We are trying to get a pure culture of Verticillium dahliae on PDA, but after sometime green color colonies of some other spores appear on the plate, when seen under microscope, the size is quite smaller than those of V.dahliae? Anybody experience the same problem?
Use better microbiological technique when pouring your plates. Are you adding any antibiotics to your medium?
Then the contaminants are either coming from the air or in with your V. dahliae culture. To narrow down the source, try incubating a plate with no V. dahliae in it.
In order to pure culture, use single spore technique. Use 2%agar and dispense spore suspension. Incubate the plates and next day use microscope and mark germinated single spore. Transfer it to pda
One possible problem with B.A.'s answer is that some contaminant spores are so tiny that you might not be able to see them under the dissecting scope because they can't be distinguished from particulate matter in the agar.
The best way to avoid contaminants is to follow basic procedure while handling your culture for getting pure culture. Ensure that your materials are sterilized and also use antbiotics..
Hmmm, and what sort of antibiotic do you suggest for controlling Aspergillus or Penicillium, while allowing growth of Verticillium, Matthew Baite?
In order to get a pure culture of Verticillium dahlia you have to follow microbiological basic procedure while handling your culture, ensure that your materials are sterilized. If you are purifying a V. dahliae contaminated with Cladosporium, Penicillium or Trichoderma (green spores) you should work under the stereomicroscope (40x) and select those colony areas that are not affected by these contaminants and transfer to 2% water agar plates supplemented with broad spectrum antibiotics(Chloramphenicol, vancomycin or streptomycine) to avoid bacterial contamination. Later when you obtain V. dahliae colonies without contaminants you should use the single spore technique for strains purification. Thesessteps gave me good results when I purified a strains collection of V. dahliae and V. albo-atrum .
Try to make single spore cultures. If conidia of both, V.dhaliae and the contaminant are similar, you can take several spores individually and transfer them to a single PDA plate each.
It sounds like you might have a mycoparasite infecting your culture. This is not uncommon. These can often be cleaned up by transferring to water agar and isolating single spores or transferring single hyphal tips to fresh media. The water agar shows more diffuse growth than PDA so can help in finding uncontaminated sectors and individual hyphal tips.
you can used the single spore technique its powerful and very success
thanks to all of you for your valuable suggestions, i have serially diluted the spores and have cultured on PDA, i will pick a single colony and will further process, it
This procedure can work, but it is much better to plate the conidial suspension onto Water agar, and after a few hours transfer individual germinated conidia dissecting them under stereo-microscope. Using PDA and allowing colony growth until visible, could not ensure bacterial-free colonies.
I agree with Juan. Also if you allow the colonies to grow up before picking, you might still have the contaminant present - i.e. a mixed colony.
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