I am planning to study the gene expression of only those fungal cells which are capable of penetrating the plant cells. So I need a gentle but effective method for removing non-infecting fungal cells without damaging the plant tissue. Any suggestions?
If you want to study only gene expression of fungi, why care about the plant?
I would just washed the leaves and spin down the fungi ...
Hope it helps.
Do you think just a water wash will remove the fungal hyphae on the leaves?
I have found some protocols suggesting ethanol wash or wash with sodium hypochlorite. Any experience with that?
I have no experience with that, but I would assume that adding a little bit of detergent (SDS or Triton), and maybe some reducing agent as DTT would increase the efficiency of fungi removal. But one of the most important thing will be controls. Use non infected plants, and maybe even a different species.
Wait, you want fungi that can penetrate the plant cells? do you want fungi that are already in symbiosis?
I'm confused about the aim of the experiment ...
Wash the leaf with 90% ethanol with the help of a swab.Sodium hypochlorite can also be used but that may damage other fungal cells also
Fernando : No these cells are not in symbiosis just infecting the plants. Thanks for your suggestions. Will certainly try them to see if they do the needful. Thanks!
Bijendra: Haven't tried swabbing the leaves...may be that'll work. thanks!
surface sterlization with sodium hypochlorite , I think this will do the trick.
First of all, vortex the leaves in sterile distilled water and that will get rid of all the loosely attached (non-penetrating) fungal cells. After wash the leaves with either 70% ethanol or 5% Sodium hypochlorite to make sure they're surface sterilized. Another option would be to use gentle detergent after vortexing and get rid of all the still attached fungal cells.
Time and vigor of vortexing is -of course dependent- on the type of leaf plant.
good luck!
Hi,
This could be an alterantive.
I have heard recently that chlor-dioxide (ClO2) might be suitable for surface-disinfection.
It is a very effective and stabilised active ingredient of certain commercial products (e.g. SOLUMIUM DENTAL) used by dentists and/or for drinking water disinfection, or in food-industry. It is soluble in water, not harmful to human, nor to plant cells when used in a proper concentration (might be necessery to determine the best of concentration for your particular plant species/parts experimentally ). As I heard, just submerge/keep the plant part in the solution for a short while, then let it evaporate and dry in the air (no residues) . No need for rinsing.
Cheers,
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