Kafatos:Extraction of fungal spores

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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

Malt-agar plates:

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

Potato Dextrose agar plates:

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

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