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Basic Pract Cover 2009
16/1/09
12:01
Page 3
Basic Practical
Microbiology
A Manual
Society for General Microbiology (SGM)
Basic Pract Cover 2009
16/1/09
12:01
Page 2
The Society for General Microbiology (SGM) is a learned society with over 5,000
members worldwide who work in universities, industry and research institutes
...
It produces and distributes
a wide range of resources to support microbiology teaching in schools and
colleges across all key stages and post-16
...
SGM has a
charitable status
...
microbiologyonline
...
uk or contact: Education
Department, SGM, Marlborough House, Basingstoke Road, Spencers
Wood, Reading RG7 1AG, UK (Tel
...
ac
...
Basic Pract Book 2006
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Page 1
Contents
About this resource
Inside front cover
Part 1: The Basics
An introduction to microbiology, aseptic technique and safety
Preparation
Safety guidelines
Risk assessment
Good microbiological laboratory practice (GMLP)
Spillage management
Aerosols
1
2
3
3
3
Resources
Equipment
Apparatus
Materials
4
5
5
Media, sterilisation and disinfection
Preparation of culture media
Pouring a plate
Storage of media
Sterilisation vs disinfection
Sterilisation using the autoclave/pressure cooker
Sterilisation of equipment and materials
Choice, preparation and use of disinfectants
6
6
6
6
7
7
7
Inoculation and other aseptic procedures
Essential points
Using a wire loop
Using a pipette
Flaming the neck of bottles and test tubes
Working with bacteria and yeast
Streak plate
Pour plate
Using a spreader
Spread plate
Working with moulds
11
12
13
14
15
Incubation
16
In conclusion: clearing up
17
8
8
9
10
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Page 2
Essential methods for maintaining, preparing and using cultures
Obtaining suitable cultures
Pure cultures
Maintaining stock cultures
Checking cultures for contamination
Preventing contamination of cultures and the environment
Aseptic transfer of cultures and sterile solutions
Preparing cultures for class use
18
18
18
18
19
19
19
Part 2: Microbiology in Action
Practical activities
1
...
Microscopy
Using the microscope
Stained preparations
Making a smear
A simple stain
A differential stain: Gram’s staining method
22
23
23
24
24
Appendices
1
...
Safe micro-organisms
3
...
Suppliers of cultures and equipment
5
...
Preparing serial dilutions
26
31
37
38
39
40
21
Basic Pract Book 2006
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Page 1
Part 1: The Basics
An introduction to microbiology, aseptic
technique and safety
As well as causing a familiar range of diseases in animals and plants and problems in food spoilage and deterioration
of other materials, microbes are also our ‘invisible allies’
...
The teaching of such an important subject as microbiology cannot be achieved
effectively without enhancing the theory with ‘hands on’ experience in the laboratory
...
This manual has been written for a right-handed person
...
Activities involving micro-organisms are controlled by the Control of
Substances Hazardous to Health (COSHH) Regulations and teachers and technicians have a duty under the Health and
Safety at Work Act to comply with any safety instructions given by their employers
...
2, Topics in Safety, 3rd edition (ASE, 2001), Microbiology: an HMI Guide (DES, 1990) and Safety in Science
Education (DfEE, 1996)
...
Planning ahead is essential when embarking on practical microbiology investigations
...
Preparation and sterilisation of equipment and culture media
...
Inoculation of the media with the prepared culture
...
Sterilisation and safe disposal of all cultures and decontamination of all contaminated equipment
...
For model risk assessments, adaptations to model risk assessments and factors which need to be considered when
contemplating carrying out practical work that is not covered by a model risk assessment, see CLEAPSS Laboratory
Handbook (revised 2001), section 15
...
2 and CLEAPSS Guide L169, Managing Risk Assessment in Science 1997
...
teacher
demonstration
Composition of culture media
Possibility of selecting for growth of pathogens
Volume of cultures
Increased risk with increase in volume of liquid culture
Laboratory facilities
Suitability for level of practical microbiological work
Equipment
Adequate for purpose
Incubation conditions
Possibility of selecting for growth of pathogens
Disposal procedures
Ensures elimination of risk to others
Expertise of technicians and teachers
Competence and level of training in techniques and procedures
appropriate to level of practical work
Student age and discipline
Appropriate to level of practical work; confidence in class discipline
Sources of competent advice
ASE*, CLEAPSS*, MISAC, NCBE, SSERC* (*members only)
Useful check list
CLEAPSS Laboratory Handbook (2006), section 15
...
34–37
Essential reference
Topics in Safety, 3rd edition (ASE, 2001), Topic 15 or
CLEAPSS Laboratory Handbook (2006), section 15
...
[Appendix 1: Safety guidelines]
[Appendix 2: Safe micro-organisms]
2
© 2006 SGM
Basic Practical Microbiology – A Manual
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Good microbiological laboratory practice (GMLP)
Training in GMLP is aimed at developing
proficiency in containing any uncontrolled
spread of microbes in order to protect:
practical investigations from becoming
contaminated with microbes from external
sources
the operators (students, teachers and technicians) from the very small possibility
of infection
...
g
...
)
It is important to arrange the workplace carefully to ensure safe and effective operations
...
Suggested components:
beaker for making fresh disinfectant
disposable gloves
dustpan
paper towel/cloth
autoclave/roasting bag
Spillages of cultures must be reported immediately to the teacher
or technician to be dealt with quickly
...
Spilled cultures and surrounding
debris (e
...
glass, cotton wool plugs), if any, must not be touched with
unprotected hands
...
Spill debris should then
be swept into a dustpan using paper towels
...
g
...
The dustpan must be
decontaminated either by autoclaving or by soaking (at least 24 hours)
in hypochlorite (sodium chlorate I)
...
It should be swept carefully into a suitable container, autoclaved and
disposed of in a puncture proof container
...
Splashes on
the skin should be treated as soon as possible; washing thoroughly
with soap and hot water should be sufficient, but if necessary the skin
can be disinfected
...
The risk of spillages
occurring is lessened by using cultures grown
on agar instead of in liquid media whenever
possible
...
The
risk is minimised by adhering to GMLP with
special attention to the correct use of pipettes
(see Inoculation and other aseptic procedures
page 8)
...
g
...
g
...
Before sterilisation, ensure ingredients are
completely dissolved, using heat if necessary
...
Normally allow 15–20 cm3 medium per Petri dish
...
Agar slopes are prepared in test tubes or Universal/McCartney bottles by allowing sterile molten cooled medium to
solidify in a sloped position
...
[Appendix 4: Suppliers of cultures and equipment]
Pouring a plate
Step 4
1
...
2
...
3
...
4
...
5
...
6
...
7
...
The base of the plate must be covered, agar must not touch the lid of the plate and the surface must be smooth with
no bubbles
...
