the host bacterium is lysed, the prophage no longer exists.
To recap, see Capture fig. 12.

bacterial cell
receptor

Capture fig. 12
Prophage and lytic pathways.

phage
attachment to host cell
and injection of phage DNA
phage DNA circularizes

bacterial DNA

integration of phage DNA


into bacterial DNA

synthesis of viral proteins


needed for formation of
new viruses

cell division

rapid replication of phage


DNA and its packaging into
complete viruses

induction event

cell lysis releases a large


number of new viruses

integrated phage DNA replicates along with bacterial DNA


PROPHAGE PATHWAY

LYTIC PATHWAY
Part1 Capture 21

Part C. FYI #2: Bacteriophage Lifestyles

The Big Picture


Making good decisions about your approach to
studying the phage you isolate depends on being
familiar with the basics of phage biology. The
investigative process that you are beginning is more
than an exercise in laboratory steps and procedures.
Though these steps are important, you are embarking
on a collaboration with your instructor, your fellow
students, and SEA participants across the country
to further phage biology and genomics. When you
consider the basic science behind your experiments,
you will find that you are more productive in the
laboratory and that you are in a position to contribute
more to the process and to your team of scientists.
You should, therefore, ask yourself why? as often
as you ask what next?
If you have other questions or want to learn more
about phage biology, see Appendix 2 in this manual
for websites, references, and other materials.

Part1 Capture C.22

TAB
GOES
HERE

YouAre
AreHere
Here
You
COLLECT ENVIRONMENTAL SAMPLE

ISOLATE PHAGE FROM


ENVIRONMENTAL SAMPLE

TAME
PURIFY THE PHAGE

CONFIRM THE PLAQUE VIA


SPOT TEST

PHAGE-TITER ASSAY OR
PLAQUE STREAK PROTOCOL

PURIFY THE PHAGE

CONFIRM THE PLAQUE VIA


SPOT TEST

Repeat 3x

PHAGE-TITER ASSAY OR
PLAQUE STREAK PROTOCOL

PICK SINGLE PLAQUE


CHARACTERIZE GROWTH RATE,
PLAQUE MORPHOLOGY, TITER

Repeat 3x

CHARACTERIZE THE PHAGE

Rev. May 2011

ii

Tame

Contents
Part A. Purify the Phage

Objective

Supplies (for students)

F1 Overview

Equipment (for students)

Procedure

The Spot Test


Key Words and Concepts

Supplies and Equipment

13 Notes

F2 Implementation Timeline

14 The Big Picture

Questions

1 Overview

Objective

Supplies (for students)

Equipment (for students)

Procedure: Using a Pipette Tip to Pick


a Single Putative Plaque (or Plaques) into

100 L of Phage Buffer (PB)

2 Procedure: Preparing a Bacterial


Lawn in Top Agar

3 Procedure: Spotting Putative Plaques


and Buffer Control onto a Labeled Plate

Procedure: Checking Spot Plate for Plaques


4 Questions

F3 Procedural Notes

Plaque Streak Protocol

F4 Overview

Key Words and Concepts

Supplies and Equipment


F5 Implementation Timeline

5 Objective

Supplies (for students)

Equipment (for students)

Procedure

8 Notes

F9 Procedural Notes

Final Plaque Purification

F10 Overview
Key Words and Concepts
Supplies and Equipment
F11 Implementation Timeline
15 Overview

Objective

Supplies (for students)

Equipment (for students)


16 Procedure: Harvesting a Plate Lysate

Procedure: Preparing a Bacterial Lawn in TA

17 Procedure: Performing a Spot Test on the



Phage Lysate
18 Notes
19 Decision Tree #2
20 Questions
F12 Procedural Notes
Part B. Make Phage Stocks

Empirical Testing of Phage Lysates

F13 Overview
Key Words and Concepts

F6 Procedural Notes

Supplies and Equipment

F14 Implementation Timeline

The Phage-Titer Assay

F7 Overview
Key Words and Concepts
Supplies and Equipment

21 Overview

Objective

Supplies (for students)

F8 Implementation Timeline

9 Overview

continued
iii

Tame

Contents (continued)

Equipment (for students)

Procedure: Preparing Your Dilutions


22 Procedure: Performing the Empirical Assay
24 Procedure: Analyzing the Data
Notes
Questions
F15 Procedural Notes

The 10-Plate Phage Infection and Harvest

F16 Overview
Key Words and Concepts
Supplies and Equipment
F17 Implementation Timeline
25 Overview

Objective

Supplies (for students)

26 Equipment (for students)


Procedure: Setting Up Your Work Area
Procedure: Performing the Large-Plate Infection
27 Procedure: The Next Day, Harvesting your Phage
29 Procedure: Titering Your Phage
Notes
Questions
F18 Procedural Notes

iv

Part A. Purify the Phage: The Spot Test

Overview
This is a relatively quick assay used to determine whether
spots on a bacterial lawn are actually plaques. Because
plaques can vary in size, morphology, and effects on the host
bacterial cell (see pictures in Appendix I. Troubleshooting
Guide), it can be easy to mistake an air bubble or other anomaly in the top agar for a plaque. If the plaque or plaques are
suspect, a spot assay can be performed to determine whether
or not phages are present. If you are reasonably certain that
plaques are present on a sample plate, the plaque streak protocol or phage-titer assay should be done (see Decision Tree
#1) instead of the spot test. Students will spot putative phages onto a bacterial lawn in top agar. If the original sample is a
true plaque, students will see plaques or clearing within the
spot area(s) and can then move on to the phage-titer assay.
During this procedure, students will become familiar with the
basic skills they need to determine whether they have obtained an actual plaque containing a phage or phages.
Key Words and Concepts
Plaque, spot test, aseptic technique, sterile field.

Supplies
For each student (provided in
kits):
A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (2 to
10) and a tube rack
A 5-mL sterile disposable pipette
and pipettor
Supplies and reagents for
each student (provided by SEA
laboratories and prepared by
instructors):
50 mL of phage buffer (PB)
0
 .5 mL of M. smegmatis culture (in
a 10-mL sterile culture tube)
50 mL of top agar (TA) melted and
stored at 55C
An agar plate
Each student will also need
access to
A 100- or 200-L and a 20-L micropipettor and tips
An automated pipettor for larger
volumes
A vortexer

Equipment
37C incubator
A microwave oven
55C water bath

Part1 Tame F1

Part A. Purify the Phage: The Spot Test

Implementation Timeline1

LAB
Aliquot

0.5 mL of
saturated M. smegmatis
into enough 10-mL
culture tubes for the
class.

t1D
Prep

1X TA; store
at 55C.

Prewarm agar plates


to RT.

t2D
Prep PB.

t3D
Prep 2X TA.

t4D
Prep saturated
M. smegmatis culture
for plaque assays.

Part1 Tame F2

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part A. Purify the Phage: The Spot Test

Overview
This is a relatively quick assay used to determine whether spots on a bacterial
lawn are actually plaques. Because plaques can vary in size, morphology, and
effects on the host bacterial cell (see pictures in Appendix I. Troubleshooting Guide), it can be easy to mistake an air bubble or other anomaly in the
top agar for a plaque. If the plaque or plaques are suspect, a spot assay can
be performed to determine whether or not phages are present. If the original sample is a true plaque, you will see plaques or clearing within the spot
area(s).

Objective
The objective of this procedure is to determine whether putative plaques contain phage(s).

Supplies










A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (2 to 10) and a tube rack
A 5-mL sterile disposable pipette and pipettor
50 mL of phage buffer (PB)
0.5 mL of M. smegmatis culture (in a 10-mL sterile culture tube)
50 mL of top agar (TA) melted and stored at 55C
An agar plate
A 100- or 200-L and a 20-L micropipettor and tips
An automated pipettor for larger volumes
A vortexer

Equipment


37C incubator
A microwave oven
55C water bath

Procedure: Using a Pipette Tip to Pick a Single Putative


Plaque (or Plaques) into 100 L of Phage Buffer (PB)

Read all the way


through each
procedure carefully
before setting up
your bench.

A. Prepare your work area.





Remove clutter.
Wipe the bench with disinfectant (allow to air-dry).
Start your flame.
Arrange your supplies.

B. Label possible plaques; label and prepare tubes.


1. Aseptically aliquot 100 L of PB into microcentrifuge tubes (one tube for
each potential phage).
2. Label plaques on agar plate(s) by drawing a small circle (on the bottom of
the Petri dish!) around the area with a labeling pen. Designate all plaques
(numbers, letters, or other) with your labeling pen.

All tubes should


remain closed except
when transfers are
being made!

Part1 Tame 1

Part A. Purify the Phage: The Spot Test

It is possible that you will obtain more than one plate with putative
plaques. If this happens, be sure to label all your putative plaques with
a plate designation that makes it easy for you to determine from which
sample each plaque was obtained.

3. Label each microcentrifuge tube with the same designation(s) as that of


the plaque(s).
C. List any putative plaques in your laboratory notebook and note the
characteristics (morphology, size, cloudy versus clear, etc.) of each.
D. Pick putative plaques.
1. Place a sterile 100-L tip onto a 100- or 200-L micropipettor.
2. Open the lid of the Petri dish and keep it in your hand (do not invert
remember the sterile field!).
3. Touch the center of the putative plaque with the end of the tip.
4. Return the cover to the Petri dish.
These samples should
be stored at 4C
(refrigerated) until
the presence of
phage is verified.

5. Place the end of the tip into the PB in the corresponding microcentrifuge
tube and gently tap the tip on the wall of the tube. Discard the tip.
6. Mix the tube well by vortexing.
7. Repeat steps 1 through 6 for each plaque.

Procedure: Preparing a Bacterial Lawn in Top Agar


A. On your bench, you should have



A 0.5-mL aliquot of M. smegmatis in a 10-mL culture tube


An agar plate
Top agar (TA) from the 55C water bath2
A 5-mL sterile pipette and pipettor

B. Draw a grid onto the bottom of the agar plate (see Tame fig. 1).

Tame fig. 1
A grid drawn on the bottom of
a Petri dish.

C. Label the grid with each phage designation, and save a section for the
negative (buffer-only) control.
Part1 Tame 2

Part A. Purify the Phage: The Spot Test

D. Prepare a TA-bacteria plate.


1. Using aseptic technique, aspirate (suck up) 4.5 mL of TA into a 5-mL
sterile pipette.
2. Transfer the TA to 0.5 mL of bacteria and immediately draw the TA back
into the same pipette.
3. Add the TA-bacteria mixture to the labeled agar plate.
4. Swirl gently to spread the mixture.
5. Allow to solidify completely. This will take at least 10 minutes.

Procedure: Spotting Putative Plaques and Buffer Control


onto a Labeled Plate
A. On your bench, you should have




The labeled TA-bacteria agar plate


All possible plaque samples in 100 L of PB in labeled microcentrifuge
tubes
A tube with 100 L of PB only (the negative control)
A 20-L micropipettor
A box of sterile 20-L micropipettor tips

B. Spot control and samples onto plate.


1. Set the micropipettor to 5 L.
2. Aseptically transfer 5 L of PB to the negative-control block on the grid.
3. Transfer 5 L of all putative phage samples onto the corresponding
blocks on the grid. Allow the droplets to soak into the agar for a few
seconds, until no apparent liquid remains on the agar.

Do not touch the pipette tip to the agar! Hold the tip slightly above the
agar, and push the droplet out slowly to avoid splattering.
Avoid making bubbles because they can burst and scatter any phages
that may be present across the plate.
Labels on the bottom of the Petri dish are mirror images (i.e., they will
appear backwards) of your labeling scheme once you turn the plate over.
Be sure to spot your sample in the right place!

4. Invert the plates and place into the 37C incubator overnight.

Procedure: Checking Spot Plate for Plaques


The next day, check spot plate for plaques. If you see clearing zones, or spots,
in any of your grid areas, you have picked a genuine plaque!3

The TA must be
completely solidified
before any sample is
spotted onto
the plate!

Do not discard the


microcentrifuge
tubes containing
your samples. These
should be stored at
4C (refrigerated)
after the spot test
until the presence
of phage is verified.

Before deciding whether you have any plaques, you must make certain
your negative control has neither clear zones nor spots!
Part1 Tame 3

Part A. Purify the Phage: The Spot Test

A. By referring to the grid, identify the origin of the phage sample.
Use the refrigerated sample for the phage-titer assay (see page Tame A.9).

Food
for Thought
Is there a correlation between
negative or positive spot
test results and the original
notes you made on plaque
morphology, size, etc.?

B. In your laboratory notebook, for each putative plaque tested, be sure to
record the following:


whether the spot test was positive or negative


plaque size and morphology on the spot plate
the number of plaques in the spot

Questions
1 What are the advantages and disadvantages of performing the spot test
rather than the phage-titer assay (next section)?

Tame fig. 2
Results of Lees spot test.

2. Lees spot test produced the results shown in Tame fig. 2. What conclusions
can be drawn from these results?

P1

CONTROL

P2
P5
P4

P3

3. What are some possible explanations of different plaque morphologies


from the same original sample? In other words, if you spotted a putative
sample that originally appeared to be a clear plaque and, on the spot plate,
observe cloudy plaques or a variety of plaque sizes, what could be the
reason(s) for this?

Part1 Tame 4

Part A. Purify the Phage: The Spot Test

Procedural Notes
1.
You may want to obtain a count of the number of plates, phage buffer aliquots, and bacterial aliquots
needed for each class by surveying the students before they leave for the day. This will give you an
estimate (most likely an underestimate) of what to prepare for the next meeting period and will, more
importantly, require the students to think ahead to the next steps. Questions to ask include, how many
environmental samples do you plan to obtain, and how many of you will be performing the phage-titer
assay? You should anticipate that approximately 20% of the students will need to perform a spot test
rather than the more involved phage-titer assay early in the course.
2.
Students should label aliquots of TA in the 55C water bath with their initials and the date they opened
their bottle. If there is less than 10 mL of TA remaining in the bottle, the bottle should be rinsed out
and discarded (or washed, autoclaved, and reused). If, however, there is enough TA for additional
procedures, the bottles can remain stored at 55C for 1 to 2 weeks. Do not store at room temperature
or 4C and remelt in the microwave, because the CaCl2 will precipitate. If a student has a contaminated TA bottle, it will be apparent at the next meeting time. A contaminated bottle can be removed and
used as an example of a contaminated solution and, of course, discarded.
3.
Students should be encouraged to keep bringing environmental samples to class until they begin the
process of purifying a specific phage. This is true even when students are absolutely sure that they
have isolated a phage and progress into the spot test or their first titration assay! Incidents of certainty
often turn out to be contamination, air bubbles, or some other anomaly.

Part1 Tame F3

Part A. Purify the Phage: Plaque Streak Protocol

Overview
The plaque-streak assay is used to purify populations of
phages from samples with putatively heterogeneous populations. This technique is very similar to streaking for single
bacterial colonies. Students perform this assay after they are
relatively sure that they have actual plaques from either direct plating or the enrichment procedure. If they are unsure,
they should perform the spot test outlined in the preceding
section.
Briefly, the students will streak plates in duplicate for each
phage, overlay each streaked plate with top agar (TA) containing the host bacteria, and incubate at 37C overnight.
After 24 hours of incubation (48 hours, if needed), students
will check the plates for single isolated plaques and note
whether plaque morphology is consistent. Students will
then pick isolated plaques and repeat the procedure. The
plaque-streak method should be repeated at least twice more
to ensure that only one phage has been isolated (see Decision
Tree #1). Each time the method is repeated, students should
note the plaque morphology(ies) and attempt to pick a single
plaque for streaking. This procedure will, therefore, be performed by each student at least three times.

Supplies
For each student (provided
in kits):



A laboratory notebook
A labeling pen
Sterile wooden sticks
5-mL sterile disposable pipettes (3
per phage tested)

Supplies and reagents for


each student (provided by SEA
laboratories and prepared by
instructors):
0.5 mL of M. smegmatis culture in
a 10-mL culture tube (3 per phage
tested)
15 mL of top agar (TA) melted and
stored at 55C
Agar plates (3 per phage tested)
Each student will also need
access to
An automated pipettor for larger
volumes

Equipment
37C incubator
A microwave oven
55C water bath

During this exercise, students will become familiar with the


basic skills of streaking plates as a method for purifying microorganisms.
Key Words and Concepts
Plaque streaking, plaque morphology.

Part1 Tame F4

Part A Purify the Phage: Plaque Streak Protocol

Implementation Timeline

LAB

t1D

Aliquot

0.5 mL of
saturated M. smegmatis
into enough 10-mL
culture tubes for class.
Prewarm

agar plates
to RT .

P
 rep 1X TA; store
at 55oC

t3D
Prep 2X TA

t4D
P
 rep saturated M. smegmatis culture for plaque
assays.

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part1 Tame F5

Part A. Purify the Phage: Plaque Streak Protocol

Objective
The objective of this procedure is to purify a single phage population from
samples with potentially mixed phage populations.1

Supplies



A laboratory notebook
A labeling pen
5-mL sterile disposable pipettes (3 per phage tested)
0.5 mL of M. smegmatis culture in a 10-mL culture tube
(3 per phage tested)
Top agar (TA) melted and stored at 55C (15 mL per phage tested)
Agar plates (3 per phage tested)
Sterile wooden sticks2
An automated pipettor for larger volumes

Equipment


37C incubator
A microwave oven
55C water bath

Procedure
A. Prepare your work area.



Remove clutter.
Wipe the bench with disinfectant (allow to air-dry).
Start your flame.
Arrange your supplies.

B. Label plaques and prepare plates.


1. Label the plaque you intend to pick from a plate containing plaques (or
putative plaques) by drawing a small circle around the area with a labeling pen. Designate all picked plaques with your labeling pen.
2. Label each plate with the plaque designation, including a plate for your
negative control.
C. For each plaque being purified, note the morphology and size in your
laboratory notebook.
D. Prepare your negative control plate.3
1. Using aseptic technique, remove a sterile wooden stick from its packaging.
2. Open the Petri dish labeled Negative Control.4
3. Gently streak back and forth across the top third of the agar plate (see
Tame fig. 3), without lifting the stick from the agar.
Part1 Tame 5

Part A. Purify the Phage: Plaque Streak Protocol

4. Return the cover to the Petri dish and discard the wooden stick.
5. Carefully remove a new wooden stick.
6. Open the lid of the Petri dish. Beginning in the area streaked in the
previous step, streak the adjacent un-streaked portion (~1/3) of the agar
making sure to overlap only on the first few strokes.
7. Repeat steps 4-6 for the remaining un-streaked portion of the plate.
Tame fig. 3
Streaking for isolation of a
phage from a mixed population.
The center of a well-isolated
plaque is touched with a sterile
wooden stick. On the first
streak, this stick is used to streak
an area of roughly one-third on
a fresh agar plate. A new stick is
then used to streak an adjacent
un-streaked area (1/3) of the
plate on the second streak, making sure to overlap the original
streaked area with only the first
few strokes. Again, a new stick
is used for the third streak to
make a few overlapping strokes
of the second streak. The area
where the third streak was made
is the most dilute and is designated by the red X.

It is a good idea to
streak the phage
plaques in duplicate
until you have
mastered the technique. This will help
to ensure that you
get an isolated single
plaque every time.

E. Pick plaques and streak on a fresh agar plate.


1. Using aseptic technique, remove a sterile wooden stick from its
packaging.
2. Open the Petri dish with the (putative) plaque and keep it in your hand
(do not invert-remember the sterile field!).
3. Touch the center of a plaque with the end of the stick.
4. Return the cover to the Petri dish.
5. Open the lid of the Petri dish (to be streaked) with the corresponding
label.
6. Perform steps 3-7 in section D above.
7. Indicate the most dilute area of the plate with a marker.

Part1 Tame 6

Part A. Purify the Phage: Plaque Streak Protocol

F. Add top agar and bacteria.


1. Add 4.5 mL of TA to 0.5mL aliquot of M. smegmatis.
2. Immediately, bring the mixture back up into the serological pipet.
3. Carefully, dispense the TA/bacteria mixture slowly onto the most dilute
(marked) area of the streaked plate (see Tame fig. 4).

Do not swirl the plate.

4. Allow the mixture to spread across the plate from the most dilute point
to the more concentrated areas by gently tilting or tapping the plate.
5. Allow the TA to harden.
Tame fig. 4
Plating bacterial cells and top
agar on a plate streaked with
phage. The mixture of cells and
top agar is added to the plate
at the most dilute point and
allowed to spread to the more
concentrated regions of the
plate without swirling.

Part1 Tame 7

Part A. Purify the Phage: Plaque Streak Protocol

G. Incubate plates at 37C.


1. After the TA has solidified, invert the plates rapidly (but gently).
2. Put all inverted plates in a 37C incubator.

H. Check plates for plaques.


1. Check plates for plaques after 24 hours (see Tame fig. 5).
Food
for Thought
Why is three the minimum
number of times you would
streak out individual plaques
for isolation? Why would
you do additional rounds of
purification?

2. Be sure to ascertain that there are no plaques on the negative-control


plate.
3. If you do not have plaques, allow the plate(s) to incubate an additional
24-48 hours.
4. Repeat this procedure at least two times (more if necessary)until you are
relatively certain you have a pure phage population.

Tame fig. 5
Results of a plate streaked with
phage and topped with bacteria/
top agar after incubation. The
most concentrated area of the
plate can be completely cleared
by the phage while the more
dilute parts of the plate yield
individual plaques. The X
indicates where the TA/bacteria
was added to the plate.

Notes
Some samples may include multiple phages that are difficult to isolate and
purify. It is extremely important that the final plaque purification contain
only a single phage. Continue to pick well isolated plaques as you purify your
phage. For phages that are particularly difficult to isolate, The Phage-Titer
Assay (Tame page 9) can also be used (See Procedural Note #1 in this section).

Part1 Tame 8

Part A. Purify the Phage: Plaque Streak Protocol

Procedural Notes
1.
This protocol can be used for several iterations of phage purification before doing serial dilutions
to titer and further purify the phage, and for the generation of the MTL (medium titer lysate). Serial
dilutions can also be used sequentially to obtain a pure phage isolation, but the streaking method
outlined here saves time and supplies.
2.
Sterile wooden sticks are used in this protocol due to their size and ease of manipulation. Sterile
loops or toothpicks can also be used to streak out plaques. It should be noted, however, that any
implement that requires the user to reach directly over the plate (e.g. a sterile toothpick) can increase
the possibility of contamination.
3.
You may want to have the students prepare a positive control plate, especially if they are not sure
they have a true plaque. For this, supply the students with known phage plaques (D29 or one of your
choosing) and have the students perform the steps outlined in part E.
4.
If students have more than one phage to purify, there is no need for additional negative or positive controls as long as the same solutions are being used throughout (i.e., only one batch of TA per
student). Also, once students are familiar with the appearance of plaques from their initial positive
controls (or results), there is no need to continue using a positive control with each iteration of purification.

Part1 Tame F6

Part A. Purify the Phage: The Phage-Titer Assay

Overview
This assay is the basis for determining the concentration of
phage particles in a given solution and conducting successive rounds of purification of an individual phage. Students
perform it once they are certain they are observing an actual
plaque on the bacterial lawn. After obtaining a pure phage
population using the phage streak protocol, students will
perform serial 10-fold dilutions of each phage, incubate each
concentration with bacteria, and plate out the infected bacterial cells in top agar. They will check the plates at 24 hours
(and 48 hours, if needed) and count the number of plaques
on each plate. They will then calculate the number of plaqueforming units per milliliter of original phage sample. This is
called the titer. Once a titer has been established, students
will use the plate exhibiting a web pattern to make a
Medium Titer Lysate (or MTL). Note that students may need
to repeat this protocol one or two times to obtain an accurate
titer.
During this exercise, students will become familiar with the
basic skills of counting phage and calculating phage concentration, and with the prerequisites for isolating large numbers of phage.
Key Words and Concepts
Plaque titer, serial dilutions, plaque morphology, plaqueforming unit (pfu), pfu/mL.

