I am looking for a protocol for studying the bacterial-fungal interactions via microscopy. which staining technique would be the best and which culturing method would yield the most optimum results?
The paper in the attachment might give you some useful information. You could also contact the authors if you require more specific information. Good luck with your project!
Regards, Christian
I think for staining bacteria protocol will be EMB agar plates where you can easily differentiate between the gram positive and gram negative bacteria based on growth and follow by Christian gram staining .For fungi protocol be the same .For viewing the bacteria and fungal you can use phasecontrast microscope.That will be better.
Hi Shubhangi,
It, surely, depends on your strains. But we've optimized a very straight forward staining technique with Calcoflour white and Syto9 for intrahyphal bacteria. In above mentioned paper, FM4-64 was used to stain the fungal membrane, since the endocytosis was investigated. It might be helpful for you to use the combination if CFW and Syto9 as decribed in the paper. Brief note: do not incubate Syto9 longer than 5 or 10 minutes. Then you will get unspesific staining.
Good luck
Zerrin
Dear Zerrin,
Thank you so much for the helpful input and advice. I am actually trying to study the hyphal colonization by bacteria, do you think this staining would work for that too? I stained the bacteria and fungus with DAPI but it wasn't that good.
Thanking you
Shubhangi
Dear Shubhangi,
Combination of CFW and Syto9 would work perfectly for any differential staining of filamentous fungi and Gram negative bacteria as my personal experience. Especially for hyphal colonization! I also now cases that people succeeded with yeast and Gram positive bacteria as well. DAPI requires longer incubation times and you cannot differentiate fungus vs bacterium, as -I suppose- you had experienced, too. I recommend using combinations of dyes for fungal membrane or wall, and anything specific to bacteria (even FISH).
Best,
Zerrin
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