5/8/98

A user in our lab has started a project that requires the staining of many

TEM grids, dozens, or more, at a time. She is really in mass production

mode and is frustrated by keeping track of drops in dishes.



She saw a commercial automatic grid stainer ($10K, choke) and thought we

should get it. That's kind of pricey for me to consider without some other

feedback.



I would like to help her out. I am not sure most users in our lab (a fairly

low volume central campus, general EM lab) would ever need an automatic

stainer. Maybe you could give me some ideas about how useful and practical

they are for routine use.



If they are not what we need, is there anything I could get to help her

with this project? How useful are the little gizmos in some of the

catalogs? What is your favorite? Have you tried any of them and been happy,

or not?



Thanks



Jonathan Krupp

Microscopy and Imaging Lab

University of California

Santa Cruz, CA 95064

(408) 459-2477

FAX (408) 429-0146

jmkrupp@cats.ucsc.edu

I take rings-shaped slices of BEEM capsules(slices

are about 4-5 mm high); cut perpendicular slits, evenly spaced,

along one side of the ring, leaving room on the other side for pinching

the ring ,which opens the slits. Then you place grids into the slits,

grabbing just the rim of the grid. When you let go of the pinching

fingers, the grids are held in place. It takes a bit of practice to

get the slits spaced right and to learn how to get several grids

into the slits without losing the first ones you put in. Then I

stain using 10ml beakers, submersing the grid rings in the stain,

and rinsing by bobbing the rings up and down in water, holding

the rings with tweezers. You can blot excess water between the wet grids

with points of filter paper.It takes more stain but I think the results

are cleaner. Sometimes I produce a "lucky" ring that will hold

6 grids for me!



Julie Gross

Dept. of Anatomy

UCONN Health Center

Farmington, CT 06029

jgross@neuron.uchc.edu

I use the SynapTek grid staining system, also, to stain dozens of grids. To

overcome the problem of turbulence created in the staining pipettes, a

clever person who preceeded me melted down the pipette tip, presumably with

a Bunsen burner, to create a larger opening at the tip's end.



Missy Josephson

Eleanor Josephson

Department of Anatomy MC-3405

263 Farmington Ave.

Farmington, CT 06030-3405

ejosephs@neuron.uchc.edu

Some of the gizmos are very useful and inwexpensive. The one I like to use is

the Synap Tek GridStick kit, available from Ted Pella, for around $25. Kit

includes 5 gridstick bars which hold 11 grids each in a simple pipet. Only small

volumes of stains are required, almost no contact with air during staining, can

do rinses with them, easy to use and they don't make a mess. I don't use the

flow-limiting plugs that come with them, tho. Just be careful to not intake or

expell liquids too fast, to avoid turbulent flow. These work great for staining

ultra-thin epoxy or acrylic sections, not sure sure about thick sections.



Another kit that allows you to process even more grids simultaneously, is the

Hiraoka Grid Staining Kit, available from Polysciences. Its does up to 40 grids

at once, and uses a trough for stains over which you invert a plastic square

with slots in that hold the edge of the grids by pressure. I've not used it as I

never have to do that many grids at a time, care is required to avoid a mess,

but any careful, patient, motivated person who needs to stain lots of grids

would be able to use it successfully.



I have no financial interest in Pella or Polysciences, just a satisfied user of

these two products.



I'd like to hear about what others use as alternatives to the ol' classical

method of drops on Parafilm surrounded by a ring of sodium hydroxide pellets,

inside of a covered Petri dish.







Gib Ahlstrand, Minnesota Micoscopy Society Newsletter Editor

Electron Optical Facility, University of Minnesota, Dept. Plant Pathology

495 Borlaug Hall, St. Paul, MN. USA. 55108 (612)625-8249

612-625-9728 FAX, giba@puccini.crl.umn.edu



Jon,

We have an LKB ultrostainer in our lab, I think now supplied by Leica.

We cannot recommend it enough!, especially in a multiuser facility like

ours, (and yours). Can stain 38-40 grids at a time, low contamination of

sections, no exposure to nasty chemicals and very easy to use.

The small plates which come with the stainers only hold about 20 grids, we

actually buy one from another supplier which holds 40, which is better for

us (Hiraoka staining plate).

The only problem sometimes, especially with our end of the world is supply

of the stain bags. But over i your neck of the woods, it probably isnt too

bad.

We calcualted I think, that in the first 2 years of use, it had saved about

6months worth of staining time!



Hope this is of some help, as I mentioned, if you can get one, and you have

lots of grids, go for it! OUrs is about 10-12 years old now, and have had

minimal troubles with it. Not like some of the newer equipment we have

recently purchased in our lab!



All the best,



Rich.



richard.lander@stonebow.otago.ac.nz

Richard Lander

Electron Microscope Technician

South Campus Electron Microscope Unit

Otago School of Medical Sciences

P.O. Box 913

Dunedin

New Zealand.

Tel. National 03 479 7301 Fax. National 03 479 7254

We use the flexible plastic piece from the Hiraoka kit, but put a

large puddle of stain over the sections rather than inverting the

holder over the square dish of stain.

For washing we support the plastic by a wire frame over a

large beaker, and dribble water over it through a Pasteur pipette

connected through a hose to a water container.





Sally Stowe





----------------------------------------------------------------------

Dr Sally Stowe |Email: stowe@rsbs.anu.edu.au

Facility Coordinator |Post: Box 475

ANU Electron Microscopy Unit |ANUEMU (RSBS)

Ph 61 (0)2 6249 2743 |Australian National Univ.

FAX 61 6 249 4891 |Canberra, Australia 2601

http://online.anu.edu.au/EMU/home.htm

We also use the UltroStainer and are very happy with it. It is now about 5 years old and receives daily use. Although we did have a problem with it while it was relatively new, it has not given us any problems since. It is reliable, clean, and almost maintenance free. Several years ago there was problems with the stain bags, but the company seems to have fixed that now. One caution about autostainers, they do stain both sides of the grid. And I am not convinced that this is the cheapest way to proceed.



Peter Steele

STEELEP@allkids.org

I also concur with Sally's Hiraoka kit, which we also use in the same

manner. Except that to wash the grids, we first dump the stain, and

then grab the plastic with locking hemostats and then vigorously shake

it up and down in water through 3 beakers of water, 60 times per beaker.

In order to ensure that the sections don't float off, we first dry the

sections on the grids for 5 minutes in a drying oven. Then there is no

force in the universe which can remove the sections.



For the Lead Citrate stain, we use a glass petri dish with 4 KOH pellets

to remove the CO2. We hold the Hiraoka a bit higher by supporting it on

a plastic frame used for paraffin embedding.



The method works well.



Garry

GBurgess@exchange.hsc.mb.ca

Jon-

there used to be two type of multi stainers on the market, I've used:

1) Hiroka stainer- basicly it is a soft piece of plastic with small slits

cut in it, when you bend the plastic the holes open and release grids,

when flat holes close and hold grids. flip it upside down in staining

solution ... and usually all grids stay in and get stained.

2) Polysciences (I think) multi grid stainer, basicly a grid box with

holes in the back side of the holder (inner part) and holes on the front

side of the cover (outer part) these work very well, and can probably be

made quite easily with the appropriate drill bits and some grid boxes...



good luck

-Mike

MIKE ROCK

merock@du.edu

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