BY154 Laboratory handbook

Introduction to infection science

School of Applied Science

Biology and Biomedical Sciences Division

University of Brighton
 Practical 1

BY154
Complement lysis of sheep red blood cells laboratory protocol

INTRODUCTION:
In this practical, we will test the Complement’s haemolytic activity on sheep red
blood cells (SRBC).
The aim of this practical is to provide an opportunity to learn and practice a number
of basic lab techniques, such as:
• Performing dilutions and serial dilutions
• Using a spectrophotometer to read absorbance
• Making a graph using results collected from the experiment

Serial dilutions of complement are added to a volume of SRBC and haemolysis is

assessed measuring absorbance.

HEALTH and SAFETY:

Lab coats, safety glasses and gloves must be worn at all times. If you do not comply
you will be asked to leave the laboratory.
If you feel unwell at any point during the practical please notify the laboratory
supervisor immediately.
BY154 Complement lysis of sheep red blood cells laboratory protocol

I. Sensitization of SRBC with anti-SRBC antibody

1. You are provided with an Eppendorf tube containing 1ml of SRBC. Pellet the cells by spinning
the tube at 5900rpm in a micro centrifuge for 5 minutes. Be sure that the centrifuge is
balanced. This will form a loose pellet.
2. Remove the tubes from the centrifuge, and carefully pipette off the clear supernatant without
disturbing the pellet.
3. Pipette 9.9 ml of ‘Antibody buffer’ in a universal tube and add 100µl of the SRBCs pellet. This
will result in a 1% SRBC suspension.

II. Complement serial dilution

Starting from a stock solution of Complement, make serial doubling dilutions into LP3 tubes, using PBS to
dilute.

1. Label a set of 6 LP3 glass tubes 1-6:

1. Neat
2. 1:2
3. 1:4
4. 1:8
5. 1:16
6. 1:32

To make ‘1:2 serial dilutions’ of complement:

• pipette 1ml of stock complement into tube 1 (i.e. ‘neat’)

• pipette 1ml of PBS buffer in all remaining tubes
• pipette 1ml of stock complement into tube 2. This will make a 1:2 dilution.
• Gently mix by pipetting up-down
• take 1 ml from tube 2 (1:2) and add to tube 3 (1:4)
• Repeat the same procedure up to tube 6 (1:32)
 III. Complement haemolysis

1. Label a set of Eppendorf tubes 1-8

2. Add to each tube 500µl of sensitised SRBC
3. Into tube 1, add 500 µl of neat complement (tube1)
4. Into tube 2, add 500 µl of 1:2 complement (dilution tube 2)
5. Continue all the way to tube 6, where you will add 500 µl of 1:32 complement
6. Into tube 7, add 500 µl of water. This will be the positive lysis control
7. Into tube 8, add 500 µl of PBS buffer. This will be the ‘blank’ control
8. Incubate the tubes at RT for 20minutes, inverting the tubes occasionally
9. Spin tubes at 10,000 rpm for 3 minutes.

 Observe the haemolysis in the tubes and record the highest dilution of complement at which
you observe full lysis. This is the minimum amount of complement required to lyse all of the
tested volume of SRBC and it is called Minimum Haemolytic Dose (MHD). MHD can be used
as ‘indicator system’ for complement fixation assays in clinical screenings (see lecture notes).
 Keep a record of what you can observe.

IV. Quantification of haemolysis.

1. Set up the spectrophotometer to read at 540nm as instructed

2. Into a plastic cuvette, add 900 µl of PBS buffer and calibrate the
spectrophotometer to baseline
3. Careful to avoid the pellet, transfer 900 µl of each haemolysis tube in a plastic
cuvette and read their absorbance
4. Read the absorbance of the haemoglobin released into the supernatant
5. Record all readings in the table below

V. Make a graph

1. Read the absorbance for each sample and record the OD in the table below.
2. From each sample OD, subtract the value of the Blank absorbance (i.e.
spontaneous lysis, tube 8). The ‘total lysis OD’ is selected from the tube where you
observe full lysis (i.e. no pellet). Note that this may be found in the highest
concentration of complement, rather than in the control tube with water.
3. Calculate the ‘% lysis’ for each dilution using the equation:

% lysis = (OD test / OD total lysis) x 100

 OD control lysis

OD Blank

OD of sample OD of sample % lysis

– Blank OD

4. Using the graph paper provided, plot the % lysis (on the vertical axis, y) against the
serum dilution (on the horizontal axis, x)

5. Using the graph, estimate the dilution of serum required to lyse 50% of the
sensitised SRBC. This is called the CH50 and is a measure of the functional ability
of serum complement to lyse SRBC pre-coated with rabbit anti-SRBC antibody
(haemolysin).
1. Describe the term Complement
2. How does the complement lyse SRBC?
3. Why do SRBC need to be ‘sensitized’ di Antibody?
4. What are the control tubes of this experiment and why?
5. What other control could you have used?
6. Which class of antibody is best at mediating complement haemolysis? Why?
BY154
Blood film and blood groups laboratory protocol

INTRODUCTION:

The aim of this practical is to provide an opportunity to apply a number of

the techniques that were covered in the lectures. The focus is on the
techniques themselves and the quality of results obtained.

The areas of investigation are:

1. Prepare and stain a blood film.
2. Identify the various WBC using a critically focused microscope.
3. Create labelled diagrams for each WBC’s morphology.
4. Describe the function for each WBC.
5. Perform a blood group agglutination test.
6. Interpret the blood group reactions.

Bi o l o g i c a l Handle blood, blood products and sharps with great caution.

Hazard Dispose in contaminate waste bins and sharps bins.

HEALTH and SAFETY:

Lab coats, safety glasses and gloves must be worn at all times. If you do not
comply you will be asked to leave the laboratory.

If you feel unwell at any point during the practical please notify the laboratory
supervisor immediately.

If you are using your own blood you must only attempt this once.
1. Prepare and stain blood film METHOD
1. Mix the sample of blood well by gently inverting and flicking the bottom of the tube until the blood
is fully re-suspended.
2. Place a small drop of blood 0.5 cm from the edge of a glass slide closet to the frosted end.
3. Place a second slide — spreader in front of the drop on the slide at 45o angle.
4. Hold the spreader between the thumb and index finger at the lower end of the spreader for better control.
5. Draw the spreader back along the slide towards the drop of blood. As the spreader touches the drop, the
blood will spread out across the edge of the spreader.
6. Push the spreader forward in a rapid, uniform and smooth motion. By applying the right pressure, altering
the angle between slide and spreader and varying the speed of spreading will affect the thickness of the film.
7. Allow the film to dry in air for approximately 5 minutes.
8. Label the film with your full name and date of practical.

Stain Blood Film

1. Ensure blood slide is fully air dried.
2. Use forceps to fully immerse blood film in coplin jars that contain the staining solutions
3. Allow the slides to drip off well after each staining step.
Stain 1 – Dip slide 5 times; drain excess stain
Stain 2 – Dip slide 3 times; drain excess stain
Stain 3 – Dip slide 6 times; drain excess stain
Stain 4 – Dip slide 20 times
4. Dry the back of the slide with tissue paper, place the slide vertically with tissue underneath the slide and
leave to dry.
2. Critically focus the microscope

METHOD
1. Set the eyepiece lenses to your interpupillary distance. You should have a single circular field of vision. If
not readjust.
2. Move objective lens to 10x lens.
3. Place the dried slide on the mechanical stage. Make sure the slide is placed with stained blood facing up.
4. Use the coarse adjustment to bring the stage as far as it go up towards the 10x lens
5. Under low power (10x) objective focus the image using the coarse to lower the stage.
6. Use the stage control to locate best area is in-between the body and the tail of the blood smear and find the
area of ideal thickness i.e., the RBC should not be overlapping.
7. Switch to the 40x objective.
3. Create labelled diagrams for each WBC’s morphology

METHOD
1. Use a pencil and draw only the lines you see. No shading or colouring.
2. Each circular diagram (field of view) draw one type WBC and one RBC to compare the sizes.
3. Record the magnification, name of the cell and date of observation below the circular diagram.

