9/24/97

After each step of the UA stain and the LC stain, I rinse the

copper grids by immersion. I have four changes of water and "dip" the

grid about 40 times in each change. I am interested in changing that to

the procedure that rinses the grid by "flooding" it using a syringe or

whatever. Can someone share with me exactly how that is done ... what

type of water ... etc. Also ... do the sections ever wash off the grid

when rinsing that way??

Thanks in advance,

Sharron Chism HT (ASCP)

Electron Microscopy Lab

Harris Methodist Hospital

Fort Worth, Texas

SharronChism@hmhs.com

I published an article entiled as "A Modified Autowasher Device for

Rapidly Washing Large Numbers of EM-grids" in Microscopy Researdh and

Technique vol 26:177-179 (1993). Here I just copy the summary part for

you:

This device consists of a siphon system and 5 or 10 grid disks, modified

from the previous mode (Chen, 1973), for large numbers of grids with

ultrathin sections. This method improves the ease of assembling grids

onto the grids disk and also requires much less stain solution. This

system only takes 5 min for one single stain washing, at a maximum of 100

grids, and also avoids stain contamination. The grid disk can also be used

for immunocytochemistry work and for critical point drying of grids with

biological specimens.

Ming Chen

mingchen@gpu.srv.ualberta.ca

place copper grids, section side down, on drops of UA and LC. Inbetween

stain and after I grasp edge of copper grid and flood by dripping from top

of forcep for approx 10 sec each grid. I use Distilled, Sterile water to

rinse. any reason for change. I know several techs that rinse as you do,

but felt there needed to be changed. I've been staining as above for over

22 years.

tflore@lsumc.edu

Flores, Teresa

whatever type of aid (apparatus) you will use for staining your ultrathins:

concerning water quality: if possible, use UHQ (ultrahigh-purified water) or at least bi- (better triple-)distilled water from a quartz-glass distilling apparatus (most conveniently in your own lab!). My/our story:

our quartz-glass bi-distilling apparatus which at that time had a life-span of nearly 12 years (in which time we got no bigger problems at all) had broken down (heating wire burned through). Therefore we thought to overcome the problem in ordering >>A.bidest.sterile, "non pyrogenic" << via our hospital pharmacy (+/- every time freshly produced, etc.). In fact we got worsest stainings of our sections due to precipitates never seen before in such an amount and shapes. We thought this to be a possible source with respect to our handling in washing the grids, unclean glass ware, syringes, etc.... or just more dust in the lab or else; nothing of that all: despite using same methods for mixing up our staining solutions as usual, we were not able to locate this source of junk on the grids! When I asked at our pharmacy, how they produce their water, they showed me & told me about their apparatus: it was/is a still producing vapours by means of copper plates! An analytic chemist told me some days after, that their central analytic lab got their own distilling (UHQ) machine because of too high copper-contents of the bidistilled water of the former source. Since that info I used only their UHQ (coming along in clean glassware, most preferably quartz glass) with no precipitate-problems any more, hoping to get my old quartzglass still as soon as possible from repairing.

Hope this adds another aspect in "hunting our elephantine precipitates",

Best regards

Wolfgang MUSS

SALZBURG/Austria, Europe

W.Muss@lkasbg.gv.at

if you don't like the dip method try the dilution method, because any

flooding method with the syringe or whatever may destroy your sections,

try allowing the grid to float to the bottom of a small beaker (10-20 ml)

repeat 3-5 times, pour off the excess water to retrieve the grid, and

rember to pick it up by the edge of the grid.

-MR

MIKE ROCK

merock@du.edu

Rinse grids easily this way: Find a source of freshly, freshly, (not a

typo) distilled water. (The collecting jars for the water must be

maintained cleanly, as well as the tubing, and the water must not "sit"

for days in the jars). If you must use deionized water be aware that it

may not be as clean. That is, deionized water may be filtered through a

3 micrometer filter and then through a 1 micrometer filter. If you then

apply a 0.22micrometer filter before use, the water may still not be

really clean enough for TEM. Please be aware of all these possibilities

if you see miscellaneous dirt on your sections. What most of us do not

realize graphically is that a particle which passes through a 0o.22

micrometer filter may be really huge at a 15x mag.

At any rate, fill a clean syringe with good quality water. Attach a 0.22

micrometer filter which you have cleaned by running through it (at a

previous time) about 15cc of boiling water. (some filters contain dust

aquired during the manufacturing process). When rinsing grids allow

genrous quantities of water to run down the forceps and over the grids.

Blot with dustless filter paper.

If we have a lot of grids to do, we use the Hiroka Staining Kit. This

works really well and is easy to use, especially if one only loads the

center three or four rows of the pad.

Most important - always pay attention to your water! It has to be as

clean as you can get it.

Bye,

Hildy

HILDEGARD CROWLEY

hcrowley@du.edu