thanks in advance.

Rajesh Patel
rpatel@umdmj.ufl.edu

Hi Rajesh
We have routinely prepared Aspergillus small plugs for TEM using an overnight fixation in 2.5%GA in phosphate buffer, followed by several buffer washes and postfixation in 0.5 - 1% osmium tetroxide for 1 hour (all at 4C). After a distilled-water rinse, en bloc staining/fixation with 0.5% uranyl acetate in 30% acetone (30 min), followed by graded acetone series, infiltrated with 50% Spur's resin for 4 hs and overnight in full resin.
Two questions to the forum, though:

1. It is interesting that the cultures will float until (approx) the 50% acetone step. Although osmium vapour will fix those exposed areas, I wonder about the role played by GA (or even U Ac) when it comes to conidiophores and conidia. The results are good anyway.

2. I have know users to favour the use of an equal-parts mixture of GA and osmium at fairly high concentrations for SEM. How widespread is the use of this protocol? Given the instantaneous cross-reactivity between the two fixatives, I really wonder about its usefulness.

Any comments?

James Wesley-Smith
wesleysm@biology.und.ac.za

Aspergillus freezes quite nicely. If you have the set-up for plunge freezing and freeze substitution I would recommend that procedure over regular chemical fixation. I can send you the protocol if you want to consider fs.

Best regards,

Beth Richardson
: beth@dogwood.botany.uga.edu