Currently we are working on the cultivation of Aspergillus oryzae in shake flask liquid culture. To inoculate the cultures, a certain concentration of spores in the inoculum is wanted (Spore count at the start of cultivation: 2*10^5 spores/mL).
Sadly, the concentration of spores we get from the cultivation on potato-dextrose-agar and czapek-dox-agar is way too low (T=30 °C, t= 5-7days) to reach the wanted spore count at the start of cultivation. The whole agar plate is covered in mycelium, but no significant spore production occures.
Procedure: Flood the plate with 10 mL 0,1% Tween 80, lightly scrub the surface with a drigalski spatula, swirl the suspension and transfer into a falcon tube. Cell counting in a hemacytometer under a microscope.
Any ideas how to improve the spore production?
Hi Marius Tölle
I am currently maintaining several Aspergillus species in the PDA medium. They are highly spore-forming isolates. According to my observations, their sporulation is highly obtainable in PDA medium over czapek-dox-agar. I hope you can subculture your isolate in multiple plates, maybe triplicate plates and harvest the spores. According to my understanding, your ultimate objective is only to make your initial spore concentration 2*10^5 spores/mL and not to obtain it from a single culture plate. Therefore I do not see it wrong to harvest spores from several plates and adjust the final spore concentration to your requirement
Hi again, Marius Tölle
The following paper talks about how to enhance the culture media composition for improving the sporulation of certain fungal isolates. I hope you will find some ideas from this paper to improve your PDA medium to facilitate sporulation further if multiple plate culturing is not the method for you.
https://www.hindawi.com/journals/bmri/2011/487329/
Hello Sandhya Jayasekara ,
thanks for the link, I will certainly take a look at the options in this paper!
You are right, cultivating in triplicates and concentrating the solution via centrifugation has been the way we go about this problem, for the future I'd just like to reduce the materials and steps used and be a bit less wasteful and more sustainable.
Dear Marius, if it's possible, try to cultivate A. oryzae on Czapek-Dox agar with yeast autolysate (CYA), yeast extract with supplements (YES) and malt extract agar (MEA). Also you can try to lower temperature, it could affect sporulation as well.
Dear, Marius Tölle,,
Aspergillus oryzae can grow and sporulate well on the medium Potato Dextrose Agar. You can test it.
The fungus Aspergillus oryzae is one of the important fungi which produces toxins affecting on the human health.
It also can be grown on Czapek-Dox Agar medium and Malt Extract Agar medium.
Thanks for all the suggestions, I tried all of them out, but the spore production on any conventional solid media (MEA, YES, PDA, CDA, CYA) was still very low.
But the problem is solved: The addition of 3 and 6% (6% worked quicker) of sodium chloride to PDA did the trick and after 7-10 days at 25 °C there is significant spore production in the range of 10^8 to 10^9 spores/mL.
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