9/3/98

Does anyone know how long you can keep Formvar in solution? I need to make

perfect films. I have a lot of Formvar in Ethylene Dichloride, but it has

been on the shelf unopened since 1990. Can I use it?

Sally Shrom

sally@retina.anatomy.upenn.edu

to light, you tend to form HCl molecules in the dichlorethane weakening the

formvar.

Hendrik O. Colijn

colijn.1@osu.edu

Formvar solutions deterioate due to atmospheric moisture and age. This

causes holes. If not holes, then "measles" (light areas in the film). It

is not worth ruining sections because one has used old Formvar solution.

Buy or make new from powder. Make a few films and check in the TEM for

stability, thickness, etc., before making a thousand.

hcrowley@odin.cair.du.edu

I use Formvar in Cloroform solution in a well stopped glass ( 100 ml,

0.25%). I am using the solution at 1 year and obtained excellent films.

With a pen, mark the level of the solution and if it evapore, refill

with Cloroform. The Butvar give excellent films too and it easy to make

very fine films.

Rinaldo Pires dos Santos

UFRGS - Dept. of Botany - Lab. of Plant Anatomy

Porto Alegre - RS - Brazil

e-mail: rinaldop@botanica.ufrgs.br

We store formvar for up to three years so far. Normally we make

small amounts an use it till it is finished. We add Molecular sieve

to keep it dry and store at ~4 Deg. C to reduce evaporation.

Mr. S H Coetzee Tell: (011) 716 2419

Electron Microscope Unit Fax: (011) 339 3407

Private bag X3 E-mail: Stephan@gecko.biol.wits.ac.za

Wits

Johannesburg

2050

is. In the back of my mind I seem to recall hearing that it is

polyvinylformal, but I'm not certain that is correct. I have just

consulted two polymer scientists in our department, and neither one of them

knows what it is, nor could they find a chemical formula for it in their

reference books.

If anyone knows what it is, I'd like to know too.

Wilbur C. Bigelow, Prof. Emeritus

Materials Sci. & Engr., University of Michigan

Ann Arbor, MI 48109-2136 e-mail: bigelow@umich.edu;

Fx:734-763-4788; Ph:734-764-3321

Formvar. In the book 'Techniques for Electron Microscopy' D. H. Kay, Ed.,

Blackwell Scientific, 1965 I find a statement indicating that Formvar is

Polyvinyl Formal (p. 60)

In the book 'Polymer Chemistry' by M. P. Stevens, Oxford Univ. Press,

1990, p.302, I find that the reaction of vinyl alcohol with butyl aldehyde

produces a polymer called polyvinyl butyral. By analogy, if vinyl alcohol

were reacted with formaldehyde (HCHO) one might assume it would produce

polyvinyl formal. If this is so, AND IT IS ONLY A GUESS, then by analogy

the chemical formula for the repeating unit in the polymer chain might be:

CH2

/ \

-[CH2-CH CH]-

| |

O O

\ /

CH2

I hope this formula survives the process of being transmitted across the

internet. This word processer is not ideal for writing organic chemical

formulas.

Wilbur C. Bigelow, Prof. Emeritus

Materials Sci. & Engr., University of Michigan

Ann Arbor, MI 48109-2136 e-mail: bigelow@umich.edu;

Fx:734-763-4788; Ph:734-764-3321

According to the free sample, yes, I said free sample, I got from

Monsanto, Formvar is a "polymer from polyvinyl alcohol and formaldehyde

as as copolymer with polyvinyl acetate". If that is not enough

information you could call Monsanto in St. Louis. I believe that it was

originally developed to coat copper wire. Note that are several

different types of Formvar. I think the type us EM folks use is 15/95

but I could be wrong.

Geoff McAuliffe, Ph.D.

Neuroscience and Cell Biology

Robert Wood Johnson Medical School

675 Hoes Lane Piscataway, NJ 08854

voice: (732)-235-4583; fax -4029 e-mail: mcauliff@umdnj.edu

overhead transformer manufacturing firm and they buy the stuff by the

barrels for coating the wire (not just copper). It is a different

grade and formulation, otherwise I would've been tempted to never buy

the stuff again after purchasing a 55 gal drum of it...

jpshield@arches.uga.edu

John Shields

Can people tell me their favourite method for adjusting thickness of formvar

films. I'm trying concentration variation but without much success. If

concentration variation is the method of choice what are suitable % ranges

to try.

Chris Gilpin

cgilpin@fs1.sem.man.ac.uk

1. Dipping method: 0.25 - 1%

2. Drop on water method: 1 - 2.5%

3. Increase thickness in method 1 by a multiple dipping process.

Charles Duvic, Chemist

LADD RESEARCH

13 Dorset Lane

Williston, VT 05495

TEL 1-800-451-3406 (US) or 1-802-878-6711 (FROM ANYWHERE)

fAX 1-802-878-8074

e-mail ladres@worldnet.att.net

web site http://www.msa.microscopy.com/SM/LADD

The way we do this is by taking a graduated cylinder with a constricted end

that fits a glass slide (to conserve solution), filling it to the proper

depth with the formvar solution, and then capping the cylinder to let the

atmosphere inside become saturated with the evaporating solvent. The slide

is dipped into the formvar, then lifted out and left dangling in the

saturated atmosphere for varying amounts of time. This allows the formvar

to drain off the glass surface without drying out. The longer the drain

time, the thinner the film.

A paper clamp on a piece of wire is all you need to hold the slide, and the

wire allows the tube to remain mostly capped as you pull the slide up out

of the solution. Crude, but it works.

