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School of Life Sciences

BMS1071M: HEALTH AND DISEASE

Practical Protocols 2014 - 2015

Practical Coordinator: Dr Nicola Crewe (Tel 01522 837364), JBL1W10

Email: ncrewe@lincoln.ac.uk

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CONTENTS 2

SAFETY NOTES 3

PRACTICAL ONE

Determination of the efficacy of hand washing 7

Determination of the level of microbial contamination in the atmosphere 8

Determination of the antibiotic susceptibility of E. coli 8

Subculture of the mycelial mat of hyphal fungi 8

Subculture of a unicellular yeast 9

Broth culture of a mycelia fungus 9

Fungal identification 9

PRACTICAL TWO

Bacteriophage plaque assay 13

PRACTICAL THREE

Food and Water microbiology 15

PRACTICAL FOUR

Determination of the growth curve of E. coli 29

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SAFETY NOTES

Safety and Code of Practice for working in the laboratory

Laboratories can be dangerous places in which to work. It is important that you

know the hazards that exist in the laboratory so that you can work safely and you

are not a danger to yourself, or your colleagues.

Hazards in the microbiology laboratory can be assigned to the following categories:

Microbiological

Chemical

Physical

The code of practice is an important document that relates to safe working practice

in the laboratory. It is essential that you are familiar with and understand the details

of this document. Some of the items relate to laboratories in general, others are

rather more specific to microbiology.

Regulations known as the Control of Substances Hazardous to Health regulations

(COSHH) came into force on the 1st October 1989, and were updated in 1999 in

relation to biological sciences. These regulations govern experimental work carried

out in laboratories. In order to comply with these regulations it is essential that you

follow the code of practice, take careful note of any specific risks associated with

handling either microorganisms or chemicals in the laboratory, and understand how

these risks are prevented or adequately controlled.

Safe working will be emphasised throughout these sessions in microbiology. You

should pay particular attention to elements of safety and aseptic technique

highlighted during the practicals. Make notes on these points as you go along.

Don’t hesitate to ask for clarification or explanation of any aspects you

are doubtful about.

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The Code of Practice for the Microbiology Laboratory

The following code of practice has been produced as a result of the assessment of

risks associated with working in the microbiology laboratory to comply with

COSHH regulations.

This Code of Practice identifies mandatory conditions for working in the

laboratory and must be complied with for each practical.

1. A laboratory coat must be worn. No one may engage in laboratory work

without an adequate lab coat.

2. Lab coats used in the microbiology lab must be of the designated type –

identified by a green flash on the pocket. Personal lab coats must not be

used.

3. Safety glasses must be worn whenever chemicals or reagents are handled or

boiling operations carried out.

4. Students must wash their hands when contamination is suspected, when

viable materials have been handled and before leaving the laboratory.

5. No hand to mouth operations are allowed e.g. eating or pencil chewing.

6. Mouth pipetting is prohibited.

7. Operations involving the possibility of wound infection e.g. use of scalpels or

hypodermics should be avoided or carried out with extreme caution.

8. Disposable infectious materials, e.g. used Petri dishes containing agar, must be

discarded into the metal bins provided. These plates will be autoclaved and

disposed of by the technical staff. Used plates must not be left lying around

in the laboratory or in incubators.

9. Broken glass must be disposed of in the cardboard container specially

designed for this purpose and not in the ordinary waste bin. These waste

receptacles are normally labelled ‘sharps’.

10. Spillage of cultures must be reported to the lecturer in charge. The spillage

must be covered with disinfectant. Use 10% hypochlorite if available – wear

safety glasses and rubber gloves and avoid splashing clothes. Allow

reasonable contact time i.e. 2 minutes and then mop up.

11. Swab your bench area with alcohol at the end of the practical period. Ensure

all naked flames have been extinguished before carrying out this procedure.

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12. Make sure you are familiar with the location of fire extinguishers and

eyewash bottles for use in an emergency.

13. Avoid producing aerosols or clouds of micro-organisms. Operations

producing aerosols include:

a. Splashing or frothing cultures during pipetting and plating out

b. ‘twanging’ of wires and loops

c. any procedure involving film bursting

d. ‘sizzling’ of materials on loops

e. spillage of liquid cultures

f. breakage of culture bottles.

14. Every effort must be made to become familiar with and skilled in the use of

aseptic techniques.

15. All open wounds must be covered with a waterproof dressing before starting

work in the laboratory. This includes minor cuts. If in doubt seek advice

from your tutor.

16. Accidents, no matter how minor, must be reported to your tutor

immediately.

