6/23/99

To all,

Any tips, info., papers about the use of bacitracin when negative staining

would be greatly appreciated.

Thanks,

Kim

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Kimberly A. Riddle

Florida State University tel: 850.644.6519

Biological Science Imaging Resource

119 Bio Unit I, 4370 fax: 850.644.0481

Tallahassee, FL 32306 riddle@bio.fsu.edu

Hi Kim,

I don't know where it was published ( I think in J. of Ultrastructure

Research) but if you search by authors: Dr. Harry L. Malech & John P.

Albert. "Negative Staining of Protein Macromolecules: A Simple Rapid

Method" One of the slickest negative stains I've ever done. If all else

fails I have a copy that I can send you.

Best,

Al Coritz

Cryobiology Product Manager, Ventana-RMC

acoritz@ventanamed.com

Dear Kim,

I have appended some references for using bacitracin during negative

staining. 'Hope you would find them useful. We use bactracin as a wetting

agent routinely and get excellent results (see "Negative Staining" under

"Gallery" in our web site (http://www.cimc.cornell.edu). Please get in

touch with me if you have any questions.

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

TI: MORPHOLOGICAL DESCRIPTION OF SURFACE STRUCTURES ON

STRAIN B41 OF BOVINE

ENTEROTOXIGENIC ESCHERICHIA-COLI BEARING BOTH K99 AND F41

ANTIGENS

AU: DUCHET-SUCHAUX-M. BERTIN-A. DUBRAY-G.

SO: J GEN MICROBIOL

134 (PART 4). 1988. 983-996.

AB: In order to describe morphologically the structures on the cell surface

of bovine enterotoxigenic Escherichia coli, variants of reference

strain B41 (K99+F41+) either negative for K99 and positive for F41

antigens (variants B41A, B41*C), or phenotypically negative for both

antigens (variants B41B1, B41B2, B41*CB), and a transconjugant

harbouring the K99 plasmid and expressing the K99 adhesin

[transconjugant B41 .times. H510a:H510(2)] were examined by

transmission electron microscopy using negative staining. Several

negative staining procedures were tested for strain B41 and variant

B41A: direct harvesting of strains into ammonium molybdate (2% w/v),

with bacitracin (50 .mu.g ml-1) as wetting agent, gave the best

results. Three morphologically distinct structures on the cell surface

could be identified in cultures grown on Minca medium. Firstly, thin,

filamentous, flexible fibrillar structures, presenting a helical

structure and a mean diameter of approximately 3 nm, were recognized as

K99 fimbriae, since they were present on strain B41 and on

transconjugant H510(2), but not on K99-negative variants nor on the

recipient strain H510a. Secondly, coil-like structures with a diameter

of about 17-20 nm were observed on strain B41 and on variants B41A and

B41*C. These structures appeared to consist of two more curled

filaments (diameter 3 nm) joined to coil on themselves into dense

spirals. They were very rare in variants B41B1 and B41B2 and were

absent on variant B41*CB and on a transconjugate B41* .times. B41*CB,

which had reacquired the K99 plasmid and which again exhibited K99

fimbriae. Strains B41 and variant B41A grown on 37.degree. C for 24 h

on sheep-blood agar exhibited coiled structures like those seen on

Minca medium. In contrast, after growth at 18.degree. C for 48 h (which

inhibits the synthesis of F41 antigen), coiled structures were no

longer expressed on the cell surface of strain B41 and of variants B41A

and B41*C. Thus the presence of coiled structures correlated with the

expression of F41 antigen in strains and variants, which suggests that

F41 had a coiled morphology. Finally, straight fimbriae (diameter 6.5-7

nm) were observed on the cell surface of every strain and variant.

Their expression on the cell surface was enhanced by several

subcultures in static broth, and it was inhibited by subculture on

agar, but not by culture at 18.degree. C after serial subcultures in

static broth. These facts indicated that the straight fimbriae could be

common fimbriae, and excluded their being F41 structures.

TI: NEGATIVE STAINING OF PROTEIN MACRO MOLECULES A SIMPLE

RAPID METHOD

AU: MALECH-H-L. ALBERT-J-P.

SO: J ULTRASTRUCT RES.69 (2). 1979. * EN * 191-195.

AB: A simple negative stain technique is described which is suitable for

high-resolution imaging of protein molecules in the range of 105-106

daltons. Protein solutions mixed with phosphotungstate stain containing

bacitracin are applied to cleaned naked 400- or 500-mesh Cu grids

resulting in the formation of thin films that are stable in the

electron beam. Since no additional support film is present, the stain

films are very thin and provide unusually high resolution images of

protein molecules. The method is easy and relatively artifact-free

compared to other high-resolution negative stain methods.