How to use Dehydrated Culture Media

How to use Dehydrated Culture Media

Preparation

Dissolving the dehydrated culture medium

Dehydrated culture media should always be prepared with clean, freshly distilled or completely demineralized water which gives a neutral reaction.

Rinse the clean containers to be used (usually conical flasks) thoroughly with completely demineralized water to remove any traces of other substances (e.g. detergents). The vessels should be so large that the reconstituted culture media can be thoroughly shaken. If possible, do not use more than 1-2 liters per vessel. Weigh out the desired quantity of the dehydrated culture medium, mix it with approximately half the required volume of water and shake vigorously until a homogeneous suspension is obtained. Add the remaining water being careful to rinse down any material adhering to the walls of the vessel.

All culture media are heat-sensitive.

Do not heat them any longer than necessary.

Culture media without agar-agar or gelatin can usually be dissolved in cold water or only require gentle heating. Use should be made of this fact to ensure that the medium is prepared under mild conditions.

Culture media containing agar or gelatin must be heated in order to dissolve them completely. Heating should be carried out in a boiling water bath or free-flowing steam (e.g. in a steam pot or autoclave without excess pressure). Culture media which are not subsequently autoclaved must be checked for complete dissolution, this is achieved when the viscous solution flows smoothly and if no agar particles can be seen sticking to the walls of the vessel after shaking.

In the case of some culture media turbidity is essential (e.g. bismuth sulphite agar). The insoluble components should then be distributed as finely as possible to ensure that the turbidity is homogeneous.

If, in the case of agar- or gelatin-containing media, a volume of more than 2 liters per vessel is required, the medium should be dissolved under mild conditions in the following manner:

-	make a slurry by mixing the dehydrated culture medium with about 1/10 of the total volume of water specified, allow to swell for 15 minutes
-	bring the rest of the water to the boil
-	stir the slurry into the boiling water
-	heat until the medium dissolves.

pH adjustment

The pH value of our reconstituted dehydrated culture media prepared with neutral water should be identical with the prescribed values at a temperature of 25°C.
It is advisable, however, (particularly if older material is being used) to check the pH and correct it if necessary.

The pH value depends very much on the composition of the culture medium, the temperature at which the pH is measured and the treatment which the culture medium has been subjected to during reconstitution (dissolving, sterilization). The pH should therefore be measured after sterilization; it is best determined with a pH-meter (taking care to compensate for temperature when standardizing the electrode) or with special indicator test strips pH 4.0-7.0 (Cat. No. 9542) and
pH 6.5-10.0 (Cat. No. 9543).

pH measurement and correction of the pH should be carried out at 25°C with solid as well as liquid culture media. The pH should be adjusted to the value specified, if necessary. The pH should be corrected by adding 1N or 1/10N hydrochloric acid or sodium hydroxide solution to a sample of known volume taken from the reconstituted culture medium (e.g. 50 ml). The volume of acid or alkali added to the sample can then be used to calculate the quantity necessary to adjust the pH of the remaining prepared culture medium. It therefore should be kept liquid during the pH measurement of the sample, in order to be adjusted if necessary.

	How to correct the pH:
1.	Sterilize the reconstituted culture medium
2.	Remove a sample under sterile conditions
3.	Measure the pH of this sample and if necessary adjust to the correct value by titration
4.	Add the volume of hydrochloric acid or sodium hydroxide solution, that has been calculated from the titration to the reconstituted culture medium under sterile conditions. (The hydrochloric acid and sodium hydroxide can be sterilized by filtration through a glass or special membrane filter).

Sterilization

Before sterilization is carried out, the culture medium should be divided into smaller portions, for example poured into containers which are to be used in the test (but not Petri dishes).

If not otherwise stated in the directions, sterilization should be performed in an autoclave for 15 minutes at 121°C. This does not include the time required for heating up and cooling which depends on the apparatus and the volume of medium used. This information must be obtained from the manufacturer of the autoclave. Complete sterility can only be guaranteed if the steam chamber and the vessels are completely degassed. This is achieved by passing a larger amount of free-flowing steam through the autoclave with the valve open at the beginning of the heating up phase. After sterilization and pressure equalization the vessels should be removed from the autoclave immediately and cooled rapidly (e.g. to the temperature used for pouring) to minimize exposure of the culture medium to heat. The vessels should be cooled under cold, running water.

High temperatures and prolonged heating lead to deterioration in the quality of the culture medium.

Autoclaving is the most reliable way of sterilizing culture. If an autoclave is not available, the culture media can be heated in a household pressure cooker as an emergency solution.

Pouring the plates

The culture media should be poured into plates at about 45-55°C to avoid the formation of condensed water in the lids of the Petri dishes. The medium should be swirled before pouring to ensure that it is evenly mixed. Air bubbles in the plates can be removed by briefly fanning them with the luminous flame of a Bunsen burner. Wet agar surfaces promote microbial swarming and colony liquefaction, they can be dried by heating at 30-40°C in the incubator before inoculation is performed. The base of the Petri dish is inverted and placed on the edge of the lid.

Drying takes about 20-30 minutes or somewhat less if an incubator with circulating air is used.

Acidic culture media

Agar culture media whose pH value is below 6.0 must be prepared under very mild conditions as agar-agar is hydrolyzed when heated in an acid medium and the gel stability of the culture medium thus declines. Reliquefaction should therefore be avoided. If it is impossible to prepare the medium under appropriate conditions or reliquefaction cannot be avoided, Agar-agar (Cat. No. 1614) can be added to the culture medium before dissolution. Approximately 5.0 g/liter is generally sufficient.