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Preparation and Storage of Culture Media

Contents

Precautions - dehydrated media
Light
Humidity
Temperature and time

Preparation of dehydrated media
Reconstitution of dehydrated media
Sterilization of culture media
Sterilization checks
Overheating effects
Table of faults and possible causes in media sterilization

Culture media must be stored at the specified temperature, under specified conditions and not longer 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 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.

All prepared culture media and their components should be stored away from light and exposure to direct sunlight should be avoided at all times.

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.

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.

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.

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.

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.

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.

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.

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 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.

Wrong pH value

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.

Turbidity, precipitation.

Poor quality water or containers. Overheating or prolonged storage at 50°C. pH value incorrect. Incomplete solution.

Darkening.

Overheating, incomplete solution or pH drift. Presence of phosphate in addition of glucose or other sugars and agar.

Soft gel.

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.

Poor bacterial growth.

Prolonged and excessive heating, incomplete solution. Inhibitory substances in water or containers. Darkening and pH drift.

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.

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.

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.

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.

There are a few products which contain toxic substances and these must be treated with care.

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.

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)