If they are not going to be used straight away they need to
be stored inside sealed plastic bags to prevent the agar from drying out
...
Media in vessels closed by cotton wool plugs/plastic caps that are stored for future use will be subject
to evaporation at room temperature; avoid wastage by using screw cap bottles
...
Once melted, agar can be kept molten in a water bath at ca 50 °C until
it is ready to be used
...
Sterilisation vs disinfection
Sterilisation means the complete destruction of all the micro-organisms including spores, from an object or
environment
...
Disinfection is the destruction, inhibition or removal of microbes that may cause disease or other problems, e
...
spoilage
...
6
© 2006 SGM
Basic Practical Microbiology – A Manual
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Sterilisation using the autoclave/pressure cooker
The principle of sterilisation in an autoclave or pressure cooker is that steam under pressure is used to produce a
temperature of 121 °C which if held for 15 minutes will kill all micro-organisms, including bacterial endospores
...
Empty glassware and glass (not plastic!) pipettes and Petri dishes
Either: hot air oven, wrapped in either greaseproof paper or aluminium and held at 160 °C for 2 hours, allowing
additional time for items to come to temperature (and cool down!)
...
Note: plastic Petri dishes are supplied in already sterilised packs; packs of sterile plastic pipettes are also available
but cost may be a consideration
...
Glass spreaders and metal forceps
Flaming in alcohol (70 % IDA)
...
The choice is now much more
straightforward as the range available from suppliers has decreased
...
p
...
(0
...
Disinfectants for use at working strength should be freshly prepared from full strength stock or powder form
...
g
...
Use working strength hypochlorite on day of preparation
...
Operations must not be started until all requirements are within immediate reach and must be completed as
quickly as possible
...
On being opened, the neck of a test tube or bottle must be immediately warmed by flaming so that any air
movement is outwards and the vessel held as near as possible to the horizontal
...
The parts of sterile pipettes that will be put into cultures or sterile vessels must not be touched or allowed to come in
contact with other non-sterile surfaces, e
...
clothing, the surface of the working area, the outside of test tubes/bottles
...
They must be heated to red hot to make
sure that any contaminating bacterial spores are destroyed
...
This leaves the little finger free to take hold of the cotton wool
plug/screw cap of a test tube/bottle
...
1
...
This is the cool area of the flame
...
Draw the rest of the wire upwards slowly up into the hottest
region of the flame, (immediately above the light blue cone)
...
Hold there until it is red hot
...
Ensure the full length of the wire receives adequate heating
...
Allow to cool then use immediately
...
Do not put the loop down or wave it around
...
To correct the
problem, first ensure that the loop has been
sterilised and then reshape the loop with
forceps
...
7
...
Step 1
8
Step 2
© 2006 SGM
Step 3
Step 4
Step 5
Basic Practical Microbiology – A Manual
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Using a pipette
Step 1a
Step 1b
Step 2
Step 3
Step 4
Step 6
Sterile graduated or dropping (Pasteur)
pipettes are used to transfer cultures, sterile
media and sterile solutions
...
Remove the pipette from its container/
wrapper by the end that contains a cotton
wool plug, taking care to touch no more
than the amount necessary to take a firm
hold
...
Fit the teat
...
Hold the pipette barrel as you would a pen
but do not grasp the teat
...
4
...
5
...
The pipette tip must remain beneath
the liquid surface while taking up liquid
to avoid the introduction of air bubbles
which may cause ‘spitting’ and, consequently, aerosol formation when liquid is
expelled
...
Immediately after use put the now
contaminated pipette into a nearby
discard pot of disinfectant
...
Basic Practical Microbiology – A Manual
Hints
A leaking pipette is caused by either a faulty or ill-fitting teat or fibres
from the cotton wool plug between the teat and pipette
...
Converting a Pasteur
pipette by attaching
a syringe barrel
Commercial dispensing
systems are available such as
measuring Pasteur pipettes
...
Loosen the cap of the bottle so that it can be removed easily
...
Lift the bottle/test tube with the left hand
...
Remove the cap of the bottle/cotton wool plug with the little finger
of the right hand
...
)
4
...
5
...
6
...
g
...
(Turn the bottle, not the cap
...
Either marker pens or self-adhesive labels are suitable
...
Douse the
flames by immediately covering with a dry cloth, not by blowing or
soaking in water
...
This involves the progressive dilution of an inoculum of bacteria or yeast
over the surface of solidified agar medium in a Petri dish in such a way that colonies grow well separated from each other
...
1
...
2
...
B
3
...
4
...
A
5
...
C
6
...
D
7
...
At all times, hold the loop as still as possible
...
Flame neck of the bottle/test tube
...
Replace the cap/cotton wool plug on the bottle/test tube using the little finger
...
A streak plate
10
...
11
...
Hints
12
...
13
...
Turn the dish through 90°
anticlockwise
...
With the cooled loop streak the plate from area ‘A’ across the
surface of the agar in three or four parallel lines (‘B’)
...
15
...
16
...
Turn the dish through 90°
anticlockwise again and streak from ‘B’ across the surface of the
agar in three or four parallel lines (‘C’)
...
Remove the loop and close the Petri dish
...
Flame the loop and allow to cool
...
19
...
Flame the loop
...
Seal and incubate the plate in an inverted position
...
Basic Practical Microbiology – A Manual
Label the half of the dish that contains
medium; use abbreviations and keep them to
the edge of the plate so as not to interfere
with the later observation of colonies
...
Either marker pens or selfadhesive labels are suitable
...
e
...
The
second method is best reserved for older
students working in a relatively dust and
draught-free laboratory; it is the one used
by professional microbiologists
...
Molten, cooled agar medium in a test tube or bottle, is then poured into the Petri dish containing the inoculum
...
Pour plates allow micro-organisms to grow both on the surface and within the medium
...
If the dilution and volume of the inoculum, usually 1 cm3, are known, the viable count of the sample, i
...
the number
of bacteria or clumps of bacteria, per cm3 can be determined
...
[Appendix 6: Preparing serial dilutions]
Inoculation using a Pasteur pipette
1
...
2
...
3
...
4
...
5
...
6
...
Do not squeeze the teat bulb of the pipette after it is in the broth as this could cause bubbles and possibly
aerosols
...
Remove the pipette and flame the neck of the bottle/test tube again, before replacing the cap/cotton wool plug
...
Place bottle/test tube on bench
...
Step 1
Inoculating the Petri dish
1
...
Replace the lid
...
Put the pipette into a discard pot
...
12
© 2006 SGM
Basic Practical Microbiology – A Manual
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Pouring the plate
Step 2
Step 3
Step 4
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
The base of the plate must be covered, agar
must not touch the lid of the plate and the
surface must be smooth with no bubbles
...
Ensure that the temperature of the molten agar is cool enough for
mixing with the culture
...
