Spore sonication

[Michael Lichten]

L.J. Latus writes

>This sporulation soup is then treated with glucouronidase 
>(1:10 dilution of a crude glucouronidase extract, 15 minutes at 37oC) and 
>sonicated (5 seconds at 50% output, using a Branson Sonifier 250).  The yeast 
>are then plated in triplicate on different selective media.  I have only played
>with this a bit but so far none of the yeast have survived the treatment; I do 
>not even observe colonies on non-selective media.  I suspect that I am blowing 
>the yeast to bits.  Does anybody have any advice, particularly on tetrad 
>sonication protocols?  

You may very well be shattering them--50% output sounds a bit high.  What 
do they look like under the scope?  I sonicate tetrads at a much lower 
power output in 0.1% Tween 80, to prevent clumping.  However, you might 
want to see if the gluculase itself is killing your spores--I've found that 
some lab isolates are very sensitive to gluculase, and are in fact killed 
before stationary phase diploids!

The best advice on working out sonication conditions is to start out at low 
power, gradually increase it, and _constantly_ check the condition of your 
spores under the microscope.  Keeping everything on ice will also help 
preserve viability.  It _is_ possible to work out conditions with most 
strains where you have >95% single spores and >50% viability.

Good luck!

Michael Lichten
lichten at helix.nih.gov