Microorganisms can be classified according to the hazard they represent to the

laboratory worker, the community in general and methods required for their

containment (Categorisation of Pathogens According to Hazard and Categories of

Containment. Report of the Advisory Committee on Dangerous Pathogens. Health

and Safety Executive 1995.) All labelled organisms used in this laboratory belong to

Hazard groups 1 and 2. Handling of these groups requires strict adherence to the

code of practice set out above.

Hazard group 1 organisms are unlikely to cause human disease.

Hazard group 2 organisms may cause human disease and could be a hazard to

laboratory workers, but are unlikely to spread in the community.

Handling of pathogens belonging to Hazard groups 3 and 4 require special methods

of containment. Advice on handling any organisms in these categories must be

sought from the Health and Safety Executive.

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During any microbiology practical work it is best to assume that the

organisms you are handling are capable of causing illness and disease.

You must wear a correctly fastened Howie lab coat at all times. If you need

to leave the lab for any reason, you should wash your hands before leaving,

then remove your lab coat in the designated area, and then wash your hands

again before leaving the locker room.

We do not recommend wearing gloves for microbiological work, as they can

cause serious injury if they come into contact with the Bunsen flame. They

can also reduce your ability to carry out the fine manipulations needed for

microbiology. If you have a cut on your hands then you should apply a

plaster before carrying out any work. If you have several open wounds on

your hands then it is probably more practical to wear gloves, but remain

vigilant at all times.

Goggles are generally not used in the microbiology lab, as they can restrict

your eyesight, and can be dangerous around Bunsen flames. Any time you

are required to carry out liquid manipulations, do so slowly and carefully to

avoid splashes.

It is also important to keep microorganisms away from your lab book, as you

will take this away from the lab and therefore you do not want to

contaminate it. Long hair must always be tied back, and headscarves or hats

must not come into contact with the Bunsen flame, or touch any

microbiological material.

It is very important to label all of your cultures correctly, and put them in the

correct place for incubation at the end of the practical. Agar plates should be

incubated with the agar at the top, so that any moisture that is formed during

incubation will drip into the lid of the Petri dish, not onto the culture. Any

cultures that have not been labelled, or are not on the tray for incubation at

the end of the practical will be disposed of.

At the end of the practical you should tidy your bench, throw any

microbiological waste into the relevant autoclave bucket, and finally swab

down your bench with alcohol to destroy any contaminating microbes.

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PRACTICAL ONE – MYCOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Introduction

In this practical you will be looking at environmental contamination in the laboratory,

and the effects of hand washing on microbial load. You will also be introduced to

working with fungi safely within the laboratory. The vast majority of microbiological

laboratory work is bacteriology, but it is important to know how to handle fungi

safely.

Health and safety requirements are the same for other microbiology practicals – if

necessary, remind yourself by re-reading the first few pages of this booklet.

In this practical we will be handling spore forming fungi which create fine spores, and

in susceptible people can cause allergic reactions. When manipulating these cultures,

keep your movements slow and methodical to avoid creating a large cloud of spores.

If you are allergic to fungi / antibiotics then it is advisable to wear a facemask during

this part of the practical.

You will be using a scalpel during one of the procedures in this practical. It is crucial

to be extra vigilant when carrying out this step, as not only could you hurt yourself

with the blade, there is the extra risk of microbial infection in the open wound.

Determination of the efficacy of hand washing

‘It is well-documented that one of the most important measures for preventing the

spread of pathogens is effective hand washing’ (CDC, 2010). However, the

technique and equipment used for hand washing can greatly affect its efficacy.

Individually, take a single nutrient agar plate, and draw a line on the base of the Petri

dish to divide the plate in two. Label the plate as usual, but also include ‘unwashed’

on one half and ‘washed’ on the other. Open the lid of the plate, and touch your

fingertips to the surface of the agar on the ‘unwashed’ side. Wash and dry your

hands, and then touch your fingertips to the surface of the agar on the ‘washed’ side.

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Different soaps are available, so compare their effectiveness within your group.

Your plates will be incubated at 30oC for two days.

Determination of the level of microbial contamination in the atmosphere

Contamination of experiments with microbes is a major problem within labs. This is

particularly the case within tissue culture labs, and can result in entire labs being

closed for weeks for fumigation. In the microbiology lab, we can reduce the risks by

effective use of a Bunsen burner, but there needs to be an effective cleaning

programme in place as well.

In a pair, take one MEA plate and one nutrient agar plate. Label them fully, and then

place them, with the lid off, in the laboratory. Leave them for as long as possible

within the confines of the laboratory session, and then before you leave, replace the

lids and make a note of the length of time the plates have been exposed for. The

nutrient agar plate will be incubated at 30oC for 2 days, while the MEA plate will be

incubated at 25oC for 4 days.

