Hello,
There are a lot of suggestions on preparing a spore suspension from a fungal culture, however, it is typically for making inoculum, so it doesn't really matter whether the agitation (using a glass rod) is done consistently.
I have F. oxysporum and P. irregulare growing on petri dishes at 5 pH levels, how would I prepare a spore suspension from each plate in such a way where the method isn't biased? They're all colonies of varying diameter. I plan to then count the spores using a haemocytometer.
I was thinking of cutting agar plugs from the centre of the colony to the perimeter (to account for spore variation at different parts of the colony), and then agitating it in a vortex mixer with a fixed volume of water for a fixed amount of time. As some colonies are bigger than others on different treatments, and therefore would have more agar plugs to the perimeter, I was thinking I could adjust the final count to spores per plug. Would this work?
Dear Vikesh,
First of all, does P. irregulare correspond to Pythium irregulare, or anything else? Secondly, the aim of your study is not completely clear from the text. The method of preparation highly depends on the expected results.
Are you going to compare the spore production of two species (per colony square unit or...)? Why?
Thirdly, many details remain unclear. What colonies vary in size? You mean, colonies of different species, or colonies of the same species? In what conditions are you growing them? Are that conditions optimal for both species?
Any case, the spores of different species vary in size, weight, shape. They may have hydrophobic/hydrophilic cell walls, thus you may need various methods of suspension preparation adjusted for the species you study.
Is your aim to prepare inoculum or to assess the sporulation rate of the fungi?If you want to assess the sporulation rate of fungi, you can either:
1. grow on plate, then put water plus a few drops of tween 20 onto the plate, then use a hockey stick to detach d spores from mycelia then collect all the liquid from the plate, then do spore count, maybe you can express the sporulation by how many spores produced per area of colony
2. try culturing in broth culture, then you dont have to worry about different colony diameter
Maria V. Kozlova
Sorry for the lack of detail. Yup, I'm looking at Pythium irregulare and Fusarium oxysporum, and the aim is to see if different pH levels affect the growth and sporulation of both fungi. I'm not comparing the fungi across species, rather I'm comparing the colonies grown at different pH (which is what I meant by different colony sizes). Hence, I'd need to account for the variation in colony sizes at different pH when I count the spores (I'm guessing smaller colonies would output less spores overall, but per unit area that might not be the case). The medium I am using is PDA.
Vikki Wong what I'm concerned about with that method is the detachment process may not be too replicable? in that I may scrape more or less spores between plates. Why is why I might try using agar plugs and a vortex machine so the agitation is roughly the same between all plates. I'd like to use a broth, but I don't believe my lab has enough heated stir plates for 40 replicates.
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