Supplies
For each student (provided
in kits):
A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (5 per
phage tested) and tube rack
5-mL sterile disposable pipettes (5
per phage tested)
Supplies and reagents for
each student (provided by SEA
laboratories and prepared by
instructors):
50 mL of phage buffer (PB)
0
 .5 mL of M. smegmatis culture in
a 10-mL culture tube (5 per phage
tested)
50 mL of top agar (TA) melted and
stored at 55C
Agar plates (5 per phage tested)
Each student will also need
access to
A 100- or 200-L and a 20-L micropipettor and tips
An automated pipettor for larger
volumes
A vortexer

Equipment
37C incubator
A microwave oven
55C water bath

Part1 Tame F7

Part A. Purify the Phage: The Phage-Titer Assay

Implementation Timeline

LAB
Aliquot

0.5 mL of
saturated M. smegmatis
into enough 10-mL
culture tubes for the
class.

t1D
Prep 1X TA; store

Prewarm agar plates


to RT.

at 55C.

t2D
Prep PB.

t3D
Prep 2X TA.

t4D
Prep saturated
M. smegmatis culture
for plaque assays.

Part1 Tame F8

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part A. Purify the Phage: The Phage-Titer Assay

Overview
This assay is the basis for determining the concentration of phage particles
in a given solution and conducting successive rounds of purification of an
individual phage. This assay is performed once you are certain you are observing an actual plaque on the bacterial lawn. After a series of dilutions and
platings, you will count the number of plaques on each plate and calculate
the number of plaque-forming units per milliliter of original phage sample.
This is called the titer. Once a titer has been established, and you are sure
that you have a pure phage population, you can move on to Final Plaque
Purification (Tame, page 15).

Objective
The objective of this procedure is to determine the concentration of plaqueforming units (pfu) in a given solution and to become comfortable with titer
calculations.

Supplies





A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (5 per phage tested) and tube rack
5-mL sterile disposable pipettes (5 per phage tested)
50 mL of phage buffer (PB)
0.5 mL of M. smegmatis culture in a 10-mL culture tube
(5 per phage tested)
50 mL of top agar (TA) melted and stored at 55C
Agar plates (5 per phage tested)
A 100- or 200-L and a 20-L micropipettor and tips
An automated pipettor for larger volumes
A vortexer

Equipment


37C incubator
A microwave oven
55C water bath

Procedure
A. Prepare your work area.



Remove clutter.
Wipe the bench with disinfectant (allow to air-dry).
Start your flame.
Arrange your supplies.

B. Label plaques; label and prepare tubes.


1. Aseptically aliquot 100 L of phage buffer (PB) into microcentrifuge
tubes (one per plaque to be tested).
Part1 Tame 9

Part A. Purify the Phage: The Phage-Titer Assay

2. Label plaques on agar plate(s) by drawing a small circle (on the bottom of
the Petri dish!) around the area with a labeling pen. Designate all plaques
(numbers, letters, or other) with your labeling pen.
3. Label each microcentrifuge tube with the plaques designation.
C. For any plaques, note the morphology, size, and whether clear or
cloudy in your laboratory notebook.
D. Pick plaques.
1. Place a sterile tip onto a micropipettor.
2. Open the lid of the Petri dish and keep it in your hand (do not invert
remember the sterile field!).
3. Touch the center of the putative plaque with the end of the tip.
4. Return the cover to the Petri dish.
5. Place the end of the tip into the PB in the corresponding microcentrifuge
tube and gently tap the tip on the wall of the tube.
6. Discard the tip.
7. Mix the tube well by vortexing. This is your neat, or undiluted, phage
sample, also referred to as the 10 0 plaque sample.

8. Add 0 or 100 to the label of this tube.


9. Repeat steps 1 through 8 for each plaque.
Avoid transfer
errors. Use two racks
and move tubes to a
different rack after
you add the sample.

E. Perform serial 10-fold dilutions.


For each putative phage, using aseptic technique in every step:
1. Arrange four microcentrifuge tubes in a tube rack and label
them 1, 2, 3, and 4.
2. Add 90 L of PB to each tube.
3. Add 10 L of your 100 (undiluted) phage sample to the 1 tube and vortex well. (See Tame fig. 6.)

To prevent the sample from spraying out of the tube, gently tap the
capped tube on the counter. This forces the fluid to the bottom of the
tube, rather than onto the sides or top.

4. The 1 tube is your 101 (or 1:10) dilution.


5. Add 10 L of the 1 tube to the 2 tube and vortex well.
6. Continue each successive dilution until you get to the 4 tube.

Part1 Tame 10

Part A. Purify the Phage: The Phage-Titer Assay

10 L

10 L

10 L

10 L

10 0

10-1

10-2

10-3

10-4

100 L

90 L

90 L

90 L

90 L

Tame fig. 6
The serial dilution of phage preparations for a titer. A volume of 10 L of
the undiluted phage sample (100) in
100 L of phage buffer is transferred into 90 L of PB in the 101
tube. After the tube is mixed, 10 L
of the 101 sample is transferred
into the 102 tube, and the tube is
mixed. This serial dilution continues until the desired dilutions
are obtained. The last tube will
contain a final volume of 100 L.

F. Infect M. smegmatis cultures with diluted phage solutions.


For each putative phage, using aseptic technique in every step:
1. Label culture tubes (containing 0.5 mL of M. smegmatis) with the
phage designation (e.g., A1) and each dilution (101). For a set of
serial dilutions from 101 to 104 plus a negative control, you will
need five tubes.
2. Infect 0.5 mL of M. smegmatis in the appropriately labeled
culture tubes with 10 L of the 101, 102, 103, and 104 dilutions.
3. Add 10 L of PB to the negative-control tube.

Food
for Thought
Why is the undiluted sample
designated 10 0?

4. Allow the phage to infect the bacteria for 15 to 30 minutes.


Be sure to record the length of time allowed for infection in your
laboratory notebook.
G. A
 dd 4.5 mL of top agar (TA) and plate each sample (including the
negative control).1
1. Label each of the five agar plates (at room temperature) with sample
or control information plus the date and your initials.
2. Remove a 50-mL bottle of top agar (TA) from the 55C water bath.
3. For each sample, including the negative-control tube:
a. Use a sterile 5-mL pipette attached to a pipettor to aseptically
transfer 4.5 mL of TA to the culture tube (containing the bacteria).
b. Immediately pull the mixture back up into the pipette. Try to
avoid bubbles.
c. The TA should not sit in the pipette for more than a few moments,
because the agar will begin to solidify.
d. Transfer the entire mixture to the appropriately labeled plate and discard the pipette.

The neat, or
undiluted, phage
sample (100) and the
dilutions should be
stored at 4C
(refrigerated) until
the phage titer is
calculated. All tubes
must be well labeled
and easily identified.

Part1 Tame 11

Part A. Purify the Phage: The Phage-Titer Assay

e. Swirl the plate gently to spread the mixture evenly on top of the agar
plate.
f. Let the plates sit, undisturbed, for at least 20 minutes.

Use a fresh, sterile pipette for each sample!

H. Incubate plates at 37C.


1. After the TA has solidified, invert the plates rapidly (but gently).
2. Put all inverted plates in a 37C incubator.
I. Check plates for plaques.
1. Check for plaques after 24 hours (see Tame fig. 7).
2. Be sure to ascertain that there are no plaques on the negative-
control plate.
Tame fig. 7
Representative appearances of
plates from serial dilutions of a
phage lysate. A. No lysis.
B. Discrete plaques, pfu can be
counted. C. A web pattern plate.
Notice the wispy M. smegmatis
growth on this plate, in which
most of the M. smegmatis has
been lysed. D. Complete lysis.
With total bacterial lysis, it is
impossible to determine the
point (number of pfu) at which
this occurred.

3. If you have plaques, proceed to step J. If there are no plaques on your
sample plates, or if plaques are small or difficult to see, check them again
at 48 hours. If you have plaques at 48 hours, proceed to step J.
J. F
 or each plaque tested, count the number of plaques on the plate and
calculate the titer.
1. Count the number of plaques on a plate with a range of 20 to 200
plaques.
2. Record in your laboratory notebook the name of the putative phage, the
dilution plate, the volume tested (in this case, 10 L), and the number of
plaques on the plate. A suggested template is shown in Tame table 1.

Part1 Tame 12

Part A. Purify the Phage: The Phage-Titer Assay

Date:
Phage designation

Number of pfu

Dilution

Sample volume (L)

A1

78

10

A2

62

10

B4

106

10

E7

93

10

Tame table 1
Recording titer data for calculating pfu/mL.

3. Calculate your pfu/mL (plaque-forming units per milliliter) according to


the formula:
Titer (pfu/mL) = (pfu/#L) (1000 L/mL) dilution factor*
*For a 10 3 dilution, the dilution is 1000 (1000-fold); the dilution
factor is 1000 (or 10 3).

For example: Using Phage A1 in Tame table 1,


pfu/mL = 78 pfu/10 L 1000 L/mL 1000

= (7.8 1000 1000) pfu/mL

= 7.8 1,000,000 pfu/mL

= 7.8 106 pfu/mL

Do this calculation for each sample listed in Tame table 1.

Notes
Some plates may include multiple phages that are difficult to isolate and
purify. It is extremely important that the final plaque purification contain
only a single phage. Continue to pick isolated plaques, make dilutions,
infect M. smegmatis, plate samples, and incubate overnight until plaque
morphologies and other characteristics remain consistent. Three to six
repeats of this protocol may be required to isolate an individual phage.
Once you are sure that your phage is pure, you should name it using these
phage naming guidelines:




The name should be 3 to 10 characters long.


It must contain only letters and numbers (AZ, az, 09); other spaces
and symbols ($, #, @, !, etc.) are not permitted.
The first character of the name must be a letter and not a number.
Short, nonsensical names are discouraged (e.g., r7B).
The name should be polite and not offensive in any way.

After naming your phage, proceed to the next section, Final Plaque
Purification.

Part1 Tame 13

Part A. Purify the Phage: The Phage-Titer Assay

The Big Picture


In laboratories where serial-dilution assays are routinely performed, scientists count plates with 20 to
200 pfu (plaque-forming units), as shown in Tame
fig. 4, plate B. Counting more than 200 plaques takes
a long time, and counting fewer than 20 plaques
is less accurate (and less replicable) than counting
higher numbers. This is because the counting error
is proportional to the square root of the number
counted, so the greater the number that you count,
the smaller the error. For example, the uncertainty
in a count of 16 is 4 plaques with an error of 25%.
The uncertainty of 169 plaques is 13, or 7.7%.

Questions
1. Why do you think that the plaques are called plaque-forming units, or pfu,
rather than phage particles?
2. What are other applications of serial dilution outside the phage world?

Part1 Tame 14

Part A. Purify the Phage: The Phage-Titer Assay

Procedural Notes
1.

If students have more than one phage to titer, there is no need for additional negative controls as long as the same solutions are being used throughout (i.e., only one batch of TA
and PB per student). Each student should perform at least one negative control, however.

2.

The phage titer assay is only an assay if the titer (in pfu/mL) is calculated. If students
perform a serial dilution as a purification step, it is not an assay until they calculate their
titer. We recommend that students calculate the titer for each round of serial dilution and
purification of their phage.

Part1 Tame F9

Part A. Purify the Phage: Final Plaque Purification

Overview
Once a student has gone through at least three iterations
of the plaque streak assay and the phage-titer assay, and is
reasonably sure that each plaque on a given lawn is representative of only one phage, he or she is ready to accomplish
two things. The first is to use PB to flood a lawn containing
phage-infected bacteria on TA and then use the resulting
solution to purify higher numbers of filter-sterilized phage.
The second is to titer that phage-containing solution for use
in determining the approximate range needed for an empirical determination of the number of pfu required for lysis of a
bacterial lawn on a standard agar plate.
Students will perform the 10-plate harvest from an infection
of bacterial lawns on 10 agar plates. It is imperative, therefore, that there is little or no doubt that students have a pure
phage and that they know the titer of their phage preparation. With this information, they can do the empirical test
knowing the range of phage concentrations with which to
assay bacterial lysis.
During this exercise, students will become familiar with their
phages plaque morphology, size, and other identifying characteristics. By the end of it, students will have a phage lysate
of relatively high titer for which they will have determined
the titer in pfu/mL and have a fairly good idea of the size of
an average plaque.
Students will have to decide whether to titer their lysates
with a spot test or a phage-titer assay. The former uses fewer
supplies and takes less time; the phage-titer assay is more
accurate but requires more time and materials. Students
should also note whether the plaques vary in size or morphology as a function of their density on a bacteria-containing
plate. If students require more information, they may want to
perform the phage-titer assay. Regardless, they should be allowed to make a decision based on the characteristics of their
particular phage.

Supplies1
For each student (provided
in kits):
A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (11)
and a tube rack
5-mL sterile disposable pipettes
(1 to 10)



5-mL syringe
0.22-m syringe filter
A sterile 15-mL tube
A ruler
Supplies and reagents for
each student (provided by SEA
laboratories and prepared by
instructors):

10 mL of phage buffer (PB)


0
 .5 mL of M. smegmatis culture in a
10-mL culture tube (1 to 10)
5 to 50 mL of top agar (TA) melted
and stored at 55C
Agar plates (1 to 10)
Each student will also need
access to
A 200-L and a 20-L micropipettor
and tips
An automated pipette-aid for larger
volumes
A vortexer

Equipment
37C incubator
A microwave oven
55C water bath

Key Words and Concepts


Phage titer, plaque-forming unit (pfu), pfu/mL, pure phage

Part1 Tame F10

Part A. Purify the Phage: Final Plaque Purification

Implementation Timeline

LAB
Aliquot 0.5 mL of
saturated M. smegmatis
into enough 10-mL
culture tubes for the

t1D

class.
Prewarm agar plates

Prep 1X TA; store

to RT.

at 55C.

t2D
Prep PB

t3D
. Prep 2X TA.

t4D
Prep saturated
M. smegmatis culture
for plaque assays.

Part1 Tame F11

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part A. Purify the Phage: Final Plaque Purification

Overview
Once you have gone through at least three iterations of the plaque assay and
are reasonably sure that each plaque on a given lawn is representative of
only one phage, you are ready to accomplish two things. The first is to use
PB to flood a lawn containing phage-infected bacteria on TA and then use
the resulting solution to purify higher numbers of filter-sterilized phage. The
second is to titer that phage-containing solution for use in determining the
approximate range needed for an empirical determination of the number of
pfu required for lysis of a bacterial lawn on a standard agar plate.

Objective
The objective of this procedure is to isolate enough pure phage stock that
phage titers can be empirically tested as outlined in Tame B. Empirical Testing of Phage Lysates. This stock lysate, sometimes called the MTL for Medium-Titer-Lysate , will be used to make a large batch of pure phage (called the
HTL for High Titer Lysate). The HTL will then be used for the remainder of
your experiments.

Supplies














A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (11) and a tube rack
5-mL sterile disposable pipettes (1 to 10)
5-mL syringe
0.22-m syringe filter
A sterile 15-mL tube
A ruler
10 mL of phage buffer (PB)
0.5 mL of M. smegmatis culture in a 10-mL culture tube (1 to 10)
5 to 50 mL of top agar (TA) melted and stored at 55C
Agar plates (1 to 10)
A 200-L and a 20-L micropipettor and tips
An automated pipettor for larger volumes
A vortexer

Equipment


37C incubator
A microwave oven
55C water bath

Part1 Tame 15

Part A. Purify the Phage: Final Plaque Purification

Procedure: Harvesting a Plate Lysate


A. Prepare your work area.
Remember the
sterile field, and
remember to keep
the microcentrifuge
tube capped unless
transferring sample!
The filtrate and tube
are sterile and should
be manipulated using
aseptic technique.

B. Choose a plate to harvest and add PB.2


1. Choose the plate from your most recent titer in which you are sure you
have a single phage population and in which the bacterial lawn is nearly
cleared (see Tame fig. 7, plate C).
2. Add 8 mL of PB and swirl gently.
3. Let the plate sit for 2 to 4 hours at room temperature. Alternatively, the
plate can be stored overnight at 4C.
C. Filter-sterilize the plate lysate.
1. Invert the flooded plate (lid side down) and allow the lysate to pool in the
lid.
2. Label the sterile microcentrifuge tubes you will be using.
3. Retrieve a 0.22-m filter.
4. Open the packaging but do not remove the filter from the packaging.
5. Using a tuberculin (5-mL) syringe, aspirate the PB from the lid.

This harvested lysate


is sometimes called
the MTL for Medium-Titer-Lysate.

6. Place the syringe into the top of the filter and remove the filter from
the package.
7. Pushing the plunger, dispense the liquid through the filter into the
appropriately labeled sterile 15-mL tube.
8. Immediately cap the tube.
9. Discard the syringe and the filter.

Procedure: Preparing a Bacterial Lawn in TA


A. On your bench, you should have



A 0.5-mL aliquot of M. smegmatis in a 10-mL culture tube


An agar plate
Top agar (TA) from the 55C water bath
A 5.0-mL pipette and pipettor

B. Draw a grid onto the bottom of the agar plate.


Label the grid with designations for phage dilutions from 101 to 1010 and a
negative (buffer-only) control (see Tame fig. 8).
C. Prepare the TA-bacteria plate.
1. Using aseptic technique, aspirate 4.5 mL of TA into a 5-mL sterile
pipette.
2. Transfer the TA to 0.5 mL of bacteria.
Part1 Tame 16

Part A. Purify the Phage: Final Plaque Purification

Tame fig. 8
Plate labeled for a high-dilution
spot test.
10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

10-9

10-10

NEGATIVE
CONTROL

3. Immediately draw the TA back into the same pipette.


4. Add the TA-bacteria mixture to the labeled agar plate.
5. Swirl gently to spread the mixture.
6. Allow to solidify completely. This will take at least 10 minutes.

Procedure: Performing a Spot Test on the Phage Lysate


A. Perform 10-fold serial dilutions from 100 to 10-10.
1. Arrange 10 microcentrifuge tubes in a tube rack and label them 1
through 10.
2. Add 90 L of PB to each tube.
3. Add 10 L of your 100 (undiluted) phage sample to the 1 tube and
vortex well.
4. The 1 tube is your 101 (or 1:10) dilution.
5. Using a fresh sterile tip, add 10 L of the 1 tube to the 2 tube
and vortex well.
6. Using another sterile tip, add 10 L of the 2 tube to the 3 tube
and vortex well.
7. Continue to do successive dilutions, each with a fresh sterile pipette tip,
until you get to the 10 tube.

This is where you have a big decision to make. If you are unsure of any
aspect of phage purification or plaque morphology, you may want to perform a plate titer (as in the previous section) on all of your dilutions from
10-1 to 10-10.

B. Spot control and samples onto plate.3


1. Set a micropipettor to 5 L.
2. Aseptically transfer 5 L of PB to the negative-control block on the grid.
3. Transfer 5 L of all phage dilution samples onto the corresponding
blocks on the grid.
Part1 Tame 17

Part A. Purify the Phage: Final Plaque Purification

4. Allow the droplets to soak into the agar until no apparent liquid remains
on the agar.
5. Invert the plates and place into the 37C incubator.
6. Incubate overnight.
C. Count the number of plaques and calculate the titer.
1. After the overnight incubation, count the number of plaques on
each plate.
a. For the 5-L spot test, find the sector on the grid that has 5 to
50 plaques and count those plaques.
When exponential
numbers are
multiplied, the
exponents are
added together.
For example,
105 104 = 109.

b. For the phage-titer assay (one plate per 10 L of each dilution),


find the dilution that yields 20 to 200 plaques per plate and count
those plaques.
2. Record in your laboratory notebook the count, original volume,
and dilution factor.
3. Calculate your pfu/mL (plaque-forming units per milliliter) according
to the following formula and example.
Titer (pfu/mL) = (pfu/#L) (1000 L/mL) dilution factor*
*For a 10 3 dilution, the dilution is 1000 (1000-fold); the dilution
factor is 1000 (or 10 3).
For example: 16 plaques were identified on a plate that received
a 5-L spot of a 104 dilution.
pfu/mL = 16 pfu/5 L 1000 L/mL 104

= (3.2 103 104) pfu/mL

= 3.2 107 pfu/mL

4. Record in your laboratory notebook the following:


a. the titer of your phage lysate,
b. the appearance of your plaques (you can also insert a picture), and
c. the approximate size of your plaques (diameter in millimeters). This
will help you estimate the number of individual pfu that you will need
for complete lysis of bacteria on an agar plate.

Notes
Now that you have a pure phage at relatively high titer, you will be able to
use this lysate for the empirical assays in the next section. You will infect
larger numbers of bacteria to obtain a pure and highly concentrated solution
of phage particles. This high-titer phage stock will be used for all subsequent
procedures, including DNA isolation and electron microscopy (See Decision
Tree #2, page Tame 19).

Part1 Tame 18

Decision Tree #2
From Pure Phage to High-Titer Phage Stocks

?
perform empirical test with
phage lysate

Is there a distinct web pattern?

YES

NO

RECALCULATE DILUTIONS

Is there a distinct web pattern on all


the plates?

PERFORM 10-PLATE INFECTION

FLOOD PLATES WITH PB


HARVEST PHAGE
TITER

YES

NO

Do you have at least 10 mL of lysate with


a titer of 107 pfu/mL?

YES

NO

PROCEED TO DNAISOLATION STEP


Part1 Tame 19

Part A. Purify the Phage: Final Plaque Purification

Questions
1. What are some of the properties of individual phages that would lead to higher
or lower titers in a given phage preparation?
2. Think of an experiment to perform if you continue to obtain more than one
morphology in your phage preparations (e.g., some plaques are large and clear,
and some are much smaller and cloudy). How would you determine that an individual phage yielded two different morphologies? What would be a possible
mechanism by which one phage could yield two different morphologies?
3. In one to two sentences, describe how each of the following might look:

Part1 Tame 20

a. Doing a titer procedure and forgetting to add the bacteria

b. Contaminated bacteria

c. PB contaminated with phage

d. PB contaminated with bacteria or fungus

e. Doing a spot test and forgetting to add the top agar

f. Using top agar that is not completely melted

Part A. Purify the Phage: Final Plaque Purification

Procedural Notes
1.
The supply requirements for this exercise depend on whether each student will perform the spot test or
the phage-titer assay on the range of dilutions to be tested (1001010). You may want to err on the side
of caution and prepare supplies under the assumption that all students will use the phage-titer assay.
2.
Generally speaking, titers from older plates will be lower than titers from plates prepared more recently. The phages tend to readsorb onto the agar or onto host bacterial cells and are more difficult to
extract from plates that are more than a few days old. Ideally, students will add PB to their samples the
day the plaques first appear. They must still wait at least 2 hours for the phage to diffuse into the PB
before filter sterilization, but the titer will be a magnitude, or more, higher than if they wait for several
days. Filter-sterilized lysates can be stored at 4C.
3.
The obvious advantage of doing a spot assay on a serial dilution to 1010 is that many fewer plates are
required. If students need a better pictureboth literally and figurativelyof their phage, they may
want to infect separate M. smegmatis cultures and plate out each in TA. This will also give a more precise titer. It is most important, however, that the phage is pure and the morphology (or morphologies)
is consistent from procedure to procedure.

Part1 Tame F12

Part B. Make Phage Stocks: Empirical Testing


of Phage Lysates

Overview
Students will use their phage-lysate titer numbers and the size
of their phage to logically determine a range of dilutions to test
for almost complete lysis of a bacterial lawn grown in TA on 10
agar plates. The phrase almost complete is important because
students will attempt to form a web of small wisps of bacterial
growth on the agar plates, which indicates efficient lysis that
has stopped short of ablating all bacterial growth.
Students may have to perform this protocol more than once.
They should be allowed to make educated and logical guesses
about the number of phage required to form the web on the
large plates. Using the empirical assay, students may be able to
accurately determine the number of pfu to add for web formation, but it is likely that they will have to tweak their numbers
for the best results. This can happen for a variety of reasons.
Sometimes, students will need to try a range of pfu because
plaque size varies with different numbers of bacteria and
phages present, lytic phages behave differently from temperate
ones, and morphologies of different phages can alter the
appearance of the web.
During this exercise, students will be able to build on the
knowledge they already have about their phage (such as the
behavior of the phage under different growth conditions). It is
important to emphasize that small details may be significant,
and students should document them carefully in their laboratory notebooks. By the end of this exercise, students will have
a good estimate of the dilutions they will have to make, and the
volumes that they must use, for the 10-plate infection at the
beginning of the next section. To encourage the development of
students as research scientists, we recommend that they each
be allowed to analyze their own data and, with appropriate rationale, make procedural decisions based on the characteristics
of their particular phage.

Supplies
For each student (provided in
kits):
A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (6 to
10) and tube rack
10-mL sterile disposable pipettes (6)
A ruler
Supplies and reagents for
each student (provided by SEA
laboratories and prepared by
instructors):
10 mL of phage buffer (PB)
1
 .0 mL of M. smegmatis culture in a
10-mL culture tube (6)
At least 10 mL of the same culture
held in reserve
30 mL of top agar (TA) melted and
stored at 55C
Agar plates (6)
Each student will also need
access to
A 200-L and a 20-L micropipettor
and tips
An automated pipettor for larger
volumes
A vortexer

Equipment
37C incubator
A microwave oven
55C water bath

Key Words and Concepts


Empirical assay, web lysis, serial dilutions.

Part1 Tame F13

Part B. Make Phage Stocks: Empirical Testing of Phage Lysates

Implementation Timeline

LAB
Aliquot

0.5 mL of
saturated M. smegmatis
into enough 10-mL
culture tubes for
the class.

t1D
Prep

1X TA; store
at 55C.

Prewarm agar plates


to RT.

t2D
Prep PB.

t3D
Prep 2X TA.


t4D
Prep saturated
M. smegmatis culture
for plaque assays.

Part1 Tame F14

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part B. Make Phage Stocks: Empirical Testing


of Phage Lysates

Overview
You will use your phage-lysate titer numbers and the size of your phage to
logically determine a range of dilutions to test for almost complete lysis of a
bacterial lawn grown in TA on 10 agar plates.

Objective
The objective of this procedure is to determine the dilutions and volumes
required to form web patterns of lysis on bacterial lawns grown in top agar
(TA). This will lead to a 10-plate infection for the purpose of large-scale phage
purification.

Supplies












A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (6 to 10) and tube rack
10-mL sterile disposable pipettes (6)
A ruler
10 mL of phage buffer (PB)
1.0 mL of M. smegmatis culture in a 10-mL culture tube (6)
At least 10 mL of the same culture held in reserve
30 mL of top agar (TA) melted and stored at 55C
Agar plates (6)
A 200-L and a 20-L micropipettor and tips
An automated pipettor for larger volumes
A vortexer

Equipment


37C incubator
A microwave oven
55C water bath

Procedure: Preparing Your Dilutions


A. Calculate the approximate number of pfu that will be required for
complete bacterial lysis on an agar plate.
There are a couple of ways to do this:
1. On the basis of plaque size:
a. Use a ruler to estimate the diameter (mm) of both the average plaque
on your plate and the bottom of your plate.
b. Calculate the radii of the plaque and of the plate using this relationship: radius (mm) = 0.5 diameter.
c. Calculate the areas of the plaque and the plate using this: Area (mm2)
= r 2, where r = radius.

Part1 Tame 21

Part B. Make Phage Stocks: Empirical Testing of Phage Lysates

d. Estimate the approximate number of pfu needed to cover the agar plate:
pfu
=
max
web

Areaagar plate (mm2)


Areaplaque (mm2)

2. On the basis of lysis of a previous titer plate: If you performed the plate
titer on your phage lysate, you may have seen a plate in which the bacteria were completely, or almost completely, lysed (see plate C in Tame
fig. 7). If this is the case, youll need to add the same number of pfu to get
a similar pattern of bacterial lysis on multiple plates.

These calculations are estimates.


Volumes, the age of
bacterial cultures,
changes in growth
conditions, and a
variety of other
factors will affect
the lysis patterns of
a given phage. Make
a guesstimate about
the best concentration to use. Test
a range of plaqueforming units to
determine the best
way to obtain the
desired bacterial
lysis pattern.

B. Calculate the dilutions you will need to make for an infection that will
yield an optimal webbing pattern on an agar plate.
For example:
1. Your lysate titer is 5 106 pfu/mL and you estimate that you will need
3000 pfu for full lysis of your plate.
2. Use this formula: Volume (mL)phage stock = (pfudesired)/(pfu/mLphage stock)
For this example, (3000 pfu/5,000,000 pfu/mL) = 0.0006 mL, or 0.6 L.
3. You therefore would perform serial dilutions (using PB) to 102. This
solution now has 5 104 (50,000) pfu/mL.
4. You would then recalculate the volume needed as:
(3000/50,000 mL) = 0.06 mL (60 L)
5. To attempt complete lysis of bacteria on an agar plate, you would,
therefore, add 60 L of a 102 dilution.
C. Determine the range of pfu you would like to test in the empirical assay.
Continuing with the example above, you would test either

two 2-fold dilutions above and below your calculated pfu (750, 1500,
3000, 6000, and 12,000 pfu) or

two 5-fold dilutions above and below your calculated pfu (120, 600,
3000, 15,000, and 75,000 pfu).

D. Determine the concentrations and volumes needed for each of the


desired lysate additions.
You can do these calculations using one of the formulas above.

Procedure: Performing the Empirical Assay


A. Prepare your work area.
Remove clutter and wipe down your workbench. You should have


Part1 Tame 22

Sterile 10-mL culture tubes containing 0.5 mL of M. smegmatis (6)


Agar plates (6), labeled and prewarmed to 37C

Part B. Make Phage Stocks: Empirical Testing of Phage Lysates

Sterile microcentrifuge tubes (for dilutions)


PB
A 20-L micropipettor
A 200-L micropipettor
Sterile micropipettor tips
5-mL serological pipettes (6)
30 mL of 55C TA (remove from 55C water bath right before use)

B. Perform serial 10-fold (or other) dilutions as determined by your


calculations.
1. Arrange microcentrifuge tubes in a tube rack and label them with the
appropriate dilution designations (e.g., 1 through 7).
2. Perform serial dilutions using a fresh sterile tip for each volume transfer.
3. Carefully outline in your laboratory notebook the way you performed the
dilutions and the volume you will use for each infection.
C. Infect M. smegmatis cultures with diluted phage solutions.1
1. Label the six culture tubes (each containing 0.5 mL of M. smegmatis)
with designations corresponding to your dilution tubes.2
2. Label one tube as the negative control. This tube will contain a lawn
of bacteria with PB only (no phage). It will be a control for no lysis. It
should also contain 0.5 mL of M. smegmatis culture.3
3. Infect each sample tube with the dilution and volume specified by your
calculations.
4. Allow the phage to infect the bacteria for 20 to 30 minutes. Be sure
to record the length of time allowed for infection in your laboratory
notebook. Take careful note of all of the conditions of the infection
(time allowed to infect, the culture of M. smegmatis used, volumes and
dilutions used, etc.) so that you can replicate these parameters when
you do the 10-plate infection.
D. Add 4.5 mL of top agar (TA) and plate each sample (including the
negative control).
1. Label each of the six agar plates (prewarmed to 37C). Return these
plates to the 37C incubator. Remove the plates immediately before you
add the top agar to the samples.
2. Remove a 30-mL bottle of TA from the 55C water bath.
3. For each sample, including the negative-control tube, add the top agar
and plate as before.
4. Let the plates sit, undisturbed, for at least 30 to 40 minutes or until the
top agar has solidified.
E. Incubate the plates at 37C.
Part1 Tame 23

Part B. Make Phage Stocks: Empirical Testing of Phage Lysates

Procedure: Analyzing the Data


The next day, determine which volume-dilution combination yielded the
best web pattern of phage-infected M. smegmatis growth (see Tame fig. 7).
The combination of lysate volume and lysate dilution that yields the best
web pattern along with a minimal presence of M. smegmatis will be used
for the subsequent 10-plate infection.
If you do not have a clear web pattern, repeat the empirical test using
different lysate volume and dilution combinations. Increase or decrease
your phage concentration depending on your results.

Notes
Before starting the 10-plate infection, you must be confident with the volumedilution combination of lysate that yields a web pattern on an agar plate. To
standardize the conditions, the same subculture of M. smegmatis must be
used throughout the empirical test and 10-plate infection.
The 10-plate infection (see next section) should also be done very soon
(within 2 to 3 days) because the age of the bacterial culture can also affect
infection efficiency and plaque size.

Questions
1. What are the reasons for performing the empirical test?
2. Why not calculate the area of a plaque and infect 10 plates immediately?
3. In addition to human error, list as many different parameters that can vary
from one infection to the next that may influence a typical titer procedure.
4. Why do you think that a web pattern is better than complete lysis for isolating high numbers of phage particles?

Part1 Tame 24

Part B. Make Phage Stocks: Empirical Testing of Phage Lysates

Procedural Notes
1.
You should prepare a large quantity of M. smegmatis for this protocol. Keep in mind that bacterial
stocks can be kept at room temperature for up to a week after the laboratory session. This is important
because students need to use the same bacterial stock for their 10-plate infection as was used for
these empirical tests. In other words, each student will need a minimum of 3.0 mL of bacteria and six
agar plates for the empirical test. If successful, the students will require an additional 10 mL, at least,
of the same bacterial culture (and 20 more agar plates) for the large-scale infection and subsequent
titration of their phage harvest. For each 10 students performing this assay and moving on to the
10-plate infection, therefore, approximately 150 mL of bacterial culture will be required.
2.
The yield of pfu from an empirical infection can vary greatly depending on the age of the lysate and
the bacterial culture used. This is one of the reasons that the empirical test must be performed for
each phage. Rarely do the calculations outlined yield the exact number of pfu required for an acceptable web pattern. Students will likely learn this the hard way!
3.
This is a two-purpose control. First, using PB only for the bacterial infection will determine whether the
PB or the bacterial cultures are contaminated in any way. This concept should be familiar to students
by this point. Assuming no contamination, students will also need an example of a pristine bacterial lawn. This is especially true if students have isolated a temperate phage. If a temperate phage is
used to infect a lawn of bacteria, lysogens will likely grow. When this happens, the resultant bacterial
growth may look as though there is no infection or there are no plaques. The uninfected lawn will,
in a side-by-side comparison, make it clear that phage are present. In other words, unless the plates
can be directly compared, it can be difficult to determine the difference between secondary growth of
lysogens and complete lack of lysis.

Part1 Tame F15

Part B. Make Phage Stocks: The 10-Plate Phage


Infection and Harvest

Overview
Students will use their phage-lysate dilution and volume that
yielded the best web pattern to perform a 10-fold (volume)
infection to spread on 10 agar plates. The purpose of this
infection and harvest is to obtain large absolute numbers of
phage (>1010) for later DNA-isolation protocols. This rather
bulky protocol yields consistently high numbers of phage
regardless of individual phage properties or phenotypes. If
the 10 plates have good web patterns, they will be flooded
with phage buffer and allowed to sit for several hours (or
overnight) so that phage can diffuse into the PB. The PB-containing phage will be aspirated from the plates, spun down to
pellet cell debris (the phages remain in solution), and filtersterilized. A phage titer will be performed, and the harvest
will be considered successful if there are >109 pfu/mL.
The information recorded for a successful web formation
in the previous empirical test should be followed to the letter
in the 10-plate infection protocol. Further, the same bacterial
culture should be used, and this protocol should be performed
within 2 to 3 days of the empirical test.
Web formation is important for maximum yield. At the same
time that the phage are infecting bacterial cells, other (uninfected) bacteria are replicating. A web pattern ensures that
there are enough phage to almost completely lyse a growing
bacterial lawn. Complete lysis indicates only that enough
phage were present to infect and lyse all the bacteria on the
plate. If fewer phage are present, each generation of phage
released by lysis can go on to infect new bacterial progeny. A
good web pattern increases the yield of phage significantly.

Supplies
For each student (provided in
kits):






A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (11)
5-mL sterile disposable pipettes (11)
25-mL sterile disposable pipettes (16)
Sterile 50-mL conical tubes (2)
Filter-sterilization unit, 0.22-m pore
size and 100-mL volume
Supplies and reagents for
each student (provided by SEA
laboratories and prepared by
instructors):1, 2

12.0 mL of M. smegmatis culture


(the same as used in the previous
empirical tests)
60-mL bottles of top agar (TA)
melted and stored at 55C (2)
Agar plates (11)
100 mL of phage buffer (PB)
Each student will also need
access to
A 200-L and a 20-L micropipettor
and tips
An automated pipettor for large
volumes
A vortexer

Equipment
37C incubator
A microwave oven
55C water bath
High-speed (e.g., Sorvall) centrifuge

By the end of this exercise, students will have harvested


large numbers of their phage and will know whether their
titer is high enough (>10 mL of >109 pfu/mL) to warrant
further purification of their 10-plate lysate. If students have a
high-titer lysate, they will continue to the DNA-isolation step.
Key Words and Concepts
High-titer lysate, web lysis, serial dilutions.

Part1 Tame F16

Part B. Make Phage Stocks: The 10-Plate Phage Infection and Harvest

Implementation Timeline

LAB
Aliquot

0.5 mL of
saturated M. smegmatis
into enough 10-mL
culture tubes for the
class.

t1D
Prep

1X TA; store
at 55C.

Prewarm agar plates


to RT.

t2D
Prep PB.

t3D
Prep 2X TA.

t4D
Prep saturated
M. smegmatis culture
for plaque assays.

Part1 Tame F17

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part B. Make Phage Stocks: The 10-Plate Phage


Infection and Harvest

Overview
You will use your phage-lysate dilution and volume that yielded the best web
pattern to perform a 10-fold (volume) infection to spread on 10 agar plates.
The purpose of this infection and harvest is to obtain large absolute numbers
of phage (>1010) for later DNA-isolation protocols. If the 10 plates have good
web patterns, they will be flooded with phage buffer and allowed to sit for
several hours (or overnight) so that phage can diffuse into the PB. The PBcontaining phage will be aspirated from the plates, spun down to pellet cell
debris (the phages remain in solution), and filter-sterilized. A phage titer will
be performed, and the harvest will be considered successful if there are >109
pfu/mL.
The information recorded for a successful web formation in the previous
empirical test should be followed to the letter in the 10-plate infection protocol. Further, the same bacterial culture should be used, and this protocol
should be performed within 2 to 3 days of the empirical test.
Web formation is important for maximum yield. At the same time that the
phage are infecting bacterial cells, other (uninfected) bacteria are replicating.
A web pattern ensures that there are enough phage to almost completely lyse
a growing bacterial lawn. Complete lysis indicates only that enough phage
were present to infect and lyse all the bacteria on the plate. If fewer phage are
present, each generation of phage released by lysis can go on to infect new
bacterial progeny. A good web pattern increases the yield of phage significantly.

Objective
The objective of this procedure is to obtain a high-titer phage lysate with
high enough phage concentrations that students can progress to the DNAisolation protocol.

Supplies













A laboratory notebook
A labeling pen
Sterile microcentrifuge tubes (11)
5-mL sterile disposable pipettes (11)
25-mL sterile disposable pipettes (16)
Sterile 50-mL conical tubes (2)
Filter-sterilization unit, 0.22-m pore size and 100-mL volume
12.0 mL of M. smegmatis culture (the same as used in the previous
empirical tests)
60-mL bottles of top agar (TA) melted and stored at 55C (2)
Agar plates (11)
100 mL of phage buffer (PB)
A 200-L and a 20-L micropipettor and tips
An automated pipettor for large volumes
A vortexer

Part1 Tame 25

Part B. Make Phage Stocks: The 10-Plate Phage Infection and Harvest

Equipment



37C incubator
A microwave oven
55C water bath
High-speed (e.g., Sorvall) centrifuge

Procedure: Setting Up Your Work Area


A. Prepare your workbench.
Remove clutter and wipe down your workbench. You should have





A flask containing 5.0 mL of M. smegmatis


A culture tube containing 0.5 mL of the same M. smegmatis culture
A micropipettor and sterile tips (to add your phage)
25-mL serological pipettes (2) and an automated pipettor
60 mL of 55C TA (remove from 55C water bath right before use)
Agar plates (11), prewarmed to 37C and labeled (remove right before use)

B. Using the volume-dilution combination that yielded the best web pattern in the empirical test, calculate the volume and dilution of lysate
necessary for a 10-plate infection.
M
 ultiply the volume of phage used to create a web on one plate by 10 to get
the volume needed for all 10 plates. For example, if 60 L of a 105 dilution
of lysate formed an acceptable web pattern on the test plate, then you will
need 60 10 = 600 L for all 10 plates. Instead of using 600 L of a 105
dilution, you could use 60 L of a 104 dilution for the infection.

Procedure: Performing the Large-Plate Infection

Once the plates are infected, you must return the following day to flood
the plates and the day after that to harvest and prepare the lysates. This
is not a protocol to start on a Friday if you are not planning to work in the
lab over the weekend!

A. Infect the M. smegmatis.


1. In a 100-mL flask, infect 5.0 mL of M. smegmatis with the appropriate
volume and dilution of lysate.
2. Swirl gently to mix the bacteria and lysate well.
3. Allow phage to infect for the same amount of time used to infect for the
empirical test (usually 20 to 30 minutes).

Part1 Tame 26

Part B. Make Phage Stocks: The 10-Plate Phage Infection and Harvest

B. Add TA.
1. TA must be between 55C and 60C (use directly from water bath; it
should be very warm to the touch but not hot enough to burn).
2. Add 45 mL of TA to the flask that contains the infected M. smegmatis.
3. Swirl gently to mix, avoiding bubbles.
C. Immediately plate 5-mL aliquots onto each of the 10 prewarmed
agar plates.
1. Using an automated pipettor and a 25-mL pipette, pull up 25 mL of the
mixture at a time.
2. Dispense 5 mL per agar plate (five plates per 25-mL pipette load). Swirl
plates gently to cover the agar plate completely with the TA mixture. To
reduce contamination, change the pipette after dispensing the 25 mL.
D. Prepare a control plate with 0.5 mL of bacteria plus 4.5 mL of TA.
E. Allow plates to harden and then invert and incubate at 37C overnight.

This harvesting step must be done within


24 hours of the initial infection.

Procedure: The Next Day, Harvesting Your Phage


A. Prepare your work area.
Remove clutter and wipe down your workbench. You should have








10 infected plates with web-pattern lysis


100 mL of PB
25-mL serological pipettes (14) and an automated pipettor
50-mL conical tubes (2)
A filter-sterilization unit, 0.22-m pore size and 100-mL volume
Sterile microcentrifuge tubes (for titer dilutions)
A 20-L and a 200-L micropipettor and tips
5-mL serological pipettes (10)
Aliquots of 0.5 mL of saturated M. smegmatis culture in 10-mL culture
tubes (10)
60 mL of 55C TA (remove from 55C water bath right before use)

Part1 Tame 27

Part B. Make Phage Stocks: The 10-Plate Phage Infection and Harvest

B. Flood the 10 plates with PB.


Be sure that
bubbles do not come
up the pipette past
the cotton plug. This
will clog the filter in
the automated
pipettor, which
will lose suction and
stop working.

1. Using the 25-mL serological pipettes and automated pipettor, add 8 mL


of PB to each plate (use one pipette per three plates).
2. Make sure the buffer covers the entire surface of the agar.
3. Let the plates sit for 2 to 4 hours at room temperature. Alternatively, the
plates can be stored overnight at 4C.
C. Harvest the phage lysate.
1. Using the 25-mL serological pipettes and automated pipettor, remove the
PB (now containing phage) from the agar plates. Keep in mind that some
of the PB may have seeped under the agar. Remove the PB as outlined in
Tame fig. 9.

Tame fig. 9
Removing PB-containing phage
from an agar plate. Go through
the agar with the pipette and,
while tilting the plate up at a
slight angle, aspirate the PB
from the bottom of the plate. Be
very careful not to spill lysate
on the bench top, which could
contaminate other samples with
your phage.

2. Pool the PB-phage into a 50-mL conical tube.


3. Balance and centrifuge the tubes at 2500g for 20 minutes to pellet cell debris.
This harvested lysate
is sometimes called
the HTL for HighTiter-Lysate. The
HTL will be used for
DNA purification..

Part1 Tame 28

4. Transfer the supernatant into a 50-mL filter-sterilization unit.


5. Using suction, filter-sterilize your lysate.
6. Turn off the suction.
7. Using aseptic technique, place the lid on the bottom reservoir of the
filter-sterilization unit.
8. Label the tube containing your lysate for storage at 4C. You will
use this lysate for the titer determination below.

Part B. Make Phage Stocks: The 10-Plate Phage Infection and Harvest

Be careful not to spill your lysate onto countertops or equipment.


High-titer lysate spills can contaminate the work of others!
Save your lysate. If the titer is high enough, you will use this lysate
for the preparation of electron microscopy grids and visualization of
your phage. You will also use this lysate for the preparation of phage
genomic DNA.

Procedure: Titering Your Phage


A Perform serial dilutions on a sample from the pooled and sterilized lysate.
B Use 10 L of these dilutions to infect 0.5-mL aliquots of M. smegmatis;
let sit for 20 to 30 minutes.
C. Add TA to each sample and spread onto prewarmed agar plates.
D. Count plaques the following day, and, if the titer is >108 pfu/mL,
continue to the DNA-purification protocol.

Notes
Before starting the 10-plate infection, you must be confident about the volume-dilution combination of lysate that yields a web pattern on an agar plate.
The same subculture of M. smegmatis must be used throughout the empirical
test and 10-plate infection in order to standardize the conditions. If the 10-plate

infection does not yield a good web pattern, the empirical test will have to be
repeated and the 10-plate infection performed again.
It is best to time your lab work to coordinate with your schedule. If you do
not plan well, long delays could influence your results. For example, phage
titers tend to decrease over time, even when the phage is stored at 4C.
Depending on the characteristics of your particular phage, the age of the
bacterial culture can affect the efficiency of phage binding, infection, burst
size, and other characteristics. Good planning will increase the likelihood of
a good titer and, therefore, a high yield of DNA for sequencing. It will also
increase your efficiency in the laboratory and could, potentially, save you a
lot of time.

Questions
1. Name at least three factors that would decrease the titer of a given phage
preparation.
2. Knowing what you know about your phage, what are your predictions about
the yield that you could expect from a 10-plate harvest?

Part1 Tame 29

Part B. Make Phage Stocks: The 10-Plate Phage Infection and Harvest

Procedural Notes
1.
You may want to consider preparing individual saturated M. smegmatis cultures (20 to 50 mL) for
each student. This will place the responsibility on the students for maintaining their cultures and
replicating lab conditions from the empirical test to the 10-plate infection. Keep in mind that up to an
additional 5 mL of culture is required for the titer of the filter-sterilized lysate. Larger batch cultures can
also be used, however, as long as the students can track the batch from which their cultures came and
enough is prepared for the empirical tests, the 10-plate infection, and the titer procedure. Alternatively,
you could prepare batches for groups of 2, 4, 6, etc., students. Keep in mind that each student will
require approximately 10 to 15 mL of saturated M. smegmatis culture for each iteration of the
empirical-to-10-plate-infection-to-titer cycle.
2.
Quite a bit more PB, TA, and other materials are required for this stage of the phage-purification
process. This is especially true if some, even a few, students need to repeat their empirical tests or
10-plate infections.

