Exp 2: Media Preparation, Sterilization, Plating
Introduction
Preparation of culture media
Rehydrate tablets or powder according to manufacturer’s instructions. Before sterilization,
ensure ingredients are completely dissolved, using heat if necessary. Avoid wastage by
preparing only sufficient for either immediate use (allowing extra for mistakes) or use in the
near future. Normally allow 20-25 cm3 medium/ Petri dish. Dispense in volumes appropriate
for sterilization in the autoclave/pressure cooker. Agar slopes are prepared in test tubes by
allowing sterile molten cooled medium to solidify in a sloped position. Bottles of complete,
sterile media are available from suppliers but are expensive.
Sterilization
Autoclave/pressure cooker: The principle of sterilization in an autoclave or pressure cooker is
that steam under pressure is used to produce a temperature of 121ºC which if held for 20
minutes will kill all micro-organisms including bacterial endospores.
Sterilization of equipment and materials
1. Wire loop: Heat to redness in Bunsen burner flame.
2. Empty glassware and glass (not plastic!) pipettes and Petri dishes: Either, hot air oven,
wrapped in either greaseproof paper or aluminium and held at 160ºC for 2 hours, allowing
additional time for items to come to temperature (and cool down!). Or, autoclave/pressure
cooker.
Note: plastic Petri dishes are supplied in already sterilized packs; packs of sterile plastic
pipettes are also available but cost may be a consideration.
3. Culture media and solutions: Autoclave/pressure cooker.
4. Glass/metal spreaders and metal forceps: Flaming in alcohol (70%)
Pouring a plate
1. Collect one flask of sterile molten agar from the autoclave/ hot air oven/water bath.
2. Hold the flask in the left hand; remove the cotton plug with the little finger of the right
hand.
3. Flame the neck of the flask. Transfer the flask from left hand to your right hand
4. Hold the Petri dish in your left hand and lift the lid of the Petri dish slightly with left hand
fingers and pour the sterile molten agar into the Petri dish and replace the lid.
5. Flame the neck of the flask and replace the lid.
6. Gently rotate the dish to ensure that the medium covers the plate evenly.
7. Allow the plate to solidify.
8. Seal and always incubate the plate in an inverted position.
Note: The base of the plate must be covered, agar must not touch the lid of the plate and the
surface must be smooth with no bubbles.
Storage of media
Store stocks of prepared media at room temperature away from direct sunlight; a cupboard is
satisfactory but refrigerator or cold room is ideal. Media in vessels closed by cotton wool
plugs that are stored for future use will be subject to evaporation at room temperature; avoid
wastage by using screw cap bottles. Re-melt stored agar media in boiling water bath, pressure
cooker or microwave oven. Sterile agar plates can be pre-poured and stored in well-sealed
plastic bags (media-containing base uppermost to avoid heavy condensation on lid).
Sterilization vs. Disinfection
Sterilization means the complete destruction of all the micro-organisms including spores,
from an object or environment. It is usually achieved by heat or filtration but chemicals or
radiation can be used.
Disinfection is the destruction, inhibition or removal of microbes that may cause disease or
other problems e.g. spoilage. It is usually achieved by the use of chemicals.
Disinfectants
Choice, preparation and use of disinfectants: Specific disinfectants at specified working strengths
are used for specific purposes. The choice is now much more straightforward as the range available
from suppliers has decreased.
Calculations:
Observations & Result: