The preparation of culture media from dehydrated media requires accuracy and attention to preparation. The following points are included to aid the user in successful and reproducible preparation of culture media.
DEHYDRATED MEDIA AND INGREDIENTS
- Store in a cool (15-30C), dark and dry area unless otherwise specified.
- Note date opened.
- Check expiry (applied to intact container).
- Verify that the physical characteristics of the powder are typical.
- Use high quality, low alkali borosilicate glass.
- Avoid detergent residue.
- Check for alkali or acid residue with a few drops of bromo thymol blue pH indicator (yellow is acidic; blue is alkaline).
- Use vessels at least 2-3 times the volume of medium.
- Do not used etched glassware.
- Use measuring devices, scales, pH meters, autoclaves and other equipment that are frequently and accurately calibrated..
- Use distilled or deionized water.
- pH 5.5-7.5.
- Accurately weigh the appropriate amount of dehydrated medium.
- Dissolve the medium completely.
- Agitate the medium while dissolving.
- Take care to not overheat. Note media that are very sensitive to overheating. Overheated media will frequently appear darker. Do not heat in a microwave.
- The autoclave set-temperature should be 121C.
- Routine autoclave maintenance is important. Ask manufacturer to check for "hot" and "cold" spots.
- The recommended 15 minute sterilization assumes a volume of 1 liter or less. Larger volumes may require longer cycles. Check with your autoclave manufacturer for recommended load configurations.
- Quantities of media in excess of two liters may require an extended autoclave time to achieve sterilization. Longer sterilization cycles can cause nutrient concentration changes and generation of inhibitory substances.
- Enrichments and supplements tend to be heat sensitive.
- Cool medium to 45-55C in a water bath prior to adding enrichments or supplements.
- Commercial dehydrated media are designed to fall within the specified pH range after steam sterilization. The pH tends to fall approximately 0.2 units during steam sterilization.
- For filter sterilization, adjust the pH, if necessary, prior to filtering.
- Avoid excessive pH adjustment.
- Ensure gentle mixing during despensing.
- Cool the medium to 50-55C prior to dispensing to reduce water evaporation.
- Dispense quickly.
- If using an automatic plate dispenser, dispense general purpose media before dispensing selective media.
- Immediately recover or recap tubes to reduce the chance of contamination. Leave Petri dish covers slightly open for 1-2 hours to obtain a dry surface.
- In general, store steam-sterilized plated media inverted in a plastic bag or other container in a dark refrigerator for up to 1-2 weeks.
- For media prepared in-house, each lot of every medium must be tested.
- Maintain Quality Control Organisms appropriately.
- Maintain appropriate records.
- Report deficiencies to the manufacturer.
1.Block, S. 1992. Sterilization, p. 87-103. Encyclopedia of microbiology, vol. 4. Academic Press, Inc., San Diego, CA.
2.Cote, R. J., and R. L. Gherna, 1994. Nutrition and media, p. 155-178. In P. Gerhardt, R.G. E. Murray, W.A. Wood, and N.R. Krieg (ed.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C.
3.The United States Pharmacopels (USP XXIII) and The National Formulary (NF 18). 1995. Sterilization and sterility assurance of compendial articles, p. 1976-1980. United States Pharmacopeial Convention Inc., Rockville, MD.
4. Perkins, J.J. 1969. Principles and methods of sterilization in health sciences, 2nd ed. Charles C. Thomas, Springfield, IL.
5.Leahy, T.J. 1986. Microbiology of sterilization processes. In F.J. Carleton and J.P. Agalloco (ed.), Validation of aseptic pharmaceutical processes. Marcel Dekker, Inc. New Yark, N.Y. 6.Simko, R.J. 1986, Organizing for validation. In F.J. Carleton and J.P. Agalloco (ed.), Validation of aseptic pharmaceutical processes. Marcel Dekker, Inc. New York, N.Y.
