University of Warwick
Headstart Summer School
Microbiology Lab
Practical manual
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The set of experiments over the next two days are designed to introduce you to a range of
microbiological techniques.
AIM OF DAY 1:
 Streaking agar plates for single colony isolation
AIM OF DAY 2:
 Staining of bacteria to help with identification
 Investigation of bacteria under microscopes
 Bacteria motility testing
Background
Microbiology is the study of micro-organisms. "Micro-organisms" is a vague, flexible term
used to describe free-living, single-celled organisms. This covers prokaryotes (archaea and
bacteria) and eukaryotes (protozoa, yeasts, green algae and fungi). Today, you will be
looking at bacteria.
Before you can study bacteria in the laboratory, you have to master a number of techniques
which are principally designed to make sure that you are dealing with a single strain of the
bacterium growing on a solid, or in a liquid, nutrient medium. This is referred to as a pure
culture. We will be growing our bacteria on agar pates, a solid media.
Bacteria are divided into two classes by their reaction to the Gram stain. This stain is a
valuable tool in the identification of bacteria and can also be used to evaluate the purity of a
culture. Examination of stained preparations can give you some information as to the types
of bacteria present. As an example, a pure culture should only contain one type of bacterial
cell. A mixed population would contain a mixture of cells. Equally, as all the cells in a pure
culture should have the same reaction to the Gram stain, a mixture of Gram positive and
Gram negative cells usually indicates that the culture is mixed.
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You will be provided with some of the bacteria below and you will isolate single colonies
from some of them.
Or
Escherichia coli
Staphylococcus epidermidis
Staphylococcus mutans
Pseudomonas fluorescens
Flavobacterium aquatile
Bacillus pumilis
Bacillus mycoides
Serratia marcescens
Micrococcus luteus
(E. coli)
(S. epidermis)
(S. mutans)
(P. fluoescens)
(F. aquatile)
(B. pumilis)
(B. mycoides)
(S. marcescens)
(M. luteus)
37°C
37°C
37°C
30°C
30°C
30°C
30°C
30°C
30°C
Streaking agar plates for single colony isolation
This technique is used to isolate separate colonies (single colonies) from a mixed culture
and to subculture pure cultures.
The transfer of bacteria from one plate to another (or from liquid culture to solid medium) is
carried out with a disposable inoculating loop. These are sterile and so with proper use will
not contaminate your culture. If you put the loop down, or accidently touch something with
it you should dispose of it and get a new loop. A loop that has been used should never be
placed on the bench and should be disposed of.
To streak for single colonies you need an agar plate with a culture on it plus a new plate to
streak onto.
Place the new plate LID DOWN on the bench and label the base of the petri dish with the
name of the culture, the date and your name or initials. This is essential information which
should be on every plate you use. You should never label the lid as these can be mixed up
(the base is always in contact with the agar!).
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Touch the loop to a colony of bacteria on the plate and withdraw the loop. Lift the base of
the new plate and quickly streak the loop across the plate as shown in Fig.1a.
a
c
b
d
Figure 1: Streaking bacteria onto agar plates
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Replace the base on the lid, get a new sterile loop, allow it to cool and streak from the first
area to a new area as shown on Fig. 1b and c. The subculture is usually finished off with a
streak into the centre of the plate (Fig.1d). When you have finished, dispose of the loop.
Always use a new disposable loop for streaking new bacteria.
If this operation is carried out swiftly then there is a minimum chance of the plate becoming
contaminated by airborne bacteria. Never wave the plate about in the air this will increase
the risk of contamination and always replace the lid when the streaking is done.
If the streak is done properly then tomorrow you should have areas on the plate where the
growth is thick and areas where there are visible single colonies. Essentially, this is a
dilution method and the less you touch the initial streak the better the dilution will be. You
also have to avoid putting too much into the initial streak. You don't have to be able to see
the bacteria on the loop for there to be enough to give good growth. A simple rule is if there
is a clearly visible amount on the loop then there is too much.
Streaking from a liquid culture involves the same steps. To remove liquid from a flask,
remove the bung with the little finger of the right hand whilst holding the sterilised loop,
flame the neck of the flask and insert the loop into the liquid. Withdraw a loopful of liquid
replace the bung and put the flask down. Now streak as described above.
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DAY 2: Examination of bacteria by microscopy
It is not possible to see bacteria under the microscope without using stains or phase
contrast microscopy. The former technique uses a heat fixed smear which can create
artefacts by distorting the shape of the bacteria as they are dried and fixed. The latter
technique is useful to test for motility, although to the expert eye much information can be
obtained through the examination of wet preparations.
Preparing a Gram Stain
The majority of bacteria are either Gram negative (stain pink/red) or Gram positive (stain
purple). Different dyes and de-colourising agents can be used to perform Gram stains, the
ones described here give the best results with heat-fixed smears. Gram stains with tissue
preparations, as done in some hospital laboratories, would use different agents. The basis
of this stain is that both groups of bacteria take up crystal violet which forms a complex with
iodine. In the case of Gram-negative organisms, this complex is solubilised by the addition
of alcohol and removed. The complex in Gram-positive bacteria associates with the murein
layer of the cell wall and is less readily removed. Gram-negative organisms have to be
counter-stained with safranin in order to make them visible.
