BMS1021 PRACTICAL UNIT GUIDE

IMPORTANT INFORMATION
You MUST bring your practical manual/guide to each practical class (or
alternatively a copy of the relevant prac printed from Moodle) as you will
work-off these during the prac, and may have to hand them in at the
conclusion of your class.

Please read the Practical Notes carefully before your scheduled class and
bring with you the relevant items. Some practicals have specific requirements
for clothing etc. please be aware of these. All information can be found in this
practical manual/guide and on Moodle.
Additionally, there may be some prior reading and interactive online-sites to
visit before coming to class. Make sure you complete these as your
assessment may depend on them.

Please bring your labcoat to practical classes in Week 1, 4-6 and 8-12.

REQUIREMENTS FOR SAFE CONDUCT OF STUDENTS IN LABS

Safety in the laboratory depends upon students achieving a recognized standard of
behaviour .The following requirements shall apply to all students who use or enter the
laboratory:

Laboratory coats or surgical gowns shall be worn at all times while in the laboratory
unless otherwise stated.

Safety eyewear should be worn at all times while in the laboratory unless a risk
assessment has deemed it unnecessary.
(You will be supplied with safety glasses when necessary)

Only non-slip closed in footwear is acceptable footwear for the laboratory. Do not
enter the laboratory wearing open-toed shoes.

Long hair should be tied back at all times.

Do not handle, store or consume food or drink in the laboratory.

Do not smoke within the laboratory or associated storage areas.

Do not apply makeup while in the laboratory.

Do not mouth-pipette.

Wash skin areas which come in contact with chemicals, irrespective of

concentration. Wash hands upon leaving the laboratory.

Always report hazards, faults, incidents and injuries to the demonstrators/practical

convener.

Do not indulge in reckless behaviour in the laboratory.

Always adopt an alert attitude and be conscious of potential hazards.

Regard all substances as hazardous unless there is definite information to the

contrary.

Dispose of specialized wastes (e.g. broken glassware, syringe needles, biological

and radioactive substances) in containers designated for the particular type of
waste.

Keep all fire-escape routes completely clear at all times.

Footwear To Be Worn While Working In The Labs

All students should wear proper protective footwear (closed all round and the
foot covered totally).

Slippers, sandals, straps & high heels not permitted

PRACTICAL CLASSES
You can allocate yourself to a practical class through Allocate+.
Students only attend one session per topic, see below (note Week 4/5 and 8/9 where students attend
once in each of the 2 weeks designated).
Additionally, some students may be allocated into a Microbiology practical in Wk9 or 10 (instead of
Wk11 and 12). This means students may have 2 pracs for different topics in the same week. This is
unavoidable due to location capacity and student numbers.
Clayton Map http://www.monash.edu.au/assets/pdf/about/who/clayton-campus-map.pdf

Week

Practical Class / Topics

Venue

Assessment%

Week 1

Practical 1
Microscopy

22 Rainforest Walk

3.5%

Practical 2
Biochemistry
Online

Online-only

3.5%

Essay Workshop: Finding and

referencing information for your
essay

Online-only

Practical 3
Introduction to Developmental
Biology

10 Chancellors Walk /
CG63 or
22 Rainforest Walk

Week 6

Practical 4
Histology

10 Chancellors Walk /
CG63 or
22 Rainforest Walk

Week 7

Mid-Semester Exam

Week 8

Practical 5
Metabolism

Week 2

Week 3

Week 4
Mid-semester

3.5%
Break

Week 5

10 Chancellors Walk /
CG63

3.5%

10%

3.5%
22 Rainforest Walk

Week 9

Week 10

Week 11

Week 12

Practical 6
Immunology

Practical 7A
Microbiology Part A
Practical 7B
Microbiology Part B

10 Chancellors Walk /
CG63 or
22 Rainforest Walk

12 Innovation Walk
Room G27/G38

3.5%

4%

Practical Classes Total = 25%

Mid-Semester Test = 10%
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TABLE OF PRAC
DETAILS

Assessment
type

Assessment
due dates

Submission
location

PRAC 1: Microscopy

Prac questions

At the end of
the practical

In prac

PRAC 2: Biochemistry

Online quiz

Week 3
PRAC 3: Introduction
to Developmental
Biology
PRAC 4: Histology

Available
Online
5pm 4th
March until
5pm Mon
14th Mar
Essay Workshop. No assessment
Online
Available
Online
assessment TBA 5pm Mon
21st Mar until
5pm Mon
11th April
Online activity
Available
Online
5pm Mon
11th April
until 5pm
Mon 18th
April

Week 7 - Mid-Semester Exam

PRAC 5: Metabolism
Prac questions

At the end of
the practical

In prac

PRAC 6: Immunology

Prac questions

At the end of
the practical

In prac

PRAC 7A:
Microbiology
PRAC 7B:
Microbiology

Tutor
Assessment
Tutor
assessment
&
Quiz

During prac

n/a

During prac

In prac

Toward the
end of the
practical

Assessment
marking and
return
Prac
returned
within 2
weeks
At quiz
closing time

Assessment
feedback

Prac mark/
comments
returned
within 2
weeks
Same as
above

Online
feedback

Prac
returned
within 2
weeks
Prac
returned
within 2
weeks
See below

Feedback
written on
prac

Overall
marks (Quiz
and Tutor
assessment)
within 2
weeks via
Moodle
Grades

Grades-only

Feedback
written on
prac
Online
feedback

Online
feedback

Feedback
written on
prac
See below

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PRAC 1: Microscopy
Location: 22 Rainforest Walk
Assessment for this practical will be questions to be handed in at the
end of the practical.
Assessment pages to be handed in can be found at the end of this prac and should be filled in, torn-out and
handed in at the end of the practical session.

USING THE COMPOUND LIGHT MICROSCOPE TO STUDY CELL STRUCTURE AND

ORGANISATION IN A PROTIST
Specific Learning Objectives:

1. To learn the mechanics and use of the compound light microscope

2. To become familiar with cells and tissues using the light microscope
3. To learn and apply some of the conventions associated with the drawing of biological
specimens

PRE-LABORATORY PREPARATION
1.

Read Campbell Biology

pp. 93-99 (Microscopy)

(10th Edition)

pp. 102-103 (Eukaryotic Cells)

pp. 134-136 (Water balance in cells without walls)
pp. 612-613 (Ciliates: Paramecium)

For some groovy images and information on Paramecium, go to:

http://protist.i.hosei.ac.jp/PDB/Images/Ciliophora/Paramecium/index.html
or
http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopyuk.org.uk/mag/articles/param1.html
2.

Before coming to the practical, using the textbook and these notes, make sure you understand and

are able to define the following terms

3.

cell membrane

cytoplasm

oral groove

vacuole

osmoregulation

protist

micronucleus

cilium (plural cilia)

macronucleus

Bring 1-2 sharp HB pencils, a ruler and an eraser to this practical

In Part 1 of this practical, you will learn how to use the microscope and determine the relationship between
the magnification and field of view of different microscope objectives.
In Part 2 you will examine and draw a unicellular protist, applying standard conventions for biological drawings.

Please be aware that the answers to your questions and drawing of your specimen will be due before you
leave the session.

SAFETY NOTES

Please do not wear mascara to this practical it ends up on the microscope eyepieces and is difficult
to remove

Microscopes are expensive, precision instruments please handle them with care

PART 1. INTRODUCTION TO THE COMPOUND LIGHT MICROSCOPE

The compound light microscope is perhaps the single most important instrument used in cell biology.
It can magnify objects from 40 up to 1000 times life size, allowing observation of cell and tissue
details that are not visible to the naked eye. Light from a lamp is sent through the specimen and
passes two lenses, the eyepiece and objective (refer to the diagram illustrating the microscope).
Total magnification is the result of multiplying the magnification factor of each lens. The high power
objectives (total magnification of x400 or greater) on a compound microscope mean that most tissues
must be fixed, embedded in a resin, sliced into very thin sections and stained before they can be
observed. This obviously kills the cells. Only very small and very thin, transparent specimens can be
observed whole and alive. Adjustment of the iris diaphragm alters the properties of the light passing
through the specimen, affecting brightness, resolution and contract. It is possible to monitor the
activities of living cells and to observe changes in their subcellular organisation. The light microscope
may also be used to monitor certain operations such as cell fractionation and biochemical
characterisation of cellular components.

