Hello, you could try with an homogenizer, or maybe a bit of sputasol? I think that 10 minutes into an homogenizer, could help you to separate the thallus without breaking the cells walls. I'd try to cut a portion of the agar with the thallus on and put it into a warm broth (like MHbroth or BHI broth at 37°C) and then into the homogenizer for 10 minutes. Then if you still have no results, maybe, sputasol?

I've never try anything like this but I use homogenizer for Cystic Fibrosis samples and it works brilliantly without killing bacteria...

Give it a chance!

Good luck.

Ramona.

Try Tween 40 or 80 and vortex. I can't provide you with details - experiment. 

I suggest the following buffer to disaggregate spores before inoculation:

NaCl 18 g

A. dest. ca 1800 ml

Tween 80 200 µl

A. dest. ad 2000 ml

In our hands, sonication Works really well, without loosing too much cell viability (tested with propidium iodine staining), I can provide details if you wish.

Thank you all, I tried with a sonication bath with samples in microeppendorf vials it worked partially, some samples gave a milky suspension (my intended target) whereas other do not.

Ramona,I'll try sputasol or other fluidificant agents because I have it at hand.

Hector, if you could give me details on how do you operate it would be nice.

We usually perform it in 30s cycles with resting in between on ice. We saw that after 5 cycles the viability starts to be compromised. We also use the 1.5 mL-eppendorff tubes.