Kafatos:Extraction of fungal spores

Extraction of Beauveria bassiana spores


Extraction of spores:

 * note: everything should be done under sterile conditions to avoid contamination

1. take several plates (10 cm diameter) of BB that have been growing for min. 3 weeks 2. add 10-15ml ddH2O to plate and scratch the surface with a sterile spatula 3. pour the juice over autoclaved glass wool (in a sterile glass funnel) into a centrifugatable bottle (4000 rpm) 4. wash the plate twice with 10-15 ml of ddH2O 5. centrifugate 10 min @ 4000 rpm 6. discard supernatant 7. wash pellet 2x with ddH2O  8. add 100-250 μl of ddH2O 9. count spores in Thoma (or Neubauer) counter by diluting an aliquot (x100, x1000 or more) 10. add sterile 50% glycerol to have a final conc. of 10.000.000 spores/ml in 25% glycerol in eppendorf tube 11. freeze the eppendorf tube @ -80˚C



Plating out of spores:
plate out 50 μl of 10.000.000 spores/ml on a malt-agar plate

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Malt-agar plates:
(1l medium, for ~25 plates of 10 cm dish)<BR> 1g Peptone (Select peptone GIBCO BRL Cat No. 30392-021)<BR> 20g Glucose (α-D(+)glucose monohydrate ROTH Art 6780)<BR> 20g Malt (Sigma M-0383)<BR> 15g Select agar (Invitrogen Cat No 30392-023)<BR>

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Potato Dextrose agar plates:

 * (this is an alternative to the Malt-agar plates, but I never tried them)

Infusion from potatoes 	1000 ml<BR> Glucose                20 g<BR> Agar                  	15 g<BR> Potato infusion: Boil 200 g scrubbed and sliced potatoes in 1000 ml water for 1 hour. Pass through fine sieve. Avoid new potatoes.<BR>