University of Warwick

University of Warwick

Headstart Summer School

Microbiology Lab

Practical manual

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.

You will be provided with some of the bacteria below and you will isolate single colonies from some of them.

37°C
Escherichia coli / (E. coli) / 37°C
Staphylococcus epidermidis / (S. epidermis) / 37°C
Or / Staphylococcus mutans / (S. mutans) / 30°C
Pseudomonas fluorescens / (P. fluoescens) / 30°C
Flavobacterium aquatile / (F. aquatile) / 30°C
Bacillus pumilis / (B. pumilis) / 30°C
Bacillus mycoides / (B. mycoides) / 30°C
Serratia marcescens / (S. marcescens) / 30°C
Micrococcus luteus / (M. luteus)

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!).

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.

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.

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:

·  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

·  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 / Shape/Organisation
Escherichia coli
Staphylococcus epidermidis
Staphylococcus mutans
Pseudomonas fluorescens
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.

·  Place the slide onto a microscope and initially focus using the 10x objective to find your smear

·  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:

·  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.

Table 2: Endospores and motility

Bacteria / Gram positive/negative / Are spores intra- or extracellular? / Is the bacteria motile? Describe here
Escherichia coli
Staphylococcus epidermidis
Staphylococcus mutans
Pseudomonas fluorescens
Flavobacterium aquatile
Bacillus pumilis
Bacillus mycoides
Serratia marcescens
Micrococcus luteus

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