Microscope Field of View (FOV)

Units of length
The basic unit of length used in all aspects of scientific measurement is the metre (m). In microscopy,
smaller units are used, which are:

micrometre:

1 mm = 10-3 m
1 m = 10-6 m

nanometre:

1 nm = 10-9 m

millimetre:

Determining the field of view diameters of a microscope

To estimate the actual size of an object or structure being viewed, you first need to know the diameter
of the field of view. The higher the magnification, the smaller the field of view. The diameter of
the field of view for your microscope should be in the range 1.6-2.0 mm (1600-2000 m) using the
x10 objective (i.e. total magnification x100), and approximately 0.4-0.5 mm (400-500 m) with the
x40 objective (i.e. total magnification x400). You will use an object of known size (a micrometer) as
the first specimen to calibrate the field of view.
Obtain a compound microscope by holding the microscope at the arm and under the base.
Do not tilt the microscope or the eyepieces will fall out.
Collect a micrometer slide from the slide folder. Hold the slide up to the light you should
just be able to see a small, straight, gray-black line (the micrometer), in approximately the
middle of the coverslip. The micrometer is exactly 5 mm long, and the smallest divisions in
the scale are 100 m (or 0.10 mm) wide.

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 Always start a microscope session with the lowest power objective (the x4 objective it
has a red band). With the x4 objective in position, use the coarse focus knob to position the
microscope stage some distance below the tip of the objective, then place the micrometer
slide on the microscope stage. Position the slide, using the stage adjustment knobs, so that
the micrometer is in the middle of the beam of light shining through the hole in the stage.
Watching from the side, carefully raise the microscope stage to its upper limit.
Now, looking through the eyepieces, slowly lower the stage until the micrometer comes into
focus. Using the stage/slide adjustment knob, adjust the position of the slide until the
micrometer is in the center of your field of view. When you move to a higher magnification
(i.e. changing the objective by rotating the nosepiece), the micrometer should remain visible
and you should only need to adjust the fine focus. Using the scale on the micrometer slide,
estimate the field of view (FOV) diameter and write this value (in m) into the x40 mag. box
on the first page of these notes.
Change to the x10 objective. Use the fine focus knob to focus on the micrometer.
Again, using the micrometer slide scale, estimate the FOV diameter and write this into the
x100 mag. box on the first page of these notes.
Answer Question 1. All questions and space for answers are provided at the end of
these prac notes.

Now, without altering the focus, switch to the x40 objective focus, (please take care not to
crush the objective into the micrometer slide), and measure the exact diameter of the
field of view on your microscope. This diameter will vary between microscopes and should
be measured individually for each microscope.
Answer Questions 2-5.

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PART 2. OBSERVING
ORGANISM

AND

DRAWING

LIVING

The first living cells observed using the earliest microscopes were various unicellular
microorganisms. Some unicellular eukaryotes are rather transparent, and thus need to be stained to
give contrast to the cell and its contents. Others are fast swimmers, and need to be slowed down by
immersion in methyl cellulose. In this part of the practical, you will observe and draw a ciliate protist
and use different optical methods to enhance the observation of it and its subcellular components.

Examination of Cells
View the organism at low (x100 mag.) and at high (x400 mag.) power. When examining cells, you
should look for a number of different features. For example, cell size, shape and colour are important
characteristics, along with the presence (or absence) of a cell wall, whether the nucleus is obvious,
and the presence of particular organelles. A drawing will record much of this information. An
accepted part of scientific drawings (and one that is useful for revision purposes) is to record any
useful descriptive information on the drawing or in the caption.

Paramecium, a freshwater ciliate

The unicellular organism that you will examine is Paramecium, a freshwater ciliate. The surface of
each cell is covered with hundreds of cilia arranged in longitudinal rows, which propel the organism
through water by rhythmic beating.
1. Using the pasteur pipette provided, take a small sample of Paramecium culture (do not swirl
the mixture and make sure your sample is taken from the bottom of the beaker) and place
one drop of this culture onto a clean slide. Add a small drop of methyl cellulose.
2. Using a toothpick, mix the Paramecium culture and methyl cellulose solutions together, then
carefully lower a coverslip over the mixture.
3. Touch a piece of filter paper to one side of the coverslip to remove any excess liquid.
4. Using the x4 and then the x10 objective, bring a Paramecium into focus, then switch to the
x40 objective. Close the iris diaphragm (refer to the diagram of the microscope) to better
examine the way Paramecium moves. HINT: if you are having trouble locating the
Paramecium, focus along the edge of the cover slip and you should find some there.
Observe the cilia and the slow pulsation of the contractile vacuole.

12

5. On the page provided, and using the x40 objective, draw a single Paramecium indicating
the cilia (single = cilium), the cell membrane, the oral groove, and a contractile
vacuole(s). Make sure that your drawing of the cell is at least 15 cm long (use half an A4
page) and that it conforms to scientific drawing standards. For examples of standard
conventions such as title, labels, captions, scale bar, etc., please refer to the provided
materials in the lab.

|--------------|
20 m
Figure 1. Photoimage of Paramecium.

Example of a scale bar

6. Put an appropriate scale bar on your diagram (in the style of Fig. 1 above). To do this, focus
on your specimen with the x40 objective (total magnification x400), and then use the following
procedure:
Work out the real length of the Paramecium
you are observing. To do this, estimate how
many times (lengthwise) it would fit across the
field of view. It may be easier to work this out by
placing it to one side of the field - e.g. at the
right.
Edge of field of view

Paramecium

Note: Do not draw the edge of the field of

view in your diagram.
Now divide the field of view diameter which you measured in order to answer Question 2, by
the number of lengthwise Paramecium that would fit across your field of view. This gives you
the actual size of your specimen in real life. Your answer should be in m.
For example, if the diameter of the field of view is 500 m (0.5 mm) using the x40 objective
(total magnification of x400), and the specimen fits across the field about 4 times, then this
particular Paramecium is:
500/4 = 125 m long in real life (i.e. its actual length is 125 m)
13

In Figure 1, the specimen is drawn about 10 cm long, so the ratio of actual length to drawn
length is:
125 m : 10 cm
Now you need to draw a 1 cm scale bar next to your diagram. You need to indicate what this
1 cm represents in real life (let this value = y). You can calculate the value of y from the
following ratio:
125 m : 10 cm as y m : 1 cm
Thus, 125 m / 10 cm = y m / 1 cm
So, y = (125 x 1) / 10 = 12.5 m
Thus for this drawing, a 1 cm scale bar represents 12.5 m in real life. Therefore, a 2 cm
scale bar would represent 25 m in real life. If you generate a value like 24.2 m, round up
to the nearest whole number (i.e. 25 m).
7. Observe and identify the different cellular components in Paramecium and label those that
are asked for on your drawing.
Answer Questions 6 and 7.

Once Completed
Make sure your microscope is clean. Remove the slide from the stage of the microscope. Wipe the
x40 objective with lens tissue to remove any methyl cellulose which may be on it. Remove the power
cord from the microscope and carefully return it and the microscope to their appropriate spots. Please
ensure that the micrometer slide is returned to the slide folder, and place all used slides in the waste
containers provided. Dispose of any rubbish and leave your workbench clean.

Assessment
Make sure your name, authcate ID (not ID number) and prac session are written on each page
that you are submitting. Staple your Paramecium drawing (Figure 1) and your answers to
Questions 1-7 together and submit them BEFORE YOU LEAVE the lab. Late submissions will not
be accepted.

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15

Prac 1: Microscopy prac

Name:

Authcate ID:
Prac Session (Day & Time):
PART 1
Q1. What is the diameter of the field of view on your microscope using the x10 objective (i.e.
at a magnification of x100)? [0.5 mark]

Q2. What is the diameter of the field of view on your microscope using the x40 objective (i.e.
at a magnification of x400)? [0.5 mark]

Q3. What is the ratio of magnification between the x10 and x40 objectives? [0.5 mark]

Q4. What is the ratio of the diameter of the field of view between the x10 and x40
objectives? [0.5 mark]

Q5. What do these ratios tell us about the relationship between magnification and the
diameter of the field of view? [1 mark]

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Prac 1: Microscopy Prac

Name:

Authcate ID:
Prac Session (Day & Time):
PART 2
Using the x40 objective, draw a single Paramecium indicating the cilia (single = cilium), the
cell membrane, the oral groove, and a contractile vacuole. Make sure that your drawing of the
cell is at least 15 cm long (use half an A4 page) and that it conforms to scientific drawing
standards. For examples of standard conventions such as title, labels, captions, scale bar, etc.,
please refer to the materials available in the lab. [5 marks]
Note: Do not draw the edge of the field of view in your diagram.