Part1 Tame F18

TAB
GOES
HERE

You
Are
Here
You
Are
Here
COLLECT ENVIRONMENTAL SAMPLE

ISOLATE PHAGE FROM


ENVIRONMENTAL SAMPLE

PURIFY THE PHAGE

DISSECT
CHARACTERIZE THE PHAGE

Molecular

Structural

ASSESS HEAD SIZE &TAIL


LENGTH VIA ELECTRON
MICROSCOPY

ISOLATE & PURIFY DNA

ASSESS DNA QUALITY,


QUANTITY, & SIZE

ELECTROPHORESE WITH
MASS STANDARDS

RESTRICT WITH ENDONUCLEASES


(RESTRICTION ENZYMES)

ELECTROPHORESE WITH
SIZE STANDARDS

COMPARE RESTRICTION
MAP WITH THOSE OF OTHER
KNOWN MYCOBACTERIOPHAGES

SEND THE PHAGE FOR


SEQUENCE ANALYSIS

Rev. May 2011

Dissect

Contents
Part A. Analyze Phage Using Electron
Microscopy

Equipment (for students)

F1 Overview

10 The Big Picture

Key Words and Concepts

Supplies and Equipment

F2 Implementation Timeline

1 Overview

Objective

Supplies (for students)

Equipment (for students)

Procedure

3 Questions

F3 Procedural Notes
Part B. Isolate and Purify Phage
Genomic DNA
F4 Overview

Key Words and Concepts

Supplies and Equipment

F5 Implementation Timeline

4 Overview

Objective

Supplies (for students)

Equipment (for students)


5 Procedure

7 Questions

F6 Procedural Notes
Part C. Restrict and Analyze Phage
Genomic DNA
F7 Overview

Key Words and Concepts

Supplies and Equipment

F8 Implementation Timeline

8 Overview

Objective

Supplies (for students)

ii

9 Procedure

14 Notes

Questions

15 Decision Tree #3
F9 Procedural Notes
Part D. Evaluate Genomic DNA Quality and
Send DNA to Sequencing Center
F10 Overview

Key Words and Concepts

Supplies and Equipment

F11 Implementation Timeline


16 Overview

Objective

Supplies (for students)

Equipment (for students)

Procedure

18 Questions
19 Decision Tree #4
F12 Procedural Notes

Part A. Analyze Phage Using Electron Microscopy

Overview
The objective of this series of procedures is to observe each
phage using electron microscopy (EM). You need to establish
which EM facility you will be using and how this facility will
want you to send samples. If the facility would like prepared
and stained grids, the procedure below is a straightforward
and relatively simple one to follow. Variables include the concentration of phage to be placed on the grid, staining time,
and any preparation of the grid required by your facility.
For this analysis, students will be working with uranyl
acetate, a very toxic compound for which you should have
the MSDS file on hand (from the suppliers website or as a
hard copy that came with the chemical). Students should
wear gloves, and exposure should be minimized. The grids
are 200-to-400-mesh carbon-stabilized, formvar-coated
copper.1 They are placed onto the edge of double-sided disks
or sticky tape for all manipulations. Students will also need
very small forceps, called fine-point capillary tweezers, to
work with the 3.0-mm-diameter grids. The phage are allowed
to settle onto the grid, excess phage are removed in a series
of washes, and the grid is stained with uranyl acetate. After
the grid has dried, it can be placed either back into the grid
holder (in which the grids were received) or into a beem
capsule for individual transport or mailing.

Supplies
For each student (provided
in kits):
A Petri dish
Latex or vinyl gloves
Supplies and reagents for
each student (provided by SEA
laboratories and prepared by
instructors):
A plastic-faced paper (diaper)
Parafilm, 5 x 5 cm (1 piece)
Double-sided disks (PELCO Tabs) or
sticky tape
Sterile distilled or filtered H2O
Fine-point capillary tweezers
(1 pair)
An EM grid
Whatman #3 filter paper
Each student will also need
access to
A micropipettor and tips
F
 iltered 1% uranyl acetate solution
(100 L)

Equipment
An electron microscope

It is important to have a scale bar on each electron micrograph. The area of the capsid (in nanometers) correlates
with the size of the phage genome it contains. Likewise, the
length of the tail will correlate with the size (in base pairs) of
the tape-measure gene found in all mycobacteriophages. It is
also important to note that two phages that are very similar
in appearance can have vastly different genome organization.
We cannot overstate the importance of this procedure in
allowing students to experience the joy of discovery science.
This is the first time that they can truly see the phage.
With electron microscopy, the students will see a life-form
that has likely never been seen before and that they,
themselves, have isolated.
Key Words and Concepts
Electron microscopy, magnification, sample staining.
Part1 Dissect F1

Part A. Analyze Phage Using Electron Microscopy

Implementation Timeline

LAB
Dilute uranyl acetate to
1.0%.
Dispense materials.

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part1 Dissect F2

Part A. Analyze Phage Using Electron Microscopy

Overview
The objective of this series of procedures is to observe each phage using
electron microscopy (EM). The area of the capsid (in nanometers) correlates
with the size of the phage genome it contains. Likewise, the length of the tail
will correlate with the size (in base pairs) of the tape-measure gene found in
all mycobacteriophages. It is important to note that two phages that are very
similar in appearance can have vastly different genome organization.

Objective
To observe individual phages using electron microscopy.

Supplies










A Petri dish
Latex or vinyl gloves
A plastic-faced paper (diaper)
Parafilm, 5 5 cm (1 piece)
Double-sided disks (PELCO Tabs) or sticky tape
Sterile distilled or filtered H2O
Fine-point capillary tweezers (1 pair)
An EM grid
Whatman #3 filter paper
A micropipettor and tips
Filtered 1% uranyl acetate solution (100 L)

Equipment

An electron microscope

Procedure
A. Prepare your phage sample.
1. Aseptically transfer 0.5-1.0mL of your high-titer lysate into a sterile microcentrifuge tube.

Do NOT dilute your high titer lysate before the high-speed centrifugation
step!

2. Balance the tube(s) and centrifuge for one hour at 4oC and at 10,000g.
3. Using the micropipettor and sterile tip, carefully remove all but 20-50
L of the supernatant without scraping the bottom of the tube with the
pipette tip or otherwise disturbing the pellet.
4. Add 100 L of fresh phage buffer and mix gently using the pipette tip.
5. Store this concentrated sample at 4oC for at least one hour, but no more
than 48 hours, before staining to allow the phage pellet to completely
dissolve.
Part1 Dissect 1

Part A. Analyze Phage Using Electron Microscopy

Handle the EM grids only with forceps and only by the edges.

B. Prepare your work area.


1. Put on a fresh pair of latex or vinyl gloves.
2. Cover the designated lab counter2 with the plastic-faced paper to create
a clean area.
3. Remove the cover paper from a 5 5-cm piece of parafilm, and place the
parafilm into the lid of a Petri dish.
4. Using tweezers, place a PELCO Tab or small (1-to-2-cm) piece of doublesided tape onto the parafilm in the lid of the Petri dish.
5. Remove the liner from the tape or tab to expose the adhesive.
6. Using the pointed tweezers, remove a fresh grid from a box of unused
grids, touching only the very edge of the grid.
7. Place the grid, shiny side UP, on the edge of the tab or double-sided tape
so that only the very edge of the grid (no more that 0.5 mm) is touching
the adhesive.
C. Mount and stain your phage.
1. Using a micropipettor, place 10 L of your phage preparation3 onto the
grid without touching the tip to the grid itself.
2. Allow the phage to adsorb (or settle and attach) onto the grid for at least
2 minutes. The approximate times are indicated in Dissect table 1.4
Dissect table 1
Approximate times that phage
should settle onto the EM grid
based on phage titer.

Phage titer

Approximate time

10 --10

5-7 minutes

108

5 minutes

10

2 minutes

3. Using a small (2- to 3-cm) wedge of Whatman #3 filter paper or a low-lint


laboratory wipe, wick off the excess fluid (see Dissect fig. 1).

Work quickly (and SAFELY). Do NOT allow the grid to dry out!!

4. Wash the grid two times by this method:


a. Carefully pipette 10 L of sterile water onto the grid.
b. Allow to sit for 2 minutes.
c. Wick off water using a fresh wedge of filter paper or a corner of a
laboratory wipe.5
Part1 Dissect 2

Part A. Analyze Phage Using Electron Microscopy

Dissect fig. 1
Wicking liquid off a grid. Touch
only the side of the grid and with
only a corner of the filter paper or
low-lint laboratory wipe. This will
prevent contamination of the grid
with paper fibers.

5. Add 10 L of 1.0% uranyl acetate6 to the grid.


6. Allow to stain for 2 minutes.
7. Wick off the excess stain and allow the grid to air-dry.

Uranyl acetate is a very toxic compound. You should wear gloves


throughout this procedure and when working in any area where this
material has been used.

D. Observe your phage.7


U
 se the bottom of the Petri dish to cover your stained phage grids and take
it directly to your EM facility. If you are planning to send the phage to a
facility, prepare the grid or samples according to the specifications of the
facility and send as directed.

Questions
1. Why cant you see phages with a standard microscope? What is the resolution
of an electron microscope compared with a light microscope?
2. What can you learn from your observation of an electron micrograph of your
phage? What could the size of the capsid tell you? What could the length of the
tail tell you?
3. If two phages look very similar by electron microscopy, would you predict that
they will have similar genomes? Why?

Part1 Dissect 3

Part A. Analyze Phage Using Electron Microscopy

Procedural Notes
1.
Several grid types will work well. An example is catalog number 01810 from Ted Pella, Inc. These grids
have a formvar coating to optimize phage binding. Important: Grids need to be fresh! Opened grid
boxes have a limited (< 3-month) shelf life.
2.
Because of the toxicity of the stain, a countertop or hood area in the laboratory should be designated
as the area to be used exclusively for staining EM grids.
3.
Though this volume is best for loading phage onto the grid, the phage concentration is the most
important consideration of the staining protocol. Though the student may be able to see phage of low
titer by this method, the best results will be obtained with a lysate or phage preparation with a titer
108 pfu/mL. Do not allow the phage to settle for longer than 10 minutes, because the sample might
evaporate. A dry grid is difficult to stain.
4.
These times are approximate. You may want to consider having students vary the time with a given
phage titer or dilute the phage and stain two or three dilutions for the same time period.
5.
It is extremely important that the grid not dry out, especially between the last water wash and the addition of the stain. The capsid proteins will destabilize in water (as there is no salt available) and the
capsid will collapse and/or burst open if the grid dries out.
6.
If you have difficulty obtaining or disposing of uranyl acetate, a filtered 2% solution of ammonium
molybdate can also be used for staining. The procedure is the same: A 2-minute settling time (for hightiter phages), two 2-minute washes, and a 2-minute stain. Alternative staining protocols are available
on the SEA Wiki.
7.
Positively stained capsids (i.e., the phage heads look black because the stain has permeated the
capsid) should not be used when describing the size or the morphology of the phage. Positive staining and/or capsid collapse can be caused by allowing the grid to dry out (see Note #5 above) or by
mechanical damage, e.g., vortexing or pipetting the sample too vigorously. Finally, best results are
obtained when FRESH lysates are used for the EM staining experiments.

Part1 Dissect F3

Part B. Isolate and Purify Phage Genomic DNA

Overview
Students will isolate and purify phage genomic DNA from
the phage head, which is virtually all protein. Any residual
bacterial cell debris and macromolecules (including capsid
proteins and bacterial proteins) must be removed. Of all of
the bacteria-derived macromolecules, the bacterial nucleic
acids, both DNA and RNA, are the most important ones to
be removed from solution before the protective coat proteins
of the phage head are disrupted. Removing them will reduce
the potential complexities of the restriction and sequence
analyses.
The initial step is to incubate the intact phage in a solution
containing both DNase I and RNase A, two nucleases with
nonspecific DNA and RNA hydrolytic activities, respectively.
After the phage is precipitated from the polynucleotide
solution, the coat proteins and enzymes are denatured
(unfolded and/or inactivated) by the buffer that contains
the resin. The denaturant in the buffer is guanidinium
thiocyanate. While the proteins are being denatured, the
phage genomic DNA will bind to the resin. Upon removal
of the denatured proteins and salts from the resin with an
aqueous alcohol, isopropranol, the phage genomic DNA is
eluted (removed) from the column with hot (80C) buffer
and is then ready for analysis.
Key Words and Concepts
Macromolecules, denaturation, resin.

Supplies
Supplies and reagents for
each student (provided by SEA
laboratories and prepared or
dispensed by instructors):
A sterile serological pipette (5-mL)
Isopropanol (80%) (2 mL/10 mL
lysate)
Phage precipitant (4 mL/10 mL
lysate)
Nuclease Mix (40 L/10 mL lysate)
Promega DNA Clean Up Kit
1.5-mL microcentrifuge tubes (6)
A 15-mL clear screw-capped tube
(e.g., Oak Ridge tube)
Sterile ddH2O (0.5 mL/10 mL lysate)
TE pre-warmed to 80oC
Gloves (latex or vinyl)
Each student will also need
access to
A micropipettor and tips
A refrigerator or ice

Equipment






A high-speed centrifuge
A microcentrifuge
80oC heating block or water bath
37oC water bath
A refrigerator (or ice)
20oC freezer
A spectrophotometer, Nanodrop or
fluorometer (and accessories)
for DNA quantification

Part1 Dissect F4

Part B. Isolate and Purify Phage Genomic DNA

Implementation Timeline

LAB
Prep 80% isopropanol.
Store at RT in the hood.

t1D

P
 lace the DNA Clean Up
Resin tube at 37oC to warm.

Prep the Nuclease Mix.

t2D
Prep the phage
precipitant.
A
 llow to stir
overnight at RT.

D
 ispense the Nuclease
Mix in 120-L aliquots
and store at 20oC.
D
 ispense the phage precipitant in 5-mL aliquots
and store at RT.
H
 eat either a heating
block or water bath to
80oC.

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part1 Dissect F5

Part B. Isolate and Purify Phage Genomic DNA

Overview
You will isolate and purify phage genomic DNA from the phage head, which
is virtually all protein. Any residual bacterial cell debris and macromolecules
(including capsid proteins and bacterial proteins) must be removed. Of all of
the bacteria-derived macromolecules, the bacterial nucleic acids, both DNA
and RNA, are the most important ones to be removed from solution before the
protective coat proteins of the phage head are disrupted. Removing them will
reduce the potential complexities of the restriction and sequence analyses.
The initial step is to incubate the intact phage in a solution containing two
enzymes. These enzymes, DNase I and RNase A, are nucleases with nonspecific DNA and RNA hydrolytic activities, respectively. Nucleases are enzymes
that disrupt the structure of nucleic acids. After the phage is precipitated
from the polynucleotide solution, the coat proteins and enzymes are denatured (unfolded and/or inactivated) by the buffer that contains the resin.
While the proteins are being denatured, the phage genomic DNA will bind to
the resin. Upon removal of the denatured proteins and salts from the resin
with an aqueous alcohol, isopropranol, the phage genomic DNA is eluted (removed) from the column with hot (80C) buffer and is then ready for analysis.

Objective
The primary objective is to isolate and purify phage genomic DNA in sufficiently high amounts for restriction analysis and sequencing protocols.

Supplies

A sterile serological pipette (5-mL)


Isopropanol1 (80%) (2 mL/10 mL lysate)
Phage precipitant (4 mL/10 mL lysate)
Nuclease Mix (40 L/10 mL lysate)
Promega DNA Clean Up Kit2
1.5-mL microcentrifuge tubes (6)
A 15-mL clear screw-capped tube (e.g., Oak Ridge tube)
Syringes, 3-mL or 5-mL
Sterile ddH2O (0.5 mL/10 mL lysate)
TE pre-warmed to 80oC
Gloves (latex or vinyl)

Equipment






A high-speed centrifuge
A microcentrifuge
80oC heating block or water bath
37oC water bath
A refrigerator (or ice)
20oC freezer
A spectrophotometer, Nanodrop, or fluorometer (and accessories) for
DNA quantification

Part1 Dissect 4

Part B. Isolate and Purify Phage Genomic DNA

Procedure
A. Degrade bacterial DNA that is in 10 mL of phage lysate.3
If your lysate has a
titer>109 pfu/mL, you
can dilute it (e.g., 1:2
or 1:10) with sterile
phage buffer.

1. Transfer 10 mL of the filter-steriled phage lysate into a Oak Ridge tube.


Store the remaining lysate at 4oC.
2. Put on gloves.
3. Add 40 L Nuclease Mix, screw on cap, and mix gently but thoroughly by
repeated inversions. Do not vortex!

When handling the Nuclease Mix, you must wear gloves and work in
a hood to avoid contaminating other solutions with the mix. After adding
Nuclease Mix to your sample, cap the tubes and discard your gloves
before returning to your work area. Otherwise, the enzymes can
contaminate all DNA-containing solutions and ruin your sample.

4. Incubate at 37oC for 30 minutes.


5. Let sit undisturbed at room temperature (RT) for 1 hour.
Remove as much
phage precipitant
solution as possible
without disturbing
the phage pellet.
Failure to do so will
interfere with or
clog the column and
significantly reduce
your DNA yield.

B. Precipitate phage particles.


1. Put on a fresh pair of gloves.
2. Using a 5-mL pipette, add 4.0 mL of phage precipitant solution to the
nuclease-treated lysate, and cap the tube.
3. Mix gently but thoroughly by inversion.
4. Incubate 30 minutes on ice or, for maximum yield, overnight at 4oC.
5. Place the tube in a high-speed centrifuge and spin at 10,000g for
20 minutes.
6. Decant the supernatant being careful not to disturb the pellet. Drain
excess liquid from the pellet by inverting for 2 to 3 minutes on a paper
towel. Discard the paper towel and your gloves.
C. Re-suspend the phage pellet.
1. Add 0.5 mL of sterile ddH2O to the pellet.
2. Gently re-suspend the pellet by pipetting up and down.
3. Allow to sit at room temperature for 5-10 minutes (no more than 10 minutes).

Food
for Thought
Why wouldnt you want to
let the resuspended phage
particles sit too long in
solution?

Part1 Dissect 5

D. Obtain the DNA-purification kit from your instructor.


E. Uncoat the phage genomic DNA.
1. Put on a fresh pair of gloves.

The resin contains guanidinium thiocyanate, a chemical


that denatures proteins. Do not get it on your skin!

Part B. Isolate and Purify Phage Genomic DNA

2. Add 2 mL of pre-warmed (37oC) DNA Clean Up Resin.


3. Uncoat the phage particles by gently pipetting up and down. Gently swirl
to mix.
F. Isolate the phage genomic DNA.
1. You will need two columns for this procedure. Attach one column
supplied with the kit to a syringe. Repeat with the second column.
2. Apply 1.25 mL of water-resinphage-genomic-DNA solution to each column using a pipette.
3. Use the syringe plunger to push this solution through the column.
4. Wash salts and proteins off DNA. For each column:




Pushing the solution


through sometimes
requires a great deal
of elbow grease.

a. Remove the column from the syringe.


b. Remove the plunger from the syringe.
c. Re-attach the syringe to the column.
d. Add 2 mL of 80% isopropanol to the column.
e. Push the isopropanol through the column with the plunger.

5. Dry each column:


a. Remove the column.
b. Centrifuge the column for 5 minutes at maximum speed to remove
the isopropanol (do this in a clean microcentrifuge tube that has had
its lid removed).
c. Transfer the column to a clean, lidless microcentrifuge tube.
d. Centrifuge for 1 minute at maximum speed to remove the residual
alcohol.
6. Elute the phage genomic DNA from each column:

Do not allow the TE to cool below 80C before it is applied to the column.

a. Transfer the column to a clean, autoclaved, properly labeled microcentrifuge tube.


b. Rapidly apply 50L of pre-warmed, 80oC TE to the resin in the column.
Let the TE sit on the column 30 to 60 seconds to dissolve the DNA.
c. Centrifuge for 1 minute to elute your purified phage genomic DNA.
7. Combine the DNA samples into a single tube.
8. Store your DNA at 4oC until you are ready to quantify and analyze it.
G. Determine the concentration of your DNA.
U
 sing a spectrophotometer (fluorometer, or Nanodrop) and a protocol from
your instructor, quantify your DNA.

Part1 Dissect 6

Part B. Isolate and Purify Phage Genomic DNA

Questions
1. Drew, who is incredibly conscientious and cares deeply about the environment,
decided it would be best to use the same pair of gloves throughout todays
procedure. Why is this, although good for the environment, a pretty bad idea
for todays procedure?
2. An aqueous alcohol solution is used to wash the salts from the column.
Why not just use straight alcohol?

Part1 Dissect 7

Part B. Isolate and Purify Phage Genomic DNA

Procedural Notes
1.
Make the 80% isopropanol using a fresh bottle of reagent-grade isopropanol that has been stored at
room temperature. As alcohols absorb water from the air, their concentration changes. The phage
genomic DNA sticks to the resin under conditions of high salt or high alcohol. The DNA will come off
the column in solutions that lack salt or alcohol. This means that if the isopropanol is not actually 80%,
the DNA will be washed off the column at this step and thrown away.
2.
It is a good idea to read the Promega Kit insert. You will note that the binding capacity of each column
is 12-15g of DNA under ideal conditions. Overloading the column can be as bad or worse than
under-loading, in terms of DNA yield.
3.
Students must use their filter-sterilized phage lysates for this step; any other sample will clog the
column. If there is doubt that the sample was filter-sterilized before storage or if particulate matter is
apparent, repeat the filter-sterilization step. If lysate clogs the sterile filter, centrifuge the lysate for 20
minutes at 10,000xg, decant the supernatant into a clean 50-mL screw-cap tube, and refilter.

Part1 Dissect F6

Part C. Restrict and Analyze Phage Genomic DNA

Overview
The objective is to use restriction analysis of genomic DNA
to help identify a unique mycobacteriophage that can be sent
for sequence and subsequent bioinformatic analysis. The
general strategy is to digestthat is, cut, or restrictthe
DNA into fragments of manageable size and to separate the
fragments using agarose gel electrophoresis.
Students will add a tracking dye to the samples that will
help determine how long the gel should be run. The dye is
a mixture containing bromophenol blue (BPB; a dark blue
color) and xylene cyanol (XC; a medium sky blue). The BPB
runs at a rate that is approximately equal to a 500-base-pair
(bp) DNA fragment; XCs migration is similar to a 4000-bp
fragment. The gel should be run long enough to get good
resolution of the DNA fragments (bands). Generally, gels are
run with DNA standards until the BPB is near the bottom
edge of the gel. Once the electrophoresis is complete,
students photograph the gel to document and record the
results. The results should be compared with restriction
patterns of previously characterized mycobacteriophages that
are on file at the SEA website. Samples with unique patterns
will be prime candidates for further characterization.
Key Words and Concepts
Restriction analysis, agarose gel electrophoresis, nucleic
acids, proteins, carbohydrates.

Supplies
For each student (provided in kits):
Microcentrifuge-tube floaters
Latex or vinyl gloves
Supplies and reagents for
each student (provided by SEA
laboratories and prepared by
instructors):
Restriction enzymes (BamHI, ClaI,
EcoRI, HaeIII, and HindIII)
Restriction enzyme buffers
ddH2O (autoclaved)
10X bovine serum albumin (BSA)
1-kb DNA Ladder
Seakem LE agarose gel powder
1X TBE stock buffer
Ethidium bromide (EtBr, 10 mg/mL)
Tracking dye
Each student will also need
access to
A micropipettor and tips

Equipment



A microcentrifuge
37oC water bath
A microwave oven
Horizontal midigel (approximately 20 x
20 cm) apparatus with combs
Power supply
Photography equipment and
supplies

Part1 Dissect F7

Part C. Restrict and Analyze Phage Genomic DNA

Implementation Timeline

LAB

 ilute and aliquot the


D
following:
1
 00X BSA to 10X with
ddH2O.
1
 0X TBE to 1X with
ddH2O.

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part1 Dissect F8

Part C. Restrict and Analyze Phage Genomic DNA

Overview
The objective is to use restriction analysis of genomic DNA to help identify
a unique mycobacteriophage that can be sent for sequence and subsequent
bioinformatic analysis. The general strategy is to digestthat is, cut, or restrictthe DNA into fragments of manageable size and to separate the fragments using agarose gel electrophoresis.
You will add a tracking dye to the samples that will help determine how long
the gel should be run. The dye is a mixture containing bromophenol blue
(BPB; a dark blue color) and xylene cyanol (XC; a medium sky blue). The BPB
runs at a rate that is approximately equal to a 500-base-pair (bp) DNA fragment; XCs migration is similar to a 4000-bp fragment. The gel should be run
long enough to get good resolution of the DNA fragments (bands). Generally,
gels are run with DNA standards until the BPB is near the bottom edge of the
gel. Once the electrophoresis is complete, a photograph of the gel should be
taken to document and record the results. Samples with unique patterns will
be prime candidates for further characterization.

