LABORATORY PROCEDURES FOR MICROORGANISMS
Appendix
M/1998/2.03 Appendix 1
Preparation and Storage of Culture Media
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Description
Precautions - dehydrated media
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Catalogues Humidity
Temperature and time
Browse Preparation of dehydrated media
catalogues Reconstitution of dehydrated media
Sterilization of culture media
Collections Sterilization checks
Overheating effects
Table of faults and possible causes in media sterilization
Prices
Preparation of sterilized media
Guidelines
CELL LINES Storage of prepared media
PLANT CELL LINES
PLANT VIRUSES
PLASMIDS Precautions in the use and disposal of prepared media
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MICROORGANISMS Precautions - dehydrated media
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PROCEDURES User-laboratory quality control tests on prepared media
CATALOGUE
Search Web Culture media must be stored at the specified temperature, under specified conditions and not longer
Site than the shelf-life periods appropriate to each product. The storage conditions and expiry date of each
product are shown on the labels or product inserts but the following general rules will help to ensure
Contacts that they are kept in an optimum environment. When storing products note the shelf life expiry dates on
the labels and use the products in order of their lot/batch numbers.
FAQ Light
Site Map All prepared culture media and their components should be stored away from light and exposure to
direct sunlight should be avoided at all times.
Humidity
Sealed glass and plastic containers are unaffected by normal laboratory humidity. Opened containers
of dehydrated powders will be affected by high humidity. Hot, steamy media preparation rooms are not
suitable environments to store containers of culture media; particularly containers which are frequently
opened and closed. An adjacent cold room or an adequate storage cupboard are preferable storage
areas.
Temperature and time
The temperature storage conditions of culture media and their components vary widely. The following
product groupings will help to differentiate the various requirements.
Culture Media: Sealed, unopened containers should be stored at room temperature 15-20°C. Opened
containers should have the cap or lid carefully and securely replaced. It is important that opened
containers are stored in a dry atmosphere at room temperature. Shelf life 1 to 5 years.
Prepared Broth Media: Store at 2-8°C. Do not allow the products to freeze. Shelf life 6 months to 2
years.
Prepared Plates of Culture Media: Poured plates of agar media are especially vulnerable to infection,
dehydration and chemical degradation. Aseptic preparation and storage are essential to protect plates
from microbial infection. Water losses on storage can be minimised by impermeable wrapping and/or
storage at 2-8°C. Chemical degradation e.g. oxidation or antimicrobial loss, can be retarded by
protection from light, heat and dehydration.
It is important, however, to monitor the storage of prepared plates by quality control tests so that any
deterioration can be detected and the storage period accurately determined. Simple weighing tests of
fresh and stored plates will determine the rate of moisture loss. Weight loss greater than 5% will
indicate a significant loss of water.
Gas Generating Kits: Store at 2-8°C in a dry place. Do not store these kits at a higher temperature for
long periods. Shelf life 3 years.
Sterile Reagents: Store at 2-8°C, except Horse Serum store at -20 to +8°C.
Susceptibility Discs: Store at -20°C but keep working stock at 2-8°C. Shelf life 1 to 2 years.
Preparation of dehydrated media
Dehydrated media are hygroscopic and are sensitive to moisture, heat and light. They are adversely
affected by drastic changes in temperature e.g. hot/cold cycling temperatures which may occur
between day and night laboratory temperatures in winter.
Storage conditions are usually indicated on the product label and should be followed.
1 Write on the label the date of receipt in the laboratory.
2 Store as indicated on the label; usually below 25°C in a dry area, away from direct sunlight,
autoclaves, drying ovens or other heat sources.
3 Check expiry date on the label, some media have significantly shorter shelf-lives than others.
4 Use stock in lot/batch number order. Do not open a new bottle until the previous bottle has
been emptied. Note on the label the date the container is first opened. After use, make sure the
container is tightly closed and return it to the designated storage area.
5 Order the medium in an appropriate size of container and in a quantity which accords to
normal use requirements. A medium in a large container which has been opened many times
will deteriorate on storage. Discard the medium if the powder is not free flowing, if the colour
has changed or if it appears abnormal in any way.