17

Prac 1: Microscopy Prac

Name:

Authcate ID:
Prac Session (Day & Time):
Q6. Which organelle in the Paramecium regulates its water content? What would you expect
to happen to the cell if this organelle was inoperative? (Hint: Paramecium lives in
freshwater) [2 marks]

Q7. Cilia allow the Paramecium to move through water. Do these cilia move randomly or in a
coordinated manner? Describe how the cilia move in relation to each other to allow for
movement. [2 marks]

ASSESSMENT SUMMARY
Activity

Marks

Questions 1-7

Drawing of Paramecium

Total

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PRAC 2: Biochemistry
This is a self-directed online practical go to Moodle for details
Assessment for this practical will be the completion of a quiz on Moodle.

Available 5pm Friday 4th March until 5pm Mon 14th March

Ionisation of aspartic acid. Protein function often relies on specific charged groups to
catalyse reactions and as a result each protein will have an optimal pH at which it can
function. The separation of amino acids and proteins often relies in differences in their
molecular charge. In order to achieve a separation of two amino acids of similar size,
conditions must be found under which they have different molecular charges.
These will be studied by looking at the ionisation of the amino acid aspartic acid at different
pHs.
There will be a self-directed learning exercise available on Moodle.

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21

Essay Workshop:
Location: Online-only
Finding and referencing information for your essay:
The activities that are part of this workshop are an important part of your
Biomedical Science degree. Once you understand and apply this information,
you will find that starting, researching and writing scientific essays will come
more easily.
This workshop will require completing two sets of benchmark activities:
one in week 3 and one in week 6.
The activities are built around skills needed to complete the 15% Essay for
BMS1021. They have been prepared by library staff including Tomas Zahora
and Penelope Presta.

Topics covered will include:

How do I source information online? (week 3)

What library resources can I use for the essay? (week 3)
How do I use and properly reference scientific articles? (weeks 3 and
6)
How do I organise my essay? (week 6)
How do I avoid common essay writing mistakes? (week 6)

All necessary information is included in the workshop activities. No

additional reading or preparation is needed.

See Moodle for more details.

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PRAC 3: Introduction to Developmental Biology

Location: 10 Chancellors Walk (CG63) or 22 Rainforest Walk (Friday
only)
Assessment for this practical will be the completion of an online Moodle
assessment TBA.
Available 5pm Mon 21st March until 5pm Mon 11th April

EXPLORING THE BASIS OF MALE INFERTILITY

Infertility or subfertility is a common problem in Australia, with approximately 15% of couples
experiencing difficulties conceiving. This has been addressed by the introduction of artificial
reproductive technologies (ART) including in vitro fertilisation (IVF) and intracytoplasmic sperm
injection (ICSI). In Australia, more than one percent of births are now the result of ART. However,
these techniques cannot address all causes of infertility, particularly in cases where the underlying
defect is unknown. Further research is required to identify the underlying causes of infertility and
develop new individualised treatment options.
Male fertility is also an important consideration for industries where breeding of animals is
important either for agricultural purposes or for other purposes (for example selective breeding).
Artificial insemination can be used which requires the assessment of the quality of semen.
In this practical we will examine the process of spermatogenesis, which is responsible for the
continual production of sperm, and the characteristics of sperm that are often examined in fertility
testing.

PRACTICAL OBJECTIVES
1. To visualise the different cell types within the seminiferous tubules and the progressive steps in
the process of spermatogenesis.
2. To examine sperm and identify the structure and function of different regions of mature
spermatozoa.
3. To investigate the role of a secreted growth factor from Sertoli cells in male fertility.
4. To analyse a porcine semen sample and measure sperm concentration, motility, morphology
and viability.
A worksheet will be provided in class.

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25

PRAC 4: Histology
Location: 10 Chancellors Walk (CG63) or 22 Rainforest Walk (Friday
only)
Assessment for this practical will be the completion of an online (Moodle)
assessment. Available 5pm Mon 11th April until 5pm Mon 18th April
The take home message from this practical is to recognise that organs are made up of the
4 primary tissue types.
Activities will include microscope work, hands-on activity, group work and an opportunity to
present student group-work. The prac will be led by experienced demonstrators from the
Anatomy & Developmental Biology Department as has been designed to be a fun and
engaging class, to help you apply information learned in lectures.

Please bring to class:

Your histology lecture notes, any histology textbooks, iPads/personal laptops/smart phones
(there will be computers available in class on request).
Dont forget your labcoat.

Background reading:
Chapters in Histology books such as Kerr Functional Histology 1st or 2nd edition, or
Wheaters Functional Histology or any other basic histology texts. Focus on the 4 primary
tissue types.
Revision of lecture notes may also be useful.

Worksheets/other will be provided in class.

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27

PRAC 5: Metabolism
Location: 22 Rainforest Walk
You MUST check your week and lab allocations for this class.
If you need to change, please see
Yardenah Brickman or Christopher Wilson
in the 1st Year Biology Teaching Laboratory,
23 Rainforest Walk, BEFORE missing your lab.
Space is limited so
DO NOT assume that there will be space if you turn up to a session
which is different to the one allocated to you.
Assessment for this practical will be questions to be handed in at the
end of the practical.
Assessment pages to be handed in can be found at the end of this prac and should be filled in, torn-out and
handed in at the end of the practical session.

CELL METABOLISM: AMYLASE ACTIVITY IN GERMINATING BARLEY

Specific Learning Objectives:

To extract an active enzyme from biological material and use it to catalyse a

specific biochemical reaction

To quantify the amount of enzyme activity present in the sample

To correlate enzyme activity with metabolic function during development

To identify the product of the enzyme-catalysed reaction

28

PRE-LABORATORY PREPARATION
1. Read the following pages in Campbell et al. (2015), Biology 10th Edn (Australian version):
pp. 871
pp. 68-75
pp. 151-154
pp. 167

Seed germination
Carbohydrates
Enzymes
Overview of cellular respiration

2. Use the textbook and these notes to make sure that you understand and can
define the following key words:
amylase
ATP
biosynthesis
control
dissacharide

enzyme reaction
glucose
glycolysis
hydrolysis
maltose

metabolism
pH buffer
polysaccharide
product
reaction rate

starch
substrate

INTRODUCTION
Amylase is an enzyme that catalyses the chemical reaction that breaks down starch (a
polysaccharide) into smaller disaccharide units which can be further broken down to
glucose, the universal energy source. Different forms of this enzyme exist in plants and
animals. In our own digestive system it is produced by salivary glands in the mouth and by
the pancreas. In plants it plays a major role in making energy stored in seeds available for
the development of seedlings. Germination of seeds starts when water is taken up
(imbibition) and passes through the embryo (Figure 1). This results in gibberellic acid, a
plant hormone, activating DNA that codes for the enzyme amylase. The amylase is shipped
into interior of the endosperm (food store), where it hydrolyses the (l - 4) glycosidic bonds
between pairs of glucose units to release the disaccharide maltose, which is transported to
the embryo. Maltose is then further hydrolysed to single glucose molecules by a second
enzyme called glucosidase. Glucose can enter the glycolytic pathways of cellular
respiration where it is utilized for producing ATP and carbon molecules for biosynthesis. As
the embryo divides the seed expands, the seed coat ruptures and the radicle, the first part
of the new seedling, emerges, completing the process of germination.

Figure 1. Activation of -amylase production during germination in a barley seed.

29

As the seedling grows, by the time the endosperm is completely used up the seedling should have
reached a stage where its energy and carbon requirements can be met by photosynthesis.

