LAB

3: Phage Titration

Introduction:
Viruses are non-living agents that infect all life forms. Different viruses have different, but very specific, host
targets. For a virus to multiply, it must invade a host cell and direct the hosts metabolic machinery to produce
viral enzymes and components. Viruses that are specific to infecting bacteria are called bacteriophage (or
simply, phage). More specifically, bacteriophage are obligate intracellular parasites that multiply inside bacteria
by making use of some or all of the hosts biosynthetic machinery.

In industry, phage can be used for studying transduction (gene transfer in bacteria), to serve as models for
animal viruses, and medical applications such as identification of bacteria (phage typing).

There are many different types of bacteriophage, each with distinct and defining characteristics. T4 and Lambda
() are two examples of a class of bacteriophage. A T4 phage structure is composed of a head (capsid), tail, tail
fibres, and base plate. The capsid contains the genetic information (dsDNA); the tail fibres and base plate are
used for attachment to the host; the tail, which is covered by a contractile sheath, is the region through which
the genetic information is transferred into the host.

Phage can have two different life cycles: lytic and lysogenic. Lytic, or virulent phage, phage multiply within
bacteria and kill the cell by lysis. During this infection the virus uses the host cell machinery to further replicate
and thus creates more virus particles. The general stages of the lytic cycle are:

1. Adsorption - attachment to cell surface receptors (chance encounter no active movement)
2. Penetration only DNA enters
3. Transcription of viral DNA by the host cell machinery
4. Replication and protein synthesis (phage induced proteins)
5. Assembly or maturation to form intact phage
6. Release due to phage induced lysozyme production

Among the virulent phage is the T4-phage (T1, T2, T3, T4, T5, T6, and T7), a group of double-stranded DNA
viruses. Also included in this class are a series of single-stranded RNA phages (R17, MS2, f2, M12, fr, and Q) and
a group of single-stranded DNA phages (fd, f1, and M13). All of these phage form clear plaques on sensitive
strains.

Lysogenic, or temperate phage, phage can either multiply via the lytic cycle or enter a quiescent state in the
bacterial cell. Once the phage has infected the host cell, the phage DNA is integrated into the host
chromosome, thereby becoming referred to as prophage. In this quiescent state expression of most phage
genes are repressed. The infected bacterium is called a lysogen. While the prophage state can be indefinite, a
lysogenic cell can be induced to produce mature virus particles (e.g. by exposure to UV light). This process is
called phage induction and it is the conversion of lysogenic to lytic state.

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Experimental Design:

In this experiment you will be performing a Plaque Assay.

1. Plaque Assay (phage titration)
Plague assay is a method used to enumerate lytic phage (Figure 2). A plaque is a clear area that results
from the lysis of bacteria. Each plaque arises from a single infectious phage. The infectious particle that gives
rise to a plaque is called a pfu (plaque forming unit).

The one-step growth curve can be studied and quantified by performing a plaque assay. In a plaque
assay, a dilution of a suspension containing the virus material is mixed in a small amount of melted agar with the
sensitive host bacteria, and the mixture poured on the surface of a nutrient agar plate. The host bacteria, which
have been spread uniformly throughout the top agar layer, begin to grow, and after overnight incubation form a
lawn of confluent growth. In the mixture, each virus particle attaches (adsorbs) to and infects a single cell.
Inside the cell, the virus reproduces and after 15-60 minutes the cell lyses, thereby releasing several hundred
new virus particles into the medium. The released virus particles can spread to adjacent cells in the agar, infect
them, be reproduced, and again lead to lysis and release. Bacterial cells that have been lysed by the phage form
plaques in the lawn of bacterial cells. The plaques are approximately 1-2mm in diameter. The size of the plaque
formed depends on the virus, the host, and conditions of culture.

Virulent phage kills virtually all the infected bacteria and form clear plaques. However, a percentage of
the cells infected by a temperate phage become lysogenic (and thus immune to super-infection) and survive.
The growth of lysogens results in the formation of turbid plaques.

Figure 2 Plaque Assay - Adsorption, infection and lysis of bacterial cells by bacteriophage to form plaques.
Plague assay is a method used to enumerate lytic phage. A plaque is a clear area that results from the lysis of
bacteria. Each plaque arises from a single infectious phage. The infectious particle that gives rise to a plaque is
called a pfu (plaque forming unit).