To avoid contamination ensure:
that the water in the water bath is at the right depth
the bottles are kept an upright position
that the outsides of the bottles are wiped before they are used
Using a spreader
Sterile spreaders are used to distribute inoculum over the surface of already prepared agar plates
...
Dry the surface of agar plates
by either incubating the plates for several hours, e
...
overnight, beforehand or put them in a hot air oven (ca 55–60 °C) for
30–60 minutes with the two halves separated and the inner surfaces directed downwards
...
They can
also be sterilised by flaming with alcohol
...
Dip the lower end of the spreader into a small volume of alcohol
(70 % IDA) contained in a vessel with a lid (either a screw cap or
aluminium foil)
...
Pass quickly through a Bunsen burner flame to ignite the alcohol;
the alcohol will burn and sterilise the glass
...
Remove the spreader from the flame and allow the alcohol to burn off
...
Do not put the spreader down on the bench
...
Keep the alcohol beaker covered and away
from the Bunsen flame
...
This means that they can be used to test the
sensitivity of bacteria to many antimicrobial substances, for example
mouthwashes, garlic, disinfectants and antibiotics
...
If
the dilution and volume of the inoculum, usually 0
...
e
...
The dilutions chosen must be
appropriate to produce between 30 and 100 separate countable colonies
...
Loosen the cap of the bottle/test tube
containing the broth culture
...
Remove a sterile Pasteur pipette from its
container and attach the bulb held in the
right hand
...
Hold a sterile pipette in the right hand
and the bottle/test tube containing the
broth culture in the left
...
Remove the cap/cotton wool plug of the
bottle/test tube with the little finger of
the right hand and flame the neck
...
With the pipette, remove a small amount
of broth
...
Flame the neck of the bottle/test tube and
replace the cap/plug
...
With the left hand, partially lift the lid of
the Petri dish containing the solid nutrient
medium
...
Place a few drops of culture onto the
surface about 0
...
14
© 2006 SGM
Basic Practical Microbiology – A Manual
Basic Pract Book 2006
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9
...
Steps 7–8
Step 10
Step 11
Steps 12–13
10
...
11
...
12
...
13
...
Make sure the entire agar surface
is covered
...
14
...
15
...
16
...
17
...
Hint
The calibrated drop (Miles & Misra) method for colony counts of pure cultures of bacteria and yeast is a more economical
method than pour and spread plates
...
g
...
02 cm3; (2) many drops (six or more) can be put on one plate
...
g
...
Working with moulds
It is sometimes appropriate to prepare a mould inoculum as a spore suspension (particular care is necessary to prevent
them from escaping into the air), but often the inoculum is a portion of the mycelium taken with a loop or straight wire
with the end few millimetres bent at a right angle
...
This can be avoided by placing the Petri dish on the working surface lid down, lifting the base
(containing medium) vertically above the lid and introducing the inoculum upwards onto the centre of the downwardsfacing agar surface with a bent wire
...
Labelling a plate
For guidance on incubation temperatures see Appendix 1: Safety
guidelines
...
Agar plates must be incubated with the medium-containing half (base)
of the Petri dish uppermost otherwise condensation will occur on the
lid and drip onto the culture
...
Taping a plate
The advantages of incubators are that they may be set at a range of
temperatures and reduce the possibility of cultures being interfered
with or accidentally discarded
...
The temperature of an incubator varies from the set temperature,
oscillating by several degrees in the course of use
...
g
...
They should be used with distilled or deionised
water to prevent corrosion and emptied and dried for storage
...
This can occur in an incubator, at room
temperature and even in a refrigerator
...
If cultures have been used
the benches must be swabbed with disinfectant (VirKon; see Choice,
preparation and use of disinfectants page 7)
...
Discard
containers must be carefully and securely packed and never overloaded
...
Pouring disinfectant on the bench
Cultures and contaminated paper towels, gloves, etc
...
Slides, pipettes and Pasteur pipettes must be discarded in the appropriate containers of hypochlorite (sodium chlorate 1) (see Choice,
preparation and use of disinfectants page 7)
...
Swabbing with disinfectant
Never discard sharp or broken items in a way which would endanger
anyone (see Spillage management page 3)
...
Care must be taken that glass is adequately packaged to prevent injury
...
Basic Practical Microbiology – A Manual
Washing hands
© 2006 SGM
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Page 18
Essential methods for
maintaining, preparing and
using cultures
Obtaining suitable cultures
Micro-organisms on the list approved for use in schools and colleges (see Preparation page 2) present minimum
risk given observance of GMLP
...
Ensure that the current version of the list is consulted because recommendations are altered from time to time
with changes in experience and assessment of the risks
...
Isolation of cultures from the environment may be conducted if appropriate to the level of work (i
...
Level 1,
Level 2 or Level 3; see Appendix 1: Safety guidelines)
...
This
skill is crucial for reasons of safety and for maintaining the scientific integrity of an investigation
...
Maintaining stock cultures
It may be convenient to maintain a stock of a pure culture instead of re-purchasing it when needed
...
Be prepared to transfer cultures
four times a year to maintain viability
...
Slope cultures in
screw cap bottles are preferred because the screw cap reduces evaporation and drying out and cannot be accidentally
knocked off (cf
...
Slope cultures are preferred to broth (i
...
liquid medium) cultures because the
first sign of contamination is much more readily noticed on an agar surface
...
Incubate at an appropriate
temperature until there is good growth
...
As soon as there is adequate growth, the cultures may be stored in a refrigerator, but never one in which human
foodstuffs are kept
...
Keep on the lookout for contamination
...
There should be uniformity of colony form and cell form (and consistency with the
appearance of the original culture!)
...
If a culture becomes contaminated, it is not advisable to try to remedy the situation by taking an inoculum from a
single colony from a streak plate of the mixed culture because of the possibility of (1) not being able to distinguish
between the colony forms of the contaminant and the original culture, and (2) culturing a variant of the original
culture that does not behave as the original culture did
...
The necessary movements of air in and
gaseous products out are not prevented and the gaps between the cotton wool fibres are even wide enough for
micro-organisms to pass through
...
The cotton wool must remain dry
because this filtration property is lost if the cotton wool becomes moist – hence the use of non-absorbent cotton wool
...
Aseptic
technique cannot be maintained with poorly made plugs; working surfaces, floors and cultures may become
contaminated and students may become understandably (but avoidably) frustrated and lose interest
...
Making a streak plate is a basic procedure that tests several skills and serves several purposes
...
The choice of loop or pipette for transfers between test tubes and screw cap bottles depends on whether they contain
agar slopes, liquid media or sterile solutions
...
g
...
Preparing cultures for class use
Microbial cultures cannot be taken from a shelf and instantly be ready for use
...
The key is to transfer cultures several times
in advance to ensure that they are growing well and are presented as young, fully active cultures on the day of the
practical class
...
It is usual to grow moulds on the surface of an agar medium, allowing an incubation period of from several
days to a week
...
e
...