SAFETY NOTES
Lab coats must be worn. Enclosed footwear must be worn in the lab.
Steam from boiling water baths can burn you must wear the safety gloves

provided.
BEFORE YOU START YOUR EXPERIMENT:

Make some predictions as to what level of amylase activity you expect to see in each
life stage of the barley. Would all stages show presence of amylase activity? Would
some stages have more or less amylase activity than others?

EXPERIMENTAL PROCEDURE
The specific aims of this practical session are to:
1. Determine and compare the mass-specific amylase activity among dormant barley seeds,
germinating barley seeds and barley seedlings by measuring their rates of starch
hydrolysis;
2. Interpret these results in terms of the metabolic role of amylase during plant development;
3. Identify maltose as the product of starch hydrolysis by amylase.
Work in pairs throughout this practical. Refer to the flowchart on the last page of
these prac notes for an overview of the experimental procedure.

Part 1 Preparation of amylase extract from germinating barley

1.1 You have been provided with 10 germinating barley seeds (germinants, 3 days old). Using
paper towel, pat the germinants dry, then weigh and
record the total weight of the 10 germinants.
Weight of germinants = ______ g
1.2 Crush the 10 germinants to a fine paste with a mortar and pestle.
1.3 Slowly add 10 mL of buffer and continue crushing and mixing for 3 minutes to extract the
amylase into solution.
1.4 Filter this solution through a tea strainer into the 100 mL beaker. Pour this amylase extract
into the measuring cylinder, record the volume, and then return the solution to the
beaker.

Volume of amylase extract = ______ mL

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1.5 Make a five-fold dilution of the amylase extract by auto-pipetting 20 mL of buffer into a
measuring cylinder and then adding 5 mL of the amylase extract (use the 5 mL pipette) to
make the total volume 25 mL. Do not be concerned if the volume on the measuring cylinder is
not 25mL. The volume on the measuring cylinder is not as accurate as the auto pipette
dispenser. Mix well. This is your diluted amylase extract.
Note: Save the excess amylase extract in case you make a mistake!
1.6 Prepare a control extract by adding 5 mL of the diluted amylase extract to a test tube and
place it in the boiling water bath for 10 minutes. Whilst waiting, start on Part 2. When the 10
minutes have elapsed, remove the test tube from the boiling water bath and leave it to cool at
room temperature.
Part 2 Determination of amylase activity in germinating barley by measuring the rate of
starch hydrolysis
The rate of starch hydrolysis can be determined by making up a reaction mixture containing starch
in a buffer of appropriate pH. Amylase extract is added, and the samples are tested with starch
indicator solution at constant time intervals. Starch indicator solution (iodine) gives a blue colour
when bound to starch, but does not give a colour reaction with maltose. The colour intensity
decreases as the reaction proceeds, until no colour develops when all the starch has been
hydrolysed. This is called the achromic point, and the time taken to reach this point gives a measure
of the rate of the reaction catalysed by amylase. If the amount of starch at the start of the reaction is
known, then it is possible to calculate the activity of amylase as the amount of starch hydrolysed/unit
time/g barley tissue. Carry out the following experiment to determine the activity of amylase per mass
of germinating barley tissue.
2.1 Place one drop of iodine into each of 21 labelled wells on the ceramic test plates. Ensure the
volume of each drop is approximately equal for each well.
2.2 Add 5 mL of buffer and 1 mL of 0.5% starch solution to a test tube, and mix well. This is
referred to as the reaction mixture.
2.3 Using a disposable pasteur pipette, add a single drop of this reaction mixture to a drop of
the iodine in the well labelled T (for test) on the test plate. It should turn blue/black, indicating
the presence of starch in the reaction mixture.
Now that you are familiar with the colour change reaction that occurs when starch and
iodine are mixed together, you are ready to start your experiment.
2.4 Thoroughly remix the diluted amylase extract and then add 1 mL diluted amylase extract
to the reaction mixture in the test tube (time = 0 min), mix well. This is the amylase reaction
mixture.
You have combined amylase with a starch/buffer solution think about what is NOW
occurring in this tube and immediately do the step below!
2.5 Starting with well number 0, at 1 minute intervals add a drop of the amylase reaction
mixture (use the disposable Pasteur pipette) to sequential wells until the achromic point is
reached.

2.6 Record the time taken to reach the achromic point. This should be between 5 and 20
minutes. If not, review your experimental procedure and ask a demonstrator for help.

31

Please note the following:

If the achromic point is reached in less than 5 minutes, repeat the experiment but
add less amylase extract and additional buffer to maintain the total volume of the
reaction mixture at 7 mL. Ensure to record the volume of amylase extract used.

If the achromic point is more than 20 minutes, repeat the experiment but add more
amylase extract and less buffer to maintain the total volume of the reaction mixture at
7 mL. Ensure to record the volume of amylase extract used.

When the achromic point has been reached, save the amylase reaction mixture for the
determination of maltose in Part 3.
2.7 Repeat the experiment using the identical volumes of buffer and starch that gave a
satisfactory reaction rate for your germinants, but replace the diluted amylase extract with the
same volume of boiled control extract.
Answer Question's 1 and 2. All questions and space for answers are provided at the
end of these prac notes.
2.8 Calculate the activity of amylase in the germinating barley. You will notice that the formula for
this is not directly given to you. Part of the assessment is that you work it out yourself.
To make this calculation, you will need the following information:

The concentration of the starch solution = 0.5%, i.e. 500 mg/100 mL

The time taken to reach the achromic point
The volume of diluted amylase extract added to the reaction tube
The total volume of the filtered, non-diluted amylase extract
The total weight of the barley seeds used to make the extract

Also note that the unit of amylase activity is mg starch hydrolysed/min/g barley tissue.
Suggested approach:

Calculate the amount of starch added to the reaction tube.

Calculate the average amount of starch hydrolysed/min to reach the achromic point.
Assume that all the amylase in the barley grains was washed into the filtered
extract. Knowing the volume of diluted amylase extract added to the reaction tube,
the total volume of filtrate, the filtrate dilution factor and the mass of barley tissue, it
is possible to calculate the activity as mg starch hydrolysed/min/g barley tissue.

Answer Question 3.

Part 3 Identifying maltose as the product of starch hydrolysis by amylase

Benedict's reagent gives a red-orange precipitate of cuprous oxide when boiled with maltose. This
reaction does not occur with starch. Carry out the following:

32

3.1 Using your saved reaction tube for maltose determination, add 2 mL of this amylase reaction
mixture to a test tube containing 2 mL of Benedict's reagent.
3.2 Prepare a control by adding 5 mL of buffer to 1 mL of 0.5% starch solution in a clean test
tube. Mix well, and then transfer 2 mL of this into a new test tube containing 2 mL of
Benedict's reagent. This is your control mixture. Note that for this experiment both the control
and experimental tubes should contain equal volumes of solution (4 mL).
3.3 Place the two Benedict's reagent tubes in the boiling water bath for 10 minutes, then examine
them for the presence of cuprous oxide precipitate.
Answer Question 4.

Part 4 Presence of amylase activity during barley development

4.1 Following the same procedure used with the germinating barley in Part 1 and Part 2, prepare
extracts of 10 dormant seeds (0 days old) and/or 10 barley seedlings (7 days old) as advised
by your demonstrator. If necessary, include the use of a boiled control.
Between preparations, take care to clean the mortar, pestle, tea strainer, pipettes and
measuring cylinder to avoid cross contamination of the extracts.
4.2 Determine the amylase activity of the extract(s) using the procedures outlined in Part 2.
Answer Question's 5-7.

Once Completed
Before leaving the laboratory, empty the tubes containing Benedicts solution into the toxic waste
container. All other tubes can be emptied into the sink and placed in the appropriate tubs. All mortars
and pestles, beakers, measuring cylinders, and ceramic plates are to be rinsed clean and left to
drain on the trays provided. Clean up any rubbish, wipe away any spills on your bench and leave
your work area clean.

Assessment
Make sure your name, authcate (not ID number) and prac session are written on each page
that you are submitting. Staple your pages containing your answers to Questions 1-7 together
and submit them BEFORE YOU LEAVE the lab. Late submissions will not be accepted.