4. Describe the function for each WBC.

Title: Cheek Cell

Magnification: x400
Description and Function Cheek cells scraped,
fixed and stained on a slide. These cells are thin
and flat line the line the inner surface of the
cheek. The reason they a thin and flat is to create
thick layers so that the cheek lining is flexible,
strong and smooth.
 Title

Magnification

Description and Function

Title

Magnification
Description and Function
4. Perform a blood group agglutination test.

ABO and Rh D Group Typing

1. Lay out four microscope slides and label each slide A, B, AB and D respectively.
2. Place one drop of anti-A serum at end of microscope slide
3. On the next slide place one drop of anti-B serum.
4. On the third slide place one drop of anti-AB serum.
5. On the fourth slide place one drop of anti-D serum.
6. Place a small drop of the patient’s blood next to each antisera.
7. Use a stirrer to gently mix the anti-sera with the patient’s blood. Avoid
spreading drops over a large area as this will cause the sera to dry out.
8. Gently rock the slide aid and visualise the agglutination.
9. Record whether agglutination — clumping, occurs with anti-A, anti-B, both or
neither and interpret the ABO group of the patient’s blood sample.

Agglutination (Yes or No) Blood Group

use correct terminology

Anti-A Anti-B Anti-AB Anti-D e.g.,
anti-sera anti-sera anti-sera anti-sera AB Rh D positive

6
Microbiology Practical Handbook

Introduction
This manual is a working laboratory methods workbook for the 1st year microbiology
practical classes in your BSc programme at the University of Brighton.

It contains information about health and safety in the laboratory and standard laboratory
techniques. Please make sure you have a copy of this each time you come to a microbiology
session in each year of your studies. If you do a practical project in microbiology, you may
also find this manual useful.

You can easily add additional material and keep everything together, not least at the practical
exam for this module. You can add documents for subsequent microbiology practicals in
years 2 and 3.

Safety in the Laboratory and Good Laboratory Practice (GLP)

The bacteria in the lab classes are real and may cause infections if mishandled. Behave
carefully and sensibly in the laboratory at all times and always follow any instructions given
by any member of university staff. This is to ensure your own safety and that of other people
in the laboratory.

Please note and observe all of the following points explained here, which comprise
Good Laboratory Practice:

• Always wear appropriate personal protective equipment (PPE)

In the microbiology laboratory classes you will use white coat held in the microbiology lab.,
i.e do not use your own white lab coat. This way bacteria are not transferred out of the
laboratory.

You should bring your own safety glasses to all the practical sessions in case they are
required.
You will be told when it is necessary to wear disposal gloves or safety glasses. Long hair
must be tied or clipped back at all times.

• Always dress appropriately for laboratory sessions and in particular wear

sensible shoes
Please do not wear high heels or open toe sandals as you might catch your shoes and fall over
or spill chemicals or infectious fluids onto your feet. Do not wear your best clothes in case
they are caught by spillages of infectious agents or chemicals and do not wear clothes which
cannot be fully protected by a laboratory coat. Wear only minimal jewellery – so no rings
with very large stones, no dangly earrings and no watches which cannot be fully covered up
by the laboratory coat sleeves - in case they get stuck in laboratory equipment and then have
to be decontaminated.

• Bags, coats and books must not be brought into the laboratory
Please leave all bags, coats, books and anything not required during the practical class in your
locker. This is to avoid contamination of your belongings and also to avoid a large pile of
bags and coats creating an obstruction within the laboratory.

• No eating, drinking, smoking or applying cosmetics in the laboratory

7
This includes chewing gum and drinking bottled water. Do not bring any food or drink into
the laboratory (even if you are planning to consume it later) to avoid contamination. This also
includes putting pens, pencils and fingers in or near your mouth – you do not want to ingest
microorganisms.

• Always assume that all biological fluids and samples contain infectious agents
and that all cultures may contain human pathogens
This means that you must treat every plate or broth culture, every sample and any pre-
prepared slides (including fixed and stained slides) with the utmost care at all times.

