Gremenniella isolation

["David Brayford ", IMI]

I don't think there is a "quick and easy" way with this fungus because its 
apothecia
tend to accumulate debris and hence potential contaminants.  What I did some 
years
back was to pick off each apothecium, push it into the surface of a plate of 
water agar and
move it around in the agar with sterile needles so as to scrape off as much 
dirt, algae,
etc. as possible.  Then put the fruit body into a drop of streptomycin 
solution on an alcohol-flamed microscope slide.  Let the fruit body swell 
and open for a couple of
minutes, then using a sterile scalpel carefully scrape out a small bit of 
the ascal layer
and place it into a drop of water on the surface of a plate of tap water 
agar + streptomycin.
 Use a flamed glass spreader to smear the ascal suspension over the agar 
surface.
Incubate overnight and then look for germinating ascospores.  These can be 
picked off
using a tungsten needle sharpened in molten sodium nitrite and then 
transferred to
slopes of V8 agar (or whatever).

This may sound complicated, but it's not bad once you get into the swing of 
it.  The main
thing is to get rid of as much debris as possible by rolling around in the 
agar, and then use
antibiotic to supress bacteria.  At least the ascospores have a distinctive 
shape, so you
know what to isolate!  If you incubate your apothecia at low temperature for 
a longish
time, the ascospores sometimes germinate in-situ and form conidia, which can 
be a
problem.  I recommend isolating from apothecia when they are as fresh as 
possible.

Good luck.

Dave Brayford
International Mycological Institute
d.brayford @cabi.org

 --------------------------------------------------------------
I have difficulties in obtaining single ascospore from apothecium collected
in the field. I am working with fungus Gremmeniella abietina (Ascomycete) 
and
need to screen a large number of single spore isolates. Could anyone there
advise me some easy and quick methods of obtaining single spore isolates?
Any suggestion and help will be appreciated.  Thank you in advance!

Xiao-Ru Wang