Let me know if you have any questions. Hope it helps.

Randy Tindall

Electron Microscope Laboratory

Box 3EML

New Mexico State University

Las Cruces, NM 88003

rtindell@nmsu (work)

I just copy the method from the lab manual which I wrote for my TEM course

as follows:

-clean the slide with clean lens paper.

-dip slide into formvar (0.3 % w/v solution in chloroform).

-draw back the slide slowly (the faster the motion, the thicker the film)

and dry the slide vertically (about 1 min).

-cut the membrane parallel to the edge of the slide with a razor blade.

-dip the slide ( with scored side up ) into water slowly at a 30 degrees

angle and let the slide sink down to the bottom of the deep dish. The film

should float off onto the water.

-place grids (polished side up) on the floating film.

-a piece of paper or parafillm is dropped onto the film.

-pick up the paper and leave it in a dust-free Petri dish.

The thickness of film can be judged by the light reflection as the method

for thin sections. Grey or silver-greay film are good for coating.

Ming Chen

mingchen@gpu.srv.ualberta.ca

(w/w). (I use only ethylene dichloride; it seems to give better separation

of film from the slide).

The thickness of film depends on the speed of withdrawal of the slide from

the solution; the faster, the thicker film. To make thin film, withdraw the

slide from the solution by SLOW and STEADY motion; but NOT out of the jar

yet. Drain excess Formvar solution by holding the slide in the vapor above

the solution for 15 seconds. This will help you getting more uniform

thickness of the film.

Seung-Geuk Shin

SUNY-ESF at Syracuse

sgshin@syr.edu

the thickness of plastic coatings. This is the sort of gadget that you would

use to separate organic solvent layers by draining them through a glass tap

at the bottom. There is a type which has a cylindrical chamber which is just

the right height and diameter to adequately cover a standard 76mm x 26mm

glass slide. The modification was to take off the narrow neck (apparently a

fairly straight-forward glass cutting exercise) so that slides could easily

be placed in the chamber of the funnel.

The technique was to put the slide in the funnel and cover with the plastic

solution. Let it settle to avoid any lumps or bubbles and then turn the

glass valve at the bottom and drain the plastic into its stock bottle or a

beaker. The beauty of this is that you can vary the flow rate of the plastic

off the slide (fast for thick and slow for thin) which is much easier than

controlling the rate of removal of a slide from the plastic. This often

means that you could get much thinner and more even layers with careful

selection of plastic, solvent and concentration (yes you do need to

experiment). You can even get thicker layers by pulling the tap out to

rapidly drain off the plastic.

Of course, do this in a fume hood.

Malcolm Haswell

Electron Microscopy

School of Health Sciences

Fleming Building

University of Sunderland

SUNDERLAND SR1 3SD

Tel (0191) 515 2872

e-mail: malcolm.haswell@sunderland.ac.uk

sectioning for years on large single hole grids using a very simple

technique which made the problem of film thickness and wrinkles a very minor one.

Even though this method does not deal directly with ways of determining

film thickness, it may help you out with the specific problem at hand. I do

not remember who originally gave me the idea for this method but I was

not the developer. It goes as follows:

1) Have your machine shop cut some thin pieces of plexiglass into the

size of glass slides. At one end, drill about a dozen holes, roughly 5mm

in diameter in an area about the size of a formvar film cast on glass

slides. These slides will serve as your template for holding your films.

2) Cast the formvar films on glass slides just like you normally do.

Usually a good silver film, not grey, will work fine. I routinely used

0.2% in ethylene dichloride when casting by immersing the slide into the

solution in a small jar, etc. We now use a film caster which lets us hold

the slide in the dichlorothane vapors after lowering the formvar solution

level. I would have to redetermine correct percentage and timing since this

method tends to give you thinner films consistantly.

3) Float the film off the glass slide and pick it up with the

plexiglass slide so the film covers the holes. Then draw the water out of the

holes by pressing the plastic slide down onto filter paper, or using small

pieces of filter paper and capillary action to draw the water out of

individual holes. Even thin films will hold nicely over the holes in the slide.

Store slides until needed.

4) Next, cut your sections using a block diameter that is fairly

similar to the size of the slit in the grid. Pick up the sections on UNCOATED

grids by gently lowering the grid to the surface of the knife boat. I put

the dull side down on the premise that the rough surface would grab the

film better during step 6. The surface tension of the water will hold the

sections in the grid opening. Transfer the grid to a droplet of water

until you are finished sectioning.

5) Now transfer the grid + sections + water droplet to a drop of stain.

Allow the section to stain, then wash by transferring through a series

of droplets of clean water. Continue to post-stain if desired and wash the

same way. Never let the grid dry.

6) Final step is to transfer the grid to a film suspended over the

hole in a plexiglass slide and let it dry down. The sections will now be

stuck to the film with NO wrinkles and minimum breakage. Also there was

minimum problem with stain percipitation, but you must use very clean water.

When ready to view, just punch out around the grid with the tip of your

forceps, grab the grid and insert into the mciroscope.

Believe me....the sections will still be there at the end!!!

I found that as long as the sections cover a substantial portion of

the open area of the grid, carbon coating was not essential. I used to do

50-100 grids worth of serial sections without loosing any. The films on the

plastic slides would hold for months so I could make a lot and store

until needed. The method really works...do give it a try.

Debby Sherman, Manager Phone: 765-494-6666

Microscopy Center in Agriculture FAX: 765-494-5896

Dept. of Botany & Plant Pathology E-mail: sherman@btny.purdue.edu

Purdue University

1057 Whistler Building

West Lafayette, IN 47907-1057