Determination of the antibiotic susceptibility of E. coli

Antibiotic resistance is becoming a major problem in the community, and many

research groups are focussing on alternative solutions to the problems. Within the

medical microbiology lab, one of the most important tests is to determine the

antibiotic resistance profile of an organism so that an effective treatment can be

prescribed.

Prepare one spread plate of E. coli on Mueller-Hinton agar, using the overnight

culture on your bench. Leave the plates to dry for a short length of time, and then

add antibiotic discs to the surface of the plate using the dispenser. Your plates will

be incubated at 37oC overnight.

Subculture of the mycelial mat of hyphal fungi

Individually, take a malt extract agar (MEA) plate and mark the base of the plate with

your initials, the date and the organism you will be culturing. Using a sterile scalpel,

cut a small section of agar that is covered in hyphae from either culture, and transfer

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it to the surface of the fresh MEA plate. Incubate the plate in the inverted position,

with the agar at the bottom. Mycelial fungi are the only organism that should be

incubated in this manner. Your plates will be incubated at 25oC for 3-4 days.

Subculture of a unicellular yeast

Saccharomyces spp. are the yeast species that are used for many of the fermentation

processes that create food such as beer and bread. Yeasts are cultured using the

same methods as those used for bacteria, in that streak, spread and pour plates can

all be prepared from a yeast culture.

Individually, take a malt extract agar (MEA) plate and mark the base of the plate with

your initials, the date, the organism you will be culturing. Using a sterile wire loop,

pick up some yeast cells from the Saccharomyces culture, and use these cells to

create a streak plate. The yeast colonies are quite tough to break apart with a loop,

but you will have picked up enough cells to start a fresh culture by this process.

Your plates will be incubated at 25oC for 4 days.

You should also prepare a Gram stain of the yeast cells for microscopical

observation. Yeast cells viewed in isolation from bacterial cultures are frequently

mistaken for Gram positive cocci, but careful measurement (by calibration of the

eyepiece graticule) and careful observation (which may yield visual clues such as the

presence of budding cells) will prevent this occurrence.

Broth culture of a mycelial fungus

When mycelia fungi are growth in a shaken broth culture they forms pellets. The

higher the initial concentration of inoculating spores within the growth medium, the

smaller these pellets become, to the extent that eventually they will form a

filamentous mass. If fungal spores are inoculated into a broth, but the broth is not

shaken during incubation, a mycelial mat will form on the surface – similar to that

seen after growth on solid medium.

Look at the fungal broth cultures that have been prepared for you, and make some

notes in your lab book on the method.

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Fungal identification

When hyphal fungi are grown on a solid medium, they produce growth patterns that

are very characteristic to their species. The colour of the mould is often distinctive,

as is the way it spreads across the surface of the agar, and the length of time it takes

to grow. The types of spores and hyphae it produces are also very indicative of the

classification of the organism.

Examine the plate cultures of the fungi provided under the dissection microscope.

Look carefully at the mycelial structure and the hyphae, and make some quick

sketches in your lab book. You should also be able to see the spores at the surface

of the culture.

The types of spores that a fungal culture produces are strongly identifying. In order

to observe the spores in detail, we will be carrying out a lactophenol cotton blue

stain, as described on the next page. Observe the slide under the microscope and

make some notes in your lab book. Carry out this procedure slowly in order to

reduce the number of spores released into the atmosphere.

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FOLLOW-UP INFORMATION

Determination of the efficacy of hand washing

Count the number of colonies present on the plate before and after hand washing.

Record the results on the chart in the laboratory. Do the different soaps have

different effects on the number of microorganisms present? Does there appear to

be a difference between the types of colony present before and after hand washing?

Determination of the level of microbial contamination in the atmosphere

Count the number of organisms on each type of plate. Is there higher bacterial or

fungal contamination? Calculate the number of live cells that landed on your plates

in h-1m-2.

Determination of the antibiotic susceptibility of E. coli

Observe and measure the zones of clearing around the different antibiotic discs, and

determine whether the organism is sensitive or resistant to each type of antibiotic.

Which antibiotic would you use to treat a patient who presented with an infection

caused by this organism?

Subculture of the mycelial mat of hyphal fungi

Observe how the mycelia have grown from the section of agar you subcultured.

Subculture of a unicellular yeast

Observe the streak plate of yeast to determine the effectiveness of your technique.

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PRACTICAL TWO – BACTERIOPHAGE MANIPULATION

Introduction

The practical today will provide you with an opportunity to work with bacteriophage

culturing within the lab. The bacteriophage poses no risk to human health, although

alongside it we will be manipulating Escherichia coli and therefore full bacteriological

safety procedures should be used.

