10/9/98

I am having a problem with Spurr resin. The

blocks are coming out very brittle. I am careful

when I weigh out the components and polymerize at

65C. What else would cause brittle blocks?

Another problem is that staining with 1% T blue

with 1% borax is very poor. Any suggestions would

be of help. Thanks.

--

Regards,

Gregory Rudomen

Technical Specialist

University Microscopy Imaging Center

State University of New York at Stony Brook

516-444-3126

Greg@umic.sunysb.edu

For example, I once allowed some specimen vials to remain open

overnight in a rotator - to enhance penetration. Bad idea.

Result: brittle blocks that shattered during trimming. Even traces

of moisture in Spurr's will cause this. In ordinary epoxies, this is

not a problem (Epon for example could be left open overnight

or several days without this problem developing).

John J. Bozzola, Ph.D., Director

Center for Electron Microscopy

Neckers Building, Room 146 - B Wing

Southern Illinois University

Carbondale, IL 62901

U.S.A.

Phone: 618-453-3730

Fax: 618-453-2665

Email: bozzola@siu.edu

Web: http://www.siu.edu/departments/shops/cem.html

uses), or one exposed to moisture (If you keep the bottles in a low

temperature for extended shelf life, let it warm before opening the bottle).

With bottles out of the box, I had no problem.

In my opinion, Spurr's has relatively poor cutting properties, even when the

block itself is not brittle. I usually successfully mix Spurr's (complete

homogeneous mixture including DMAE) with Epox 812 (complete homogeneous

mixture including DMP-30) to improve cutting properties, with minimal

sacrifice in Spurr's excellent viscosity. It's been very cooperative for

plant tissues.

Hello,

I apologize for providing incomplete information.

I usually mix them 1 to 1 by weight. But you can reduce the amount of Epox

812 mixture, if low viscosity is important, as in freeze-dried tissues. It's

been tested primarily on plant tissues, fixed chemically or lipophillized,

and worked well for both. It worked on some chemically fixed animal tissues

(small intestine & liver from a frog). I don't have much experience with

animal tissues and I am not sure about lipophillized animal tissues.

But, I can say generally Spurr's provides better infiltration, while Epox

812 offers better cutting properties (via better adherence to the tissue).

There has been no negative effect of mixing two, as far as I know. Thus, I

guess any tissue, which are not incompatible with Epox or Spurr's, can be

safely embedded in the mixture of the two.

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

I also forgot including the staining method with toluidine blue in the

previous message. If it is to stain 'thick section' for preliminary

observation, place a drop of your 1% toluidine onto the section, heat it on

a 70 C-hot plate until you see thin dry edge around the stain, wash the

stain gently with squirts of water.

For microautoradiographic purpose, I use following method. This applies to 1

um-sections affixed on a gelatin-coated slide.

1) Dip the slides in 10% paraformaldehyde for 1-2 min.

2) Rinse the slides in distilled water for 1 min (or rinse twice for 30 sec.

each).

3) Stain sections by dipping the slide for 1/2 to 1 min in 0.05%-0.1%

toluidine blue prepared in 0.2M Na-phosphate buffer (pH 7.0). (Stain may

precipitate over the time; shake and refilter it.)

4) Wash the slide in running water quietly for about 2 min or shorter period

(prolonged washing will result very faint staining).

5) Air-dry the slide in vertical position.

Seung-Geuk Shin

SUNY-ESF at Syracuse

sgshin@syr.edu

sgshin@syr.edu

relatively short shelf life (I usually replace mine after about 6 months or

a year). Also the anhydride hardener - NSA - is particularly prone to

absorbing moisture so if a stock bottle has been opened for a long period

and I have a problem I try an unopened one. In any event I always label

bottles with the date of arrival in the lab and the date of opening.

Some moisture may be driven out of the resin during polymerisation but not

if you seal the lids on polythene BEEM capsules.

How good is your temperature measurement? We have an old oven with a dial

setting that needs to be checked if anyone else has used it and often the

only way is to start polymerisation a couple of hours before I leave the lab

on a night, so that I can confirm the polymerisation temperature.

I hope this helps.

Malcolm Haswell

Electron Microscopy

School of Health Sciences

Fleming Building

University of Sunderland

SUNDERLAND SR1 3SD

malcolm.haswell@sunderland.ac.uk

Here is the recipe for Spurr's + Epox 812, I use.

Basically, it is 1:1 mixture (by weight) of Spurr's and Epox 812.

Each resin mixture must be completely homogenized, including accelerator,

before combining the two.

1. Prepare Spurr's in a 100 ml disposable beaker.

(Based on Spurr's original formula)

VCD -------- 10 g

DER 736 ---- 6 g

NSA -------- 26 g

DMAE ------- 0.4 g

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

Total ------ 42.4 g

2. Prepare Epox 812 in a 50 ml disposable beaker.

(Based on Luft's original formular, except for reduced DMP-30)

Epox 812 (WPE = 155*) --------- 25 g

DDSA (MW = 266) -------------- 14 g

NMA (MW = 178) -------------- 11 g

DMP-30 ----------------------- 0.5 g

*If WPE is different, proportions of each component should be recalculated.

Calculations can be found at

http://www.emsdiasum.com/ems/techdata/68.html).

3. To make 1:1 mixture, add 42.4 g of Epox 812 mixture into the 100 ml

beaker containing Spurr's and mix throughly. Amount of Epox mixture can be

reduced for hard-to-infiltrate tissues.)

4. Infiltration for plant tissues.

Transitional solvent Resin Mixture Infiltration

(propylene oxide or Time (with

diethyl ehter) rotation or occasional swirring)

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

3 part 1 part 2 hrs.

2 2 2 hrs.

1 3 4 hrs.

0 4 4 hrs.

0 4 6 hrs.

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

5. Cure blocks at 60 C for 48 hrs.

Regards,

Seung-Geuk Shin

sgshin@mailbox.syr.edu