Take a heat-fixed smear and Gram stain as follows:
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Flood the slide with crystal violet and leave for one minute
Pour off the stain and flood the slide with iodine (Gram’s diluent). Leave for no
more than 20 seconds
Pour off the iodine and wash with ethanol
This is the critical de-colourising step and the point where most Gram stains go
wrong. The slide should be repeatedly flooded with alcohol and rocked from side to
side. If Gram negative organisms are present the stain will leach out of the cells and
the alcohol will turn blue. The de-colourising step should continue until no more blue
colour leaches from the cells. However, be warned that even Gram positive cells will
turn Gram negative if this step is carried out for too long!
Wash the slide under a gentle stream of tap water, gently shake off the water
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Flood the slide with the counter stain, safranin, and leave for one minute
Wash off the safranin with tap water, dry by gentle blotting and passing through the
Bunsen flame. It is essential that the smear is completely dry before it is examined in
the microscope.
Place the slide onto a microscope and initially focus using the 10x objective to find
your smear
Place a drop of oil directly on the smear and examine it under direct light using the
oil immersion (100x) objective of the microscope.
Control organisms (essential!): Gram negative: Pseudomonas & Gram positive:
Micrococcus.
Examining Gram stains under the microscope
The stained preparations should be examined using a 100x oil immersion objective
(Reference Section 19.3.1), but first focus using the 10x objective. Record the colour
(Gram negative – pink/red; Gram positive – purple; and the shape and organisation
of the cells for each culture stained.
Table 1:Gram staining
Bacteria
Gram positive/negative
Escherichia coli
Staphylococcus
epidermidis
Staphylococcus
mutans
Pseudomonas
fluorescens
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Shape/Organisation
Flavobacterium
aquatile
Bacillus pumilis
Bacillus
mycoides
Serratia
marcescens
Micrococcus
luteus
Other methods to investigate bacteria: The Endospore stain
Endospores are found in Bacillus species and in some other Gram-positive bacteria. They
often appear as white patches in Gram-stained cells or as bright inclusions within cells
under phase contrast. They do not stain readily because they are impervious to stains that
are applied gently.
Demonstrators will have prepared endospore stains ahead of the session. Investigate these
under the microscope.
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Place the slide onto a microscope and initially focus using the 10x objective to find
your smear
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Then examined under oil immersion (100x objective) with the oil being placed
directly on the smear.
Intracellular spores appear either as green inclusions inside a pink cell, extracellular
spores appear as green stained structures close to but outside the cells. The green
colour is often very pale but using a longer staining period on the beaker of boiling
water will improve the contrast between the pink cell and green spore.
Record the results in table 2.
For reference, the method they used to stain the endospores is below.
Prepare a heat-fixed smear as detailed above and stain as follows:
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Place the slide with the smear uppermost, over a beaker of boiling water, resting it on the small glass staining
racks provided;
When water droplets have condensed on the underside of the slide (this takes at least 2 mins), flood the slide
with a 5% aqueous solution of malachite green and leave it for at least 90 seconds. It is important that the water
continues to boil and, more importantly that the malachite green does not dry out. If necessary add a couple
more drops of stain during the staining process;
Remove the slide from the beaker of boiling water and wash with cold water. This removes the malachite green
stain from the cells but not from the spores. It is important to remove any crystals of malachite green which may
have formed on the slide (often these need to be scraped off with a loop);
Counter stain with safranin (not on the boiling water beaker) for 1 minute;
Wash with tap water, dry by blotting and passing through the Bunsen flame.
Motility tests
There are two methods used to determine bacterial motility depending on whether the cells
have been grown in liquid culture or on solid media. The samples are examined under
phase contrast (phase plug 2) with the 40x objective. The bacteria should be visible as
black or grey organisms against a lighter background. Motility is a highly aerobic process in
aerobic bacteria, and ceases very quickly in these preparations as oxygen is rapidly
consumed under the cover slip. Therefore, it is essential that these preparations are
examined quickly. Alternatively find an air bubble or the edge of the coverslip where
oxygen should still be available as it diffuses into the liquid from the air.
Motility is characterised by movement of bacteria in a non-random fashion. Bacteria often
'run' for a short period of time then tumble before moving off in another direction. You have
to learn to distinguish between true motility and Brownian motion (as the bacteria 'shudder'
due to bombardment by water molecules) and between true motility and movement in
convection currents (set up as the liquid is heated by the microscope light source).
The motility stains will be set up by the demonstrators.
For reference the methods for motility assays are detailed below
Motility testing a liquid media culture
The motility of samples from liquid cultures is determined by placing a loopful of culture on a microscope slide
and diluting it with one or two loopfuls of sterile distilled water. Remember to flame the loop before placing it in
the distilled water or on the bench. Place a coverslip over the drop and examine under phase contrast as
quickly as possible.
Motility testing a solid media culture
The motility of samples from agar plates can be determined by preparing an emulsion of bacteria in a droplet
of sterile distilled water. You only need to use a speck of culture otherwise it becomes too dense. As a rule if
the droplet is cloudy (turbid) then there are too many bacteria in the droplet. Bacteria are not always motile
from agar plates (the organisms don't need to produce flagella as they cannot swim anywhere). If a culture
appears non-motile from an agar plate it is always best to set up a liquid culture to examine motility under
optimal conditions. Usually inoculation of 1 ml of nutrient broth in a test tube with a speck of culture and
incubation overnight at 30°C is sufficient.
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Table 2: Endospores and motility
Bacteria
Escherichia coli
Staphylococcus
epidermidis
Staphylococcus
mutans
Pseudomonas
fluorescens
Flavobacterium
aquatile
Bacillus pumilis
Bacillus
mycoides
Serratia
marcescens
Micrococcus
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Gram
positive/negative
Are spores intra- or
extracellular?
Is the bacteria motile?
Describe here
luteus
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