Objective
The objective is to digest the phage genomic DNA sample with restriction
enzymes (REs), compare its restriction pattern with those of known mycobacteriophages, and use this information to help decide whether the phage is a
viable candidate for bioinformatic analysis.

Supplies











Microcentrifuge-tube floaters
Latex or vinyl gloves
Restriction enzymes (BamHI, ClaI, EcoRI, HaeIII, and HindIII)
Restriction enzyme buffers
ddH2O (autoclaved)
10X bovine serum albumin (BSA)
1-kb DNA Ladder
Seakem LE agarose gel powder
1X TBE stock buffer
Ethidium bromide (EtBr, 10 mg/mL)
Tracking dye
A micropipettor and tips

Equipment





A microcentrifuge
37oC water bath
A microwave oven
Horizontal midigel (approximately 20 20 cm) apparatus with combs
Power supply
Photography equipment and supplies

Part1 Dissect 8

Part C. Restrict and Analyze Phage Genomic DNA

Procedure
A. Digest your phage genomic DNA.
1. Gently mix your DNA sample by either flicking the closed tube with your
finger or vortexing it on low.
2. Incubate at 65oC for 10 minutes, then quickly place the tube on ice.
Quick spin (i.e., for less than a minute).
3. Set up the reactions shown in Dissect table 2 in appropriately labeled
microcentrifuge tubes. There will be a total of six tubes. Each has a final
volume of 20 L.1
4. Mix each tube gently but well by flicking it with your finger.

Dissect table 2
Reactions to set up for digesting
phage genomic DNA.

Solution:

Tube, amount
1

10X Reaction Buffer

2 L

2 L

2 L

2 L

2 L

2 L

Phage genomic DNA

0.5 g

0.5 g

0.5 g

0.5 g

0.5 g

0.5 g

2 L

2 L

2 L

2 L

2 L

2 L

BamHI

10 U

ClaI

10 U

EcoRI

10 U

HaeIII

10 U

10X BSA

HindIII

10 U

ddH2O

to 20 L

to 20 L

to 20 L

to 20 L

to 20 L

to 20 L

5. Quick spin the tubes, then incubate in a 37 oC water bath for at


least 2 hours.
6. Quick spin the tubes and place on ice or store in the freezer until you are
ready to perform the next series of steps.
B. Electrophorese your sample.
1. Set up the gel apparatus according to your instructors specifications,
unless it has been set up for you.
2. Prepare enough 0.8% (weight/volume) agarose gel to fill the gel former.
a. Weigh out the appropriate mass of agarose powder; transfer the powder to an Erlenmeyer flask.
b. Add the appropriate volume of 1X TBE buffer to the agarose powder;
swirl to mix.
c. Heat the mixture in the microwave until it boils (usually 1 to
2 minutes).

Part1 Dissect 9

d. Using a mitt or heat-resistant glove, very carefully remove the flask


from the microwave; swirl to mix. Taking care not to splash the hot
liquid or touch the hot flask, examine the solution for small clumps,

Part C. Restrict and Analyze Phage Genomic DNA

often transparent, of agarose. If you see any clumps, return the flask
to the microwave oven and heat for a few seconds. Swirl, examine
again. If the solution appears to be uniform, allow it to cool slightly. If
the solution boils over, you must start over!
e. Check the volume of the solution. If the volume has decreased, bring it
back to the original volume using ddH2O. Swirl to mix.
f. Allow solution to cool to between 50oC and 60oC (very warm to the
touch but not hot enough to burn).
g. Once sufficiently cooled, put on latex gloves; then, add the EtBr to a
final concentration of 0.5 g/mL in the agarose solution.

Food
for Thought
EtBr is a known mutagen
and a suspected carcinogen
as well. Whats the difference between the two?

F
 or example, to prepare a 100-mL agarose gel solution containing
0.5 g/mL EtBr from a 10-mg/mL EtBr stock:
Total micrograms of EtBr needed in the 100-mL gel solution =
0.5 g/mL 100 mL = 50 g EtBr
(10 mg/mL = 10 g/L)
Total volume of EtBr stock needed = 50 g = 5 L

10 g/L

Extreme caution must be exercised when handling EtBr-containing


samples. EtBr is a mutagen that intercalates between the bases in DNA.
Wear gloves!

The Big Picture


An entire genome is very large. For example, the
human genome contains over 3 billion (3 x 109) base
pairs (bp); DNA from mycobacteriophages typically
ranges from 105 to 106 bp. These sizes are far too
big to be analyzed at one time in their entirety. DNA
sequencing takes time, special equipment, other
resources that are hard to come by or expensive,
and technical expertise that a novice researcher
might not have. A special class of enzymes known
as nucleases can be used to rapidly compare
DNA sequences.
DNA can be analyzed in a variety of ways. The general
strategy is to digest (cut, cleave, or restrict) the DNA
into fragments of manageable size using nucleases
known as restriction endonuleases or restriction
enzymes (REs). REs are bacterial enzymes that cut
double-stranded DNA (dsDNA) at specific sequences.
Once cut, the mixture of dsDNA fragments is separated, visualized, and analyzed.

Part1 Dissect 10

Part C. Restrict and Analyze Phage Genomic DNA

h. Pour the agarose-containing 0.5-g/mL EtBr onto the prepared gel


plate being careful not to introduce bubbles into the solution. Immediately insert the comb.
i. Allow the gel solution to cool and harden (approximately 20 to 30
minutes). During the cooling time, prepare the samples for electrophoresis. Once the gel has hardened, either transfer to electrophoresis
chamber that contains 1X TBE buffer or pour enough 1X TBE buffer
onto the gel to cover the gels surface completely so that the agarose
does not dry out. (For the appropriate step, ask your instructor.)
3. Prepare the DNA standard by placing the following in a microcentrifuge
tube:


0.5 g 1-kb DNA Ladder,


1 L 10X Restriction Enzyme Buffer, and
ddH2O to 10 L.

Mix gently. Quick spin.


4. Add 2 L of tracking dye to each of your reaction tubes (Tubes 1 to 6)
and to the 1-kb Ladder. Mix gently. Quick spin.
5. Place all samples in a 65oC heating block or water bath for 5 minutes.
Immediately place on ice to cool. Quick spin.
6. If you have not yet transferred the gel into the electrophoresis chamber
containing buffer (1X TBE), do that now. The gel should be oriented such
that the wells are closest to the cathode (black electrode). Very gently
remove the comb from the gel.

Watch the gel


for 1 to 2 minutes
to ensure that the
dye runs out of
the wells and
toward the anode.

7. Using a fresh tip on your micropipettor for each sample, carefully load
the gel with 10 L of each sample in the following order (left to right):
1-kb Ladder, Tube 1 (undigested DNA), Tube 2 (BamHI),
Tube 6 (HindIII).
8. Plug the electrodes into the appropriate slots on the power supply. Turn
on the power supply and set to run at 100V.
9. Electrophorese until bromophenol blue (BPB) is within 1 to 2 cm of the
end of the gel closest to the anode (red electrode). This will take approximately 30 to 45 minutes. Turn off the power supply.
C. Photograph the gel.
1. Carefully remove the gel plate from the electrophoresis chamber.
2. Following your instructors directions, photograph your gel.

!
Part1 Dissect 11

Ultraviolet light must be used to visualize the DNA fragments (bands) in


the gel. UV burns retinas and skin. Remember to wear eye and face
protection and clothes that protect skin.

Part C. Restrict and Analyze Phage Genomic DNA

10.0
4.0 kb

Dissect fig. 2
A properly labeled illustration
of an agarose gel containing
restricted DNA.

HindIII

HaeIII

EcoRI

Clal

BamHI

Undigested
DNA

kb Ladder

3. Use a permanent marker to label your gel photograph as indicated in


Dissect fig. 2. Include the date and your initials.

3.0 kb
2.0 kb
1.5 kb

Food
for Thought

1.0 kb

Why do the shorter fragments


move through the gel faster?

Your name
Date
Phages name

0.5 kb

D. C
 ompare the results from your gel with those for other mycobacteriophages in the SEA website to ascertain whether you have discovered
a potentially unique phage.
T
 he relationship between size and distance migrated is logarithmic and can
be used to estimate the size of the fragments generated with each restriction enzyme.
1. For each restriction enzyme tested, estimate the length of each visible
fragment.
a. For the molecular weight markers:

It is possible that
the longer fragments will not be well
resolved on your gel.
Use data only from
fragments that are
easily identifiable.
Use the sample gel
shown in Dissect
fig. 2 as a guide for
identifying the fragments in the standard.

i. Open a spreadsheet program on your computer and label the columns Fragment length (bp), Log fragment length (log bp), and
Distance migrated (cm).
Part1 Dissect 12

Part C. Restrict and Analyze Phage Genomic DNA

ii. Enter into your spreadsheet the size (bp) of the DNA fragment using
the fragment lengths for the appropriate molecular weight markers
indicated in Dissect table 3.
Fragment length (bp)

Dissect table 3
Format for recording data on
spreadsheet according to
DNA-fragment size.

Log fragment length


(log bp)

Distance migrated
(cm)

10,108
9,180
8,144
7,126
6,036
5,090
4,072
3,054
2,036
1,636
1,018
506
396
344
298
220
201
154

iii. Use the appropriate features of your spreadsheet program to fill in


the values of the second column (log bp).
iv. Use the features of your spreadsheet program to plot a graph of
log of fragment length (bp, y-axis) versus distance migrated
(cm, x-axis). Determine the slope (m) and y-intercept (b) of the
generated line.
b. For the restriction-enzyme-generated fragments:
i. Use a ruler to measure the distance (cm) migrated by each fragment, measuring from the bottom of the well to the center of the
visible fragment. Enter this information into the spreadsheet.
ii. Estimate the log of the size of each fragment using the equation of
a straight line, y = mx + b, in the third column under each restriction enzyme in Dissect table 4.
iii. Use your spreadsheet to calculate the estimated size of each generated fragment (take the anti-log (i.e., 10 y) of the y value). This is the
fourth column under each restriction enzyme in Dissect table 4.

Part1 Dissect 13

Part C. Restrict and Analyze Phage Genomic DNA

Name of Restriction Enzyme 1


Fragment
number

Distance
migrated
(cm)

log fragment
length

Fragment
length
(bp)

Name of Restriction Enzyme 2


Fragment
number

Distance
migrated
(cm)

log fragment
length

Etc.

Dissect table 4

Fragment
length
(bp)

2. Access the phage genomic DNA restriction analysis data at the SEA website (www.hhmi.org/sea). Record in your notebook the differences and
similarities between the results generated from the restriction analysis
of your phage genomic DNA and of the phages at the SEA website. These
data will help you decide whether your phage is a good candidate for the
genomics portion of the course.
E. U
 sing Decision Tree #3 as a guide, decide whether your phage is a
viable candidate to submit for sequencing and subsequent bioinformatic analysis, and prepare to present your argument to your colleagues.
It is possible that not all of the phages isolated by your class will advance to
the next procedure.

Notes
To choose the sample to be sent for sequencing, you can, as a class, add
criteria to those listed in Decision Tree #3.
As an individual, you are encouraged to lobby for your phage, but your
argument must be based on the scientific guidelines outlined in Decision
Tree #3 and any additional criteria you have come up with.

Questions
1. The photograph of Jesses gel looked exactly like the photograph of the gel
depicted in step C.3 (Dissect fig. 2).

a. Should Jesses sample be selected for sequencing? Explain.

b. From what you can see in the figure, are there any restriction enzymes that
do not cut Jesses phage genomic DNA? Explain.

2. Cam added 0.40 g agarose to 50 mL 1X TBE buffer. Will this result in a


0.8% gel? Explain.

Part1 Dissect 14

Decision Tree #3
From DNA to Informatics

?
Phage lysate

Is the titer high enough to obtain sufficient genomic DNA (usually >107pfu/mL)?
Isolate genomic DNA

NO

YES

Is the DNA yield high enough?


(>10 g TOTAL)

Restriction analysis and


electrophoresis

YES

NO

Does the undigested sample run as a


single band?

NO

YES

Is the restriction pattern unique?

ARCHIVE
SAMPLE

Part1 Dissect 15

NO

MAYBE

YES

SEQUENCING AND
INFORMATICS CANDIDATE

Part C. Restrict and Analyze Phage Genomic DNA

Procedural Notes
1.
Students must be made aware that the units listed in the table vary. Consequently, calculations must
be made regarding the appropriate volume of phage genomic DNA needed to have 0.5 g of sample
in each digest. Also, although most restriction enzymes are provided at a concentration of 10 units/L,
some are not. Therefore, you should tell students either the actual enzyme concentration of each
restriction enzyme or where to find the information.
2.
For the undigested phage-DNA control, students can use any salt-containing Reaction Buffer.

Part1 Dissect F9

Part D. Evaluate Genomic DNA Quality and


Send DNA to Sequencing Center

Overview
The purpose of this protocol is to assess the quality of the genomic DNA samples your students have selected and then to
use that information to choose which genomic DNA sample
to send to your assigned sequencing center. The procedure
has been standardized so that, regardless of the sequencing
center used, they can determine whether the DNA is of sufficient quantity and quality for sequencing. The procedure
entails running an agarose gel against the standards provided by the SEA laboratory and sending an image of the gel,
along with other information to your center for approval.
Please note: send only approved samples for sequence analysis. After the class has evaluated the samples and chosen
the phage(s) for sequencing, you will send an e-mail to your
sequencing center. You will be informed of your sequencing
facility when you receive your QC DNA Standard Kit from the
SEA laboratory. The e-mail should include the following:
1. An image of the gel saved as either a TIFF, JPG, or PDFfile. (The file type will depend on your sequencing center.
You will be informed of the type when you receive information regarding your sequencing center and contact
person).

 he DNA must not be shipped until approval is received from the


T
sequencing facility.

Supplies
Supplies and reagents (provided
by SEA laboratories and prepared by instructors):
Microcentrifuge tubes (enough for
DNA samples and standards)
DNA Standard Kit containing:
> Marker 2 Lambda HindIII Size
Standard (50 ng/L)
> Lambda DNA Mass Standards
(15, 31, 63, 125, 250,
500 ng/5 L; 25 L each)
Nuclease-free ddH2O
Agarose (ultra pure)
Ethidium bromide (EtBr; ultra pure,
10 mg/mL stock)
Loading dye (6X)1
Glycerol
TAE buffer (50X)
Each student will also need
access to
A micropipettor (20-L) and tips
Ice

Equipment
A microcentrifuge
An agarose gel electrophoresis apparatus with a 1.5-mm comb
Power supply and electrodes
Photography equipment and
supplies
65OC heating block or water bath

2. An electronic version (available on the SEA Wiki) of the


completed NGRI QC Report form.
The protocol must be followed exactly as written.
Key Words and Concepts
Quality control.

Part1 Dissect F10

Part D. Evaluate Genomic DNA Quality and Send DNA to Sequencing Center

Implementation Timeline

LAB
Dilute the following:
50X

TAE to 1X with
ddH2O.
6X
 loading dye to 1X
with ddH2O and glycerol.

LAB: the day of the lab; t1D: the day before the lab; t2D: two days before the lab; etc.

Part1 Dissect F11

Part D. Evaluate Genomic DNA Quality and


Send DNA to Sequencing Center

Objective
The purpose of this protocol is to assess the quality of the genomic DNA samples selected as candidates for sequencing. The response from your assigned
sequencing center, in addition to the criteria listed in Decision Tree #3, must
be used to decide which sample to send.2

Supplies









Microcentrifuge tubes
Nuclease-free ddH2O
Seakem LE agarose gel powder
1X TAE buffer
Ethidium bromide (EtBr, 10 mg/mL)
A micropipettor (20-L) and tips
Loading dye
Glycerol
Ice
QC DNA Standard Kit containing Marker 2 Lambda HindIII Size Standard and Lambda DNA Mass Standards (15, 31, 63, 125, 250, 500 ng/5
L)

Equipment




A microcentrifuge
An agarose gel electrophoresis apparatus with a 1.5-mm comb
Power supply and electrodes
Photography equipment and supplies
65OC heating block or water bath

Procedure
A. Prepare a 1% agarose gel in 1X TAE buffer containing 0.15-g/mL EtBr.

Although this is a slightly different electrophoresis protocol from


that used for restriction analysis, the same precautions must be
taken when handling the ethidium bromide and to limit exposure
to ultraviolet radiation.

B. Prepare the genomic DNA sample for electrophoresis.


1. Place your genomic DNA sample in the 65oC heating block or water bath
for 5 minutes. Immediately place on ice for 1-2 minutes. Quick spin.
2. Transfer 1 L of your DNA to a clean microcentrifuge tube.
3. Bring the volume to 5 L with 1X loading dye.

Part1 Dissect 16

Part D. Evaluate Genomic DNA Quality and Send DNA to Sequencing Center

C. Retrieve QC DNA Standard kit from 20oC freezer. Place the samples in
a 65oC heating block or water bath for 5 minutes. Immediately place on
ice to cool. Quick spin.
D. Electrophorese the samples.
1. Fill gel chamber with 1X TAE buffer.3
2. Mix all samples by flicking the closed tubes with your fingers.
3. Quick spin all tubes.
4. Load the gel with the specified sample in the precise order indicated in
Dissect fig. 3.
5. Run the gel for approximately 40 minutes at 120V.
6. Turn off power supply.
E. Photograph the gel and save the image in the appropriate file format.
F. Analyze the genomic DNA for quality, molecular weight, and quantity
using the guidelines delineated in Decision Tree #4.
Wells

Left

5 L 15-ng Lambda standard


5 L 31-ng Lambda standard

Dissect fig. 3
The order in which the
samples should be
loaded into the
agarose gel.

5 L 63-ng Lambda standard


3 L Marker 2 Lambda
5 L your DNA
3 L Marker 2 Lambda
5 L 125-ng Lambda standard
5 L 250-ng Lambda standard
5 L 500-ng Lambda standard

Right

(+)
Part1 Dissect 17

Polarity

()

Part D. Evaluate Genomic DNA Quality and Send DNA to Sequencing Center

G. Using Decision Tree #4, determine, as a group of collaborators, the


phage you will send to your assigned sequencing center for qualitycontrol approval. For sequence submission, use the sequencing centers
decision and the information gathered from Desision Tree #3 to determine which sample should be submitted for sequencing. Only one can be
submitted for sequencing.
H. Send DNA to the sequencing center according to instructions on the
SEA website.

Questions
1. Using the information presented in Decision Tree #4, why do you think multiple
lanes of lambda DNA standards must be run on your gel?
2. Nucleases are enzymes that degrade nucleic acids. If your genomic DNA
has nuclease contamination, what type of pattern would you expect to see
on the gel?

Part1 Dissect 18

Decision Tree #4
DNA to Sequencing Center

?
Agarose gel electrophoresis

Is the estimated molecular size of the predominant band on the agarose gel >23 kbp?

ARCHIVE
SAMPLE

NO

YES

Are several low-molecular-weight bands


(RNA contamination) present on the gel?

YES

Treat with RNase I

NO

Is there evidence of DNA degradation?

(smearing evident under the predominant band)

YES

Is the smearing evident all the way down


the lane?

YES

NO

NO

Do you have at least 10 g of purified DNA?

NO
SUBMIT DNA FOR
APPROVAL

REPEAT
DNA
PREP
Part1 Dissect 19

YES

Part D. Evaluate Genomic DNA Quality and Send DNA to Sequencing Center

Procedural Notes
1.
1X loading dye is required to load the samples. To prepare 600 L of loading dye, mix 400 L of
nuclease-free ddH2O, 100 L of 100% glycerol, and 100 L of 6X loading dye. Vortex well to mix. Quick
spin.
2.
In order to conserve the DNA mass standards, HindIII- digested lambda standard can be used on the
preliminary (or test) QC gel as a means to obtaining a rough estimate of the DNAs size, quality, and
quantity.
3.
Unlike the previous electrophoresis procedure, in which students used TBE in both the gel and the
buffer chamber, in this one, TAE is used because it enhances the resolution of larger DNA fragments.

Part1 Dissect F12

TAB
GOES
HERE

Glossary for Part 1

ablate. To remove or destroy.


adsorb (noun adsorption). To take up and hold;
adhesion in a thin layer of molecules to the surfaces of solid bodies or liquids with which they are
in contact.
agar. A gelatinous colloidal extract of a red alga
used in culturing media.
agarose. A polysaccharide derived from agar.
agarose gel electrophoresis. A method used
to separate DNA, RNA, or protein molecules
by size; a technique in which an electric field is
applied to move charged molecules through an
agarose matrix.
aliquot. To dispense a solution in exact fractions
or quantities.
amino acid. A relatively small organic molecule
consisting of an amino group on one end and
a carboxyl group at the other; the protein
building block.
analyte. A substance or chemical constituent that
is determined in an analytical procedure.
anode. A positively charged electrode; the
electrode toward which nucleic acids migrate in
an electric field.
aseptic technique. A method used to maintain
sterility and prevent contamination by unwanted
organisms.
assay. A procedure for measuring a property or
concentration of an analyte.
autoclave. To sterilize using superheated steam
and high pressure; cdjc, an apparatus for doing
the aforementioned.
autonomous replication. Capable of independent
reproduction.
bacteriophage (also phage or phages). A virus
that infects bacteria.
biodegradable. Capable of being broken down in
nature.
bioinformatics. The field of science in which
biology, computer science, and information technology merge to form a single discipline. The
ultimate goal of the field is to enable the discovery
of new biological insights as well as to create a

global perspective from which unifying principles


in biology can be discerned.
biological database. A large, organized body
of persistent data, usually associated with computerized software designed to update, query, and
retrieve components of the data stored within
the system.
capsid. The protein shell of a virus.
carcinogen. A substance or agent that causes
cancer.
cloning. The process of creating an identical copy
of something.
codon. A specific sequence of three consecutive
nucleotides that is part of the genetic code; it
either specifies a particular amino acid or it starts
or stops protein synthesis.
contaminate (noun contamination). To infect.
contractile. Capable of contracting.
cos. Abbreviation for cohesive.
covalent bond. A chemical bond formed by the
sharing of electrons between atoms.
culture (e.g., bacteria culture). Bacteria grown
in a prepared medium.
ddH2O. Abbreviation for double-distilled water.
In the SEA project, water should be double-distilled at a minimum. Filtered water (e.g., Millipore)
is ideal. Regardless of water availability, consistency of preparation is essential (i.e., always use
the same water).
ddNTP. Abbreviation for dideoxynucleotide
triphosphate.
decant. To draw off without disturbing the
sediment or lower layers.
denaturation (protein/DNA). To modify the
molecular structure, especially by heat, chemicals,
UV radiation, or force, so as to destroy or diminish
some of the original properties and/or biological
function.
deoxynucleotide triphosphate. A building block
used in deoxyribonucleic acid (DNA) synthesis.
desiccate (noun desiccation). To dry up;
dehydrate.
Part1 Glossary 1

Glossary for Part 1

dideoxynucleotide triphosphate. A modified


DNA building block that prevents nucleic acid
chain elongation during synthesis; a chemical
used as a chain terminator during DNA synthesis.
digest. To break down by chemical action using
enzymes.
disinfectant. A chemical that destroys harmful
microorganisms.

homolog (or homologue). Have the same relative


position, value, or structure.
icosahedron. Any polyhedron having 20 faces.
incubate. To cause or aid the development of.
inoculate. To introduce a microorganism into.
isolate. To distinguish from, purify.

dNTP. Abbreviation for deoxynucleotide


triphosphate.

light microscope (or optical microscope). A


microscope that uses visible light and a system
of lenses to magnify images of small samples.

electron microscopy. A type of microscopy that


uses electrons to illuminate a specimen and create
an enlarged image.

lipids. Important components of membranes;


molecules that are generally insoluble in water and
soluble in nonpolar organic solvents.

electroporation. The process of using an electric


current to produce pores (or holes) in an organisms membrane through which molecules
can pass.

lysate. A solution containing the contents of


disrupted cells.

elute. To remove adsorbed material from an adsorbent by means of a solvent.


empirical assay or test. An assay used to determine the amount of phage lysate needed for an
optimal infection that generates a high-titer lysate.
filter sterilization. A technique used to sterilize
solutions by excluding contaminants on the basis
of size.
fission. Splitting or breaking up into parts.
flagellum (plural flagella). An elongated appendage
that projects from a cell and is the primary organ
of motion of many microorganisms.
gel electrophoresis. A technique for separating
deoxyribonucleic acid, ribonucleic acid, or protein
molecules using an electric current applied to a gel
matrix.
genome. An organisms whole hereditary information. It is encoded in the DNA (or, for some viruses,
RNA). This includes both the genes and the noncoding sequences of the DNA.
genomics. The study of an organisms entire
genome.
genotype (adverb genotypically). All or part of
the genetic constitution of an individual or group.

lyse (adjective lytic). To cause to undergo lysis.


lysis (plural lyses). Refers to the death of a
cell caused by cell membrane disruption; act of
loosening.
lysogen. Bacterium that contains a prophage.
lysogeny. The mechanism by which a nave
bacterial cell becomes infected with a prophage.
macromolecules. Large, covalently bonded
chemical structures.
magnification. The apparent enlargement of an
object by an optical instrument.
media. Liquid or semisolid solutions in which
microorganisms or cells can experience growth.
micron. A unit of measure equal to one-millionth
of a meter, or one one-thousandth of a millimeter.
morphology. The appearance of an organism (e.g.,
size, shape, color, pattern, structure).
MSDS. Abbreviation for Material Safety Data
Sheet; information accompanying chemicals
that describes toxicity, flammability, solubility,
hazards, etc.
mutagen. A physical or chemical agent that
changes the genetic information (usually DNA) of
an organism and thus increases the frequency of
mutations above the natural background level.
mutation. A significant and basic alteration; change.

