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LABORATORY PROCEDURES FOR MICROORGANISMS

Appendix

M/1998/2.03 Appendix 1


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

Preparation of sterilized media

Storage of prepared media

Precautions in the use and disposal of prepared media

Precautions - dehydrated media

User-laboratory quality control tests on prepared media


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.

Light

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 15-20C. 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 2-8C. 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 2-8C. 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 2-8C in a dry place. Do not store these kits at a higher temperature for long periods. Shelf life 3 years.

Sterile Reagents: Store at 2-8C, except Horse Serum store at -20 to +8C.

Susceptibility Discs: Store at -20C but keep working stock at 2-8C. 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 25C 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 (50C) 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 121C 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 25C. 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 121-134C 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 134C is preferable to 20 minutes at 115C.

A general instruction for sterilizing culture media in volumes up to one litre at 121C 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 121C holding time.

Sterilization Cycle

The sterilization cycle can be divided into its four stages:

Stage 1 20-121C 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 <100-121C 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 121C 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 121C, 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 3 121-121C Holding time at the prescribed temperature

The holding time at 121C 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 121C.

The recommended holding times are:

Temperature (C) 121 126 134

Time (minutes) 20 10 3

Stage 4 121- 80C Cool-down time for the chamber to reach 80C

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 8OC 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 50C 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 121C or in some models at 134C. 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 25C. Overheating through prolonged sterilization, remelting or overlong period at 50C. 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 50C. 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 50C. 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 50C 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 50C. 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 2-8C (blood must not be frozen). Warm the blood in a 35C incubator before addition to sterile molten agar base, which has been cooled to 40-45C. 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-l6C). It is important to store all media away from light. Agar plates should be stored at 2-8C 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 (25C) 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 35-30C and 50-55C. 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
Page layout by CERDIC
Copyright CABRI, 1998
 

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