Bacteriophage plaque assay

In order to observe the lysis of cells by viruses, the two must be mixed together and

allowed to grow. In the case of bacteriophages, this means mixing the virus particles

with the bacterial cells to which they are pathogenic. However, in order to see the

effect of the virus on the cells (the formation of plaques), the bacterial cells have to

be ‘fixed’ in a location, while the virus particles have to be able to move in order to

infect the next bacterial cell. This is done by using a half-strength agar (soft agar),

which is solid enough to maintain the bacterial culture, but liquid enough that the

viruses can move. This soft agar is overlaid on a standard agar plate, which provides

the bacteria with nutrients for growth.

The first step of this protocol is to create a serial dilution of the phage lysate. This

will be done in exactly the same way as for the bacterial culture, only in smaller

volumes. Prepare the diluent tubes by adding 0.9ml of ¼ strength Ringer’s solution

to the 6 empty sterile Eppendorf tubes on your bench, using aseptic technique

throughout. Label these as 10-2 through to 10-7.

You have 0.1ml of phage lysate in an Eppendorf tube. Working close to the Bunsen

flame to reduce the chance of contamination, add 0.9ml of ¼ strength Ringer’s

solution to the tube to create a 10-1 dilution of the phage lysate. Label the tube as

such, and mix the tube thoroughly by flicking. Using a fresh pipette tip, take 0.1ml of

lysate from the 10-1 dilution, add it to the tube labelled 10-2, and mix the tube

thoroughly. Repeat this procedure, using a fresh tip each time, until the dilution

series is complete.

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You will be producing three plates from the dilution series, and the results will be

shared among the group to get data from the dilution range. Your lecturer will help

you determine which dilutions to plate. Take three nutrient agar plates and mark

the base of the plates with your initials, the date, the organism you will be culturing,

and the dilution factor.

The soft agar you will be using will set very quickly, so make sure you are ready to

prepare the mixture before removing the tube from the water bath.

Take out a single tube of soft agar. Using full aseptic technique, add 0.1ml of E. coli

broth culture to it, and then 0.1ml of the lysate dilution. Mix thoroughly using the

vortex for a maximum of 10 seconds. Pour the soft agar onto the surface of the

nutrient agar plate, and put to one side to set. Repeat this process for the

remainder of the dilution series. Make sure to use the correctly labelled nutrient

agar plate for the phage dilution you are adding to the soft agar. Your plates will be

incubated at 37oC overnight.

FOLLOW-UP INFORMATION

Bacteriophage plaque assay

Look for the presence of zones of clearing in the bacterial lawn. These are plaques

caused by lysis of the bacterial cells. Count the number of plaques on any plates that

appear to have between 30 and 300 plaques. Use these numbers to determine the

phage titre of the initial lysate.

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PRACTICAL THREE – FOOD AND WATER MICROBIOLOGY

Introduction

In this practical you will have the chance to analyse a water and food sample that you

have collected yourself from home. This practical will introduce the technique of

membrane filtration, and allow you to use a variety of indicator and selective media.

You should spend some time in this practical looking through the functions of the

different media you will be using, by reading the OXOID manual. You should also take

some time to think about the organisms you expect to isolate from the environmental

samples, and see if you can predict the results.

Food and Water microbiology

Routine sampling of food and water sources for the determination of the microbial

load is required by law. This work is carried out within a UKAS accredited

laboratory using protocols recommended by a variety of organisations, including the

Government body, the Drinking Water Inspectorate. The instructions given below

are taken from UKAS accredited laboratory protocols.

Analysis of the water sample

In samples that are expected to contain relatively low levels of micro-organisms, an

efficient way of determining the level of contamination is by membrane filtration of a

large sample volume. A clear liquid, such as water, normally contains low levels of

micro-organisms, and therefore it is routine to test 100ml of the sample to increase the

chance of observing any contamination. The technique for membrane filtration is given

below.

Mark the base of the plate with your initials, the date and the sample code. Using the

Bunsen burner, flame the funnel held in the clamp on the retort stand to sterilise it.

Then sterilise the filter base using the Bunsen burner, opening the level to pull some hot

air through the metal mesh and sterilise inside the base as well. Allow both parts to

cool for a couple of minutes.