It will save time in preparing large numbers of cultures of bacteria and yeast for the class if the inoculum is taken by
Pasteur pipette from a well-growing (i
...
turbid) broth culture
...
Basic Practical Microbiology – A Manual
© 2006 SGM
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Page 21
Part 2: Microbiology in action
Practical activities
1
...
The method involves preparing a pour
or spread plate of a test micro-organism, adding small amount of test
substance to either a well cut in the agar medium or (preferably) a
paper disc which is then placed on the agar surface
...
This is a straightforward activity that tests several practical skills and
is relevant to other aspects of biology and to everyday life
...
g
...
There is also the opportunity to think of less obvious materials, e
...
plants
and their products
...
g
...
Sterile filter paper discs
Sterile distilled/demineralised water (control)
Samples to be tested, 3 (e
...
mouthwashes, selected for a range of active ingredients)
Bunsen burner
Forceps
Alcohol (70 % IDA) in a small beaker covered in foil (Caution: flammable, should be kept covered away from
flames)
Incubator at 25–30 °C (if available)
Procedure
Aseptic technique should be used throughout
...
Mark and label four sections on the base of the Petri dish, for the three different samples and control (sterile water)
...
Using sterile forceps (flamed with alcohol and cooled) remove one filter paper disc
...
3
...
4
...
Remember to rinse and sterilise the forceps between
each sample and to open the plate for the minimum possible time
...
Seal the lid to the base with tape
...
6
...
7
...
Measure and record the size of any zones of inhibition around the filter paper
discs
...
Basic Practical Microbiology – A Manual
© 2006 SGM
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Page 22
2
...
Only
when it is done properly can the smaller end
of the diversity of life be fully appreciated and
its many uses in practical microbiology,
from aiding identification to checking for
contamination, be successfully accomplished
...
[Appendix 3: Safety resources]
Hints
Adjust the iris diaphragm to achieve optimum balance between
definition and glare
...
Re-adjust the iris diaphragm for each objective lens
...
Without
altering the focus, turn to the high power lens and then finely re-focus
...
Put one drop of immersion oil onto the
preparation; a coverslip is not required
...
Bacteria and yeast
Yeast can be seen in unstained wet mounts at magnifications ×100
...
A
magnification of ×1,000 and the use of an oil immersion objective lens for observing stained preparations are necessary
for seeing their characteristic shapes and arrangements
...
Moulds
Routine identification of moulds is based entirely on the appearance of colonies to the naked eye and of the mycelium
and spores in microscopical preparations
...
Routine identification of moulds is based entirely on the
appearance of colonies to the naked eye and of the mycelium and spores in microscopical preparations
...
Take a microscope slide and place 1 or 2
loopfuls of the sample in the centre
...
Seal each edge of the coverslip in turn with a
thin film of Vaseline from the warmed end of
a microscope slide
...
22
© 2006 SGM
Protozoa and algae are large organisms and therefore are readily
visible at a magnification of ×10 to ×100 in unstained wet mounts
...
Identification of algae and protozoa is based entirely on their
microscopical appearance
...
Protozoa are colourless and most are motile
...
For further information contact Sciento and see Appendix 4: Suppliers of
cultures and equipment
...
Aseptic technique must be observed when taking samples of a culture for
making a smear
...
A smear that
is thin and even enables the shape and arrangement of cells to be clearly seen and ensures that the staining procedure
is applied uniformly
...
g
...
g
...
g
...
The reaction of bacteria to Gram’s staining method is a consequence of differences
in the chemical structure of the bacterial cell wall and is a key feature in their identification
...
The basis of Gram’s staining method is the ability or otherwise of a cell stained with crystal violet to retain the colour
when treated with a differentiating agent, usually alcohol (although professionals sometimes use acetone)
...
Those that lose the colour, i
...
Gram-negative, are stained in
the contrasting colour of a counterstain, usually pink/red
...
Clean a plain microscope slide thoroughly using lens tissue
...
Label a microscope slide with a marker pen to record the culture being used, date and initials; this is also a useful
reminder of which side of the slide is being used
...
Flame a wire loop to ensure that no culture accidentally remains from a previous operation
...
Transfer one or two loopfuls of tap water on to the centre of the slide
...
Flame loop and allow to cool
...
Using aseptic technique, transfer a very small part of a single colony from a plate or slope of agar medium into the
tap water
...
Make a suspension of the culture in the tap water on the slide and thoroughly but gently spread it evenly over an
oval area of up to 2 cm length
...
Flame the loop
...
9
...
This is called a heat-fixed smear; it should be visible to the naked eye as a whitish area
...
The smear is now ready to be stained
...
Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray
...
Thoroughly cover the smear with stain and leave for, usually, 30 seconds
...
Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle over the sink
...
Rinse off the stain with tap water
...
Blot-dry the smear with filter/fibre-free blotting paper using firm pressure, but not sideways movements that might
remove the smear
...
Examine under oil immersion
...
When finished, dispose of slides into discard jar
...
e
...
A differential stain: Gram’s staining method
Times of the staining periods depend on the formulation of the staining solutions which are not standard in all
laboratories
...
1
...
2
...
3
...
4
...
5
...
6
...
Iodine solution acts as a ‘mordant’ (a component of a
staining procedure that helps the stain to adhere to the specimen), a crystal violet–iodine complex is formed and
the smear looks black
...
Hold the slide with forceps at a 45° angle over the sink and wash off the iodine solution with 70% IDA (not water);
continue treating with alcohol until the washings are pale violet
...
Rinse immediately with tap water
...
Put the slide back on the staining rack
...
Cover the smear with the counterstain, e
...
aqueous safranin solution, 0
...
11
...
12
...
13
...
14
...
24
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Gram-positive bacteria appear – VIOLET/PURPLE
Gram-negative bacteria appear – RED/PINK
Examples of typical Gram stain results are as follows
...
Micrococcus luteus
Staphylococcus albus
Streptococcus lactis
Yeasts appear violet and red, but this has no taxonomic significance
...
70% IDA
Crystal violet solution:
A Crystal violet
Absolute alcohol
B Ammonium oxalate
Distilled/deionised water
2
...
0 g
100 ml
Take care to make an even smear otherwise alcohol will continue to
wash the violet/purple colour from thick parts of the smear while thin
parts are being over-decolorised
...
5 % aqueous solution
Basic Practical Microbiology – A Manual
The amount of alcohol treatment (the differential stage) must be
judged carefully because over-treatment washes the crystal violet–
iodine complex from Gram-positive bacteria and they will appear to
be Gram-negative
...
0 g
2
...
Don’t despair if the stained smear is not visible to the naked eye; this
may happen with a Gram-negative reaction
...
