Help! Single ascospore isolate

[Plantpath]

In article <3bclcb$cv3 at kitten.umdc.umu.se>, Xiao-Ru.Wang at genfys.slu.se
(Xiao-Ru.Wang) writes:

<difficulty with single spore isolation (SAI).

In graduate school, I single-spored conidia and ascospores of
Cryphonectria ( then Endothia) parasitica.  Although the sexual structure
is a perithecium, perhaps the technique I used may be helpful.

Simply stated, I removed perithecia aseptically and surfaced sterilized,
then squashed the contents in  a depressed slided with a drop or 2 of
sterile water.  This slurry was aspirated into a test tube containing 10ml
sterile water.  Using an haemocytometer, I determined the initial
concentration.  Then, as all good bacteriology students learn, serial
dilutions until a desired concentration is obtained.  I used a
concentrations that ranged between 50-100 spores per ml.

I took .25 or .5ml of the final concentration and spread plated on 4.0%
water agar.  I examined the plates every 6 hours or so until I was
familiar with when the spores would germinate.  This can easily be seen by
inverting the plates under a dissecting scope and using a bright
transmitted light source.  The reflective quality of the spores and
germtubes can be distinguished from the background of the water agar.  By
spreading a known number of spores per plate, you can easily pick up
individual, developing isolates for transfer to pure culture.

I hope this is useful.

Mike
========================================================================
T. Michael Likins                               810likin at vdacs.bitnet
State Plant Pathologist                      vdacs3 at vtvm1.cc.vt.edu
VA Department of Agriculture             804/786-3515
Richmond, VA  23209                        804/371-7790 (fax)