Reconstitution of dehydrated media
Complete instructions for the preparation of culture media are given on the label of each bottle. As a
general rule it is wise to prepare one week's requirement only.
1 Always use freshly prepared distilled or deionised water. Use warm (50°C) water to hasten the
solution of the medium. Rinse all glassware with the distilled/deionised water and make sure
that the vessels are clean and free from toxic chemicals which may be absorbed on to the
surface of the glass e.g. bile salts, tellurite, selenite etc.
2 Prepare the medium in a vessel about twice the final volume of the medium to allow adequate
mixing. Follow the instructions given on the label of each product.
3 Open the culture medium container away from draughts and moisture. Avoid inhaling the
powder and prolonged skin contact. Weigh the powder quickly, accurately and without creating
'clouds of dust'. Reclose the container as soon as possible. Pour half the required volume of
distilled water in the vessel, then the weighed quantity of medium and agitate briskly for a few
minutes. Pour the rest of the distilled water down the sides of the vessel to wash any adherent
medium back into solution. This is an important step because dry culture media powder above
the level of the water may not be sterilized in the autoclave and may be a source of
contamination.
Agar-free media will usually dissolve on gentle agitation. Media containing agar should be heated to
dissolve the agar before autoclaving. Bring the medium to the boil without scorching or burning. Most
culture media will require final sterilization in an autoclave at 121°C for 20 minutes.
The pH of the dehydrated medium has been adjusted by the manufacturer so that the final pH of the
prepared medium conforms with the label specification when the medium has been cooled to 25°C. Do
not adjust the pH of dehydrated media prior to sterilization.
Sterilization of culture media
Although sterilization of culture media is best carried out in a steam autoclave at temperatures between
121-134°C it has to be recognised that damage is caused to the medium by the heating process.
Heat-treatment of complex culture media which contain peptides, sugars, minerals and metals results
in nutrient destruction, either by direct thermal degradation or by reaction between the medium
components.
Toxic products caused by chemooxidation can also be formed during heat-treatment. It is important,
therefore, to optimise the heating process so that a medium is sterile after heating but minimal damage
is caused to the ingredients of the medium. As a general rule it is accepted that short-duration, high-
temperature processes are more lethal to organisms and less chemically damaging than are longer,
lower temperature processes e.g. 3 minutes at 134°C is preferable to 20 minutes at 115°C.
A general instruction for sterilizing culture media in volumes up to one litre at 121°C for 20 minutes is
given on each label. Autoclaves vary in performance, however, and thermocouple tests using different
volumes of media should be carried out to determine the 'heat-up and 'cool-down' times. It will be
essential to do this when volumes of media greater than two litres are prepared. In order to avoid
overheating large volume units of media, the 'heat-up’ and 'cool-down' periods are normally integrated
into the 121°C holding time.
Sterilization Cycle
The sterilization cycle can be divided into its four stages:
Stage 1 20°-121°C Chamber heat-up time
The chamber heat-up time depends on the efficiency of the autoclave (air discharge/steam input) and
the size of the load in the chamber. The time required for this stage is measured with a recording probe
located in the air-discharge valve located in the base of the chamber.
Stage 2 <100°-121°C Heat penetration time of the medium container
The heat penetration time depends mainly on the volume of the individual containers, although the
shape and the heat-transfer properties of the containers may affect this stage. The time required for
the medium volume to reach 121°C is measured with thermocouples placed in the centre of the
innermost container.
Volume (ml) in glass bottles Time (min)
100 ml 19 min
500 ml 18 min
1000ml 22 min
2000 ml 27 min
5000 ml 37 min
These times assume that agar media have been dissolved before autoclaving. It is also assumed that
maximum exposure to steam is possible. Thus although the single l00 ml bottle required 12 minutes to
reach 121°C, when placed in a crate with other bottles it required 19 minutes and when placed in the
centre of stacked crates it required 30 minutes.
Stage 3 121°-121°C Holding time at the prescribed temperature
The holding time at 121°C depends on (i) the number of organisms originally present in the medium (ii)
the fractional number of an organism presumed present after heating e.g. N = 0.001 equivalent to one
bottle in every 1000 bottles heated becoming contaminated (iii) the thermal death rate constant of the
presumed organism present at 121°C.
The recommended holding times are:
Temperature (°C) 121 126 134
Time (minutes) 20 10 3
Stage 4 121°- 80°C Cool-down time for the chamber to reach 80°C
The cool-down time depends on the size of the load in the chamber and the heat loss rate from the
autoclave. Water-sprays are used to accelerate cooling in commercial sterilizers but very careful
control is required to avoid bottle fracture and the ingress of the cooling spray into the sterilized
medium. The latter problem occurs when the vacuum formed in the head-space during cooling sucks
contaminated cooling fluid up the thread of the cap and into the bottle.
Culture media autoclaves should be unlagged and of moderate chamber capacity only. Thermal locks
on the doors should prevent them opening when the chamber temperature is above 8O°C but even in
these circumstances care should be taken to avoid sudden thermal shock when removing glass bottles
of hot liquid from the autoclave. When screw-capped containers are placed in an autoclave the caps
should be a half-turn free to allow the escape of heated air. When removed from the autoclave the
containers should be allowed to cool down in a laminar airflow cabinet. Alternatively screw-capped
containers may be sterilized in a jar which is covered by a piece of felt which effectively protects the
containers from infection by air-borne microorganisms. Caps are screwed down tightly after the
contents have cooled to ambient temperature.
Sterilization checks
All autoclaves should be checked at fixed periods of time to ensure that they are functioning efficiently.
Physical measurements should be made on temperature and pressure readings, the quality of the
steam should be checked, the efficiency of the 'near-to-steam' air traps in the base of the autoclave
should be determined and the safety valves checked. Mandatory inspections of autoclaves as pressure
vessels are normally carried out annually by specialists under instructions from insurers of such
apparatus. With small laboratory autoclaves this inspection is not mandatory.
Chemical indicators will show the temperature reached or exceeded and some will indicate the time
held at the specified temperature. Under-autoclaving is usually self-evident because failure to destroy
all the bacterial spores naturally present in dehydrated media (the 'bioburden') will allow growth to take
place in the stored or incubated medium. Failure of sterilization should always be suspected when
contamination of prepared media occurs with sporing organisms. Biological indicators of sterilization
will demonstrate the ability of the autoclave to destroy bacterial spores.
Overheating effects
Overheating is a common cause of pH drift, darkening, precipitation, poor gel strength and reduced
bacteriological performance. These effects can also be produced if a concentrated 'pool' of ingredients
at the bottom of the container is heated. All culture media should be in solution before
sterilization. This will reduce the occurrence of Maillard-type reactions (non-enzymatic browning)
taking place in the medium.
Overheating effects will occur if agar media are allowed to gel in bottles and are later steamed to melt
the agar. They will also occur if molten media are held at 50°C for more than 3 hours before use. Agar
media with pH values at or below 5.0 are very sensitive to overheating in any form because the agar is
hydrolysed and the gel strength fails. It is recommended to sterilize the agar of media of a pH lower
than 5.0 separately.
Most of the difficulties in culture media sterilization occur when large unit volumes of media (>2 litres)
must be processed. The best solution to this problem is the use of a culture medium preparator. These
semi-automatic processors, made by New Brunswick and other manufacturers overcome the problem
of poor heat penetration of agar by a continuous stirring or agitation of the medium during the heating
phase. Such preparators will significantly reduce the time required for sterilization at 121°C or in some
models at 134°C. They are strongly recommended because of their high efficiency and minimal
damage to culture media.
Table of faults and possible causes in media sterilization
Fault
Wrong pH value
Possible Causes
pH test carried out above 25°C. Overheating through prolonged sterilization, remelting or overlong
period at 50°C. Incomplete solution of medium. Poor quality water or containers. Dehydrated medium
stored incorrectly or beyond the stated shelf-life.
Fault
Turbidity, precipitation.
Possible Causes
Poor quality water or containers. Overheating or prolonged storage at 50°C. pH value incorrect.
