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LABORATORY PROCEDURES FOR MICROORGANISMS

Appendix

M/1998/3.02 Appendix 5


PROCEDURE FOR ESTIMATING SURVIVAL RATES, FUNGI

1. Sporulating fungi

1.1.1 Resuscitate pellet (in case of freeze-dried material) or frozen culture in 1 ml demineralized water for 30 min at 22C.

1.1.2 Plate 200 ml of the suspension in triple on 8 ml of a suitable transparent agar- medium in a petridish (diam. 5 cm).

1.1.3 Incubate the cultures at 24C for 16 h.

1.1.4 Put a 16 mm coverslip on the agar.

1.1.5 Assess germination for 8 x 100 propagules by placing the petridish under the microscope.

1.2 When germination rates are too low, colony forming units (CFU's) are scored.

1.2.1 Plate 200 ml of the undiluted suspension and of the 10-1, 10-2, 10-3 and 10-4 dilutions on a suitable transparent agar medium.

1.2.2 Incubate plates at 24C for 16 h and grow them subsequently at 22C.

1.2.3 Record number of colonies after 3 - 6 days depending on the species.

1.2.4 Data are analysed statistically using a nested ANOVA (P=0.05) after transformation of germination percentages into arcsine (square root) values. Multiple compariosons based on Newman-Keuls (P=0.05) are used to calculate significance of differences between means.

1.2.5 When colonies are growing too fast, 0.3% v/v Solacol (Aagrunol B.V., Groningen, The Netherlands) is added to the agar medium to limit the spread of the fungal colonies.

2. Non-sporulating fungi (only cryopreserved strains)

2.1 Rins the straws, each containing 10 agar plugs with the fungus, in ethanol 96% and open with a sterilized pair of scissors.

2.2 Transfer the plugs with a sterilized transfer needle onto the suitable agar medium and distribute plugs evenly over the petridish.

2.3 Incubate plates at the appropriate conditions.

2.4 After incubation estimate growth from the plugs.


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

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