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• • Are there any guidelines for preparation and reconstitution of culture media?
  • Are there any guidelines for sterilising culture media?
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  • How should I test the pH of culture media?
  • My culture medium does not look right - what has gone wrong?
  • My culture medium is not performing correctly - what has gone wrong?
  • Should I boil media before autoclaving?
  • What are the main components of culture media?
  • What exactly is agar?
  • What grade of water should I use to prepare culture media?
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OXOID TECHNICAL SUPPORT DEPARTMENT GUIDELINES

STERILISATION GUIDELINES FOR OXOID CULTURE MEDIA

Using Autoclaves
The following considerations should be taken into account when sterilising media that are autoclaved following the standard parameters of 121°C for 15 minutes:

1. Attention to the media manufacturers’ instructions for use must be observed in order to ensure optimal performance. The time and temperature recommendations for sterilisation of Oxoid culture media are based on evidence from trials with heat resistant bacterial in volumes of up to one litre.

2. The amount of heat applied during sterilisation is much greater than is necessary for destruction of the majority of organisms likely to be encountered. Tolerances of 15 minutes ± 1 minute combined with 121°C ± 2°C are considered appropriate. Appropriate Quality Control checks should be carried out for temperatures which are outside 119 - 123°C, to ensure sterility and/or performance integrity.

3.Certain media are more susceptible to over-heating, such as those with a high sugar content, or those which contain inhibitory agents such as sodium desoxycholate or bile salts. The result of over-heating is a reduction in pH of the final medium.

4. For volumes over 1 litre, the medium should be pre-heated to dissolve the agar prior to autoclaving. It is not usually necessary to boil smaller volumes of media unless the medium contains gelatin, or the intention is to dispense the medium into final containers prior to autoclaving.

5. Suitable containers for autoclaving are wide-bottomed flasks - thin walled bottles should be avoided. Adequate headspace should be allowed in order to minimise loss of medium.

6. The sterilisation temperature refers to the autoclave chamber not to the temperature of the medium. It is important that the time taken to reach the sterilising temperature should be as short as possible. Sterilisation cycles must take into account suitable heat penetration times e.g. a one litre flask of medium should attain 121°C within 15 minutes of the chamber reaching that temperature.

7. The autoclave load and the relatively poor heat transferring properties of agar-containing media must be taken into account.

8. The autoclave temperature probe is best located in the valve or drain, as this is the coolest part of the chamber and best represents the dynamics of the heating process.

9. When validating an autoclave it is strongly recommended that the manufacturer is consulted in order to discuss the relevant validation protocols.

Reference
C.A., Oscroft & J.E.L., Correy (1991) 'Guidelines for the Preparation, Storage and Handling of Microbiological Media'. Technical Manual 33. Campden and Chorleywood Food & Drink Research Association.