REFERENCE:
1. Experiments in Molecular Genetics (Miller 2004)
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LAB 3 - BACTERIOPHAGE TITRATION & TYPING LABORATORY PROCEDURES

PART I. PHAGE TITRATION:
Plaque clearing assay using E. coli 11303
Bacteriophage T2 and T4 stock solutions diluted to 106 phage/ml 5 ml each/section

MATERIALS: (PER GROUP of 4)
5ml of O/N E.coli 11303
12 plates of TSA for titration
2 plates of TSA per class for controls (monitors)
12 tubes of soft agar (0.65%), 2.5 ml/TT, molten (50C)
2 tubes of soft agar for controls
Sterile pipettes (1 ml and 5 ml) and micropipettes (100l & 1000l)
Sterile tips (P100, P1000)
6 sterile tubes for dilutions (16mm, screw caps) + 2 tubes/ section for Control
80ml sterile PO4- buffer
6 Beakers (to create 50C bath at the bench) 1 per group
Hotplates

PREPARATION:
1. Warm the agar plates in the incubator (35C) for 15 minutes just before use (12 TSA
plates/group)
2. Create a boiling water bath at your bench using water in beaker and hot plate
3. Melt soft agar in boiling water bath and then hold in 50C water bath (12 tubes/group)
4. Record carefully the phage type and culture assigned to your group

PRECAUTIONS:

Top agar, or soft-agar, is 7g/L (~0.65%) agar. This allows phage particles to diffuse and infect
cells. Mix E.coli and phage in soft-top agar gently, but QUICKLY. Overlay and pour onto TSA
plates.
Recall, plaques are zones of lysed cells. Phage only infects actively dividing cells (i.e. cells in
exponential growth phase). Plaque size stops once cells are at stationary phase.

Work in groups of 4 students

Procedure A:

1. Obtain a 5ml aliquot of E.coli 11303. Obtain MF standard ! record on your flow-chart.

2. Obtain 6 dilution tubes with 9ml of phage buffer (PO4- buffer) in each tube. Label 6 dilution
tubes from 10-1 through 10-6.

3. Monitors: Label your 7th dilution tube CONTROL containing 9ml PO4- for your control. See Flow
Chart on Page 6.

10-1 10-2 10-3 10-4 10-5 10-6
6 tubes; 9ml PO4- (experimental)
4. Label duplicate plates of PRE-WARMED TSA from 10-2 to 10-7. Maximum of 12 plates for the
dilutions.


(10-2) (10-3) (10-4) (10-5) (10-6) (10-7)

5. Label 12 tubes of 2.5ml/test tube molten (50C) soft-top agar from 10-2 through 10-7

(10-2) (10-3) (10-4)
(10-5)
(10-6)
(10-7)

6. Label 2 TSA plates, tubes of 2.5ml molten soft-top agar for controls (monitors).

Procedure B: Inoculation of Phage and Bacteria

7. Using your experimental test-tube labeled 10-1 obtain a 1mL phage sample (106 stock culture).
Half the class will use T2 and half the class will use T4 your instructor will inform you. From
this test-tube prepare a 10-fold serial dilution of the phage suspension in sterile phage buffer to
the remaining 5 experimental test-tubes

8. Starting at the most dilute, aseptically transfer 0.1 ml of the 10-6 dilution to the 10-7 labeled
molten agar tube. Immediately proceed to step 9.
**NOTE: DO NOT LET THE MOLTEN AGAR SOLIDIFY WHILE IN THE TEST-TUBE. IF FALLING
BEHIND PLACE THE MOLTEN AGAR TUBES NOT INOCULATED BACK INTO THE WATERBATH**
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9. Add 0.1 ml of E. coli to the molten agar tube. MIX by rolling in the palms of the hands and
QUICKLY pour over the surface of the agar plate labeled 10-7 (*Once agar tubes are used please
place into autoclave bucket)

10. Using the same pipette continue the procedure (step 8 and 9) with the next lower dilution and
so on until all six dilutions have been plated.
11. Monitors: Perform 2 controls as shown in flow-chart: 1 No Phage control, 2 agar control
12. Incubate the plates for 18-24 hours at 35C

NEXT WEEK- FOLLOW-UP
1. Count the plaques.
2. Calculate the concentration of bacteriophage in the original suspension (Phage Titre) i.e.
calculate pfu/ml.

3. Flow-Chart

CONTROLS:

AGAR CONTROL (no E. coli)