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Plant or Fungal Cell Dissociation Technique?


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#1 RedTent1701

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Posted 02 July 2010 - 08:31 AM

I am working with a fungal pathogen called Batrachochytrium dendrobatidis. While technically a fungus, it has several "plant-like" characteristics. The mature cells clump together in liquid culture so that it is impossible to count them accurately. I have tried many things to dissociate them into a single cell suspension or at least to make the clumps smaller so the individual cells are easier to count. I have tried trypsin in EDTA, collagenase, vortexing, etc. with no success.

Does anyone know of any other techniques used to dissociate clumps of plant or fungal cells? Thanks!

#2 gebirgsziege

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Posted 04 July 2010 - 11:19 PM

Which cells do you want to count? Zoospores, sporangia.... ??????????? The solution of your problem is different regarding which cells you will count. I am not sure about the behaviour or Bd in culture, but theoretically the zoospores will be unified in the zoosporangia and it will be difficult to seperate them. And have you thought of staining the cells with dapi, which will allow you to count the single cells by a fluorescence signal.....

other techniques widely used for fungi (mainly moulds) are 0.01% Tween 80 solution, adding a drop of 70% EtOH to your microscopy slide or sonication.

Let me know what worked, as Bd seems to be invading Austria as well now;

Edited by gebirgsziege, 04 July 2010 - 11:24 PM.

A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#3 RedTent1701

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Posted 06 July 2010 - 07:46 AM

I want to count the sporangia. They tend to clump together with as many as 10 to 100+ cells in a clump, making them difficult to count accurately when in high numbers. I attached an image of what kind of clumps I am talking about, though these clumps are on the smaller side. I have seen much larger clumps under the microscope, but I don't have photos of any larger ones.

I tried resuspending the colonies in a 1:1 mixture with nutrient broth and 70% ethanol, then in 70% ethanol with no nutrient broth. Neither disrupted the colonies. I haven't tried your other suggestions yet.

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  • Clumps.png


#4 gebirgsziege

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Posted 06 July 2010 - 10:37 PM

You are growing your strains in liquid culture? It looks as if your sporangia are growing in these clumps....is it possible that they are attached to some kind of not well desolved ingredient of your broth.....if I remember correctly you need to include ceratin into the broth which probably is not dispersed well? So maybe you can solve the problem by sonnicating and proerly mixing the culture broth before inoculation or change the shaking speed/amount of medium/size of bottle during incubation (have a look for basics of fermentation techniques of fung and how they overcome this clumping there).

Tween or other mild detergents often helps a lot; just trie to mix your culture with it and shake for some time (half an hour) on a head over head shaker to dispens your cells
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)




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