• All cuts, grazes and other open wounds must be covered while in the laboratory
If you notice a cut, particularly on your hands once you are in the laboratory, please ask a
member of staff for a plaster to cover it

• Great care must be taken with reagents and equipment at all times
If you have not been given instructions as to how to use a piece of equipment or perform a
particular procedure always ask first. Always use racks for test tubes and where possible
bijoux bottles. If you have the Bunsen burner on, always turn the flame to yellow when you
are not using it ( so that you can see it) – but remember to change to the blue flame when
using it to sterilise loops or heat fix slides. Be careful and sensible when using the Bunsen and
be careful to avoid putting your hand through the flame.

DO NOT attempt to mix a suspension containing microorganisms by tipping up and down –

of the lid is loose, it will go everywhere! Always mix by holding the bottle in both hands and
rotate (swirl) gently from side to side.
• All spillages must be cleaned up appropriately
Please report all spillages in the laboratory, however small, to a member of staff and follow
their advice.

• All accidents must be reported to a member of staff

Please tell a member of staff immediately if you have any sort of accident or sustain any
injury in the laboratory. This is so that you can be given appropriate first aid and the accident
can be recorded.

• Electronic devices must not be used in the laboratory

This includes mobile phones, MP3 players, laptop computers, tablets, notebooks etc. This is
to avoid contamination of your equipment which you will use outside of the laboratory and
also to ensure that you are not distracted by music or games and miss any safety instructions
which you are being given.

• Laboratory books and practical schedules and all other paperwork should be
kept separately from cultures, fluids and slides as far as possible
Do not put agar plates, test tubes or slides on top of paperwork which you will then take out
of the laboratory – in case they get contaminated with an infectious agent.

• Laboratory benches must be properly cleared and cleaned once work has
finished.
Do not tip any cultures, contaminated liquids or chemical down the sink. Dispose of
contaminated pipette tips, any slides which you have made and any other sharps in the yellow
sharps box (or as instructed). Contaminated re-usable glassware, such as flasks and pipettes,
should be placed in the appropriate basket on the trolley in the laboratory (or as directed).
Dispose of contaminated plastic pipettes and loops in the disinfectant pots on your bench (or
as instructed). All contaminated plates, tissues, gloves or other items should be placed in
plastic autoclave bags marked with the biohazard sign. Do not put anything which might be

8
contaminated with an infectious agent in the black plastic bags by the sinks – these are for
hand towels from hand washing ONLY. Pre-prepared slides should be left on the bench for
collection at the end of the class. Place any plates or culture bottles which you have
inoculated during the class and which need incubation in the appropriate tray or basket as
directed – do not leave them for someone else to collect unless told to do so by a member of
staff. Clean your bench with appropriate disinfectant before leaving the laboratory.

• Hands must be washed thoroughly as the last action before leaving the
laboratory
Once you have completed all your tasks and cleared up your bench, remove your laboratory
coat and THEN wash your hands. Do not leave the laboratory without washing your hands.
Also, do not wash your hands before taking off your laboratory coat, as touching the
laboratory coat may contaminate your hands again.

If you are in doubt about anything regarding health and safety in the laboratory please
ask a member of staff.

Summary of General Safety Information for Microbiology

1. All organisms and cultures are to be treated as if potentially pathogenic.

2. Briefcases, bags and outdoor coats owned by undergraduate students must be left outside
teaching laboratories.

3. High heels, open toed shoes and sandals must not be worn in the lab.

4. A clean microbiology lab coat (one which buttons up to the neck) must be worn at all
times and this must be buttoned up: it should be free from major holes and tears. The coat
must be removed on leaving the laboratory.

5. Long hair should be tied back so that it does not fall into open culture vessels and Bunsen
burner flames.

6. Watches and jewellery should be removed to avoid damage which might result from any
decontamination procedures to which they might otherwise be subject

7. The lab bench should be swabbed with alcohol or disinfectant both before and after work
has been carried out.

8. All spillages should be dealt with immediately in the appropriate manner. Inform a
member of staff if a spillage occurs.

9. No cultures or contaminated liquids should be poured down the sink.

10. No specimens, slides, cultures or other items may be removed from the laboratory other
than for transportation to another microbiology laboratory.

11. All containers must be appropriately labelled with waterproof marker pens. Self-adhesive
labels may be used, but not ones which require licking.

