Preparation of Culture Media

CULTURE MEDIA

The survival and growth of microorganisms depend on available and a favourable growth environment. Culture media are nutrient solutions used in laboratories to grow microorganisms. For the successful Cultivation of a given microorganism, it is necessary to understand its nutritional requirements and then supply the essential nutrients in the proper form and proportion in a culture medium.

The general composition of a medium is as like

  • H-donors and acceptors (approximately 1-15 gL
  • C-source (approximately 1-20 gL)
  • N-source (approximately 0.2-2 g/L)
  • Other inorganic nutrients e.g. S, P (50 mg/L)
  • Trace elements (0.1-1 ug/L)
  • Growth factors (amino acids, purines, pyrimidines, approx 50 mg/L, vitamins occasionally 0.1-1 mg/L)
  • Solidifying agent (e.g. agar 10-20 gL)
  • Solvent (usually distilled water)
  • Buffer chemicals

INSTRUCTIONS FOR PREPARATION OF MEDIA

General outline for preparing any bacteriological media is as follows.

1. Study the composition of medium very carefully, Even slight differences in the composition of a medium can result in dramatically different growth characteristics of microorganisms. Formulations of most of media is per litre. Amounts required for small or large quantities is calculated from this, and weighed accurately.

2. Best results are obtained by adding ingredients to a dry flask, and then adding distilled water a little at a time with constant agitation to prevent the formation of lumps. Stirring rod may be used to secure an even mixture. Entire amount of distilled water is added when all ingredients are thoroughly wetted. Most ingredients of liquid/broth media are readily soluble in water at room temperature. However, few ingredients require little heat to dissolve.

3. Agar media must be heated gently (after adding agar powder) to boiling point for complete solution. This may be accomplished by heating the flask in a water bath. Agar or gelatin media must be in complete solution before being dispensed in the containers in which they are to be sterilized.

4. When water is indicated as diluent, distilled water is preferred but deionized (demineralized) water is also satisfactory.

5. It is a usual practice to adjust pH of the medium after all ingredients have dissolved, but before the addition of agar. However, pH should be checked again after agar has dissolved.

6. In some cases where interactions of components, such as metals, would cause precipitates, solutions may be prepared and occasionally sterilized separately before mixing the various solutions to prepare the complete medium.

7. The dissolved media are tubed or placed in flasks; cotton plugged or capped with metal or plastic caps and then sterilized usually either by autoclaving or by inspissation.

8. When autoclaving is indicated for sterilization, it is always at 15 pounds per square inch (psi) de (15 Ib in 2) at 121 °C for 15 minutes, unless and otherwise specified (heat-labile media can be sterilized either at low temperatures by inspissation or by filtration). One can prepare various media by following the general rules as described above.

The most common medium used in bacteriology is a nutrient medium. This medium can be used either as both or as a solid medium. The method of preparation is as follows.

PREPARATION OF NUTRIENT MEDIUM

Nutrient broth has been one of the earliest medium used in bacteriology. It is a basal mea which supports the growth of wide range of nutritionally undemanding chemoheterotront bacteria. Such a medium may be supplemented with particular nutrients or growth fac (to support the growth of nutritionally fastidious organisms) or with selectively inhibit substances (to prevent the growth of unwanted organisms).

Requirements

  1. Meat extract, peptone, NaCl, distilled water.
  2. pH paper or pH comparator.
  3. Flask, measuring cylinder, etc.
  4. Monopan balance.

Procedure

Preparation of Nutrient Broth

1. Weigh 0.3 g meat extract, 1 g peptone, and 0.5 g NaCl.

2. Mix above ingredients in the flask and add about 90 ml of distilled water; mix contents well

3. Place the flask in boiling water bath till the ingredients dissolve.

4. Allow the flask to cool and measure the initial pH either by pH paper or by pH comparator.

5. Adjust pH to 7.6 using either dilute acid or alkali.

6. Adjust the final volume to 100 mL with distilled water.

7. Distribute the broth so prepared, in tubes or in flask as desired.

8. Sterilize by autoclaving at 121 °C, at 15 lbs for 15 minutes

9. Allow the medium to cool and use.

Preparation of Nutrient Agar

1. Prepare the nutrient broth as described above up to step No. 6. Weigh and add 2.5 to 3.0 g agar to broth prepared as described above.

3. Place the flask in boiling water bath till the agar dissolves.

4. Recheck the pH of the medium (adjust it if necessary).

5. If slants are to be prepared distribute the medium in tubes. Pouring a plate between burners

6. Sterilize by autoclaving at 121 °C, at 15 psi for 15 minutes.

7. After sterilization, if slants are desired, place the tube in slanting position and allow it to cool without disturbance till it solidifies.

8. If plates are desired, do not distribute the medium in tubes instead sterilize medium directly in flask.

9. After sterilization, pour aseptically about 20 ml of the medium into sterile petri dishes and place the plates or the horizontal surface and allow it to cool.

(Before pouring the sterile medium into sterile petri dish, medium should be cooled down to 50-55 °C so as to minimize moisture deposition (water of condensation) on the lid of the petri dish)

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