8/26/97

I have to dry some bacteria grown on glass coverslips shortly for SEM

from acetone to liquid CO2 i.e. critical point drying. I would appreciate

advice in the next 24 hours about what times are recommended in the

drier.

Unfortunately, the 'customer' has used quite a few 22 x 22 mm

coverslips and I only have the original Polaron drier, without any

specially-made coverslip holders, so it looks like three will fit in gauze

baskets which I use for baby squid.

Regards - Keith Ryan

Plymouth Marine Lab., UK

KPR@wpo.nerc.ac.uk

First, do you *have* to CPD? I've used HMDS very successfully for bacteria,

in some cases retaining the "slime" sheath (that depends more on

dehydration).

5 min. changes, 100% Ac => 1:1 Ac:HMDS => 3 X 100% HMDS, dry at room temp

or 60 C

Air drying straight from acetone can work for bacteria nicely--do you have

time to experiment?

For CPD, use 3 to 5 changes of CO2, with 2 to 5 minutes soaking in CO2

between changes (time by slime coat). Your real problem is going to be

turbulence on filling and emptying the chamber, so this will have to be

done *very slowly* so has not to disturb the coverslips.

Note: this is Ed Basgal's design and idea!:

A cover-slip holder can be quickly made by cutting notches in a

polyethylene or glass tube (not tygon), just wider that the thickness of

the coverslips, so the slips fit down into the notch:

_ |_<--cover slip fitting into notch

| | |

|__|

place tube into a polyethylene syringe body or centrifuge tube that's been

fenestrated by a mad perforator. Cap both ends.

Phil

Philip Oshel

Station A

PO Box 5037

Champaign, IL 61825-5037

(217) 355-1143

oshel@ux1.cso.uiuc.edu

I have examined microorganisms in the SEM directly, in their hydrated

state with greatest success. I have used sputter coated or uncoated

powdery mildew and rust colonies, and have about 30min before the

spores dehydrate; any other treatment disturbes the delicate

structures. The minute amounts of free water involved have not caused

any problems with the vacuum or contamination; you obviously want to

be sensible about this. One thing to remember, however, is that you

will see the cells as they are, slime and all, which may obstruct

interesting features of the bacteria.

If you have time to experiment, it might be well worth trying the

simplest approach.

best wishes

Stephan Helfer

Sincerely

+-----------------------------------------------------------------

|Dr Stephan Helfer, SSO

|Senior Mycologist - MSc Course Director

|

|Royal Botanic Garden Edinburgh, Inverleith Row, EDINBURGH EH3 5LR,

|Scotland UK

|

|http://www.rbge.org.uk

|

|phone: +44 (0)131 552 7171 ext 280

| or +44 (0)131 459 0446-280 (direct digital VoiceMail line)

|fax: +44 (0)131 552 0382