Additional Information to Consider

Malt (maltose produced from barley by the action of amylase), is used commercially as the starting
material for producing beer. Dormant barley seeds are soaked in water to start germination, amylase
is produced to convert starch to maltose, and the process is then stopped by heating ("kilning") to
prevent seedling growth. The maltose is then extracted by heating in water and brewing yeast is
added to provide a glycolytic pathway, which in the absence of oxygen converts maltose to ethanol
and carbon dioxide (fermentation). The initial "malting" step is required because brewing yeasts
cannot metabolise carbohydrates larger than trisaccharides.

33

ASSESSMENT
The following work must be submitted at the end of your session.
Prac 6: Metabolic Prac

Name:

Authcate ID:
Prac Session (Day & Time):
Q1. Do you expect to reach an achromic point for the boiled control? What is the purpose
for using the boiled control in this experiment? [1 mark]

Q2. If an achromic point is reached using the boiled control, how would you use the boiled
achromic point value to correct the amylase activity that is calculated for the germinating
seeds? [1 mark]

Q3. Show your calculations for determining the activity of amylase in germinating seeds.
Include all the steps involved in your calculation. [3 marks]

34

Prac 6: Metabolic Prac

Name:

Authcate ID:
Prac Session (Day & Time):
Q4. What did you observe in both the control and the experimental tubes for Part 3? Was
this expected? What does the Benedict's test confirm for us in this experiment? [1 mark]

Q5. Draw a table showing the calculated amylase activity for each of the barley's life stages.
Do not include raw data. Note that your table should have an appropriate number and
caption and include the appropriate units of amylase activity. [2 marks]

35

Prac 6: Metabolic Prac

Name:

Authcate ID:
Prac Session (Day & Time):
Q6. Compare the observed amylase activity with the predicted amylase activity for each
stage of the barley seeds. For each stage, answer the following:

Do your observed results match your predicted results?

Why/why not?
What biological reasons explain why amylase activity would be present/absent or
higher/lower than other stages of the barley's germination process? [3 marks]

Dormant Seeds (0 days old):

Germinating Seeds (3 days old):

Seedlings (7 days old):

Prac 6: Metabolic Prac

Name:
36

Authcate ID:
Prac Session (Day & Time):
Q7. Starch and cellulose are both polymers of glucose. Why is amylase able to break down
starch into maltose, but unable to break down the cellulose in the cell wall into smaller
molecules? [1 mark]

ASSESSMENT SUMMARY
Activity

Marks

Questions 1-7

12

Total

12

37

FLOWCHART OF STEPS FOR EXPERIMENTAL PROCEDURE

Prepare your amylase extract from the barley

seeds

1.

Weigh your 10 seeds (W = _____ g)

2.

Grind up the seeds using 10 mL of buffer

3.

Filter the solution (filtrate volume = _____ mL)

4.

Make a five-fold dilution of 5 mL of the filtrate

Prepare your reaction mixture (RM =
starch and buffer)

5. Prepare a control
extract by placing 5 mL of
diluted extract in a boiling
water bath for 10 minutes
6. Mix together 5 mL of buffer and 1 mL of
0.5% starch solution. This is the reaction
mixture.

8. Take 1 mL of diluted
filtrate and add it to the
reaction mixture you are
adding the enzyme that
starts your experiment!!
Repeat steps 6-9
(in this flow chart)
with boiled diluted
amylase extract. Is
an achromic point
reached? What
does this mean?

7. Test for the presence of starch. Take a

drop of the reaction mixture and add it to a
well containing a drop of iodine do you
get a blue/black reaction? What does this
mean?

9. At the start and then at 1 minute intervals,

add 1 drop of the amylase reaction mixture to
successive wells containing iodine how long
does it take to reach the achromic point?
(Time = ___ min)

10. Calculate amylase activity = mg of starch hydrolysed min-1 g of

barley tissue -1 (less any activity detected using the boiled control).
Amount of tissue in 1 mL dilute extract is [mass/filtrate/5] g mL-1
Amount of starch hydrolysed was 5 mg in X mins = 5/X mg min-1
Hence, reaction rate = 5/X/[mass/filtrate/5] mg starch hydrolysed
min-1 g barley tissue-1

11. Keep the amylase reaction

mixture and use Benedicts
reagent to test that maltose is the
end product of the experiment.

38

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39

PRAC 6: Immunology
Location: 10 Chancellors Walk (CG63) or 22 Rainforest Walk (Friday
only)
Assessment for this practical will be questions to be handed in at the
end of the practical.
Assessment pages can be found at the end of this prac-worksheet and should be filled in,
torn-out and handed in at the end of the practical session. Ensure you include your name.

STUDY OF THE IMMUNE SYSTEM IN HEALTH AND DISEASE

Co-ordinator: A/Prof Robyn Slattery
Department of Immunology and Pathology

PRACTICAL CLASS OVERVIEW:

This class will demonstrate features of the normal immune system (blood cell types, primary and secondary
humoral immune responses) and also disorders of the immune system (immunodeficiency and
immunomalignancy).
Students will work in groups to set up the Haemagglutination assay (exercise I). There will be time during the
haemagglutination incubation period to commence the other exercises. At the end of the class, students will
review their results with their demonstrator.

EXERCISE 1. THE ANTIBODY RESPONSE BY AN ANIMAL AFTER PRIMARY AND SECONDARY

IMMUNISATION

NOTE: This experiment will be performed by the whole group and will be coordinated by your
demonstrator.
Introduction:
The two fundamental features of the immune response are antigen specificity and memory. Immunological
memory establishes a state of immunity and allows the body to be effectively prepared to resist a later
invasion by the same organism. Immune memory is the basis of vaccination programs.
In this experiment you will examine the antibody response by a rabbit after a first injection of an antigen and
the antibody response after a second injection of the same antigen. The antigen used is sheep red blood
cells, which is foreign to the rabbit.
Antigens present on the surface of red blood cells will be cross-linked by antibodies produced following
immunisation with the sheep red blood cells and this cross-linking can be detected by observing
agglutination of cells (referred to as haemagglutination) in special trays called microtitre trays. The amount of
haemagglutination that occurs is proportional to the concentration of antibodies in the serum higher
concentration of antibodies results in more haemagglutination.

Materials:
Sheep red blood cell suspension (2.5%)
Microtitre trays
Micropipette and tips
Pre-immune rabbit serum (day 0)
Rabbit serum taken on day 7, day 14, day 21, day 28 and day 100 after the first injection
Phosphate-buffered saline (PBS)

40

Procedure:
1. Examine the haemagglutination plate plan.
2. Prepare serial dilutions of the sera as follows:

Place 50 l of PBS in wells 1-12 of rows A, B, C, D and E.

Add 50 l of the day 0 serum to well 1 of row A. This constitutes a 1:2 dilution of the serum in
well 1.
o
Serially dilute the serum by transferring 50 l from well 1 to well 2, then mixing the
contents (which will give another 1:2 dilution of the serum in well 2 and therefore a 1:4
dilution of the original serum).
o
Prepare a 1:8 dilution of the serum by transferring 50 l from well 2 (1:4 dilution) and
mixing it with the PBS in well 3.
o
Continue serial dilutions to Well 11 of row A, and discard the final 50 l of solution.
o
This procedure has produced serial dilutions of serum in the range 1:2 (well 1) to 1:2048
(well 11: refer to figure below).

3. Using a separate (i.e. clean) tip for each row, dilute the other sera in Rows B-E (refer to figure), as
outlined above.
4. Add 50 l of 2.5% (v/v) SRBC suspension to each well in rows A-E, mix by tapping the plate gently,
cover with a lid, and incubate at 37C for 1 hour. This step can be carried out at room temperature,
but agglutination may take longer to occur.
5. Observe agglutination reactions. Tilt the tray slightly, view against a light background and observe
buttons at the bottom of the wells. Beyond the titration endpoint, the buttons run on tilting.
6. Determine the endpoint (i.e. dilution of last agglutination reaction) for each serum sample.

The antibody titre for the serum is the reciprocal of the endpoint. For example, if the endpoint of
haemagglutination of a serum sample is 1:128, the antibody titre is 128.
The higher the dilution of serum to give an endpoint, the higher is the antibody titre for the
undiluted serum.

7. a) Plot the antibody titres of the sera against the time after immunisation on the graph provided.
b) What conclusions can you make about the strength and duration of the antibody response to this
antigen?
c) What are the consequences of this result to immunisation procedures?