Incomplete solution.
Fault
Darkening.
Possible Causes
Overheating, incomplete solution or pH drift. Presence of phosphate in addition of glucose or other
sugars and agar.
Fault
Soft gel.
Possible Causes
Agar not in solution, poor mixing, prolonged storage at 50°C. Overheating at low pH values. Error in
weighing or overdilution with inoculum or media supplements. pH too low for agar.
Fault
Poor bacterial growth.
Possible Causes
Prolonged and excessive heating, incomplete solution. Inhibitory substances in water or containers.
Darkening and pH drift.
Preparation of sterilized media
Liquid media which are sterilized in their final containers should be cooled down to room temperature
as rapidly as possible. Screw caps should then be tightened.
Containers of agar media which have been sterilized should be placed in a 50°C water bath and the
medium dispensed as soon as it reaches this temperature, or within a maximum of 3 hours in the bath.
The medium should be mixed thoroughly, without bubble formation and aseptically dispensed into
sterile containers. Do not expose dishes of agar media to sunlight; it causes excessive condensation
on the lids and may cause the formation of inhibitory substances by photo-oxidation.
Heat-labile supplements should be added to the medium after it has cooled to 50°C. Allow the sterile
supplement to come to room temperature before adding it to the agar medium. Very cold liquids may
cause agar to gel or form transparent flakes which can easily be seen e.g. in blood enriched agar. Mix
all supplements into the medium gently and thoroughly, then distribute into the final containers as
quickly as possible.
Blood used for the preparation of blood agar should be as fresh as possible and should have been
stored at 2-8°C (blood must not be frozen). Warm the blood in a 35°C incubator before addition to
sterile molten agar base, which has been cooled to 40-45°C. Adequate mixing in a large head-space
vessel is essential to ensure aeration of the blood. Poorly oxygenated blood plates are purplish in
colour whereas properly aerated blood agar is cherry-red. Defibrinated blood is recommended for use
rather than blood containing an anticoagulant.
Storage of prepared media
The recommended shelf-life of prepared culture media varies considerably. Screw-capped bottles of
nutrient broth and agar can be stored for 6 months at low ambient temperatures (12-l6°C). It is
important to store all media away from light. Agar plates should be stored at 2-8°C in sealed containers
to avoid loss of moisture. DO NOT FREEZE.
Fresh media are better than stored media therefore avoid long storage times. Some very labile beta-
lactam selective agents have very short active lives and media containing such substances should be
used within a few days of preparation.
It is good laboratory practice to establish shelf-lives for all prepared media and date-stamp the
containers or holders accordingly.
Loss of moisture from agar plates is a common cause of poor bacteriological performance. Do not
preincubate all plates overnight as a sterility check. Only obviously wet plates require pre-inoculation
drying.
Ensure that all plates are incubated in a humid environment.
Examine prepared media before inoculation. Look for evidence of contamination, uneven filling or
bubbles on surface of agar, colour changes, haemolysis and signs of dehydration such as shrinking,
cracking and loss of volume. Discard any defective plates or tubes.
Precautions in the use and disposal of prepared media
It should be recognized that inoculation of culture media with bacteria, deliberately or accidentally,
leads to very great numbers of organisms being produced. High concentrations of any organisms are
potentially hazardous and must be disposed of safely by approved methods.
All infected specimens and inoculated culture media should be handled only by qualified personnel
who have been trained in microbiological procedures. Such staff should ensure that all specimens and
cultures under their care are properly handled and finally autoclaved before disposal. Any apparatus
used and contaminated must be safely disinfected or sterilized; this is particularly important when such
apparatus must be serviced or passed out of the laboratory.
The environment in which microbiological cultures are handled must also be taken into account. Most
countries have categories of organisms which are divided into those which may be handled in the
general microbiological laboratory, those which require special laboratory conditions and for the most
dangerous organisms a totally contained and highly protected environment is required. It may be a
criminal offence not to observe these rules and regulations. When using culture media always label or
identify the container with the specimen details before inoculation.