© 2006 SGM
25
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Page 26
Appendix 1
Safety guidelines
These notes are an updated version of the section on microbiology and biotechnology in Safety in Science Education
(DfEE, 1996) produced as a result of a safety conference of experts (including MISAC representatives) convened by
the ASE in 1997
...
General safety considerations
The nature of the growth, reproductive capacity and biochemistry of many micro-organisms makes them of great
economic, social and medical importance
...
Micro-organisms possess many obliging features that make
them ideal subjects for safe practical exercises in schools
...
Work in microbiology and biotechnology in schools is categorised into three levels which are described in outline below
...
Further detailed guidance for each is provided below
...
The organisms will be observed in the closed containers in which they were grown
...
Once a culture, prepared by pupils,
has been grown, sub-culturing or transfer of organisms from one medium to another is not normally done
...
Level 3 (L3): work where cultures of known fungi and bacteria are regularly sub-cultured or transferred
...
Teachers should
be thoroughly trained and skilled in aseptic technique
...
Non-specialist teachers should not carry out or supervise this work
...
Aerosols can be formed
whenever liquid surfaces are broken or material is crushed or ground
...
Many of the safety measures detailed below are designed to
minimise the risk of aerosol formation
...
However, a major difference is one of scale with a corresponding increase of risk with larger volumes of
micro-organisms
...
Before work with microbes is started, pupils should wash their hands with soap and water (except for L2 & L3 work
investigating microbes on unwashed hands) and cover any cuts with waterproof plasters
...
26
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Microbiology
Source of Hazard(s)
Guidance
Organisms
L1 Limited to algae, yeasts, moulds and bacteria used for culinary purposes, some moulds and
commonly-occurring bacteria where they grow naturally on decaying vegetable material
...
Where possible, organisms with unusual growth
requirements, e
...
high salt, low pH, low temperature, should be chosen but these may not grow well
on standard media
...
Containers of such cultures, once they have been incubated, must then be sealed before examination
...
Organisms may be cultured from the environment
or from body surfaces if the work is appropriate to the course and if cultures are not opened by students
...
Proficiency in aseptic technique and the ability to recognise when a culture has or has not
become contaminated are key skills in minimising risk as well as providing reasonable certainty that
the intended organism is the one that is being studied
...
L2 Agar-based culture media generally with a simple nutrient base, low pH or high salinity, but not those
which select for organisms which are potentially pathogenic to humans, for example, blood agar,
MacConkey’s agar, dung or faecal agar
...
L3 As for L2, unless strict precautions are taken to prevent any release of microbes
...
Such organisms should be subcultured and checked for purity every
3 months or so but only if aseptic technique can be guaranteed
...
Cultures, other than those requiring light for their growth and survival, are best stored in the dark at
10–15 °C
...
Media should be stored as dry powder or tablets
...
Contamination of
teachers, pupils
and students
Before beginning practical work, hands should be washed with soap and warm water, and all should be
washed again after the activities are finished
...
L3 Teachers, technicians and students should wear lab coats or aprons which can be relatively easily disinfected (as necessary) and cleaned
...
Inoculation of cultures
Inoculation should involve precautions to prevent contamination of the person and work surfaces
...
Media and Petri dishes,
etc
...
Media must not be deliberately inoculated with material likely to be sources of human pathogens
...
Arrangements
should be made to sterilise inoculating loops and spreaders before and after inoculation, and to
provide discard pots for pipettes and syringes
...
Lids of Petri dishes
should be opened only just enough to allow the inoculating tool to be introduced and for as little time
as possible
...
Incubation
L1 Incubation should be limited to ambient conditions in the classroom
...
Yeast cultures generate considerable quantities of carbon dioxide gas
...
L2/3 The upper limit for general school-based work should be 30 °C because in this temperature range, (a)
cultures of micro-organisms suitable for school use grow well and (b) although some pathogens can
grow on certain culture media, there is unlikely to be a hazard when conducting investigations using
material derived from suitable environments, e
...
, soil, water and appropriate culture media and
incubation conditions
...
coli for work with DNA
...
During
incubation, the lid of the Petri dish should be taped to the base with two or four small pieces of tape
so that the lid cannot be accidentally removed and conditions inside cannot become anaerobic
...
All spills should be reported to and dealt with by the teacher, who should record all incidents
...
Spills should be covered with towels or a cloth soaked in a suitable freshly-prepared disinfectant, preferably
one that is not appreciably degraded by contact with organic matter or, alternatively, freshly-prepared
sodium chlorate(I) (hypochlorite) solution with a concentration preferably greater than 1 %, and left for at
least 15 minutes
...
Disposable plastic
gloves should be worn
...
Contaminated skin should be carefully washed with soap and hot water
...
Clear phenolics are suitable but are no
longer so readily available to schools in sensible quantities at reasonable prices
...
Spills
To prevent breakages and spills, cultures must be centrifuged in capped, plastic tubes
...
L2 Cultures should be examined by pupils in containers which have been taped closed
...
If it is
necessary for pupils to open cultures for examination, special precautions may be necessary
...
A filter paper is placed in the lid of an
inverted agar plate and moistened with 40 % methanal solution (formalin)
...
(Take care with methanal: eye protection, gloves and use of a fume cupboard to
avoid breathing fumes are necessary)
...
Organisms cultured from body surfaces or any environmental source must be examined in unopened
containers, or killed before examination as described above
...
This is best done using a pressure
cooker or autoclave, in conjunction with autoclavable bags
...
It is very important that instructions for use of the auto
clave are followed in order to achieve and maintain sufficiently high temperatures for a long enough time
...
Such autoclaves can be used for microbiological preparations but advice on their
correct operation should be sought
...
Seals and safety valves should be checked before each use
...
Rapid cooling and the release of steam to lower the internal
pressure quickly to atmospheric pressure is dangerous because it may shatter glassware and/or cause liquid
media to boil over
...
Further information may
be sought from the advisory bodies such as MISAC
...
If, in exceptional circumstances,
chemical disinfection of cultures is contemplated prior to disposal, use a freshly-made solution of a
disinfectant that is not degraded when in contact with organic matter (see ‘Spills’ above)
...
Again it is
essential to follow disinfectant instructions carefully
...
Microwave ovens are not suitable for sterilisation of most items, though they are sometimes used for
liquefying prepared agar media
...
After sterilisation, solid cultures can be disposed of, in tied autoclave bags or similar, through the refuse
system; liquid cultures can be flushed away down the lavatory or the sink with lots of water
...
Incineration is an acceptable alternative to autoclaving
...
)
Clean glass equipment can be sterilised by dry heat in an oven (165 °C for at least 2 hours) or, in the case
of wire loops, by heating to red heat in a Bunsen burner flame
...
Biotechnology
For guidance on practical work with DNA, please consult the NCBE (www
...
reading
...
uk)
...
L1 Particularly suitable organisms include yoghurt bacteria, yeasts such as for the production of wine or
bread and some unicellular algae
...
g
...
