- Measure out the correct amount of distilled water required for the culture media preparation using a measuring cylinder. The water should be dispensed into a conical flask, beaker, tubes or bottles depending on the choice of the researcher. For example, 400 ml of distilled water is required to prepare 400 ml of nutrient agar solution; and this should be measured and dispensed into a clean conical flask or beaker.
- Measure out the appropriate amount of the powdered agar base (in grams) using a weighing balance. Most culture media powder is hygroscopic in nature, and they absorb moisture from the atmosphere when left open to form a solid mass. The container of culture media powder should always be covered to prevent this from happening.
- Dispense the measured powdered agar into the correct amount of distilled water in a conical flask or beaker. Shake the conical flask properly after adding the powdered media to the distilled water. Caution: Powdered agar should always be added to water and not the other way round. The reason for this is to ensure proper dissolution of the powdered agar and to avoid burning when heating.
- The dissolved powdered agar solution should be boiled under a Bunsen burner flame for complete dissolution; and boiling or heating should not last too long (20-30 minutes is allowable) in order to avoid burning or charring of the agar.
- Twenty mill (20 ml) each of the dissolved heated agar solution should be dispensed into clean capped bottles (for example, McCartney bottles). In some cases, heated agar solution in conical flask can be sterilized directly without dispensing into capped bottles.
- Sterilize the agar solution in the autoclave as recommended. Sterilization of culture media solution is usually carried out at 121oC for 15 mins and at 15 psi (psi=pounds per square inch). Caution: Allow the indicator of the autoclave to fall back to the zero level before opening the equipment once sterilization is complete in order to avoid injury. Always allow the pressure to fall back to normal level before opening the lid of the autoclave.
- Sterilized culture media solution should be allowed to cool to about 45-50oC before pouring plate. At this temperature, the prepared agar solution (now known as molten agar)can be poured into clean Petri dishes as the case may be; and this should be done around Bunsen burner flame which provides a sterile environment for pouring plates.
- Poured plates should be allowed on the bench for solidification or gelling of the medium. Broth or liquid culture medium does not gel because they lack agar or agar-agar (the solidifying agent in culture media).
- Poured culture media plates should be macroscopically observed for the presence of microbial growth after gelling. A sterile plate is one that is free of any form of microbial growth after pouring plate; and such plates are ready for further microbiological analysis. Caution: Prepared culture media plates should be properly labeled and stored in the refrigerator until they are ready for use.
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