Part1 Glossary 2

Glossary for Part 1

mycobacteriophages. Phages that specifically


infect different species of bacteria called
mycobacteria.

Mycobacterium (plural mycobacteria). A bacterial genus. The Latin prefix bnXd- means both
[jc\jh and lVm. In general, these bacteria have
waxy compounds in the cell wall.
Mycobacterium smegmatis (also M. smegmatis). An acid-fast bacterial species in the genus
BnXdWVXiZg^jb. It is a Gram-positive bacterium
that is generally considered nonpathogenic to
healthy humans.
myoviridae. A family of bacteriophages.
nave bacterial cell. A bacterium that has not
been infected by a phage.
negative control. An experimental sample used
to generate an outcome in which nothing happens.
nomenclature. Name or designation.
nuclease. An enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits
of nucleic acids.
nucleic acid. A macromolecule consisting of
covalently linked nucleotides. These molecules
carry genetic information or form structures
within cells.
nucleotide. An organic molecule consisting of a
sugar molecule, a nitrogenous (i.e., nitrogencontaining) base, and a phosphate group; the
building block of nucleic acids.
open reading frame. A segment of DNA between
the initiation and stop codons that potentially
encodes a protein.
orf. Abbreviation for open reading frames.
PB. Abbreviation for phage buffer.
pfu. Abbreviation for plaque-forming unit.

pilus (plural pili). Hairlike structure on the


surface of a bacterium.
plaque assay. A protocol used to determine
the number of plaque-forming units (pfu) in a
given solution.
plaque-forming unit (pfu). A viral particle that
infects a cell and propagates itself radically to form
a plaque; also called an infective center. It is used
to estimate the number of viral particles in a solution; however, all the particles are not capable of
infection, so this number is lower than the actual
number of viral particles.
plaque morphology. The way the plaque looks
(such as round or oblong, clear or cloudy, large or
small).
plaque titer. A method of enumerating (counting) phage particles on the basis of the number of
plaque-forming units per unit volume.
podoviridae. A group of bacteriophages characterized by virions with short noncontractile tails and
isometric or elongated heads (there are no known
mycobacteriophages in this group).
polymerase chain reaction. A chemical reaction in which DNA is synthesized in vitro using
repeated cycles of DNA denaturation (i.e., strand
separation), primer annealing, and extension (DNA
synthesis). The reaction employs a high-temperature DNA polymerase (an enzyme), deoxynucleotide triphosphates (building blocks), cofactors (to
make the reaction run efficiently), and buffer (to
maintain constant pH).
polynucleotide solution. Solution containing
nucleotides that have been hydrolyzed by the enzymatic activity of nucleases.
polysaccharide. A polymer consisting of covalently linked simple sugars (monosaccharide building
blocks).

phenotype (adverb phenotypically). A visible


physical characteristic or behavior.

positive control. An experimental sample used to


demonstrate the positive result of an experimental
outcome.

photoelectronmicrograph. A photograph or
similar image taken through an electron
microscope.

pristine (e.g., pristine bacterial lawn). Original


state; not spoiled, corrupted, or polluted.

Part1 Glossary 3

Glossary for Part 1

prokaryotes (prokaryotic). Usually refers to


bacteria; single-celled organisms that usually contain simple circular DNA chromosome(s), lack a
nuclear membrane, and have few organelles other
than ribosomes.
prophage. A phage genome incorporated into
bacterial DNA.
proteins. Macromolecules consisting of covalently
linked amino acids. These molecules perform a diverse array of functions and include enzymes (e.g.,
endonuclease), transport molecules (e.g., hemoglobin), and structural molecules (e.g., collagen).
putative. Tentative, supposed; assumed to exist.
quality control. Inspection for defects or errors.
reagent. A substance used because of its chemical
or biological activity.
recombination. A genetic combination not found
in either parent but present in the offspring.
replicate. Duplicate; repeat.
repress. To hold back, dampen, restrain.
repressor. A protein that binds to DNA and
prevents its transcription.
ribosome. A macromolecular structure
consisting of RNA and protein that is found in the
cytoplasm of living cells and serves as the site of
protein synthesis.
saturated. Unable to hold or contain more.
serial dilution. A common method of preparing a
series of a particular solution containing progressively lower solvent concentration. Each subsequent sample is prepared from the previous sample
of higher concentration.
sheared DNA. Deoxyribonucleic acid that has
been broken or torn into smaller pieces by the
action of applied force.
spot test (or chemical test). A qualitative or
semiquantitative procedure designed to
indicate the existence of an agent (e.g., a phage),
a chemical compound, or a chemical group with
the aid of a specific reagent.
standard microscope. HZZ light microscope.

Part1 Glossary 4

sterile field. An area devoid of microorganisms.


subculture. A culture (as of bacteria) derived from
another culture.
supernatant. The liquid portion of a mixture
from which the particles have been pelleted by
centrifugation.
temperate phage. A phage that can exist as a prophage in infected cells and that rarely causes lysis.
thermocycler. A machine that repeatedly heats
and cools samples to specific temperatures over a
defined period of time; a laboratory machine used
for the polymerase chain reaction (PCR) process.
titer. The concentration of phage particles in a
given solution.
titration. A method or the process of determining the concentration of a dissolved substance
in terms of the smallest amount of a reagent of
known concentration required to bring about a
given effect in reaction with a known volume of
the test solution.
turbidity. Thickness or cloudiness.
vortex (noun vortexer). To mix.
web lysis or web pattern. The appearance of a
threadlike pattern on the surface of an agar plate
resulting from almost complete lysis of a bacterial
lawn by phage.
wicking. The ability of a substance to draw
another substance into it.

TAB
GOES
HERE

Appendices for Part 1

Contents
Appendix 1
1 The NGRI Troubleshooting Guide
Appendix 2
9 Additional Resources for
NGRI Participants
Appendix 3
15 Archiving and Reporting Your Phage
Appendix 4
21 Format for Research Papers
Appendix 5
23 NGRI Equipment and Supplies
Appendix 6
29 Recipes for Stock Solutions,
Media, and Reagents
Appendix 7
67 Recipes for Bacterial Cultures

Appendix 1. The NGRI Troubleshooting Guide

Problem

Notes, suggestions, and solutions

Contamination of bacterial culture by

Any solution suspected of being contaminated


should be streaked out immediately. In other
words, when in doubt, streak it out! If colonies
form in 24 hours or less, they are probably not
M. smegmatis.

Phage
Bacteria
Fungus

In the meantime, always replace your supplies


with a full complement of fresh media and
reagents.

A
Pristine bacterial lawn.

B
Bacterial culture contaminated with phage.

Bacterial culture contaminated with bacteria (C)


and with fungus (D).

Contamination of a bottle of liquid medium by


Phage
Bacteria
Fungus

One control that will address several potential


points of contamination is the preparation of
a plate on which bacteria in top agar are spread
out. A 5-L spot of PB is then put onto the TA
bacterial lawn (be sure to mark the spot!). If the
spot yields plaques, the PB is contaminated with
phage. Bacterial contamination of the PB will also
be apparent (it will be cloudy). If the TA or the
bacterial cultures are contaminated with other
bacteria, phages, and/or fungi, it will be apparent
over the entire surface of the agar (rather
than in the marked-spot area).
Never underestimate the power of negative
controls!
Treat contaminated media with broad-action
disinfectant (e.g., Lysol or CiDecon) and discard.
This is likely to happen with already-opened
bottles of media that were contaminated by
previous use. When in doubt, set the medium
aside and open another bottle. If the contamination worsens over time, the medium should
be discarded.
Precipitates in media or other solutions can
appear to be contamination even though the
solutions are sterile!

Liquid medium contaminated with


phage (A), bacteria (B), and fungus (C).

Part1 Appendix 1 1

Appendix 1. The NGRI Troubleshooting Guide

Problem

Notes, suggestions, and solutions

A bottle of TA looks contaminated:

Could be precipitated CaCl2 or contamination.


Do not discard TA if there is precipitate that
might be contamination. Rather, use a new
aliquot, and leave the suspect bottle of TA
in the 55C water bath for a day. If the TA
is truly contaminated, it will be obvious by
the next day.

Top agar that looks contaminated (left)


and that is contaminated (right).

A soil sample wont settle:

Soil samples should fill about one-half the


volume of the tube. If there is too much soil
in a sample tube, it will be difficult, if not
impossible, to aspirate enough liquid to
filter-sterilize.
If this happens, rather than obtaining new
samples, the contents of the tube should be
diluted 50:50 into fresh PB and allowed
to resettle.

Too much soil in the tube (right);


correct amount (left).

If you see this:

Part1 Appendix 1 2

The TA was not added.

Appendix 1. The NGRI Troubleshooting Guide

Problem

Notes, suggestions, and solutions

If you see this:

A completely clear TA layer can be either the


result of complete lysis or a plate to which the
bacteria have not been added.
Do a range of lysate concentrations in an infection
experiment, and always prepare a negative (nophage) control.

Plates with no bacteria (right) and with bacteria


completely lysed by phage (left).

If you see this:

When the TA was added, the culture tube was


poorly mixed. Bacteria will drift to one side of
a plate, and the lawn will not be homogeneous.
Be sure to mix the culture well after TA addition.

If you see this:

The TA was not allowed to completely solidify


before the plate was inverted.
Do not move plates until the TA is completely
solid. Before inverting the Petri dishes, check the
TA by gently tipping the plate. It is a good idea
to test with the negative control! If the TA is not
solidified before the inversion of the plate, the
agar will slide or fall onto the lid of the Petri dish,
as shown in the photo.
No rule of thumb applies; the time that it takes for
the TA to completely solidify depends on many
factors, including room temperature and humidity.

Part1 Appendix 1 3

Appendix 1. The NGRI Troubleshooting Guide

Problem

Notes, suggestions, and solutions

If you see this:

Plates were allowed to solidify on a slanted


surface.
This is not a problem.

If you see this:

This is an artifact. When the top agar is poured


onto the plate, the seams (where the TA comes
together) have a different appearance.
This is not a problem.

If you see this:

A (presumably) single plaque appeared to yield


different morphologies when plated out.
If single plaques of both or all morphologies
yield the same assortment of plaque morphologies
each time, the phage is likely pure.
Pick from a plate that has fewer than 10 plaques,
where plaques are far from each other. This increases the likelihood of picking only one plaque.
Perform several rounds of purification, noting
the different morphologies yielded from each
morphology type to be sure that only one phage
has been isolated.

Part1 Appendix 1 4

Appendix 1. The NGRI Troubleshooting Guide

Problem

Notes, suggestions, and solutions

If you see this:

When performing the empirical assay, you may


see a clearing zone within a turbid plaque web
plate.
This happens relatively often, and it does not
indicate contamination. A turbid plaque morphology indicates a temperate phage. A clearing zone
within an area of turbid plaques is likely due to
a phage mutation or the fact that a prophage has
excised from the bacterial genome and entered
the lytic life cycle.
This is not a problem, but you should record the
information and take a photo.

If youre not getting


enough aspirant volume
off flooding 10-plate
preps, do this:

Much of the liquid can be under the agar.


Carefully flip up a corner of the agar or put
pipette under the agar. Do this without spilling
phage lysates, because this can contaminate
the entire bench.
Use a large pipette to avoid sucking bubbles
(and liquid) into the Pipette-Aid filter and thus
clogging the pipettor.

Part1 Appendix 1 5

Appendix 1. The NGRI Troubleshooting Guide

Problem

Notes, suggestions, and solutions

The phage lysate is viscous, or goopy, after


PEG precipitation:

Too much PEG in the phage precipitation


mixture was left behind after centrifugation.
Decant off as much PEG as possible. Let the
tubes sit upside-down to drain excess PEG.
If necessary, perform another low-speed spin
to compact the pellet, and use a Pasteur
pipette to pull off excess PEG.

A pellet that is diffuse and that precipitated


unsuccessfully (right); a compact pellet and
successful precipitation (left).

M. smegmatis culture is clumpy

Baffled ask growth (left) and clumpy,


nonbafed ask growth (right).

Part1 Appendix 1 6

The culture should not be used. THROW IT OUT.


Start a fresh culture in a baffled flask, for efficient
aeration, by inoculating 1 L of fresh medium with
no more than 100 L of a saturated culture.

Appendix 1. The NGRI Troubleshooting Guide

Problem

Notes, suggestions, and solutions

Your DNA gel looks like this:

The solution to each of these problems is


to pour another gel. Take care to load less
sample (B), prepare the gel with buffer rather
than water (C), use a lower voltage (D), and
avoid bubbles when pouring (E).

A
Ideal.

B
Samples overloaded.

C
Gel prepared with water
rather than buffer.

D
Voltage too high.

E
Bubble in lane.

Part1 Appendix 1 7

Appendix 2. Additional Resources for


NGRI Participants

For more information about bacteriophages, mycobacteriophages, and phage


biology and ecology, please see the following resources. This is certainly not
an exhaustive list, but it will give you a place to start learning more about
bacteriophages. If you find a particularly good resource that is not listed,
please notify the SEA staff, and we will add it to our list.

Websites
Phages DB
http://phagesdb.org/
American Society for Microbiology Bacteriophage Group Site:
www.asm.org/division/M/M.html
The Virology Journal All the Virology on the WWW:
www.tulane.edu/~dmsander/garryfavweb.html
Cells Alive! Oh Goodness, My E. coli Has a Virus:
www.cellsalive.com/phage.htm
The Bacteriophage Ecology Group:
www.mansfield.ohio-state.edu/~sabedon

Books
Birge EA. 2006. Bacterial and Bacteriophage Genetics. New York: Springer.
(ISBN 0387239197.)
Cairns J, Stent GS, and Watson JD, eds. 2007. Phage and the Origins of
Molecular Biology, the Centennial Edition. New York: Cold Spring Harbor
Laboratory Press. (ISBN 0879695951.)
Calendar R, ed. 2006. The Bacteriophages. 2nd ed. New York: Oxford
University Press. (ISBN 0195148509.)
Kutter E, and Sulakvelidze A. 2004. Bacteriophages: Biology and
Applications. Boca Raton, FL: CRC Press. (ISBN 0849313368.)

Book Chapter
Sarkis GJ, and Hatfull GF. 2001. Mycobacteriophages. In T Parish and NG
Stoker, eds. Methods in Molecular Biology, Vol. 101: Mycobacteria Protocols.
Totowa, NJ: Humana Press. (ISBN 0896034712.)

Primary Literature and Reviews


Hanauer DI, Jacobs-Sera D, Pedulla ML, Cresawn SG, Hendrix RW, and
Hatfull GF. 2006. Inquiry learning. Teaching scientific inquiry. Science
314(5807): 188081. (PMID: 17185586.)

Part1 Appendix 2 9

Appendix 2. Additional Resources for NGRI Participants

Hatfull GF, Pedulla ML, Jacobs-Sera D, Cichon PM, Foley A, Ford ME,
Gonda RM, Houtz JM, Hryckowian AJ, Kelchner VA, Namburi S, Pajcini KV,
Popovich MG, Schleicher DT, Simanek BZ, Smith AL, Zdanowicz GM, Kumar
V, Peebles CL, Jacobs WR Jr, Lawrence JG, and Hendrix RW. 2006. Exploring
the mycobacteriophage metaproteome: phage genomics as an educational
platform. PLoS Genetics 2(6): e92. Epub 2006, June 9. (PMID: 16789831.)
Bacteriophages are the most abundant forms of life in the biosphere
and carry genomes characterized by high genetic diversity and mosaic
architectures. The complete sequences of 30 mycobacteriophage genomes
show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357
proteins belonging to 1,536 phamilies of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total
number of phamilies in the mycobacteriophage population. These phamilies
contain 2.19 proteins on average; more than half (774) of them contain just
a single protein sequence. Only six phamilies have representatives in more
than half of the 30 genomes, and only threeencoding tape-measure proteins, lysins, and minor tail proteinsare present in all 30 phages, although
these phamilies are themselves highly modular, such that no single amino
acid sequence element is present in all 30 mycobacteriophage genomes. Of
the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to
previously reported proteins, reflecting the enormous genetic diversity of
the entire phage population. The abundance and diversity of phages, the
simplicity of phage isolation, and the relatively small size of phage genomes
support bacteriophage isolation and comparative genomic analysis as
a highly suitable platform for discovery-based education.
McNerney R. 1999. TB: the return of the phage. A review of fifty years
of mycobacteriophage research. Int J Tuberc Lung Dis 3(3): 17984.
(PMID: 10094316.)
The first mycobacteriophage was isolated in 1947, and since that time,
more than 250 of these viruses have been identified. Phages have made a
significant contribution to our knowledge of mycobacteria over the past
50 years, and following the development of typing techniques in the
1960s and 1970s, they were widely used in epidemiological studies of
tuberculosis. Unfortunately, attempts to use lytic phages therapeutically
during tuberculosis infection have so far failed to elicit cure in experimentally infected animals. During the past decade, phages have become
important in molecular studies of mycobacteria, both in terms of studying
phage biology and as tools in recombinant DNA technology, thus facilitating
the investigation of mycobacterial pathogenesis. Today their potential
as diagnostics reagents is also being realized with the development of
exciting new techniques for rapid bacterial detection and drug susceptibility testing. This review outlines the history of these remarkable organisms,
from their discovery 50 years ago to the current developments in rapid
diagnostic techniques.

Part1 Appendix 2 10

Appendix 2. Additional Resources for NGRI Participants

McNerney R, and Traor H. 2005. Mycobacteriophage and their application


to disease control. J Appl Microbiol 99(2): 22333. (PMID: 16033452.)
The resurgence of tuberculosis and emergence of drug-resistant disease
has stimulated fresh research into mycobacteriophage. Studies are under
way to develop phage-based tools for therapeutic and diagnostic use.
Previous attempts at mycobacteriophage therapy in experimentally infected
animals were not successful and alternative strategies of phage delivery
that enable killing of intracellular bacteria are required. Replication of
mycobacteriophage provides a simple means of detecting viable bacteria and
good progress has been made toward the development of new phage-based
diagnostic tools. When screening isolates for resistance to the major anti
tuberculosis drug rifampicin phage-based tests have been shown to have
high sensitivity. For the diagnosis of pulmonary tuberculosis evaluation
studies indicate that current phage tests are not as sensitive as traditional
culture methods. Further trials are needed to determine whether they might
have a role in the detection of smear negative tuberculosis. A second generation of phage tests are under development following the construction of
luciferase reporter phage. Preliminary data suggests they may offer rapid
detection of mycobacteria and simple screening for drug resistance. The
potential of mycobacteriophage to detect and treat other mycobacterial diseases remains largely unexplored.
Pal C, Maci MD, Oliver A, Schachar I, and Buckling A. 2007. Coevolution
with viruses drives the evolution of bacterial mutation rates. Nature
450(7172): 107981. (PMID: 18059461.)
Bacteria with greatly elevated mutation rates (mutators) are frequently
found in natural and laboratory populations, and are often associated with
clinical infections. Although mutators may increase adaptability to novel
environmental conditions, they are also prone to the accumulation of dele
terious mutations. The long-term maintenance of high bacterial mutation
rates is therefore likely to be driven by rapidly changing selection p
ressures,
in addition to the possible slow transition rate by point mutation from
mutators to non-mutators. One of the most likely causes of rapidly changing
selection pressures is antagonistic coevolution with parasites. Here we show
whether coevolution with viral parasites could drive the evolution of bacterial mutation rates in laboratory populations of the bacterium P
seudomonas
fluorescens. After fewer than 200 bacterial generations, 25% of the populations coevolving with phages had evolved 10- to 100-fold increases in
mutation rates owing to mutations in mismatch-repair genes; no populations
evolving in the absence of phages showed any significant change in mutation
rate. Furthermore, mutator populations had a higher probability of driving
their phage populations extinct, strongly suggesting that mutators have
an advantage against phages in the coevolutionary arms race. Given their
ubiquity, bacteriophages may play an important role in the evolution of
bacterial mutation rates.

Part1 Appendix 2 11

Appendix 2. Additional Resources for NGRI Participants

Papke RT, and Doolittle WF. 2003. Phage evolution: new worlds of genomic
diversity. Curr Biol 13(15): R6067. (PMID: 12906814.)
A recent comparative survey of genomes of phages infecting mycobacteria
reveals a vast combinatorial network of gene rearrangements and may provide general models for pattern and process in genome evolution.
Pedulla ML, Ford ME, Houtz JM, Karthikeyan T, Wadsworth C, Lewis JA,
Jacobs-Sera D, Falbo J, Gross J, Pannunzio NR, Brucker W, Kumar V,
Kandasamy J, Keenan L, Bardarov S, Kriakov J, Lawrence JG, Jacobs WR,
Hendrix RW, and Hatfull GF. 2003. Origins of highly mosaic mycobacteriophage genomes. Cell 113(2): 17182. (PMID: 00928674.)
Bacteriophages are the most abundant organisms in the biosphere and
play major roles in the ecological balance of microbial life. The genomic
sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent
the largest unexplored reservoir of sequence information in the biosphere.
Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling
evidence for the role of illegitimate recombination in horizontal genetic
exchange. The promiscuity of these recombination events results in the
inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections,
and autoimmune diseases such as human lupus. While the role of phages
as vehicles of toxin genes is well established, these observations suggest
a much broader involvement of phages in bacterial virulence and the
host response to bacterial infections.
Pham TT, Jacobs-Sera D, Pedulla ML, Hendrix RW, and Hatfull GF. 2007.
Comparative genomic analysis of mycobacteriophage Tweety: evolutionary
insights and construction of compatible site-specific integration vectors for
mycobacteria. Microbiology 153(Pt. 8): 271123. (PMID: 17660435.)

Part1 Appendix 2 12

Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium


smegmatis. It has a viral morphology with an isometric head and a long
flexible tail, and forms turbid plaques from which stable lysogens can be
isolated. The Tweety genome is 58,692 bp in length, contains 109 proteincoding genes, and shows significant but interrupted nucleotide sequence
similarity with the previously described mycobacteriophages Llij, PMC, and
Che8. However, overall the genome possesses mosaic architecture, with gene
products being related to other mycobacteriophages such as Che9d, Omega,
and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the center of the genome, and a putative attP site has
been identified within a short intergenic region immediately upstream of
int. This Tweety attP-int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and
slow-growing mycobacteria through plasmid integration at a chromosomal
locus containing a tRNA(Lys) gene. These vectors are maintained well in the
absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction
of complex recombinants with genes integrated simultaneously at different
chromosomal positions.