Some examples of minimum risk organisms include
Vibrio natriegens (Beneckea natriegens), Photobacterium phosphoreum and Acetobacter aceti
...
Cultures of organisms should only be obtained from recognised suppliers, including culture
collections
...
Culture media
The solutions generally used in biotechnology work present few problems other than those associated with
quantity and the potential for contamination
...
which may be generated
...
The use of animal dung
for investigations of biogas generation is not recommended; use grass clippings inoculated with wellrotted garden compost
...
However, for yoghurt making, 43 °C may be used if hygienic preparation is followed and
for work with DNA using K12 strains of E
...
L2/3 Use of fermenters is limited to these levels
...
Vessels must be suitably vented to allow the gas to escape but prevent aerosol
formation or the entry of unwanted organisms
...
Other than for work with yeasts and small-scale biogas generation using plant material, wholly
anaerobic fermentations should not be used in schools
...
g
...
Contamination
Cultures should be started by inoculation with a significant volume of actively-growing inoculum (for
example 20 % of total volume)
...
Spills
Routines for dealing with spills are the same as for microbiology
...
All possible steps should be taken to guard against this, for example,
by using equipment within a spills tray
...
Electrical hazards
Keep all electrical leads, especially mains leads, tidy and site electrical equipment so as to minimise the
risk of water entering
...
If a fermenter cannot be
sterilised complete, add a freshly-prepared disinfectant that is not appreciably degraded by contact with
organic matter to the culture and leave for sufficient time to enable disinfection to occur before pouring
the contents into containers which can be autoclaved
...
Problems
with enzymes increase with quantity as well as variety
...
Avoid the
release of powders into the air
...
Various organisations
were represented, including the ASE, CLEAPSS, SSERC, HSE, MISAC (Microbiology in Schools Advisory Committee),
Society for Applied Microbiology, Society for General Microbiology, NCBE (National Centre for Biotechnology
Education), SAPS (Science & Plants in Schools), the Wellcome Trust and the educational suppliers Philip Harris
and Blades Biological
...
One of the outcomes of the
conference is a revision of this list
...
These tables
supersede the existing lists found in the CLEAPSS Laboratory Handbook (1992), the CLEAPSS Shorter Laboratory
Handbook (2000), Microbiology: An HMI Guide for Schools and Further Education (1990, now out of print), Topics in
Safety (1988) and Safety in Science Education (1996)
...
The
lists of micro-organisms are not definitive; other organisms may be used if competent advice is obtained
...
Where possible, fungi that produce large numbers of air-borne spores should be handled before sporulation
occurs, so that the spread of spores into the air and possible risks of allergy or the triggering of asthmatic attacks are
minimised
...
It should be noted that certain species of these two fungi, previously listed as
unsuitable for use in schools, are now not thought to present such a serious risk to health, given good practice in
culture and handling
...
]
1
Categorisation of Biological Agents According to Hazard and Categories of Containment, 4th edition, 1995, Advisory
Committee on Dangerous Pathogens, HSE Books, ISBN 0717610381
...
Oxidises ethanol to ethanoic (acetic) subculturing to maintain viability
...
Agrobacterium
tumefaciens
Causes crown galls in plants; used as a DNA vector
in the genetic modification of organisms
...
Alcaligenes eutrophus
In the absence of nitrogen, it produces intracellular
granules of poly-β-hydroxybutyrate (PHB); was
used in the production of biodegradable plastics
...
Azotobacter vinelandii
A free-living nitrogen fixer, producing a
fluorescent, water-soluble pigment when grown in
iron (Fe)-limited conditions
...
Bacillus megaterium
Has very large cells; produces lipase, protease and
also PHB (see Alcaligenes); Gram-positive staining
...
Bacillus
stearothermophilus
Thermophilic species which grows at 65 °C;
produces lipase and protease
...
Grows on nutrient agar
...
Produces amylase, lipase and protease
...
Cellulomonas sp
...
Grows on nutrient agar but also used with agar
containing carboxymethylcellulose
...
A photosynthetic, anaerobic bacterium
...
Erwinia carotovora
(=E
...
Useful for studies of Koch’s
postulates
...
Escherichia coli*
K12 strain: general-purpose, Gram-negative bacterium
...
B strain: susceptible to T4 bacteriophage
...
Grows best at 20 °C
...
Lactobacillus sp
...
bulgaricus is used in the production of yoghurt
...
Leuconostoc
mesenteroides
Converts sucrose to dextran: used as a blood plasma Requires special medium as for Lactobacillus
...
Methylophilus
methylotrophus
Requires methanol as energy source; was used for
the production of ‘Pruteen’ single-cell protein
...
Micrococcus luteus
(=Sarcina lutea)
Produces yellow colonies; useful in the isolation of
the bacterium from impure cultures
...
General-purpose, Gram-positive bacterium
...
Photobacterium
phosphoreum
Actively-growing, aerated cultures show
bioluminescence; grows in saline conditions
...
Pseudomonas fluorescens Produces a fluorescent pigment in the medium
...
Rhizobium
leguminosarum
A symbiotic, nitrogen fixer; stimulates the
formation of nodules on the roots of legumes
...
Grows on yeast malt agar; some authorities
recommend buffering with chalk to maintain
viability
...
Also
grows aerobically in the dark
...
32
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Bacterium
Educational use/interest/suitability
Ease of use/maintenance
Spirillum serpens
Of morphological interest
...
Staphylococcus albus
(epidermidis)‡
A general-purpose, Gram-positive bacterium,
producing white colonies
...
Streptococcus
(=Enterococcus) faecalis
Of morphological interest, forming pairs or chains
of cocci
...
Streptococcus
(=Lactococcus) lactis
Of morphological interest, forming pairs or chains
of cocci
...
Can grow on nutrient agar with added glucose;
some authorities recommend buffering with chalk
to maintain viability
...
Grows at 50 °C
...
Streptomyces griseus
Responsible for the earthy odour of soil
...
Produces streptomycin
...
Thiobacillus ferrooxidans Involved in the bacterial leaching of sulphurcontaining coal
...
Demonstrates bacterial leaching of coal samples
containing pyritic sulphur
...
Vibrio natriegens§
(=Beneckea natriegens)
Requires medium containing sodium chloride
...
Prone,
however, to thermal shock with a sudden drop in
temperature
...
Reputable suppliers should ensure that safe strains are provided
...
‡This organism has been known to infect debilitated individuals and those taking immunosuppressive drugs
...
§A well-known supplier currently lists an unspecified species of Vibrio because of its morphological interest
...
natriegens
...
This
bacterium should only be used in establishments that have containment facilities suitable for work with Hazard Group 2 microorganisms
...
Grows on compost containing well-rotten horse
manure; available as growing ‘kits’
...
Produces rhizomorphs
...
Some authorities
recommend carrot agar
...
Produces
abundant, easily-dispersed spores – may become a
major laboratory contaminant!
Grows on Czapek Dox yeast agar
...
Aspergillus niger*
Useful for studies of the influence of magnesium
on growth and the development of spore colour
...
Produces abundant, easily-dispersed spores - may
become a major laboratory contaminant!
Requires special sporulation medium for
investigations
...
Also produces protease
...
Grows on malt agar; add starch (or protein) for
investigations
...
Useful for studies of Koch’s postulates with fruit,
vegetables and Pelargonium spp
...
Used in ELISA protocols
...
Botrytis fabae
Causes disease in bean plants
...
Candida utilis
Simulates behaviour of pathogenic Candida spp
...
Grows on malt agar or glucose nutrient agar
...
Can be grown on V8 medium but survives well
just on double thickness wall paper, coated with a
flour paste
...
Grows on horse dung
...
Requires special medium
...
Can be grown on V8 medium
...
Produces sickle-cellshaped spores, a red pigment and pectinase
...
Fusarium solani
Digests cellulose; macroconidia have a sickle shape
...
Helminthosporium avenae A pathogen of oats
...
Kluyveromyces lactis
A yeast, isolated from cheese and dairy products
...
Ferments lactose and used to convert dairy products
to lactose-free forms
...
Leptosphaeria maculans
For studies of disease in Brassica plants
...
Monilinia (=Sclerotinia)
fructigena
For studies of brown rot in apples
...
Grows on malt agar or potato dextrose agar
...
Grows on malt agar
...
heterothallic + and – strains and zygospore production
...
For sporangia
(asexual), mating types and amylase production
...
Myrothecium verucaria
For studies of cellulose decomposition, but
Chaetomium globosum is preferred
...
Neurospora crassa*
Red bread mould
...
Can be used in studies of genetics
...
Penicillium chrysogenum* Produces penicillin; useful for comparative growth Grows on malt agar, though some authorities
inhibition studies in liquid media or when inoculated indicate that it thrives better on liquid media
...
Produces yellow pigment
...
Grows on malt agar
...
Penicillium notatum*
Produces penicillin; useful for comparative growth Grows on malt agar
...
34
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Fungus
Educational use/interest/suitability
Ease of use/maintenance
Penicillium roqueforti*
Does not produce penicillin; the familiar mould of
blue-veined cheese
...
Penicillium wortmanii*
Produce wortmin rather than penicillin
...
Phaffia rhodozyma
A fermenting red yeast
...
Grows on yeast malt agar
...
Grows on malt agar
...
Thought to produce pectinase
...
Phytophthora infestans†
Causes potato blight
...
Can be grown on V8 medium
...
Useful for studies of Koch’s postulates
...
Pleurotus ostreatus
Edible oyster cap mushroom
...
Grows on cornmeal agar
...
Grows on potato dextrose agar, Czapek Dox yeast
agar and other fungal media
...
Useful for studies Grows on potato dextrose agar and other fungal
of the linear growth of fungi
...
Rhizopus stolonifer
Produces rhizoids
...
Grows on potato dextrose agar, potato carrot agar,
Czapek Dox yeast agar and other fungal media
...
On sycamore leaves, it forms ‘tar’
spot lesions, the number or diameter of which can
be compared at different sites
...
Saccharomyces
cerevisiae
Valuable for work in baking and brewing, showing Grows on malt agar or glucose nutrient agar
...
Saccharomyces diastaticus Able to grow on starch by producing glucoamylase
...
Saccharomyces
ellipsoideus
Used in fermentations to produce wine; can tolerate Grows on malt agar
...
Saprolegnia litoralis†
Parasitic on animals
...
Good
illustration of asexual and sexual stages
...
Good for
Grows on malt agar
...
for cell counts
...
Sordaria brevicollis
For studies of fungal genetics, including inheritance Requires special medium for crosses between
of spore colour and crossing over in meiosis
...
Sordaria fimicola
For studies of fungal genetics, including inheritance Grows on cornmeal, malt and other agars but may
of spore colour and crossing over in meiosis
...
White-spore strain may not always grow normally
on standard cornmeal agar
...
Found on leaf surfaces
...
Grows on malt, yeast malt and glucose nutrient
agar but laboratory cultures may not be needed
...
Grows on malt agar
...
*Possible risk of allergy/asthma if large numbers of spores are inhaled
...
Basic Practical Microbiology – A Manual
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Page 36
Viruses
These are rarely used in schools and colleges but a selected list of those which might be considered is given below
...
coli)
Cucumber Mosaic Virus
Potato Virus X
Potato Virus Y (not the virulent strain)
Tobacco Mosaic Virus
Turnip Mosaic Virus
Algae, protozoa (including slime moulds) and lichens
Though some protozoa are known to be pathogenic, the species quoted for experimental work in recent science projects
and those obtained from schools’ suppliers or derived from hay infusions, together with species of algae and lichens,
are acceptable for use in schools
...
Some fungi previously considered unsuitable have been reinstated in the list of selected
organisms now that it is thought that they do not present a major risk, given good practice
...
Publications
This book is recommended as containing the most up-to-date and detailed information on the safe use of micro-organisms in
schools:
Topics in Safety, ASE*, 3rd ed
...
, £25
...
Chapter 15 – Microbiology & biotechnology – contains detailed information, includes risks associated with each procedure;
levels of work, list of suitable and unsuitable micro-organisms, steam sterilisation, sub-culturing and transfer work at level 3
...
Borrows et al
...
(Scottish ed
...
, £6
...
General safety guidance
...
Microbiological Techniques CD1: An Interactive Manual, SSERC, 2006
...
Safeguards in the School Laboratory, ASE*, 11th ed
...
, £18
...
Health & Safety legislation, managing safety, GLP, use of pressure vessels, section on biological hazards includes microbiology/
biotechnology
...
, £14
...
Includes legislation, safety management in the science department, risk assessments, emergency procedures, first aid, resources,
laboratory design, GLP, use of chemicals, use of electricity
...
4 on microbiology is inaccurate and has been
superseded by Chapter 15 in the new edition (2001) of Topics in Safety (see above)
...
ed
...
, n
...
– Ideas for investigations
...
Tools, Techniques and Assessment in Biology, John Adds et al
...
, £14
...
There is an excellent chapter
on microscopy and observation
...
orders: 0171 873 9090
Nelson, www
...
co
...
ac
...
microbiologyonline
...
uk/misac)
CLEAPSS, Brunel University, Uxbridge UB8 3PH (Tel
...
cleapss
...
uk) – available only to members and
associates of CLEAPSS
SSERC, 2 Pitreavie Court, South Pitreavie Business Park, Dunfermline KY11 8UB (Tel
...
sserc
...
uk)
Basic Practical Microbiology – A Manual
© 2006 SGM
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Page 38
Appendix 4
Suppliers of cultures and equipment to
schools and colleges
The following companies supply cultures and/or equipment for practical microbiology in schools and colleges
...