EXERCISE 2. CELLS OF THE IMMUNE SYSTEM

Cells of the immune system are produced in the bone marrow and then circulate in the blood. In this exercise
you will identify the cell types in a human blood film and prepare a differential white cell count.
Method
1. Examine the prepared human blood film stained with Leishmans reagent under the microscope.
Start by using the low power objective to select an area containing a suitable number of white
cells (usually just back from the tail of the film), then move to the x40 objective for identification
of cell types (see table below).

41

2. Perform a differential white cell count by counting at least 100 white cells (ignore the red cells)
and determining the percentage of lymphocytes, monocytes, neutrophils, eosinophils and
basophils.

Morphology of human blood cell types

Cell Type

Morphology

Red cells (erythrocytes)

The most common blood cell type;

stained pink with paler centre due to
biconcave disc shape; no nucleus

Neutrophil

The most common white blood cell type

(40-75%); 2-5 lobed nucleus; cytoplasm
pale lilac with fine granules

Eosinophil

1-6% of white cells; bilobed nucleus;

cytoplasm contains large red granules

Basophil

<1% of white cells bilobed nucleus

obscured by large purple cytoplasmic
granules

Monocyte

2-10% of white cells; the largest white

cell; indented nucleus (not always seen
when settled on slide) grey-blue grainy
cytoplasm

Lymphocyte

20-45% of white cells; dark blue round

nucleus; thin clear blue rim of cytoplasm

EXERCISE 3: DISORDERS OF THE IMMUNE SYSTEM

Lymphocyte subsets such as T and B cells cannot be distinguished by morphology (i.e. in a blood film, T and
B cells look the same). However, they do have different markers on their cell membranes which can be
detected by using labelled antibodies against these markers. For example, the marker CD3 is found on T
cells but not B cells, and CD19 is on B cells only. A common technique for examining a patients blood for
abnormalities in the proportion of lymphocyte subsets is to stain blood cells with fluorescent-labelled
antibodies against these cell membrane markers and then examine the stained cells using a flow cytometer.
In this instrument, a thin stream of the stained cell suspension is passed in front of a laser beam so that cells
are in single file. On passing the laser, any cells which have bound the fluorescent-labelled antibody will emit
fluorescence which is detected by a photomultiplier. The flow cytometer can analyse thousands of cells per
second (quicker than counting cells down the microscope!) and generates data on the proportion of
lymphocytes expressing a particular marker. Diagnosis of immunodeficiencies or immunomalignancies is
routinely performed in this manner.

42

1. In this exercise, you are provided with flow cytometry data for a normal blood sample and for three
patient blood samples:

one is from a patient with HIV infection,

one is from a patient with a B cell leukemia,

one is from a normal patient.

Which results fit which patient?

43

PRAC 7: Immunology Prac

STUDENT NAME: .STUDENT NO:.

STUDENT AUTHCATE USERNAME: .. SESSION TIME .

BMS1021 PRACTICAL 7: STUDY OF THE IMMUNE SYSTEM IN HEALTH AND DISEASE

This answer sheet is to be handed in to your demonstrator at the conclusion of the practical and
will be used in your assessment.
EXERCISE 1:
1. Draw the observed pattern in the haemagglutination assay in the plate plan below: (+ indicates
agglutination - indicates no agglutination)
[3 marks]

2. Answer to question 7 (a) of your worksheet. Plot the time course of the antibody response to primary
secondary immunisation on the graph below (dont forget to label the y-axis):
[5 marks]

44

PRAC 7: Immunology Prac

STUDENT NAME: .STUDENT NO:.

STUDENT AUTHCATE USERNAME: ..

3. Answer to question 7(b) of your worksheet. What conclusions can you make about the strength and
duration of the antibody response to this antigen?
[3 marks]

4. Answer to question 7(c) of your worksheet. What are the consequences of this result to
immunisation procedures?
[3 marks]

45

PRAC 7: Immunology Prac

STUDENT NAME: .STUDENT NO:.

STUDENT AUTHCATE USERNAME: ..

EXERCISE 2:
For your own reference, in your notes draw the morphology of the different leukocytes observed.
5. Indicate in the table below the absolute number and percentage of each cell type observed. This
number will obviously be the same if you count 100 cells. Remember you do not need to count red
blood cells.
[5 marks]

Neutrophils

Lymphocytes

Monocytes

Basophils

Eosinophils

EXERCISE 3:
6. From the flow cytometry data presented to you identify which patient has which disorder and
indicate reasons for your choice
[2 marks each case, Total 6 marks]
Case A:

Disorder
Reasons

Case B:

Disorder
Reasons

Case C:

Disorder
Reason

ASSESSMENT SUMMARY
Activity
Questions 1-6

Marks
Total
/25

46

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47

PRAC 7 Part A and B: Microbiology

ASSESSMENT SUMMARY

Activity

Demo
Assessment

Quiz

Comments

Week A
marks

Week B
marks

2.5

2.5

15

Punctuality (0.5 marks)

Preparedness such as showing prior reading (1 marks)
Active involvement in the lab and tutorials (0.5 marks)
Ensuring work area is clean when you leave (0.5 marks)
Short answer questions which will be answered in 15
minutes at the end of the lab in Week 12 (or Wk10)
Total overall mark

/20 marks
Will contribute to 4% of final
mark

Students must check in advance on the notice boards in 12 Innovation Walk

(G27/28) to ensure you are in the correctly allocated practical session. Students
will not be permitted to change their times.
Assessment for this practical (see above) will be assessment from your demonstrator in both
sessions AND a quiz completed during the practical classes in the second session. Marks will be
put into your Gradebook on the BMS1021 Moodle site within 2 weeks of your practical session.

PRELIMINARY IDENTIFICATION OF UNKNOWN MICROORGANISMS

The aim of this practical is to introduce students to the concept that bacterial pathogens differ from our normal
flora because they express virulence determinants, and that the production of certain virulence associated
determinants can be used to identify a suspected pathogen.

LEARNING OUTCOMES:
At the end of this practical:
1. You should understand the biochemical basis of the differential Gram stain and the importance
of its role as a first stage identification test for unknown microorganisms.

2. You should be able to derive, transfer and grow pure cultures of microorganisms using aseptic
techniques

3. You should be familiar with tests to identify bacterial structures, including flagella, spores and
capsules and the production of enzymes such as haemolysin and catalase.

48

PRE-LAB PREPARATION
Read these notes carefully and refer to Biology (pp. 528-529) for a description and discussion of the Gram
stain. Using the textbook and these notes, make sure that you understand and can define the following key
words:
coccus

rod

differential stain

Gram positive

Gram negative

colony

aseptic techniques

pure culture

sterile

spore

virulence factor

pathogen

capsule

haemolysin

motility

prion

INTRODUCTION
Microorganisms are described as organisms that cannot be clearly seen by the unaided eye. Viruses and
bacteria, together with many algae, fungi and protozoa are classified as microorganisms and are widely
distributed in nature. Many play an important and useful role in the production of various foods, alcohol,
enzymes, vaccines, antibiotics and other products. They are also important members of our ecosystems and
play an essential part in the recycling of nutrients in the environment. They are involved in the carbon, oxygen,
nitrogen and sulphur cycles that take place in aquatic and terrestrial systems. Thus a relationship usually exists
between the types of microorganisms present in a particular habitat and the physico-chemical characteristics
of the habitat itself. For example, very different types of microorganisms grow in the water of a boiling sulphur
spring compared with those growing in contaminated refrigerated food.

While most microorganisms present on and in our body are harmless, some are able to cause disease and
these are called pathogens. Microorganisms that are consistently found and multiply at a given site are known
as the resident flora. In normal, healthy individuals they are non-pathogenic at that site, but some can act as
opportunistic pathogens if they gain access to another site, e.g. Escherichia coli is considered part of the
normal resident flora of the bowel but is a common cause of urinary tract infection. Other organisms that may
be temporarily found at a given site but do not multiply there are called transient flora. These species are
acquired by contact with the surrounding environment and/or contact with people and are potential pathogens.
Infection with pathogenic microbes can lead to life-threatening illnesses such as AIDS, malaria, cholera and
typhoid.

