11/11/98

It's me again,



So a little while ago I asked about prep'ing suspension cells for

SEM. I got a lot of helpful suggestions which I will summarize & post here

when I get the time. It ends up being that the guy grew them on gelatin

coated coverslips and chamber slides which are WAY too big for my cpd. So

I'm either going to have to try to cut or break them to make them fit.

Or I was considering HMDS, has anybody out there used HMDS for

things as delicate as cells? If you have could you e-mail me with

protocols? I have the one that works for bugs and I have the HMDS, I've

just never used it.

Any help you send my way will be greatly appreciated. I'm getting

sooo smart from y'all's ideas that my head hurts.

Going quietly into the SEM room,

Paula Sicurello

UC Berkeley

Electron Microscope Lab

psic@uclink4.berkeley.edu

phone: 510-642-2085

fax: 510-643-6207

http://biology.berkeley.edu/EML

This is the text of the article from the May '97 Microscopy Today. The U.

Florida web site and Shirley Pinchuck mentioned in Robin Cross' post will

have more information.



Phil



HMDS and Specimen Drying for SEM:



Hexamethyldisilizane (HMDS) is an excellent method of chemical

drying of hydrated specimens. There are several variables involved in its

use, the most easily controlled being the number of transitional steps from

100% ethanol (EtOH) to 100% HMDS and the drying temperature.

Fixation and dehydration are the same for both HMDS and CPD. Once

the specimen is in the final 100% ethanol, it must then be transferred to

100% HMDS through a graded series of ethanol-HMDS mixtures. This can follow

one of four basic paths:



Ratio absEtOH : HMDS starting from 100% EtOH going to 100% HMDS



1)100% E => 1:1=> 100% H

2)100% E => 2:1=> 1: 2=> 100% H

3)100% E => 2:1=> 1:1=> 1: 2=> 100% H

4)100% E => 3 :1 => 1:1=> 1: 3=> 100% H

(Extra gradations may be added as needed, for instance between the 3 :1 -

1:1 and 1:1 - 1: 3 steps)



After the final transitional step, make 3 changes in HMDS (the last

two steps can sometimes be skipped). Dry from the last 100% HMDS step, or

exchange with new 100% HMDS one final time then dry. There should be just

enough HMDS in the container to cover the specimen, any more just increases

the drying time.

Time in these steps will usually be the same as that used in the

final 100% EtOH steps. However, the time can apparently be extended will

little ill effect on the sample. Incomplete transition from EtOH to HMDS is

a worse source of problems than extended time in HMDS.

Choice of steps is basically determined by sample density, and the

permeability (to HMDS and ethanol) of the least permeable structures in the

sample. Microorganisms can usually be done with the first series, animal

tissues need the second or fourth series, and plants require the fourth

series,

or even more gradations because of their cell walls.



Drying is done at either:

25º C = room temperature 8 hr => overnight

37º C }

45º C }1=>4hr

60º C }

(Drying time by both temperature and volume of fluid.)

The higher the temperature, the shorter the drying time, but the

quality of results may also vary. Microscopic unicellular algae did best at

60º C, fish skin at room temperature, bacteria equally well at room

temperature and 60º C.

HMDS may have a significant time advantage over CPD. If more

specimens are being processed that can fit in the CPD chamber, then the

times in the transitional fluids and for drying will be much less than the

time necessary for CPD. The greater the number of samples that can be batch

processed, the greater the time advantage for HMDS.

HMDS has another advantage: if you can find containers that seal

tightly enough (HMDS likes to evaporate given any chance at all), samples

can be collected in the field, processed to 100% HMDS, then stored in vials

filled with HMDS and transported long distances from remote sites - like

from

Antarctica to Chicago, Illinois. The samples are protected by the fluid,

and at least for some specimens, so fewer artifacts than specimens stored

in fixatives or alcohol. Dried specimens are of course fragile.

HMDS is not the cure-all for specimen drying. It can introduce it's

own distorting artitfacts and some samples, biological ones in particular,

still shrink after drying as they do with CPD or freeze-drying. Some

specimens do poorly when dried from HMDS. Plant tissues in particular may

not do well, and may be better off dried by CPD. Also, if the specimen is

going to be studied for elements that are labile, or in solution, standard

fixation and dehydration methods wonít work. Cryo techniques must be used,

and if the specimen is to be examined in an unfrozen state, for example to

look at structures and elements within cavities that would be obscured by

ice if left hydrated, then the specimen must be dried by freeze-drying

methods.

All of this information is empirical. Theoretical explanations and

any modifications for particular samples are welcome!

A final note: HMDS must be used in a flume hood! A sniff of it will

clear the sinuses back to the foramen magnum.



--MT



****be famous! send in a tech tip or question***

Philip Oshel

Technical Editor, Microscopy Today

PO Box 620068

Middleton, WI 53562

(608) 833-2885

oshel@terracom.net

Shirley Pinchuck, in this lab, has done a fair amount of work using

HMDS, including comparisons of HMDS with other methods such

as CPD, cryo-SEM, etc. I will ask her to fax you her protocols as

well as an abstract of a conference presentation on some of the

comparative work.



I hope this helps.



Regards



Robin





Robin H Cross

Director : EM Unit, Rhodes University, Grahamstown, South Africa

eurc@giraffe.ru.ac.za - tel: +27 46 603 8168 - fax: +27 46 622 4377

http://www.ru.ac.za/affiliates/emu/em.htm



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