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12. All cuts, grazes, skin rashes and open sores on exposed parts of the body must be
covered.

13. Eating, chewing, drinking, smoking or the application of cosmetics is not permitted in the
lab.

14. Mouth pipetting is not permitted.

15. Use of mobile phones is not permitted

16. Use of personal music systems is not permitted

17. The bench must be swabbed with disinfectant after the lab work has finished and hands
must be washed before leaving the lab.

18. All accidents are to be reported immediately and the details recorded on an accident
report form.

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 Safe disposal of used lab ware, cultures, and contaminated
items

Under no circumstances should contaminated items be disposed of in

the waste paper bins or down the sinks.

Always use the appropriate, safe, disposal method (see table below).

If in any doubt ask a member of staff.

Item Disposal

Glass graduated pipettes (1, 2, & 10 ml Grey disinfectant pots on lab bench
etc)

Glass Pasteur pipettes Yellow sharps bin on lab bench

Swabs and spreaders Grey disinfectant pots on lab bench

Plastic pipette tips Yellow sharps bin on lab bench

Plastic cuvettes Grey disinfectant pots on lab bench

Used agar plates Autoclave bag

Used broth (liquid) cultures Grey disinfectant pots on lab bench

Contaminated glassware e.g. test tubes, Trolley provided

beakers, flasks.
(NOT glass pipettes)

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 Standard techniques in the microbiology laboratory
1. Preparation of smears from colonies grown in plate culture

2. Gram stain

3. Sub-culturing of organisms from plate culture

4. Rapid biochemical / identification tests:

i. Oxidase test
ii. Catalase test
iii. Coagulase test
iv. KOH test
v. Capsule stain

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 1. Preparation of smears from bacterial colonies grown in plate culture

i) Examine the agar plate in the region where single colonies are present and
identify a suitable, isolated colony
ii) Take a clean glass slide and label it at one end using the chinagraph pencil or
permanent marker; using a sterile loop, carefully transfer place a loopful of
water onto the slide.
iii) Gently touch the bacterial colony and then spread the organisms as thinly as
possible emulsify into the water across the slide.
Note that a tiny amount of the bacterial colony is sufficient – as long as you
have touched it with the loop, even if you cannot see any material, have
confidence that plenty of bacteria will be present!
iv) Leave the slide to dry in air.
v) Once it is completely dry (not before!) heat fix, by carefully passing the slide
through the middle of a blue Bunsen flame 2 or 3 times.
vi) Leave on the bench to cool
vii) Stain with the appropriate stain.

The smear should not be thinly spread over the slide – if you can just see the
material on the slide, it is probably about right.

Don’t forget to heat fix the smear, otherwise everything will run off the slide
during the staining procedure!

2. Gram stain

i) Place the slide on the staining rack across the sink or staining tray
ii) Cover the smear with crystal violet.
Avoid touching the slide, by holding the bottle of stain vertically about 1mm
above it and gently dropping the liquid. Leave to stain for 30 seconds.
iii) Wash with water
iv) Tip off fluid and cover the smear with Lugol’s iodine; leave for 10 seconds
v) Wash with water
vi) Tip off excess fluid
vii) Decolourise by adding 95% alcohol and count up to 10 seconds and then
quickly rinse off with water.
viii) Tip off excess fluid and add counterstain (safranin) for 30 – 60 secs.
ix) Wash with water and blot slide carefully with paper
x) Once dry add a drop of immersion oil on the thin part of the stained organisms
and examine under the microscope, using the x100 lens.
(oil and water will make the image cloudy. We don’t want that.)

Take care not to get immersion oil on the x10 or x40 objectives.

Interpretation of Gram reaction

Blue black staining organisms are Gram positive

Pale red or pink organisms are Gram negative

Bacterial Shape: what shape are the bacteria? bacilli (rods) or cocci ?

13
Look out for the presence of non-staining oval shapes (bacterial endospores). They
may be present within the vegetative cells or they may be free from the bacterial cell.

3. Subculturing organisms from a plate culture

Take an uninoculated plate and label the bottom of the petri dish – (NOT the lid,
which will become detached - with name (initials) and date and culture details.