The difference between harmless and harmful microorganisms is that the pathogens possess

virulence factors that allow them to infect and damage the host.

Some of the microorganisms present on and in our body include Staphylococcus epidermidis which is present
in our skin and nasal region and Escherichia coli in the large bowel. Many of them also play an important role
in the gastro-intestinal tract (GI tract) and are also considered to be commercially useful, for example
Lactobacillus acidophilus and Bifidobacterium bifidum which are used in yoghurt manufacture (AB yoghurt).

Microorganisms have also caused significant economic problems e.g.:

the foot and mouth disease outbreaks;

49

contamination of food for local and export markets

They can also be the cause of great community concern, e. g.:

Pollution of water by faecal contamination indicated by the presence of E. coli

Legionella spp. In cooling towers

Drug resistant bacteria in hospitals such as Methicilin Resistant Staphylococcus aureus (MRSA)

the use of microorganisms by terrorist in biological warfare, egs., Bacillus anthracis spores causing
anthrax.

Although not microorganisms, Prions which are proteins present on the surface of brain tissue can also cause
the following diseases when they undergo changes.

mad cow disease Bovine Spongiform Encephalopathy (BSE) in animals.

Chronic Wasting disease in deer.

Creutzfeldt-Jakob disease (CJD) in older people.

vCJD in young adults (attributed due to the consumption of meat from cattle with BSE)

Given that microorganisms live in all environments and proliferate in natural situations, they nearly always
occur as mixed populations in the environment. As such, in order to study microorganisms, they need to be
isolated under aseptic conditions as individual entities in pure culture so that their distinct characteristics can
be identified. A pure culture is one that has only a single type of microorganism present in the medium. There
are many methods of obtaining a pure culture. The three common methods used are the Streak plate, Pour
plate and Spread plate techniques. The spread plate and pour plate methods can also be used to count
the microorganisms present in a given sample so that effective treatment and level of contamination can be
studied.

In this practical you will perform a preliminary identification of unknown pure cultures of microorganisms
isolated from a clinical and an environmental sample. In the first week you will carry out the Gram stain
on the samples to detect any microorganisms present and identify their cellular morphological features
(Gram stain, shape, arrangement, etc.). In addition, you will subculture the microorganisms from the
samples provided for further identification. In the second week you will perform additional tests on the pure
cultures using specialised techniques to detect specific virulence associated characteristics that will aid
identification.

50

PRAC 7 Part A: Microbiology

Scenario: The Unit Coordinator of BMS 1021 at Monash University recently developed a severe, acute
respiratory tract infection after opening a letter containing a white powder.
Aim: To determine the likely cause of the Academic staff members unfortunate infection and identify
the suspicious white powder

A. Isolation of a pure culture for the preliminary identification of unknown bacteria

Materials provided (per pair)

sterile inoculating loops

Photo of BMS 1021 coordinator opening envelope and scattering white powder

one broth culture of bacteria grown from white powder (sample A)

one broth culture from a sputum sample (sample B)

four agar plates, two each of horse blood agar (HBA) and nutrient agar (NA)

One student from each pair will plate out Sample A, the other will plate out sample B.
1.

2.

Label the base of the blood and nutrient agar plates with:
(i)

Sample origin

(ii)

your name, day, bay and group number

Using a flame-sterilised inoculating loop, transfer a loopful of Sample A or B broth culture and spread
over one section of a blood plate as shown below in step (a). Proceed to spread the bacteria across
the remaining agar surface by flaming the loop and allowing it to cool between each set of streaks (bd). Drag the loop over the agar plate with the loop at an acute angle to the plate to prevent the loop
cutting into the agar..

(a)

area inoculated with loop

(b)

first set of steaks

(c)

second set of streaks

(d)

third set of streaks

(e)

final streak

(a)
(b)

(d)

(c)

3.

Repeat the procedure for Sample B using the remaining blood and nutrient agar plates

4.

Place all of your inverted plates into the labelled white boxes for incubation in air at 37C for 24 hours.
You will examine the plates in the next practical session.
51

B. Motility test for the preliminary identification of unknown bacteria

Many bacteria have the ability to swim through liquid or semi-solid medium. This motion is termed motility
and is powered by bacterial flagella. Nevertheless not all bacteria are motile and thus the property of motility
can help to identify unknown bacteria.
There are three possible methods for determining the motility:
a) Hanging drop preparation overnight culture has to be used and results could be obtained immediately
b) Craigie tube needs to be incubated and incubated
c) Mast method need to be inoculated and incubated

In this experiment you will be using the MAST method to study motility.
Materials Provided
(per pair)

sterile inoculating wire

samples A and B from part A.

four motility agar bottles (Mast motility agar)

control plate cultures of Escherichia coli (motile) and Staphylococcus epidermidis (non-motile)

1.

Label the bottles with sample origin, your name, day, bay and group number.

2.

Using aseptic technique and the inoculating wire, transfer the bacteria from the broth culture of
sample A and stab into the centre of the motility agar. Repeat with a new bottle each for sample B
and the control strains provided.

3.

Place the tubes into the 52abeled white boxes on the trolley for incubation in air at 37C for 24
hours. You will examine the tubes in the next practical session.

C. Gram stain for the preliminary identification of unknown bacteria

Bacterial cells are difficult to see under the bright field microscope even at high magnification because
there is very little contrast between the cell and the surrounding medium. To improve contrast, bacteria
can be stained to show their basic shape, size and arrangement. Differential stains are used to
distinguish between bacteria that differ from one another biochemically and physically, by their varied
reaction to a particular staining process.

The Gram stain is the most common differential staining technique and is used routinely for the preliminary
identification of unknown bacteria. Not only does it allow us to see the shape of bacterial cells easily using a
microscope, but it separates bacteria into two groups Gram positive (purple) or Gram negative (pink) based
on their cell wall structure.

52

Gram staining should be carried out on young cultures (less than 24 hours of incubation), otherwise variable
results would be achieved as some Gram positive bacterial groups take on increasingly Gram negative
character as the culture ages.

Here you will Gram stain the two test samples A and B to detect any bacteria present and determine if they
are Gram positive or Gram negative by comparing with the two controls provided.

Materials Provided (per student)

1 x microscope slide

samples A and B from part A

Gram stain reagents

control plate cultures of E. coli (Gram negative) and S. epidermidis (Gram positive)

1. Make a heat fixed smear of sample A and B (as per appendix A) and the control strains provided setting
them out on a slide as below:
Grease pencil marks*

E. coli

S. epidermidis

Sample A or B

Mark underneath the slide and not in the middle with the grease pencil
2. Perform Gram stain (as per appendix B) and record your results below
Students should also observe the Gram stain of their partners specimen

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Table 1. Gram stain reaction and appearance of cultured bacteria.

Microorganism

Gram reaction

Cell shape

Cell arrangement

Sample A

Sample B

E. coli

S. epidermidis

NOTE: When you have finished using your microscope, remove the immersion oil from the 100X
objective (and any other areas) using tissue paper.
A demonstrator must check your microscope before it is put away. This will ensure that the
microscope is clean and operational for the next user.

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PRAC 7 Part B: Microbiology

This week you will continue with the preliminary identification of bacteria from samples A and B.

A. Motility agar test

Materials provided (per pair of students)
Motility test results of samples A and B from part A and controls.
1) Examine the motility agar tubes you set up in week 1. With reference to control tubes, record any
observed motility from samples A and B in the Table below.
Expected Results
Organism

Sample A

Sample B

E. coli

S. epidermidis

Motility

B. Growth of bacteria on blood agar

Some pathogenic bacteria produce an enzyme called haemolysin that breaks down red blood cells.
Haemolysins can help bacterial survival in the host by destroying host immune cells and red blood cells (and
thus reducing the fitness of the host) but also by releasing the nutrient iron for bacterial growth.

Note the terminology for the type of haemolysis that occurs on blood plates
-haemolytic: greenish brown zone surrounding colony
-haemolytic: clear or yellowish zone surrounding colony (complete haemolysis).
Materials Provided (per pair of students)

Blood agar plates of samples A and B from Session 1.

