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M/1998/3.02 Appendix 4


1 Preparation of agar plates

1.1 Heat a suitable agar medium in a water bath to dissolve the agar. Add a stirring rod for magnetic stirring. Sterilize the medium and remove the bottle of molten agar from the autoclave. Add thermolabile substances if needed. Carefully mix the agar on a magnetic stirrer. Avoid the formation of bubbles or foam on the surfaces.

1.2 Place the hot agar bottle in a controlled water bath at 50C for 1 hour. (Alternatively the bottle may be placed in a large plastic vessel containing hot water at 50C. The agar cools quickly -temporarily warming the water further- but uniformly throughout the vessel and reaches a temperature plateau above its solidification point. For 1 litre of agar, 30 min is sufficient). With the procedures described there is no gelling around the edges of the container.

1.3 Pour about 20 ml of agar into the petri plates (90 mm diameter) corresponding to a thickness of about 30 mm. If the agar is sufficiently cooled, little condensation will form on the undersurface of the petri plate lid and less water of syneresis will be formed.

1.4 Store the plates at room temperature for at least 24 hours to remove water of condensation on the plate lids and water of syneresis on the agar. Storage in a laminar flow cabinet should be preferred.

1.5 The plates are then stored below 10C in the original plastic cover of the plates. Before use the plates are warmed to room temperature.

2 Preparation of cell suspensions

2.1 Prepare serial dilutions (1:10) of the bacterial suspension by transferring 0.5 ml suspension to 4.5 ml nutrient medium or 0.1% peptone water in tubes measuring 16 by 160 mm. Do not use distilled or tap water nor saline or buffer solution as a diluent since bacteria may die in these solutions.

(Freeze)-dried cultures are rehydrated with 0.5 ml of broth medium. Frozen cultures are transferred to 5 ml broth medium.

2.2 Pipette 0.1 ml of each dilution on the agar surface of the petri plate and spread the liquid over the plate with the help of a Drigalsky spreader.

Alternatively, one drop of each dilution is spot inoculated onto a single agar plate. Or one drop from a Pasteur pipette is transferred to an agar plate and distributed over four quadrants.

2.3 Incubate the inverted plates in an incubator at an appropriate temperature. For slow-growing organisms use closed containers for incubation.

2.4 Count the colony forming units on plates showing 30 to 300 colonies and calculate the number of cells in the original (non-diluted) suspension.

On spot inoculated plates count the colonies developing on the area of the different drops.

2.5 For recording the results use protocol M/1998/3.00 Appendix 5.01.1, M/1998/ 3.00 Appendix 5.05.1 or M/1998/3.00 Appendix 5.08.2.

Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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Copyright CABRI, 1998

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