Pick an isolated colony form the petri dish using a sterilised nichrome wire loop and
create a pool on the new culture plate. (fig 1 (i))

ii) Dra the wire out from the pool , creating 5 – 6 straight lines (fig 1 ii).

sterilise the loop in the Bunsen and allow to cool in air or place it in the new
agar plate near on the edge of the plastic wall)

iv) repeat the spreading technique two more times (Fig 1 iii and iv)

v) Finally, use the loop to create a final swirl (Fig 1 v).

vii) Incubate the cultures at appropriate temperature (e.g. 370C)

viii) The next day, examine your plate. This method should yield single, pure
colonies in the middle of the plate.

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4. Biochemical tests used in the identification of bacteria

KOH test
1. Using a sterile loop or a plastic pipette, place a drop (25µl) of 3% KOH solution
onto a clean glass slide.
2. Take about half of a single isolated colony onto a sterile loop and mix it into the
KOH.
3. As you are mixing, observe whether the material is becoming viscous and the
denatured DNA strands will cling to the loop when you lift it slowly out from the
suspension of organism and KOH (indicating that the cells have lysed). This is a
positive result.
4. If there is a viscous stringy reaction, then the organism is Gram negative;
If there is no change in consistency, indicating no lysis, then the organism is Gram
positive

Catalase test

1. Take a bijou bottle containing hydrogen peroxide (H2O2 ).

2. Using a sterile loop, take a colony or two from the agar plate and rub it into the inside of
the bijou bottle above the level of the liquid.

15
3. Make sure the lid is closed firmly and gently tilt the bottle so that the colony meets the
catalase reagent. DO NOT shake the bottle!
4. Observe for fizzing and bubbling (positive reaction)

Oxidase test

1. Take a strip of filter paper and place on a microscope slide.

2. Dissolve a small amount of the oxidase reagent (tetra nmethyl – p – phenylene
diamine HCl ) to make a c0.5% solution)

Using a sterile PLASTIC loop, take a colony from the agar plate and rub it onto the oxidase
strip.
3. Observe for 5-10 seconds for the development of a blue/ purple colour (positive reaction).
Only instantaneous reactions should be considered positive.

Alternative method

Oxidase test

1. Immediately before you want to perform the test, add the diluent (PBS or distilled water) to
the oxidase reagent paper in the bottle covered with tin foil or made with dark coloured glass.
Swirl gently to mix before use.
2. Take a clean cotton wool swab and dip it into the activated reagent.
3. Place the swab on a single isolated colony on the agar plate
4. Observe for 10 seconds for the development of a purple colour (positive reaction)
After this time, the reagent will start to change colour anyway due to effect of the light, so a
reaction which takes more than 10 seconds to appear cannot be recorded as a true positive

Staphytect test (used in place of the Coagulase test)

This test kit is used to identify Staphylococcus aureus from other staphylococci. It is
based on a antibodies raised against a protein specific to Staph aureus. The antibodies
have a blue latex beads attached to the non-binding end. When the antibodies bind to
Staph. aureus the beads clump together (positive result)

1. Mix the kit reagents before.

2. Place one drop of the TEST reagent onto one circle on the assay card and place one drop
of the CONTROL reagent on the other circle.
3. Using a sterile loop, take a few colonies from the agar plate and separately mix them into
the reagents (don’t mix up the two reagents with the same loop)
4. Gently rotate the card t mix the reagents with the organism.
5. Observe for clumping of the latex particles in the test reagent circle (‘agglutination’).
6. Ensure that there is no agglutination in the control reagent circle (right picture below).
Agglutination in the test reagent circle is a positive reaction (left in picture below), unless
there is agglutination in both circles - in which case it is a false positive.

16
Micro- pipettes

Quick Guide To Using A Micro-pipette

• Choose the correct pipette type for the volume required.

Pipette type Microlitre volume Millilitre volume

P20 2 - 20 µl 0.002 - 0.02 ml
P200 20 - 200 µl 0.02 - 0.2 ml
P1000 200 - 1000 µl 0.2 - 1 ml

It will say somewhere on the pipette which volumes it will measure

P1000 P200 P20

Figure A2.1

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• Set the pipette to the required volume by twisting the calibration wheel or top
plunger. The volume is displayed in the window.