Before purchasing cultures, please refer to Appendix 2: Safe micro-organisms to
ensure that the organism is suitable for use in schools
...
01709 377881
Fungi and bacteria; culture
Rotherham
Fax 01709 369264
media and antibiotic discs
South Yorkshire S60 1RR
Email sales@beecroft-science
...
uk
Ltd, Northern Branch
www
...
co
...
01908 221860
Fungi and bacteria; culture
Bradwell Abbey
Fax 01908 313269
media and antibiotic discs
Milton Keynes MK13 9HA
Email sales@beecroft-science
...
uk
www
...
co
...
01342 850242
Algae, protozoa, fungi and
Edenbridge
Fax 01342 850924
bacteria; culture media and
Email info@blades-bio
...
uk
antibiotic discs
Kent TN8 7DX
www
...
co
...
0118 987 3743
Limited range of cultures of
for Biotechnology
Science & Technology Centre
Fax 0118 975 0140
bacteria and fungi; items for
Earley Gate, PO Box 247
Email ncbe@reading
...
uk
microbiology and molecular
Reading RG6 6BZ
www
...
reading
...
uk
biology
Philip Harris
Hyde Buildings
Tel
...
co
...
philipharris
...
uk
Education (NCBE)
Sciento
61 Bury Old Road
Tel/Fax
...
co
...
01270 250459
Full range of cultures, media
Suppliers Ltd
Marshfield Bank
Fax 01270 250 601
and equipment for practical
Crewe
Email sales@timstar
...
uk
microbiology
Cheshire CW2 8UY
www
...
co
...
These centres are members of the United Kingdom Federation of Culture Collections (UKFCC; www
...
org)
...
Pressure cookers are less expensive, are lighter, have a shorter sterilisation cycle time and
are easier to maintain than autoclaves
...
Pressure cookers with weights to control steam pressure enable the pressure to be varied
...
Usual conditions are not less than 15 minutes exposure to pure steam at a pressure of
103 kPa (kN m2) or 15 lbf in–2, producing a temperature of 121 °C
...
Loading
Do not overload otherwise there will not be sufficient space for steam to circulate
...
Fill vessels to no more than 2/3 capacity and loosen caps of screw-capped vessels to allow for expansion
...
Steam generation
Ensure that there is more than enough water to generate steam throughout the sterilisation cycle and to prevent
boiling dry
...
Use either distilled or deionised water to prevent corrosion
...
Begin timing the holding time when air has been completely expelled and the heat has penetrated throughout the
materials to be sterilised
...
Unloading
After completion of the holding time, allow the autoclave/pressure cooker to return atmospheric pressure and
temperature before starting the opening procedure
...
After care
Clean out, empty and thoroughly dry the autoclave/pressure cooker to prevent corrosion
...
Contact CLEAPSS for further detailed guidance on using autoclaves and pressure cookers (Tel
...
org
...
cleapss
...
uk)
...
Serial dilution involves taking a sample and diluting it through a series of standard volumes of sterile diluent, e
...
distilled water or 0
...
Then a small measured volume of each dilution is used to make a series of pour or spread
plates
...
Materials
Culture of bacteria or yeast or sample of natural material
6 sterile test tubes containing 9 cm3 sterile diluent, fitted with a cap or cotton wool plug, labelled 1→6 and with the
dilution factor as shown in the diagram
Serial ‘tenfold’ dilution
12 sterile, plugged Pasteur pipettes
1 cm3 syringe barrel fitted with rubber
tubing (see page 9)
Mix and
transfer
1 cm3
Transfer
1 cm3
Transfer
1 cm3
Mix and
transfer
1 cm3
Mix and
transfer
1 cm3
Mix and
transfer
1 cm3
and
so
on
Pot of Virkon disinfectant
Procedure
1
...
1
2
...
2
3
4
5
6
10–5
100,000
10–6
1,000,000
3
...
4
...
The
volume of this tube is now 10 cm3
...
Tubes containing 9 cm3 of
sterile diluent
Culture/sample
(well mixed)
Concentration
Dilution factor
100
0
10–1
10
10–2
100
10–3
1,000
10–4
10,000
5
...
6
...
7
...
8
...
This gives a 10–2 dilution
...
Discard the pipette in disinfectant
...
Repeat this for the remaining tubes, removing 1cm3 from the previous dilution and adding it to the next 9 cm3 of
diluent
...
Plating and counting procedure
Use a known volume of each dilution to make either pour plates (page 12) or spread plates (page 14)
...
For statistical purposes, replicate plates should be prepared
...
Choose the plate that has an easily countable number (about 30–100) and carefully count every colony
...
Then calculate the number of micro-organisms in the sample:
Number of microbes/cm3 = number of colonies × dilution of sample
40
© 2006 SGM
Basic Practical Microbiology – A Manual
Basic Pract Book 2006
2/11/06
11:17 am
Page 41
Basic Pract Book 2006
2/11/06
11:17 am
Page 42
Basic Pract Cover 2009
16/1/09
12:01
Page 4
About this resource
Microbiology is a popular option for practical work in schools
...
It forms the notes for the one-day basic practical microbiology
training course which is run and accredited by the Society for General Microbiology (SGM), but can
be used as a standalone document
...
The SGM, in association with the Microbiology in Schools Advisory Committee (MISAC) has also
produced a book of 21 practical investigations which complements the manual: Practical
Microbiology for Secondary Schools is suitable for use with Key Stages 3, 4 and post-16 and the
equivalent Scottish qualifications
...
The SGM offers schools membership and runs an advice service on microbiology teaching
...
ac
...
Much information can be found on the
website: www
...
org
...
cleapss
...
uk
Brunel University, Uxbridge UB8 3PH
Microbiology in Schools Advisory Committee (MISAC) – www
...
org
...
ncbe
...
ac
...
sserc
...
uk
2 Pitreavie Court, South Pitreavie Business Park, Dunfermline KY11 8UB
Credits & Acknowledgements
The SGM gratefully acknowledges the support from the following sources:
Kath Crawford (SAPS Scotland)
John Richardson (SSERC)
Members of MISAC
John Schollar (NCBE)
John Tranter (CLEAPSS)
Editors: Dariel Burdass, John Grainger & Janet Hurst
Design and Production Editor: Ian Atherton
Photographs: Dariel Burdass & Faye Jones
Front cover illustration: TEK Image / Science Photo Library
ISBN 0 95368 383 4
Copyright
Basic Practical Microbiology – A Manual is copyright
...
Educational use: Electronic or paper copies of the resource or individual pages from it may
be made for classroom and bona fide educational uses, provided that the copies are distributed
free of charge or at the cost of reproduction and that the SGM is credited and identified as
copyright holder