Subject: Re: Help! Single ascospore isolate

From: [email-masked] (Plantpath)
Date: 28 Nov 1994 10:20:22 -0500

In article <[email-masked]>, [email-masked]
(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                               [email-masked]
State Plant Pathologist                      [email-masked]
VA Department of Agriculture             804/786-3515
Richmond, VA  23209                        804/371-7790 (fax)

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From: [email-masked] (Xiao-Ru.Wang)
Date: 28 Nov 1994 13:19:07 GMT

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