Appendix 2. Additional Resources for NGRI Participants

Rohwer F. 2003. Global phage diversity. Cell 113(2): 141. (PMID: 12705861.)
Ten new mycobacteriophage genomes presented by Pedulla et al. (2003)
show that most phage diversity remains uncharacterized. Extrapolation
suggests that less than 0.0002% of the global phage metagenome has been
sampled. The new genomes also contain a number of potential virulence factors that may be important in pathogenesis.

Part1 Appendix 2 13

Appendix 3. Archiving and Reporting Your Phage

Overview
Three sets of samples of each phage lysate must be archived. Two sets will
be sent to the Howard Hughes Medical Institute (one of which will be sent to
the University of Pittsburgh); one set will be held at your institution for your
archiving. You must enter information for all phages isolated, whether or not

they are sequenced, onto the PhagesDB database site.

Please check the SEA Wiki for the most up-to-date archiving protocol
information.

I. Create an account at PhagesDB


1. Go to http://phagesdb.org/
2. On the Account pane to the left, click Register. (See Appendix 3 fig. 1)
3. Fill out the information required to establish an account.
4. You MUST follow the link in the email you receive to activate your account (Emails are sent immediately. If you do not receive the email soon
after registering, check to make sure that no-reply@phagesdb.org is not
being filtered into a spam file by your email program).

Appendix 3 fig. 1
The Account tab. Use this to
create a new account or to log
into PhagesDB.

Part1 Appendix 3 15

Appendix 3. Archiving and Reporting Your Phage

II. Check that your phage name has not yet been used
1. Go to http://phagesdb.org/
2. On the Account pane to the left, click Login. (See Appendix 3 fig. 1)
3. Log in using your new account information.
4. Using the Data Entry Tab, pull down to Add new phage. (See Appendix 3 fig. 2)
5. Enter your phage name into the Phage Name field.
6. If you receive notification that the name has already been used, you must
modify/change the name (see Appendix 3 fig. 2).
7. Once your phage name has been decided, assign a Phage Designator as
outlined below and assemble your phage information.
Appendix 3 fig. 2
The Data Entry tab. Use this to
check on your phage name and
to enter information about your
phage.

Part1 Appendix 3 16

Appendix 3. Archiving and Reporting Your Phage

Appendix 3 fig. 3
Adding a new phage. If you enter
a phage name that has already
been used, you will receive a
notification in red.

III. Assign a Phage Designator


This will include, without spaces:



The year the phage was isolated from the soil


Your universitys four-letter designation (upper case; see the SEA Wiki
for your school designation)
The name of the phage (lower case)
The students initials (upper case)

For example: Mary Jane Davis is sending her archived sample in the spring
of 2011, but the phage was isolated in September of 2010. She goes to the
University of North Kansas and the name of her phage is Henry. The phage
designator is 2010UNKAhenryMJD.

IV. Complete your phage report on PhagesDB


1. Log in to http://phagesdb.org/ as above.
2. Using the Data Entry tab, pull down to Add new phage. (See Appendix 3 fig. 2)
3. Enter your phage name into the Phage Name field.

Part1 Appendix 3 17

Appendix 3. Archiving and Reporting Your Phage

4. Enter your Phage Designator into the SEA Designator field.


5. As directed, enter all other available information into the fields (see Appendix 3 fig. 4).
Note: Do not hit enter or Submit until you are done with your
submission. Once you are notified that your phage is added to
the database, you can edit and/or add more information using the
Modify a Phage menu from the Data Entry dropdown.
Appendix 3 fig. 4
Adding your phage to the
database. After logging in to
PhagesDB, enter all of your available phage information. As more
data and images become available, you can add these using the
Modify a Phage tab.

Part1 Appendix 3 18

Appendix 3. Archiving and Reporting Your Phage

V. Prepare to archive your samples


1. Download the Freezer Box Inventory (FBI) from the SEAWiki at:
http://www.hhmi.org/seawiki/display/EduRes/SEA+Research+Archive
2. Assemble the following materials from the archiving kit that you obtained from the SEAlab:





Sterile 75% glycerol


Freezer boxes (3 per schooltwo to send to the SEAlab, one to keep)
Sterile cryotubes (3 per phage)
Styrofoam shipping box
3 plastic bags
1 FedEx shipping label

3. Assemble from your laboratory, for each phage:



At least 300 L of high-titer phage lysate (>109 pfu/mL)


A sterile microcentrifuge tube

Note: As of Fall 2010, the SEAlab is no longer accepting DNA samples for archiving

VI. Prepare phage lysates for archiving:


1. Label three cryotubes for each phage lysate with the phage designator,
date and titer.
2. Place 300 L of lysate into a microcentrifuge tube.
NOTE: Before transfer, be sure to mix the lysate gently but well, especially if it has been stored for more than a few days.
3. Add 600 L of 75% glycerol to the lysate and mix well by gently pipetting
up and down. This yields a final glycerol concentration of 50% in PB.
4. Distribute 300 L into each of the three cryotubes.
NOTE: The titer on the tube label is the titer BEFORE glycerol addition.
5. Place one of each replicate phage sample into the same position in three
separate freezer boxes.
6. Record the phage designations and titer on the FBI sheet.
7. Refrigerate the freezer box until the box contains all of the samples.
Do NOT freeze/thaw! Once the box is ready, ship on ice to the SEAlab.

Part1 Appendix 3 19

Appendix 3. Archiving and Reporting Your Phage

VII. Send and store freezer boxes


1. Place two freezer boxes into Ziplock bags and seal.
2. Using the Styrofoam shipping box with which the archiving kit was
sent and the provided FedEx label, please mail the two cryoboxes to the
SEAlab on wet ice OR with cold packs. See shipping checklist below.
3. Prepare a sealed plastic bag containing a hard copy of the (FBI).
4. Place the bag on top of the Styrofoam insert.
NOTE: Do NOT ship archive samples on dry ice.
5. Place the other freezer box into a freezer at your institution (at 80C) for
long-term storage.
6. Double check all of the items on the Archiving Checklist below.
7. Do not forget to send an electronic version of the FBI to the SEA at
natexp@hhmi.org.

Archiving checklist:
1. Styrofoam shipping box (mailed to SEAlab):



Two freezer boxes (sealed in plastic bag) with all phage lysates
Ice and/or gel packs
One CD containing an electronic version of the freezer box inventory
A sealed plastic bag containing a hard copy of the freezer box inventory and the CD (on top of the Styrofoam insert)

NOTE: Please be sure to send a hard copy of the freezer box inventory
with the freezer box!
2. Stored at your institution:

A replicate freezer box (at 80C) with all phage lysate and DNA samples
Electronic versions of the freezer inventory file

Important notes:

Part1 Appendix 3 20

Students MUST generate a Phage Designator and use this to physically


label the tubes containing the phage lysate samples that will be archived!

An electronic copy of the Freezer Box Inventory must be sent to the SEA!
This will be stored at the SEAlab as well as sent on to Dr. Hatfulls laboratory with the replicate box of the student phage lysate samples.

The report format from previous years is available on the SEAWiki as the
Student Phage Report Template. You may use this as a guideline for accumulating information for complete phage reports, but electronic and/or
paper reports are no longer required. All of the phage information will be
permanently stored on PhagesDB.

Appendix 4. Format for Research Papers

One of the most important steps in any research project is effective communication of the findings. Work buried in a laboratory notebook is not going
to inform or inspire our collaborators or other researchers who might benefit
from the discoveries we have made. To truly contribute to our body of scientific knowledge, the content must be well organized, effectively summarized,
and soundly interpreted. Examples of properly formatted papers are on the
SEA website, www.hhmi.org/sea. Papers should be double-spaced, have 1-inch
margins, and use a font size of at least 10 points and black ink. Acceptable
font choices are Times New Roman, Arial, Helvetica, Garamond, and Courier.
Symbols can be used when appropriate.

Sections to Include in NGRI Research Papers


Title
A short descriptor (10 words or fewer) that gives the reader an idea
of the papers content.
Author
A list of all individuals who made a scientific contribution to the described
work. If there is more than one author, the primary author (the person who
did the majority of the experiments) should be listed first. The senior author
(usually the faculty member) should be the last name in the list. All other
contributors should be listed in the order of the amount of work contributed
to the publication (in order of most to least). If their contributions are equal,
they should be listed alphabetically.
Institutional address
Course name and number, department, institution, city, state, zip code.
Abstract
A summary of the data presented in the paper. This usually consists of a
short introductory statement (one to three sentences), a brief description of
the presented data, and a single sentence describing the relevance of
the presented work.
Introduction
An overview of known information that nicely summarizes the state-of-thefield as it relates to the presented work. It should be focused and concise.
A well-written introduction should guide the reader through previously published work in such a way that the work the author presents appears to be
the next logical step in the scientific process.
Materials and methods
A compilation of all of the reagents and specialized equipment used in
the described experiments and a description of all of the methods employed
to generate the data. It is acceptable to reference an already-published procedure if it was used without modification. If any modifications were made,
these must be described after the original work is referenced in sufficient
detail to allow the reader to repeat the experiment under the precise
experimental conditions used in the paper with the same reported results.
Part1 Appendix 4 21

Appendix 4. Format for Research Papers

Results
A description of each of the experiments performed with a brief statement
of rationale and the resulting data. Data can be depicted in tables, graphs,
charts, photographs, and micrographs. Some data that serve to substantiate
a point can be simply described along with the phrase data not shown.
Discussion
A summary of the presented data, interpretation of the data from each
experiment, the relationship of the presented data to other published work,
and speculation about the potential impact on the field. The discussion
should also provide a hint of future directions.
Acknowledgments
A list of people and why they are being acknowledged. Generally, people are
acknowledged for reading and commenting on drafts of the paper, providing
technical support and intellectual stimulation, etc.
Funding
Agencies that provided funds to support the described work.
References
A list of references cited. The format should be: Authors (last name and
initials, each separated by a comma). Year published. Title of article. Name
of journal volume number: pages. An example:



Summer EJ, Gonzalez CF, Bomer M, Carlile T, Embry A, Kucherka AM,


Lee J, Mebane L, Morrison WC, Mark L, King MD, LiPuma JJ, Vidaver AK,
and Young R. 2006. Divergence and mosaicism among virulent soil phages
of the Burkholderia cepacia complex. J Bacteriol 188: 25568.

Figures
Should be legible and appropriately labeled (content, axes, symbols, etc.)
and presented in the order in which they appear in the manuscript. Figure
numbers should be on all figures, and captions and legends should be
included.
Tables
Should be legible, appropriately labeled, and presented in the order in
which they appear in the manuscript. Tables do not have legends, so all the
information required to interpret the data should be included in the body
of the table. Table numbers should also be included.

Part1 Appendix 4 22

Appendix 5. NGRI Equipment and Supplies

Name

Alias

Equipment Purpose / Use

Automatic
pipettor

Pipette-Aid,
automated
pipettor

Aspirating (drawing up
by suction) large volumes

Baffled flask

Special growth
flask

Aerating and growing


bacterial cultures

Bunsen burner

Flame, burner

Generating aseptic field;


heating samples

Capillary
tweezers

Really, really
small tweezers

Manipulating EM grids

Centrifuge,
bench-top

Bench-top fuge,
bench-top

Separating substances of
different densities using
centrifugal force

Centrifuge,
high-speed

High-speed
fuge, Sorvall

Separating substances of
different densities using centrifugal force (generates more
centrifugal force than a benchtop centrifuge)

Part1 Appendix 5 23

Appendix 5. NGRI Equipment and Supplies

Name

Alias

Equipment Purpose / Use

Centrifuge,
micro

Microfuge,
Eppendorf

Centrifuging samples
contained within
microcentrifuge tubes

Electron
microscopy grid

EM grid, grid

Immobilizing samples
to be viewed by electron
microscopy

Filter, syringe
sterilization,
0.22-m

Syringe filter,
0.22-micron
syringe filter

Sterilizing small sample


volumes through a filter of
small pore size; generally
removes all but the tiniest
of organisms (e.g., phage)

Filter,
sterilization,
0.22-m, 100-mL

Filtersterilization
unit

Sterilizing larger volumes


through a filter of small pore
size; generally removes all but
the tiniest of organisms
(e.g., phage)

20

Maintaining samples at
temperatures below freezing,
generally at 20C (4F)

Freezer

Part1 Appendix 5 24

Appendix 5. NGRI Equipment and Supplies

Gel apparatus
power supply

Power supply

Supplying electrical current


to the electrophoresis
equipment via electrodes

Gel apparatus,
horizontal

Electrophoresis
setup, agarose
gel equipment

Casting agarose gel and


electrophoresing samples

Geldocumentation
photography
system

Gel doc

Visualizing and photographing bacterial growth plates


and electrophoresis gels

Maintaining a constant
temperature using dry heat

Heating block

Hood, fume

Hood, chemical
hood

Removing vapors and odors


emitted by chemicals by
ventilating them to a designated area

Incubator,
forced-air or
radiant-heat

Incubator

Maintaining bacterial
growth at a constant
temperature

Part1 Appendix 5 25

Appendix 5. NGRI Equipment and Supplies

Part1 Appendix 5 26

Name

Alias

Equipment Purpose / Use

Incubator,
shaking

Shaker, shaking
incubator

Maintaining liquid cultures


at constant temperature
with shaking (to aerate
the cultures)

Micropipettor

Pipettman,
pipettor

Dispensing small
(<1-mL) volumes

Pipette,
serological

Pipet

Dispensing volumes
ranging from 0.5 to 50 mL

Refrigerator

Fridge, 4

Cooling and maintaining


samples to 4C (39.2F)

Syringe,
tuberculin

1-mL syringe,
TB syringe

Aspirating samples (e.g.,


through a 0.22-m filter)

Transilluminator

UV light box

Visualizing ethidium-bromidestained gels

Appendix 5. NGRI Equipment and Supplies

Name

Alias

Equipment Purpose / Use

Tube,
centrifuge,
15-mL

Oak Ridge tube

Centrifuging samples
at high speed

Tube, conical,
15-mL or 50-mL

15-mL Falcon
tube or 50-mL
Falcon tube

Preparing, manipulating,
and storing samples

Tube, glass
culture, 10-mL

Culture tube

Preparing and manipulating


bacterial cultures

Tube,
microcentrifuge

Microfuge tube,
Eppendorf tube,
Eppi

Preparing and
manipulating small
(up to 1.5-mL) samples

15 mL

50 mL

Tube, Steriflip

Filter-sterilizing liquid
samples
(10-to-50-mL volumes)

Part1 Appendix 5 27

Appendix 5. NGRI Equipment and Supplies

Name

Alias

Equipment Purpose / Use

Vortexer

Genie mixer,
Vortex

Mixing samples by
gyration

Water bath

Part1 Appendix 5 28

Maintaining samples at
a constant temperature;
uses wet heat (i.e., water)

Appendix 6. Recipes for Stock Solutions, Media,


and Reagents

Contents
31 Notes about the Recipes
A. Stock Solutions for Media, Buffers,
and Reagents
34 AD supplement
35 100 mM CaCl2
36 500 mM EDTA, pH 8.0
37 40% glycerol
38 1 M MgSO4
39 50X TAE (Tris-Acetate-EDTA)
40 10X TBE (Tris-Boric Acid-EDTA)
41 1 M Tris (base), pH 7.5
42 1 M Tris-Cl, pH 7.5
43 20% Tween 80
B. Antibiotic and Antimicrobial
Stock Solutions
46 Carbenicillin (CB; 1000X)
47 Cycloheximide (CHX; 1000X)
C. Media
50 Middlebrook 7H9 liquid medium: Neat
51 Middlebrook 7H9 liquid medium: Complete
52 M
 iddlebrook 7H9 liquid medium: Complete
with Tween 80
53 10X 7H9/Glycerol
54 2X Middlebrook top agar
55 1X Middlebrook top agar
56 L-agar plates
D. Reagents
58 Nuclease mix
59 Phage buffer
60 Phage precipitation solution
61 Sodium acetate, 3 M
62 1X TAE
63 1X TBE
64 TE
65 Uranyl acetate stain
Part1 Appendix 6 29

Notes about the Recipes

About ddH2O
Water should be double-distilled at a minimum. Filtered water (e.g., Millipore)
is ideal. Regardless of water availability, consistency of preparation is essential (i.e., always use the same water).

About Labeling
Clearly label all media and solutions, and date the additions of supplements.
Sterile solutions should also be clearly labeled as such, and any autoclave
tape (indicating sterility) should be left on the bottles.

About Adjusting the pH of Solutions


Be sure to check each recipe for the proper acid or base solution that should
be used to adjust the pH (if needed) of each solution or reagent.

About Media
The Mycobacterium smegmatis strain mc 2155 grows very well in Middlebrook 7H9 complete media with or without Tween 80 supplementation. For
this reason, all M. smegmatis cultures should be grown in 7H9 broth, shaking
at 37C, for use in NGRI experiments. Top agar (TA) should also contain the
7H9 media base with the addition of agar for the growth of bacterial lawns.
In previous versions of this Resource Guide, standard 7H10 agar plates were
used for streaking bacterial colonies and for various phage assays. The 7H10
agar plates are not, however, required for the growth of M. smegmatis colonies
nor are they required for growth of bacterial lawns in TA. In this version, NGRI
students will use standard L-agar plates. L-agar plates are much more easily
prepared as compared to 7H10 agar plates. The L-agar plates are sufficient
for streaking out M. smegmatis cultures for single colonies, but single colonies
will take an extra 24-36 hours to grow. L-agar plates also yield the same titer
and similar plaque morphologies, as compared to 7H10 media, for all phages
tested in the SEAlab.

Part1 Appendix 6 31

A. Stock Solutions for Media, Buffers, and Reagents

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

AD Supplement (2 L)
Ingredient Amount

Final Concentration

NaCl

17 g

145 mM

Albumin (Fraction V)

100 g

5.0%

ddH2O

To 2 L

Dextrose

40 g

To Prepare




1. Weigh out NaCl and albumin and place in a 4-L Erlenmeyer flask.
2. While stirring, slowly add 1800 mL of ddH2O until the albumin and
NaCl are dissolved.
3. While the solution is still stirring, slowly add the dextrose.
4. Once the dextrose is dissolved, transfer the solution to a 2-L graduated cylinder,
and bring the volume to 2 L with ddH2O

To Sterilize

Filter-sterilize; do not autoclave.

Store

At 4C.

2.0%

AD Supplement (2 L)
Notes








Part1 Appendix 6 34

1. Adding the dextrose last helps keep it from recrystallizing on the bottom
of the flask.
2. Make sure all the albumin is in solution before the filter- sterilization step.
To dissolve the albumin, use a large stir bar on the highest stirring speed that
will not denature the protein (denatured protein foams or bubbles on the
surface of the solution).
3. Standard medium for the growth of mycobacteria (Middlebrook) generally
contains ADC (for albumin, dextrose, catalase) or OADC (ADC plus oleic acid).
This guide uses the recipe for ADC but without catalase, so we call it
AD supplement.

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

100 mM CaCl2 (100 mL)*


Ingredient Amount

Final Concentration

CaCl2

100 mM

1.47 g of CaCl2 2H2O or

1.11 g of CaCl2 (anhydrous)

ddH2O

To 100 mL

To Prepare

1. Weigh out CaCl2 and place in a large beaker.


2. Add 90 mL of ddH2O and stir until the CaCl2 is dissolved.
3. Transfer the solution to a graduated cylinder, and add ddH2O to 100 mL.

To Sterilize

Autoclave.

Store

At room temperature; do not refrigerate.

100 mM CaCl2 (100 mL)*


Notes

* A good rule of thumb is that CaCl2 should be added to all solutions that come

in contact with phage, and, ideally, this should happen immediately before the
solution is used. The CaCl2 will precipitate out when cooling after autoclaving
or when refrigerated.

Part1 Appendix 6 35

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

500 mM EDTA, pH 8.0


Ingredient Amount

Final Concentration

EDTA disodium salt dihydrate 186.1 g

500 mM

ddH2O

To 1 L

To Prepare



1. Weigh out EDTA and place in a large beaker.


2. Add approximately 950 mL of ddH2O and stir.
3. Adjust the pH to 8.0 with NaOH.
4. When the EDTA is dissolved, transfer the solution to a graduated cylinder,
and add ddH2O to 1 L.

To Sterilize

Autoclave in 250-mL or 500-mL bottles.

Store

At room temperature.

500 mM EDTA, pH 8.0


Notes

Part1 Appendix 6 36

1. The EDTA may not go into solution unless the pH is 7. If the EDTA
is not dissolving, the pH is likely <7.0. Add NaOH and stir until the EDTA
is dissolved. Adjust the final pH to 8.0 and the final volume to 1 L.

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

40% Glycerol (1 L)
Ingredient Amount

Final Concentration

ddH2O

To 1 L

Glycerol (also known as


glycerin or glycerine)

400 mL

To Prepare



1. Measure 500 mL ddH2O and place in a large beaker.


2. Slowly add 400 mL of glycerol while stirring.
3. When the glycerol has completely dissolved, transfer the solution
to a graduated cylinder.
4. Bring the volume to 1 L with ddH2O. Mix.

To Sterilize

Filter-sterilize or autoclave in 250-mL or 500-mL bottles.

Store

At room temperature.

40%

40% Glycerol (1 L)
Notes

Part1 Appendix 6 37

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

1 M MgSO4 (1 L)
Ingredient Amount

Final Concentration

MgSO4 (anhydrous)

120.36 g

1M

ddH2O

To 1 L

To Prepare

1. Weigh out MgSO4 and place in a large beaker containing approximately


950 mL of ddH2O, and stir until the MgSO4 is dissolved.
2. Transfer the solution to a graduated cylinder, and add ddH2O to 1 L.

To Sterilize

Autoclave in 250-mL or 500-mL bottles.

Store

At room temperature.

1 M MgSO4 (1 L)
Notes

Part1 Appendix 6 38

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

50X TAE (Tris-Acetate-EDTA)


Ingredient Amount

Final Concentration

Trizma base

242 g

2M

500 mM EDTA*

100 mL

50 mM

Glacial acetic acid

57.1 mL

1M

ddH2O

To 1 L

To Prepare




1. Weigh out Trizma base and place in a large beaker.


2. Add approximately 800 mL of ddH2O, and stir until the Trizma base is dissolved.
3. Add the EDTA stock while stirring.
4. Slowly add the glacial acetic acid while stirring.
5. Adjust the pH to 8.5 with additional glacial acetic acid (if necessary),
and transfer the solution to a graduated cylinder. Add ddH2O to 1L.

To Sterilize

No need to sterilize; do not autoclave.

Store

At room temperature.

50X TAE (Tris-Acetate-EDTA)


Notes

* The recipe for this ingredient is included in this appendix.

Part1 Appendix 6 39

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

10X TBE (TrisBoric AcidEDTA)


Ingredient Amount

Final Concentration

Trizma base

108 g

890 mM

Boric acid

55 g

EDTA disodium salt dihydrate 7.44 g

890 mM
20 mM

ddH2O

To 1 L

To Prepare


1. Weigh out Trizma base, boric acid, and EDTA and place in a large beaker.
2. Add 980 mL of ddH2O and stir until all ingredients are dissolved.
3. Adjust the pH to 8.3, and transfer the solution to a graduated cylinder.
Add ddH2O to 1 L.

To Sterilize

Autoclave.

Store

At room temperature.

10X TBE (TrisBoric AcidEDTA)


Notes

Part1 Appendix 6 40

1. This is the traditional Maniatis recipe. If precipitates form during


long-term storage, they can be dissolved by reautoclaving the solution.

