Help! Single ascospore isolate
plantpath at aol.com
Mon Nov 28 10:20:22 EST 1994
In article <3bclcb$cv3 at kitten.umdc.umu.se>, Xiao-Ru.Wang at genfys.slu.se
<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.
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)
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