I am working with both bacteria and fungi. I want to inoculate my sawdust with 10^9 cells. I can do this with bacteria by using thermal spectrophotometer 600 nm and adding 0.1% peptone water until Absorbancy is ~1.
There is a lot of skepticism about whether the same works for fungi. I assume it will work for yeasts but filamentous moulds, would I be able to put 1ml on agar plate and suck up fungi, then spin it down and add that or grow in a broth and add 1ml of the broth and spin it down. Or is there another way to get 10^9 cells for inoculum or would the spectrophotometer work?
Thanks
Hi,
You can grow your filamentous mould on suitable medium such as PDA and allow it to sporulate for 3-4 days. The spores can be removed and suspended in water with some emulsifier like T80 (just like bacterial suspension). You can count spores by direct microscopic count method and can adjust the dilution according to your requirement.
Depending on the capacity of your fungus to sporulate on solid media, you may need multiple confluent plates to get to a 10^9 spores/mL. Basically, grow your bug on an appropriate plate (SabDEX, Oatmeal, or similar) to confluency. When you're ready, add 10mL of PBS to the plate and scrape the surface of the plate, liberating the spores and subsequent hyphal material (if any) into the PBS. Filter through 2-4 layers of sterile cheesecloth (this works wonderfully for removing hyphal fragments but letting spores up to 8microns pass through easily...I have not tested on larger/smaller sized spores). Wash with another mL of sterile PBS. This will get you a relatively clean preparation of spores. Centrifuge to pellet spores and re-suspend in 5mL of sterile PBS and count using a hemocytometer. If the suspension is too dilute, simply re-centrifuge and suspend in a smaller volume, if too dense, adjust with PBS to get your desired concentration.
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