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

1 M Tris (base), pH 7.5 (1 L)


Ingredient Amount

Final Concentration

Trizma base

121.1 g

1M

ddH2O

To 1 L

To Prepare


1. Weigh out Trizma base and place in a large beaker.


2. Add approximately 975 mL of ddH2O and stir.
3. When the Trizma base is dissolved, bring the pH to 7.5 with HCl.
4. Transfer the solution to a graduated cylinder, and add ddH2O to 1 L.

To Sterilize Autoclave in 250-mL or 500-mL bottles.


Store

At room temperature.

1 M Tris (base), pH 7.5 (1 L)


Notes

Part1 Appendix 6 41

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

1 M Tris-Cl, pH 7.5 (1 L)
Ingredient Amount

Final Concentration

Tris-Cl

157.60 g

1M

ddH2O

1 L

To Prepare


1. Weigh out Tris-Cl and place in a large beaker.


2. Add approximately 975 mL of ddH2O and stir.
3. When the Tris-Cl is dissolved, bring the pH to 7.5 with NaOH.
4. Transfer the solution to a graduated cylinder, and add ddH2O to 1 L.

To Sterilize

Autoclave in 250-mL or 500-mL bottles.

Store

At room temperature.

1 M Tris-Cl, pH 7.5 (1 L)
Notes

Part1 Appendix 6 42

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

20% Tween 80 (100 mL)*


Ingredient Amount

Final Concentration

ddH2O

To 100 mL

Tween 80

20 mL

To Prepare


1. Measure 60 mL ddH2O and place in a medium-size beaker.


2. Slowly add 20 mL Tween 80 while stirring.
3. Once the Tween 80 has completely dissolved, transfer the solution to
a graduated cylinder and bring to 100 mL with ddH2O.

To Sterilize

Filter-sterilize. Do not autoclave.

Store

At room temperature.

20%

20% Tween 80 (100 mL)*


Notes

1. It may take a while for the Tween 80 to dissolve. Add the Tween 80 slowly
and alternate the beaker between the stir plate and a 55C water bath to
dissolve the Tween 80.

* Do not add Tween 80 to media if the bacteria being grown will be
used for phage isolation or propagation.

Part1 Appendix 6 43

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Part1 Appendix 6 44

B. Antibiotic and Antimicrobial Stock Solutions

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Carbenicillin (CB)
Stock Concentration 50 mg/mL (Stock = 1000X)
To Prepare



1. Weigh out 100 to 600 mg of carbenicillin powder and place


in a 10-to-15-mL tube.
2. Divide the number of milligrams of carbenicillin by 50 to determine the volume
(in milliliters) of ddH2O to add (e.g., for 477 mg of CB, add 9.5 mL ddH2O).
3. Stir or shake until dissolved.

To Sterilize

Filter-sterilize (0.22-m pore size) into a fresh sterile tube.

Store

At 4C for 60 days.

Label Stock Solution CB, 1000X, date, initials


To Prepare Final Solution 1. Add stock solution at 1:1000. For example, for 1 L of medium,

add 1 mL of stock. Label and store at 4C.

Carbenicillin (CB)
Notes


Part1 Appendix 6 46

1. Mycobacterium smegmatis is resistant to CB, so it is added to


prevent growth of other bacteria.
2. Mechanism of action: CB is semisynthetic penicillin that interferes with
bacterial cell-wall synthesis.

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Cycloheximide (CHX)
Stock Concentration 10 mg/mL (Stock = 1000X)
To Prepare




1. Weigh out 20 to 100 mg of cycloheximide powder and place in


a 10-to-15-mL tube.
2. Divide the number of milligrams of cycloheximide by 10 to determine the
volume (in milligrams) of ddH2O to add (e.g., for 46.2 mg of CHX,
add 4.6 mL ddH2O).
3. Stir or shake until dissolved.

To Sterilize

Filter-sterilize (0.22-m pore size) into a fresh sterile tube.

Store

At 4C for 60 days.

Label Stock Solution CHX, 1000X, date, initials


To Prepare Final Solution 1. Add stock solution at 1:1000. For example, for 1 L of medium,

add 1 mL of stock.
2. Label and store at 4C.

Cycloheximide (CHX)
Notes



1. CHX is added to mycobacterial media to prevent the growth of


fungi and yeast.
2. Mechanism of action: CHX inhibits protein biosynthesis in eukaryotic organisms.
It interferes with the peptidyl transferase activity of the 60S ribosome, thus
blocking translational elongation.

Part1 Appendix 6 47

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Part1 Appendix 6 48

C. Media

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Middlebrook 7H9 Liquid Medium: Neat (900 mL)


Ingredient Amount

Final Concentration

7H9 broth base

4.7 g

40% glycerol stock*

5 mL

0.2%

ddH2O

To 900 mL

To Prepare**



1. Place broth base and 850 mL of ddH2O into an Erlenmeyer flask.


2. While stirring, add the glycerol. Stir until the broth base powder is
completely dissolved.
3. Bring up to 900 mL with ddH2O in a graduated cylinder.
4. Aliquot into 90-mL, 450-mL, or 900-mL portions as needed.

To Sterilize

Autoclave.

Store

At room temperature.

Middlebrook 7H9 Liquid Medium: Neat (900 mL)


Notes

1. This medium will be diluted by the addition of 10% AD supplement (see


Middlebrook 7H9 Liquid Medium: Complete). This is why medium is dispensed
in 9/10 volumes (e.g., 450 mL).

* The recipe for this ingredient is included in this appendix.


** First, see manufacturers instructions, and use them if they differ from these.

Part1 Appendix 6 50

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Middlebrook 7H9 Liquid Medium: Complete (100 mL)*


(For bacterial growth without Tween 80)
Note: Use aseptic technique when adding all components.
Ingredient Amount

Final Concentration

7H9 liquid medium: neat** 89 mL

1X

AD supplement**

10 mL

10%

CB stock**

100 L

50 g/mL

CHX stock**

100 L

10 g/mL

100 mM CaCl2 stock**

1 mL

1 mM


To Prepare

1. All ingredients must be added to a sterile bottle or flask using aseptic


technique. The final medium cant be autoclaved or filter-sterilized.

To Sterilize

All ingredients are sterile.

Store

At 4C.

Middlebrook 7H9 Liquid Medium: Complete (100 mL)*


(For bacterial growth without Tween 80)
Notes

1. Make complete 7H9 medium as needed; refrigeration may cause the


CaCl2 to precipitate. If antimicrobials and AD supplement have been added,
the medium must be stored at 4C.

* A good rule of thumb is that CaCl2 should be added to all solutions that
come in contact with phage, and, ideally, this should happen immediately
before the solution is used. The CaCl2 will precipitate out when cooling
after autoclaving or when refrigerated.
** The recipe for this ingredient is included in this appendix.

Part1 Appendix 6 51

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Middlebrook 7H9 Liquid Medium: Complete with Tween 80*


(For bacterial growth with Tween 80)
Note: Use aseptic technique when adding all components.
Ingredient Amount

Final Concentration

7H9 liquid medium**

90 mL

1X

AD supplement**

10 mL

10%

CB stock**

100 L

50 g/mL

CHX stock**

100 L

10 g/mL

100 mM CaCl2 stock**

1 mL

1 mM

20%Tween 80 stock**

250 L

0.05%


To Prepare

1. All ingredients must be added to a sterile bottle or flask using aseptic


technique. The final medium cannot be autoclaved or filter-sterilized.

To Sterilize

All ingredients are sterile.

Store

At 4C.

Middlebrook 7H9 Liquid Medium: Complete with Tween 80*


(For bacterial growth with Tween 80)
Notes

Part1 Appendix 6 52

1. In liquid culture, Mycobacterium smegmatis tends to clump. When liquid


cultures are grown from bacterial colonies, 0.05% Tween 80 must be added
to prevent clumping.
2. This culture is then subcultured into medium without Tween 80 for use
in phage infections (see Appendix 7, Recipes for Bacterial Cultures).
3. Make complete 7H9 medium as needed; refrigeration will cause the
calcium to precipitate. If antimicrobials and AD supplement have been
added, the medium must be stored at 4C.

* Do not add Tween 80 to media if the bacteria being grown will be used
for phage isolation or propagation.
** The recipe for this ingredient is included in this appendix.

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

10X 7H9/Glycerol Media (500mL)


For Enrichment Protocol
Ingredient Amount

Final Concentration

7H9 broth base

23.5g

10X

40% Glycerol Stock*

25 mL

2.0%

ddH2O

To 500 mL

To Prepare**

Place broth base and 400mL of ddH2O into an Erlenmeyer flask. While
stirring, add the glycerol. Stir until the broth base powder is completely
dissolved. Bring up to 500mL with ddH2O in a graduated cylinder. Aliquot in
50mL or 100mL volumes as needed.

To Sterilize

Autoclave

Store

Room temperature.

10X 7H9/Glycerol Media (500mL)


For Enrichment Protocol
Notes

T
 his media, diluted for use in the enrichment protocol, will likely have a fine,
cloudy precipitate when stored. This is normal, but the solution should be
mixed, and the precipitate suspended, before use.

*The recipe for this ingredient is included in this appendix.


** First, see manufacturers instructions and use them if they differ from these

Part1 Appendix 6 53

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

2X Middlebrook Top Agar (2XTA; 1 L)


Ingredient Amount

Final Concentration

7H9 broth base

4.7 g

Agar

8 g

ddH2O

1 L

To Prepare







1. Place broth base, agar, ddH2O, and a stir bar into an Erlenmeyer flask;
put onto a stir plate.
2. Stir until the broth base powder is completely dissolved
(the agar will not dissolve until it is heated).
3. Autoclave the flask, and cool to 55C in a 55C water bath.
4. In a hood, using aseptic technique, dispense into sterile bottles using only half
the available volume (e.g., for 100 mL final, add 50 mL of 2XTA to a 100-mL
bottle). Keep any lids loose until the agar has solidified.
5. Tighten lids for storage.

To Sterilize

Autoclave

Store

At room temperature.

0.8%

2X Middlebrook Top Agar (2XTA; 1 L)


Notes

Part1 Appendix 6 54

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

1X Middlebrook Top Agar (TA; 100 mL)*


Ingredient Amount

Final Concentration

100 mM CaCl2 stock**

1 mM

1 mL

7H9 liquid medium: neat** 50 mL


2XTA**

50 mL

1X

To Prepare






1. Add 1 mL of CaCl2 stock to the 50 mL of 7H9 medium.


2. Place into a 55C water bath.
3. Melt the 2XTA in a microwave. Make sure the cap is loose! The agar must
be completely melted, so carefully swirl the bottle and check to make sure
there are no clumps. The 2XTA should come to a boil but not boil over.
4. Once the agar is completely melted, place the 2XTA into a 55C water bath.
When both solutions are at 55C, aseptically add the medium-calcium
mixture to the 2XTA.

To Sterilize

All ingredients must be added using aseptic technique.


The final medium cannot be autoclaved or filter-sterilized.

Store

In the 55C water bath 7days. Cooling to room temperature will cause
the CaCl2 to precipitate out of solution.

1X Middlebrook Top Agar (TA; 100 mL)*


Notes

1. The final agar concentration will be 0.4%.

*A good rule of thumb is that CaCl2 should be added to all solutions that
come in contact with phage, and, ideally, this should happen immediately
before the solution is used. The CaCl2 will precipitate out when cooling after
autoclaving or when refrigerated.

** The recipe for this ingredient is included in this appendix.

Part1 Appendix 6 55

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Luria Agar (L-Agar) Plates (1L)


Ingredient Amount

Final Concentration

L-Agar base

30.5 g

ddH2O

To 1L

CB stock*

1.0 mL

50 g/mL

CHX stock*

1.0 mL

10 g/mL

To Prepare **

1. Mix 30.5g of L-Agar base with 1L of ddH2O and mix well with a magnetic stir bar.
2. Heat while stirring and boil for up to 1 minute to completely dissolve the
powder.
3. Autoclave at 121C for 10 minutes.
4. Cool to 55C in a 55C water bath
5. Once the agar has cooled to 50-55C, aseptically add the CB and CHX stock
solutions.
6. Mix well by swirling, and avoid bubbles.
7. Using aseptic technique, pour agar into Petri dishes.

To Sterilize

Autoclave (see above)

Store

At 4C.

Luria Agar (L-Agar) Plates (1L)


Notes

1. Media can be autoclaved and aliquoted into sterile bottles and allowed
to cool. These bottles can be stored at room temperature and microwaved
to melt the agar. The agar solutions are cooled to 55C (as above) before
addition of antimicrobial supplements and pouring of plates.

*The recipe for this ingredient is included in this appendix.


** First, see manufacturers instructions, and use them if they differ from these.

Part1 Appendix 6 56

D. Reagents

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Nuclease Mix
Ingredient Amount

Final Concentration

NaCl

0.088 g

150 mM

ddH2O

4.25 mL

DNase I

500 L of 5-mg/mL stock

RNase A

250 L of 10-mg/mL stock

0.25 mg/mL

Glycerol

5 mL

50%

To Prepare



1. Dissolve the NaCl in 4.25 mL of ddH2O in a sterile 15-mL conical tube.


2. Add the RNase and DNase stock solutions, and mix with gentle inversion.
3. Add the glycerol slowly to a final volume of 10 mL, and mix with gentle
inversion until the solution is homogeneous.
4. Aliquot into microcentrifuge tubes.

To Sterilize

Not required.

Store

At 20C (freezer).

0.25 mg/mL

Nuclease Mix
Notes

Part1 Appendix 6 58

1. Be extra careful not to contaminate pipettes, benches, or other reagents


with this nuclease mixture, because it will degrade any DNA samples.

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Phage Buffer (PB; 1 L)*


Ingredient Amount

Final Concentration

1 M Tris stock (pH 7.5)**

10 mL

10 mM

1 M MgSO4 stock**

10 mL

10 mM

NaCl

4 g

68 mM

ddH2O

970 mL

100 mM CaCl2 stock**

10 mL

To Prepare




1. Place all ingredients except CaCl2 into an Erlenmeyer flask.


2. Stir until the NaCl is completely dissolved.
3. Autoclave in the Erlenmeyer flask. Cool to 55C in a 55C water bath.
4. After cooling, aseptically add the CaCl2 and mix well by swirling.
5. Aliquot into sterile bottles, flasks, or tubes in 50-mL or 100-mL
(or other) portions as needed.

To Sterilize

Autoclave.

Store

At room temperature.

1 mM

Phage Buffer (PB; 1 L)*


Notes

1. The CaCl2 should be added shortly before use. It will precipitate out after
autoclaving and cooling or when solutions are refrigerated.

*A good rule of thumb is that CaCl2 should be added to all solutions that
come in contact with phage, and, ideally, this should happen immediately
before the solution is used. The CaCl2 will precipitate out when cooling
after autoclaving or when refrigerated.
** The recipe for this ingredient is included in this appendix.

Part1 Appendix 6 59

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Phage Precipitation Solution


Ingredient Amount

Final Concentration

ddH2O

To 100 mL

Polyethylene glycol

30 g

30%

NaCl

19.3 g

3.3 M


To Prepare






1. Add 19.3 g NaCl and 60 mL of ddH2O to a glass bottle with a screw cap,
and swirl until the NaCl is completely dissolved.
2. Slowly add 30 g of PEG 8000. Add only a few grams at a time, alternately
swirling and heating in a microwave until all the PEG is dissolved.
3. Add sterile water to 100 mL.
4. Add a sterile magnetic stir bar and stir until homogeneous.
5. Filter sterilize.
6. Dispense into sterile bottles or tubes.

To Sterilize

Filter sterilize.

Store

At room temperature.

8000 (PEG 8000)

Phage Precipitation Solution


Notes



Part1 Appendix 6 60

1. The NaCl and the PEG 8000 are difficult to dissolve. Several iterations of
heating and stirring may be required. If there are precipitates in the solution,
do not use it! Rather, attempt to dissolve particulates by alternately heating
the solution in a microwave and swirling. Allow the solution to cool before
using. Use this solution only at room temperature.

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Sodium Acetate, 3 M
Ingredient Amount

Final Concentration

Sodium acetate trihydrate 40.8 g


ddH2O

To 100 mL

Glacial acetic acid

To pH 5.2

To Prepare



1. Weigh out the sodium acetate and place in a beaker.


2. Add approximately 80 mL of ddH2O and stir.
3. When the sodium acetate is dissolved (this will likely take 3 to 4 hours),
bring the pH to 5.2 with glacial acetic acid.
4. Transfer the solution to a graduated cylinder and add ddH2O to 100 mL.

To Sterilize

Autoclave.

Store

At room temperature.

Sodium Acetate, 3 M
Notes

Part1 Appendix 6 61

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

1X TAE
Ingredient Amount

Final Concentration

50X TAE*

20 mL

1X (40 mM Tris, 20 mM acetic acid,

and 1 mM EDTA)

ddH2O

980 mL

To Prepare

1. Dilute the 50X TAE to 1X with the volumes above (i.e., 20 and 980 mL).
There is no need to check or adjust the pH of this solution, and it does not have
to be sterilized before use.

Store

At room temperature.

1X TAE
Notes

Part1 Appendix 6 62

* 
The recipe for this ingredient is included in this appendix.

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

1X TBE
Ingredient Amount

Final Concentration

10X TBE*

100 mL

1X (89-mM Tris, 89-mM boric acid,

and 2-mM EDTA)

ddH2O

900 mL


To Prepare

1. Dilute the 10X TBE to 1X with the volumes above (i.e., 100 and 900 mL).
There is no need to check or adjust the pH of this solution, and it does not have
to be sterilized before use.

Store

At room temperature.

1X TBE
Notes

The recipe for this ingredient is included in this appendix.


* 

Part1 Appendix 6 63

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

TE (10 mM Tris-Cl, 1 mM EDTA)


Ingredient Amount

ddH2O

988 mL

1-M Tris-Cl stock (pH 7.5)*

10 mL

500 mM EDTA stock (pH 8.0)* 2 mL

Final Concentration

10 mM
1 mM


To Prepare


1. Add 950 mL of ddH2O to a large beaker or flask.


2. Add 10 mL of Tris-Cl stock and 2 mL of EDTA stock while stirring.
3. Transfer the mixture to a 1-L graduated cylinder, and bring the volume
to 1.0 L with ddH2O.

To Sterilize

Autoclave.

Store

At room temperature.

TE (10 mM Tris-Cl, 1 mM EDTA)


Notes

Part1 Appendix 6 64

The recipe for this ingredient is included in this appendix.


* 

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Uranyl Acetate Stain


Ingredient Amount

Final Concentration

Uranyl acetate

0.1 g

1% w/v (1 g in 100 mL)

ddH2O

10 mL

To Prepare

1. Weigh out the uranyl acetate and place in a beaker.


2. Add approximately 9 mL of ddH2O and stir.
3. When the uranyl acetate is dissolved, bring the final volume up to 10 mL
with ddH2O.

To Sterilize

Prepare a 0.22-m filter by rinsing it several times with ddH2O. Filter-sterilize


the solution and dispense in 1-mL aliquots.

Store

At room temperature, in the dark.

Uranyl Acetate Stain


Notes

1. This is a highly toxic compound. Always wear gloves when preparing or


working with it.

Part1 Appendix 6 65

Appendix 6. Recipes for Stock Solutions, Media, and Reagents

Part1 Appendix 6 66

Appendix 7. Recipes for Bacterial Cultures

Contents
69

 rowing Mycobacterium smegmatis mc 2155


G
from a frozen stock

70

Growing liquid stock cultures of


Mycobacterium smegmatis mc 2155 from
a single colony

71

Growing liquid cultures of Mycobacterium


smegmatis mc 2155 for use in phage
experiments

Part1 Appendix 7 67

Appendix 7. Recipes for Bacterial Cultures

Growing Mycobacterium smegmatis mc2155 from


a frozen stock
To Prepare


1. Retrieve a sample from the 80C freezer.


2. Streak out a sample for single colonies on a standard L-agar plate
(7H10 + AD supplement + CB + CHX).
3. Allow to grow for 4 to 6 days at 37C or until single colonies are 24 mm in
diameter.

Store

At room temperature or at 4C.

Growing Mycobacterium smegmatis mc2155 from


a frozen stock
Notes





1. The agar plate can be stored for weeks at room temperature or for
months at 4C.
2. It is a good idea to wrap the edges of the plate with parafilm to minimize
drying of the agar.
3. Be mindful of condensation, especially if the plate is stored at 4C. If the
agar becomes wet (and/or contaminated), the culture should be discarded
and a new plate streaked from frozen stocks.

Part1 Appendix 7 69

Appendix 7. Recipes for Bacterial Cultures

Growing liquid stock cultures of Mycobacterium


smegmatis mc2155 from a single colony*
To Prepare




1. Add 50 mL of 7H9 Complete with 0.05% Tween 80 ** to a 250-mL


bafed ask.
2. Using an inoculation loop, pick a small piece from the middle of a single
colony of M. smegmatis.
3. Aseptically transfer the bacteria from the loop into the medium in the ask.
4. Incubate, shaking (250 rpm), for at least 72 hours at 37C.

Store

At room temperature or at 4C.

Growing liquid stock cultures of Mycobacterium


smegmatis mc2155 from a single colony*
Notes











1. This is your P1FF (passage 1 from frozen) stock. This stock culture can be
stored at room temperature for several weeks. We recommend storage at 4C
for no more than 6 months. It can be aseptically aliquoted into smaller
volumes for storage (this minimizes the potential for contamination).
2. We suggest that you start P1FF stock cultures biweekly, depending on use and
the size of a given class. Regardless, because of the possibility of contamination,
a backup P1FF culture should be available at all times. It is not necessary to
start each from a frozen culture; rather, single colonies from the agar plate can
be used for as long as the plate remains uncontaminated (up to 6 months).
3. Tween 80 is added to bacterial cultures to minimize clumping during
bacterial growth.
4. The presence of Tween 80, however, will likely inhibit phage infection.
For this reason:

To grow liquid stock cultures from the P1FF stock, use 7H9 Complete
with Tween 80.

To grow large cultures for use in phage experiments, use 7H9 Complete
without Tween 80.

Do not add Tween 80 to media if the bacteria being grown will be used
* 
for phage isolation or propagation.
** The recipe for this ingredient is included in Appendix 6.

Part1 Appendix 7 70

Appendix 7. Recipes for Bacterial Cultures

Growing liquid cultures of Mycobacterium smegmatis


mc2155 for use in phage experiments
To Prepare



1. Add 50 mL of 7H9 Complete (i.e., without Tween 80) to a 250-mL baffled ask.
For larger volumes, adjust accordingly (see notes).
2. Aseptically, add a 1:1000 volume of a P1FF culture. For example, for a 50-mL
culture, add 50-L M. smegmatis.
3. Incubate, shaking (250 rpm), for at least 72 hours at 37C.

Store

At room temperature.

Growing liquid cultures of Mycobacterium smegmatis


mc2155 for use in phage experiments
Notes


















1. The volume of a culture should be approximately one-fifth the size of the ask.
For example, for 200 mL of bacterial culture, use a 1-L baffled flask. This
maximizes oxygenation and volume when the bacteria are grown on a shaker
at 37C.
2. Depending on the bacteriophage being studied, this culture can be stored
at room temperature for an extended period without inhibiting the growth
of mycobacteriophages. Though M. smegmatis will grow slowly at room
temperature, these cultures can be used for phage titration and isolation for
up to 2 weeks.
3. Tween 80 will inhibit phage infection. It is advisable to double-check that any
medium that will be used in phage experiments does not include Tween 80.
4. The addition of small (1:1000) volumes of the stock culture reduces the
likelihood of bacterial clumping in subculture. If clumping does occur, continue
incubating the culture up to an additional 48 hours.
5. Once M. smegmatis is growing homogeneously (without clumping) in broth
culture, it can be transferred to flasks for larger volumes. Limit the number of
serial transfers as much as possible. Its best to go back to the initial P1FF stock
culture. Cultures of M. smegmatis can be stored at room temperature for weeks,
or for as long as the cultures resuspend smoothly when subcultured. Cultures
with large clumps should not be used.

Part1 Appendix 7 71

Appendix 7. Recipes for Bacterial Cultures

Part1 Appendix 7 72