Gram reagents

demonstration photographs of blood plate cultures of Streptococcus mitis (-haemolytic),

Streptococcus pyogenes (-haemolytic) and Enterococcus faecalis (non-haemolytic)

1. Appearance of bacterial colonies. Briefly observe the shape (round, irregular, wrinkled), colour (white,
yellow) and surface texture (smooth or rough) of the colonies from sample A and B grown on blood
plates.

2. Compare zones of haemolysis around colonies from sample A and B with control plates and record the
result below.

3. Using an isolated colony from each plate, prepare a heat fixed film and perform a Gram stain. Examine
the slides by microscopy and record results below, in particular noting the presence of any spores which
appear as refractory (non-staining) bodies.

C. Capsule stain
Many bacteria produce an extracellular capsule that provides protection from desiccation and allows some
pathogens to evade detection by the host immune system. Capsules are usually made from polysaccharide
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and can make the surface of the colony appear mucoid (sticky). Again not all bacteria produce a capsule and
thus the presence of a capsule can help to identify unknown bacteria.

Materials Provided (per pair of students)

Nutrient agar plates of samples A and B from Session 1.

Manevals Stain, Congo red

Control plate cultures of Klebsiella pneumoniae (encapsulated) and E. coli (non-encapsulated).

For each of samples A and B and the controls, perform a Manevals stain as outlined in appendix D. Examine
the fields by microscopy and record results in the table below.

E. coli

K. pneumoniae

Sample A or B

D. Catalase test
Many bacteria also produce the enzyme catalase, which catalyses the breakdown of hydrogen peroxide to
oxygen and water. Catalases can be detected easily by adding Hydrogen peroxide to bacterial cells. Again
not all bacteria produce the catalase enzyme and detection of catalase activity can help further identification,
particularly of Gram positive organisms.

Materials Provided (per pair)

Nutrient agar plates of samples A and B from Session 1

Catalase reagents

Control plate cultures of Enterococcus sp. (catalase negative) and E. coli (catalase positive)

1. For each of sample A and B and the controls, perform a catalase test as outlined in appendix E. Record
your results in the table below.

Table 2. Characteristics of organisms present in Samples A and B (Results)

Microorganism

Motility

Haemolysis

Sample A

beta

Sample B

beta

Gram stain

Spores

Capsule

Catalase

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CONCLUSIONS
You have now completed the preliminary identification of two unknown samples of microorganisms using the
flow chart provided.

1) Based on the information below, is it possible that the BMS 1021 Coordinator was sent a virulent
organism in the envelope?

2) What is the likely cause of the BMS 1021 Coordinators respiratory infection?

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FLOW CHART TO IDENTIFY THE ORGANISMS

Gram positive bacteria

Cocci

Rods

Growth in air

Spores

+
Catalase

Staphylococcus,
Micrococcus

Growth in air

Anaerobic
Cocci

+
+
Bacillus

Streptococcus

Listeria,
Corynebacteria

Clostridium

S. pneumoniae

S. pyogenes

S. mutans

B. anthracis

B. cereus

B. subtilis

+ cocci

+ cocci

+ cocci

+ rods

+ rods

+ rods

Yes

Yes

Yes

Yes

Yes

Yes

Catalase

Capsule

Yes

Yes/No

Yes

Yes

Yes/No

Yes

Spores

No

No

No

Yes

Yes

Yes

Motility

Haemolysis

NA

NA

NA

Gram stain
Growth in air

Egg Yolk
Lecithinase

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Appendix A

Preparation of fixed films

It is essential that slides for the preparation of films of bacteria are clean and free from grease. Prior
to use, the surface of the slide should be degreased by passing through a Bunsen flame several times.
To prepare the film:
(1)

Label the slide at one end with a code for the particular colony to be sampled.

(2)

Sterilize an inoculating loop by flaming and allow to cool.

(3)

If the sample is a liquid culture, with the sterile loop transfer a loopful of the culture to the slide
and spread over about a quarter of the surface of the slide. Sterilize the loop by flaming and
proceed to step (7).

(4)

If the sample is a plate culture (solid medium), place a loopful of sterile water in the middle of
the slide.

(5)

Sterilize the inoculating loop by flaming and allow to cool.

(6)

With the sterile loop transfer a very small quantity of cells from the selected colony to the
drop of water on the slide, emulsify and spread thinly over about a quarter of the surface of
the slide. Sterilize the loop by flaming.

(7)

Allow the film to dry in air

(8)

With the film uppermost, fix the film by slowly passing the slide three or four times through the
hottest part of the Bunsen flame. (When the slide is just too hot to be borne on the back of the
hand, fixation is complete).

(9)

Allow the slide to cool before staining the film.

(10)

Remove the carbol fuchsin by washing with water, drain off the excess water.
Gram positive bacteria appear purple and Gram negative bacteria

Appendix B
Gram stain (modified)

(1)

Circle the area beneath the bacteria film with a grease pencil mark. This will be the area
within which the solutions will be applied.

(2)

With the film uppermost, pour crystal violet solution onto the slide within the grease pencil
area and allow to remain for 30 seconds.

(3)

Remove the crystal violet by washing briefly with water.

(4)

Add iodine solution and allow to remain for 30 to 60 seconds.

(5)

Remove the iodine solution by washing briefly with water.

(6)

Decolourise with 95% ethanol by holding the slide at an angle of 45 over the sink and adding
the ethanol to the top of the slide, allowing it to run down the slide across the film.

NOTE:

(a) Make sure all of the film is treated with the ethanol.
(b) Ideally, the ethanol treatment should be continued until no more colour comes out
of the film. In practice, with thin films, decolourization is usually complete within 5
seconds.
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(7)

Wash immediately with water.

(8)

Counterstain by adding dilute carbol fuchsin for 30 seconds.

(9)

Remove the carbol fuchsin by washing with water, drain off the excess water.

(10)

Air dry or blot dry the slide.

(11)

Typical Gram positive bacteria appear purple and Typical Gram negative bacteria appear pink.

Appendix C
Microscopic examination of stained films
(1)

Place a small grease pencil mark across the centre of the film or you can focus on the grease
pencil around the smear.

(2)

With the stained film in position on the stage (place the grease pencil mark across the centre
of the substage condenser), set up the microscope for use with the x10 objective lens.

(3)

Focus on to the grease pencil mark on the stained film. (Do not attempt to describe the
stained bacteria at this stage.)

(4)

Swing the x40 objective lens into position and refocus on to the grease pencil mark smear
using the fine focus knob.

(5)

Swing the x40 objective lens aside and place a drop of immersion oil on to the film directly
above the substage condenser.

(6)

Swing the x100 objective lens into position.

(7)

With the substage condenser in its highest position and the condenser iris diaphragm fully
open, focus on to the grease pencil mark and then move the slide so as to view the stained
bacteria.

Appendix D
Manevals Stain
This is a negative staining technique. The stain does not bind the bacteria but stains the background.
Therefore the capsule appears as a clear halo surrounding the cell.

(1)

Place a loopful of Congo red onto a slide and mix into it a small amount of growth from the agar
plate.

(2)

Make a well spread smear and allow to dry. DO NOT HEAT FIX

(3)

Flood the slide with Manevals stain and leave for one minute.

(4)

Rinse with water and blot dry.

(5)

Observe using oil immersion lens.

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Results
Organisms:

Red

Background:

Grey

Capsule:

Clear halo around organism (capsule is negatively stained)

Appendix E

Catalase test

(1)

With a wire loop remove cells from the agar surface and make a thick suspension in a drop of
hydrogen peroxide on a slide. Catalase activity is indicated by the appearance of bubbles of gas
(oxygen) within a minute.

NB: Media containing blood will give a false positive reaction

Appendix F
Approved Tests for the Detection of Bacillus anthracis in the U.S. Laboratory Response Network
(LRN)1
Test Procedure

Laboratory level

Preliminary testing

Gram stain (micromorphology)

Capsule (microscopic observation)

Colonial morphology

Haemolysis

Motility

Sporulation

Lysis by gamma-phage

Direct fluorescence assay (capsule specific)

Antimicrobial susceptibility testing

Advanced technology (PCR-testing)

Confirmatory tests

Molecular typing and characterization

1Protocols

for Level A tests are publicly available at www.bt.cdc.gov/agent/anthrax. Protocols for Level B are
available only to laboratories in the LRN. These laboratories include state public health laboratories and
many federal laboratories (below).

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