I have an issue with reviving mycelial/ conidial glycerol stocks from the -80 freezer. The fungi I'm working with belong to the Botryosphaeria and Neofusicoccum species.

I prepare the conidial stocks by the following protocol:

1)scraping pycnidia off agar medium , adding 0.01% Triton X-100,

2)transferring to 1.5 ml tubes and centrifuging, grinding using small pestle

3) adding glycerol to 20% volume, vortexing and directly putting in a box in -80 freezer.

Now I may have doing mistakes on this protocol for years. The main problem is that over time the viability is reduced.

Meaning that when I take the conidial stock out of the -80 freezer to transfer to agar medium often there is no growth. I know I probably have conidial suspension about 10^4 conidia/ml but I can't think what mistakes I could be doing.

Maybe when the conidial stock is freezing the conidia travel towards the bottom of the tube and that's why I often have issues with the viability of my isolates. Also, because of the frequent opening of the freezer, the temperature often goes up to -64C.

Furthermore we have a very old benchtop cooler which is broken and the tubes often partly melt when removing them from the -80 freezer. Any ideas to make a ghetto benchtop cooler?

Any input welcome!