Inoculate the medium using aseptic techniques and incubate under the appropriate conditions.
Examine the medium after incubation for evidence of microbial growth and carry out the appropriate
isolation and identification procedures.
Precautions - dehydrated media
Most of the products supplied have no known risks except those usually associated with fine powders.
However to prevent the risk of inhaling fine dust it is recommended that masks should be worn whilst
handling dehydrated media. The mask chosen should perform to the level of British Standard No.
6016. The type of mask manufactured by 3M Corporation would be suitable for this purpose.
Hazard data sheets are available for individual products.
Dehydrated culture media supplied as powders, granules or tablets should not be eaten. Powders
should not be inhaled because irritation of the upper respiratory tract may occur especially with bile salt
products. To avoid mild skin rashes prevent prolonged contact with the powder. Powdered products, if
spilled, can be swept up and disposed of in the normal way. Any residue should be washed away with
ample cold water.
Hazardous products
There are a few products which contain toxic substances and these must be treated with care.
1 Media containing Thallium salts. These products are labelled POISON.
Thallium salts are very toxic by inhalation or by ingestion and there is a danger of cumulative
effects. Products containing thallium salts must be kept away from food, drink and animal
feeding stuffs. Always wear a mask and gloves when handling the powder.
2 Media containing Sodium Azide
These products contain less than 1% sodium azide and have low toxicity. Some persons,
however, have enhanced sensitivity to azide and therefore could react to accidental exposure to
the product. Precautions must be taken to prevent ingestion or inhalation of the dust. Always
wear gloves, mask and eye protection.
Sodium azide reacts with many metals, especially copper, to produce explosive metal azides.
When washing products containing azide down sinks it is essential that sufficient water is used
to prevent the powder remaining in contact with the pipework and gulleys. The same precaution
applies to any biological solution which contains sodium azide as a preservative.
3 Sodium Biselenite. This product is labelled TOXIC.
It is corrosive on contact with skin and produces toxic effects if inhaled or ingested. Teratogenic
effects have been suggested.
4 Cycloheximide
This compound, prepared in Supplement vials, reaches a concentration which is considered to
be toxic and is labelled accordingly. However, when diluted out into the culture medium its
concentration falls below the minimum level considered to be hazardous. It is important when
reconstituting vials containing toxic levels of cycloheximide to ensure that the vial solution does
not touch the skin and to prevent the creation of aerosols which would allow the compound to
be inhaled. Protective gloves and face mask are advised when using these vials.
User-laboratory quality control tests on prepared media
Quality control tests should be carried out by the end-user laboratory to ensure that the performance
characteristics of the medium are within specification and that the methodology of medium preparation
is satisfactory.
Each lot/batch of prepared medium should be subjected to a minimal testing programme which will
ensure that it is acceptable and will demonstrate a typical bacterial performance.
1 pH value: check that the pH of the prepared medium, when tested in final form at ambient
temperature (25°C) lies within the range given on the product label. The medium should be
discarded if the pH value lies outside the specified range.
2 Sterility: a representative sample of each lot/batch of medium should be incubated for 2-5
days at 35-30°C and 50-55°C. As a general rule, for a lot of 100 or less units a 3-5% sample
should be tested. For a larger lot,10 random plates or tubes are taken. There should be no
evidence of microbial growth after incubation. Discard all sterility samples when the tests have
been completed.
3 Growth performance: test the growth support properties of the product by inoculating the
medium with appropriate stock cultures and/or fresh isolates. Use a standard inoculation
procedure and examine the quantitative and qualitative results obtained. If testing new
lots/batches of media, inoculate old and new lots in one test and compare the performance of
the two lots side by side.
4 Stability: periodically perform the above procedures on stored prepared media in order to
determine whether the storage conditions will give optimal results.
NOTE: If a medium does not perform to expectations and all the manufacturers recommendations
have been followed, then the following steps should be taken: (1) record the nature of the problem and
the method of preparation of the medium; (2) note the lot/batch number and the date it was received;
(3) call the Technical Services department of the supplier.
(reproduced, with few changes, from The Oxoid Manual, 6th edition, 1990)
Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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