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Take the forceps and dip them in the pot of alcohol (keeping the pot well away from the

Bunsen burner). Then quickly pass the forceps through the flame. This will ignite the

alcohol and sterilise the forceps. Hold the forceps NEAR TO the flame, not in it,

allowing the metal to cool slightly. Unwrap a sterile cellulose nitrate filter, and place

this onto the base of the filtration unit using the sterile forceps. Place the funnel onto

the base, and lock it into position. Pour the sample into the funnel up to the highest

black mark (which is 100ml), and allow the sample to pass through the filter. Once the

sample has completely filtered, sterilise the forceps once more, remove the funnel and

peel the cellulose nitrate filter from the base of the filtration unit. Transfer the cellulose

nitrate filter to the agar plate, gently rolling the filter down onto the surface of the agar

to make sure that no bubbles are trapped underneath. Sterilise the forceps once more.

The same filtration unit can be used for all filtrations of the same sample, however it is

important to keep them as sterile as possible and therefore all filtrations should be

carried out as close to each other as possible. In other words, ensure all your plates

are assembled and correctly labelled; your sample is close to hand, and all the

equipment you require is close by before sterilising the filtration unit.

Culturing requirements:

Agar Full name Purpose Culture method

MLSA Membrane lauryl sulphate agar

Incubate at 37oC

Isolation of

coliforms Filter 100ml

Ps CFC Pseudomonas CFC agar

Incubate at 30oC

Isolation of

Pseudomonas spp. Filter 100ml

S+B Slanetz and Bartley agar

Incubate at 37oC

Isolation of

Enterococcus spp. Filter 100ml

TSC Tryptose Sulphite Cycloserine agar

Incubate at 37oC anaerobically

Isolation of

Clostridia spp. Filter 100ml

YEA Yeast Extract agar

Incubate at 30oC General count 1 x spread plate

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Analysis of the food sample

In order to analyse the microbiological quality of a food sample, it first needs to be

made into a liquid form. This is done by preparing a known dilution of the food in ¼

strength Ringers solution in a stomacher bag. This solution is then stomached for two

minutes in order to break down the food particles as much as possible, so that the

liquid can be spread plated.

After this, the liquid can be manipulated in the same way as any culture within the

laboratory. Culture the organisms within the liquid by following the table below.

Culturing requirements:

Agar Full name Purpose Culture method

YEA Yeast extract agar

Incubate at 30oC General count 1 x spread plate

MEA Malt extract agar

Incubate at 25oC

Yeast / mould

count 1 x spread plate

MLSA Membrane lauryl sulphate agar

Incubate at 37oC

Isolation of

coliforms 1 x spread plate

BP Baird Parker agar

Incubate at 37oC

Isolation of

Staph aureus 1 x spread plate

FOLLOW-UP INFORMATION

Food and water microbiology

Observe the cultures you produced during the practical and from them draw

conclusions regarding the level and types of microbial contamination in the different

samples.

You should count the number of colonies present on each plate, and then use that data

to calculate the number of CFUs per ml or per gram in the original samples. The results

you have obtained will be discussed within the group.

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PRACTICAL FOUR – BACTERIAL GROWTH AND PARASITOLOGY

Introduction

In this practical we will be looking at the growth of a bacterial culture in broth. You will

be producing a growth curve of E. coli so that you can determine the generation time of

this organism, and therefore appreciate how quickly it can grow under ideal conditions.

This growth curve will take the entire practical to produce, so concentrate on this to

start with, and when you feel confident with what you have to do for this experiment,

you can work on the other activities.

We have provided some demonstration materials covering some alternative methods

for testing microbiological samples, such as API strips, test kits and serological

identification, which covers many of the areas that were covered within lectures.

Handling parasites is a highly specialised area, which we are not equipped to do in

this university, but we do have some display organisms for you to look at. We have

several specimens of parasites available in the lab for you to look at, both under the

microscope and in display cases. Look at these organisms, and make some notes in

your lab book.

Determination of the growth curve of E. coli

Producing a growth curve is one of the most common experiments carried out by

microbiologists during research. When you begin to work with an organism, it is

crucial to understand how quickly it can grow, so that you can plan your future

experiments effectively.

As a pair, you will be provided with a conical flask containing 30ml of a broth culture

of E. coli that was inoculated within the last two hours. You will be taking

absorbance readings of the culture every few minutes for the duration of the

practical. Follow the steps overleaf for each sample taken.

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Remove the flask containing the E. coli from the 37oC incubator.

Using a pipette, place 1ml of the broth culture into a cuvette.

Return the flask to the incubator.

Measure the absorbance at 590nm of the culture within the cuvette.

Pour the live culture into the beaker containing bleach.

Fill the cuvette with water, and pour the water into the beaker containing bleach.

Place the cuvette upside down on a piece of paper towel to dry.

Plot your results as you go along on the graph paper provided. Once the organism

has reached log phase growth, you can determine its generation time.