Plunger

Calibration wheel

Volume window

Figure A2.2

• Select the correct tip for your pipette type.

Pipette Type Tip Type

P20 Yellow tips
P200 Yellow tips
P1000 Blue tips

To use your pipette

• Place the appropriate tip firmly onto the end of the pipette

• Press down the plunger to the first stop (see figure A2.3)

• Put the tip into the solution to be sucked up

• Gently and slowly release the plunger back up to the top position. The liquid

will be sucked up. Remember to keep the pipette vertical at all times. Note:

Make sure no air bubbles are sucked up as it affects the volume

accuracy.

• To dispense your sample liquid, press the plunger all the way down to the

second stop (see figure A2.3).

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 Top Position First Stop Second stop
Figure A2.3

• Once pipetting is finished, eject the tip into a yellow sharps bin using the eject
button which is just behind the plunger.

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 BY 154 Practical Sessions
PRACTICAL 1

Gram staining, sub culturing and estimation of concentration of organisms in

a broth culture of bacteria

In this practical, you will be given cultures of bacteria in two forms – plate culture
and broth culture. You will practice preparing a smear from the culture growing
on an agar plate and then applying the Gram stain, to allow you to see the
bacterial cells under the microscope. You will then practice plating out a sample
of that organism onto a fresh agar plate using aseptic techniques. You will also
perform ‘viable count’ to estimate the concentration of bacterial cells in a broth
culture by preparing a dilution series and culturing a sample from each dilution.

1. Preparation of a smear and Gram staining.

On your bench, there are 2 agar plates which have been inoculated with bacteria.
One contains Escherichia coli and the other Staphylococcus aureus. You will be
shown how to prepare a smear from isolated colonies and how to apply Gram’s
stain to that smear. Follow the instructions in your Microbiology Manual
(techniques 1 and 2) to practice these techniques yourself.

Gram’s stain results:

Escherichia coli is Gram ………………. and ………………….shaped

Staphylococcus aureus is Gram ………………. and ………………….shaped

2. Sub –culturing of an organism onto plate culture

Use the organism which you worked with in part 1 to subculture a single colony onto
a fresh agar plate, following the instructions in your Microbiology Manual (technique
3). Make sure you choose the correct plate for your organism and keep the Bunsen
flame on blue to maintain sterile conditions around the area where you are working.
Put your plate for incubation at 370C overnight and when you come back to observe
it, make notes on how successful your attempt at subculturing was. This is also
difficult to get this right the first time, so think of ways to improve your technique.

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 • Did you achieve a pure culture with no contamination?

• Did you have single colonies growing in the middle of your plate?

3. Estimating bacterial numbers in an overnight broth culture : ‘total viable count’

To count the numbers of bacteria in a broth culture you need to create a series of
dilutions and see which plates allow you to quickly count the number of colonies
formed after incubation overnight. Each colony is assumed to arise from one single
bacterial cell and is called a colony forming unit (CFU).

Given that we know how many times we diluted the culture ( the dilution factor) and
what volume we put onto the plates (usually 100 µL) we can calculate the number of
organism in the original sample.

1. Work in the vicinity of a lit Bunsen burner with a blue flame.

2. Label agar plates (on the base, not the lid) with your initials, the date and the
dilution factor. Prepare three plates for each dilution factor
3. Place 4.5ml of sterile diluent (PBS) into each of 7 sterile tubes.
4. Swirl the sample (DO NOT tip up and down!) to ensure that all the cells are fully
suspended which is important for accurate counting.
5. Take 0.5ml (500 µL) of sample into 4.5ml diluent (tube #1, giving a 1 in 10
dilution) and mix thoroughly.
6. Using a fresh pipette tip, remove 0.5ml from tube #1 and add to tube #2 and mix
thoroughly.
7. Repeat the procedure down to tube #7.
8. For each dilution place 0.1ml (100 µL) onto the surface of an appropriate agar
plate and spread evenly across the surface using a sterile spreader. Prepare 3
plates for each dilution.
9. Incubate the labelled plates, inverted to avoid condensation dripping onto the
culture) for 24 hours at 37oC.
10. Your incubated plates will be returned to you at the next session. Select plates
with between 30-300 colonies, count and calculate the cfu/ml of the original
sample.
11. Record your results in the space below and record the concentration of bacterial
cells in the original broth culture as organisms / mL.

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 A) DILUTION SELECTED FOR CALCULATION = x 10-5

Convert to DILUTION FACTOR = x 10 +5

B) Number of colonies for each replicate :

COUNTS #1 = #2 = #3 =
MEAN =

C) VOLUME OF SAMPLE PLATED OUT =

Convert to ‘organisms per mL’ rather than number of organisms in the volume you
put onto the plates (C).

D) Concentration of bacteria in original culture = ………………

Express the final count to 2 decimal places= ………………

DON’T FORGET the units! : CFU/ml

Extra questions: how many organisms in total in the original 20 ml culture?

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 BY 154 Practical Sessions
PRACTICAL 2

Examining plate cultures and identification of bacteria

In this practical, you will be given plate cultures of four bacteria to work with.

Identify the different bacteria using some basic techniques:

Colonial morphology
Catalase production
Oxidase production
KOH reaction
Latex agglutination kit
Capsule stain

The results will allow you to identify the organisms.

The four organisms are:

Bacillus cereus
Staphyloccocus aureus
Escherichia coli
Moraxella catarrhalis

1. Examination of the bacterial colonies

Examine the colonial morphology of each of the four bacterial species.

Example features you may notice :

smell
consistency of the colonies: wet, mucoid, dry
haemolysis on blood agar
colour of the agar surrounding the colonies

Make notes about your observations in the space provided:

Bacillus cereus

Shape and colour of colonies

diameter of colonies

Other features

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Staphylococcus aureus

Shape and colour of colonies

Elevation and margin of colonies

diameter of colonies

other observations………………………………………………………………………

Escherichia coli

Shape and colour of colonies

Approximate diameter of colonies

Other observations………………………………………………………………………

Moraxella catarrhalis

Shape and colour of colonies

Elevation and margin of colonies

diameter of colonies

Other observations………………………………………………………………………

2. Biochemical tests on bacterial cultures

Carry out the four biochemical tests ( technique 4) on each of the bacteria. Record
your observations and interpretations of your findings in the space provided:

Bacillus cereus

Oxidase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

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Catalase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

Coagulase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

KOH test i) Observations

ii) Positive or negative?

iii) Explanation for result………

Staphyloccocus aureus

Oxidase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

Catalase test i) Observations

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ii) Positive or negative?

iii) Explanation for result………

Coagulase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

KOH test i) Observations

ii) Positive or negative?

iii) Explanation for result………

Escherichia coli

Oxidase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

Catalase test i) Observations

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ii) Positive or negative?

iii) Explanation for result………

Coagulase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

KOH test i) Observations

ii) Positive or negative?

iii) Explanation for result………

Moraxella catarrhalis

Oxidase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

Catalase test i) Observations

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ii) Positive or negative?

iii) Explanation for result………

Coagulase test i) Observations

ii) Positive or negative?

iii) Explanation for result………

KOH test i) Observations

ii) Positive or negative?

iii) Explanation for result………

3. Gram’s stain

Carefully prepare a smear from each of the four bacteria (technique 1) and stain with
Gram stain (technique 2) as you did last week. Record your results in the space
provided:

Bacillus cereus Gram ………………. and ………………….shaped

Staphylococcus aureus Gram ………………. and ………………….shaped

Escherichia coli Gram ………………. and ………………….shaped

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Moraxella catarrhalis Gram ………………. and ………………….shaped

Capsule stain:

A ‘negative stain’ which means the stain does not stain the target but acts by
colouring the background and allows you to see the capsule surrounding the
bacterium.

i) Place a large loopful of undiluted India ink on a slide

ii) Mix in a small amount of the bacterial colony
iii) Add a small cover slip

Capsules appear as clear zone around the organism,

4. Reading last week’s results

Read the sub culture plates and the spread plates from the viable counts which
you set up last week and record the results in the space provided in the schedule
from practical 1.

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