Warthin Starry Staining Procedure

Purpose:
This standard operating procedure (SOP) establishes the standard for staining spirochetes and other microorganisms using the Warthin Starry staining procedure.

Reagents and Materials:

Reagents

Glycine- Acetic Acid, Stock
Glycine 2.4gm
Acetic Acid 0.3ml
Distilled water 100ml

Glycine - Acetic Acid, Working
Glycine - Acetic Acid, stock 2.0ml
Distilled water 200ml

0.5% Silver Nitrate
Silver Nitrate 0 .25gm
Glycine-acetic acid, working 50ml

2% Silver Nitrate
Silver Nitrate 0 .5gm
Glycine-acetic acid, working 25ml

4% Gelatin
Gelatin 4.0gm
Glycine-Acetic Acid, working 100ml

0.1% Hydroquinone
Hydroquinone 0.05gm
Glycine-Acetic Acid, working 50ml

Silver Nitrate-Gelatin-Hydroquinone Developer
2% Silver Nitrate 10ml
4% Gelatin 25ml
0.1% Hydroquinone 15ml
Prepare immediately before use: Combine in order given making certain the solutions are mixed well after each addition.

 

Materials
Tissue slides and control slides for special stain requested.
Microwave to bake slides.
Staining rack, 20 slide capacity.
Coverslips and synthetic mounting media.

Hazards and Precautions:
See Material Safety Data Sheets.

Sample Preparations:
Paraffin tissue section slides cut at 4 microns.

Calibration and Standardization:
A tissue section slide containing spirochetes and other microorganisms is stained with each procedure.

Procedure:

1. Deparaffinize and hydrate to distilled water.

2. Place in 0.5% Silver Nitrate in a plastic coplin jar with the lid loosely applied.
Microwave at 10% power for 5 minutes. Allow the slides to remain in the solution for two more minutes.

3. Silver Nitrate-Gelatin-Hydroquinone Developer.
Microwave at 70% power for 30 seconds.
Allow the slides to remain in the solution until the sections appear brown.

4. Wash in hot running water.

5. Distilled water rinse.

6. Dehydrate, clear and coverslip.

Positive control: proliferative ileitis with Lawsonia intracellulare (Figure 1)

Results:
Spirochets: Brachyspira, Lawsonia, Leptospira - black (if well developed) (Figure 1).
Background – pale yellow to light brown.




Figure 1. Porcine proliferative ileitis with Lawsonia intracellulare (arrows) in the apical cytoplasm of hyeprtrophic enterocytes

Effective: July 1, 2004
Cherie Chapman
James R. Turk

References:

Sheehan DC, Hrapchak BR: Theory and Practice of Histotechnology, 2nd Ed, CV Mosby, St. Louis, 1980, pp. 240

Armed Forces Institute of Pathology Laboratory Methods in Histotechnology, eds. Prophet EB, Mills B, Arrington JB, Sobin LH, American Registry of pathology, Washington D.C. , 1992, pp. 214-215.

Kubo M, Nakagawa M, Kashiwazaki M, Konno S. Pathological observation on experimental swine dysentery.
Natl Inst Anim Health Q (Tokyo). 1979 Fall;19(3):83-90

Chappel RJ, Prime RW, Millar BD, Mead LJ, Jones RT, Adler B. Comparison of diagnostic procedures for porcine leptospirosis. Vet Microbiol. 1992 Feb;30(2-3):151-63.

Nietfeld JC, Franklin CL, Riley LK, Zeman DH, Groff BT. Colonization of the tracheal epithelium of pigs by filamentous bacteria resembling cilia-associated respiratory bacillus. J Vet Diagn Invest. 1995 Jul;7(3):338-42

De Brito T, Sandoval MP, Silva AG, Saad RC, Colaiacovo W. Intestinal spirochetosis: first cases reported in Brazil and the use of immunohistochemistry as an aid in histopathological diagnosis. Rev Inst Med Trop Sao Paulo. 1996 Jan-Feb;38(1):45-52. Erratum in: Rev Inst Med Trop Sao Paulo 1996 Mar-Apr;38(2):48.

Caniatti M, Crippa L, Giusti M, Mattiello S, Grilli G, Orsenigo R, Scanziani E. Cilia-associated respiratory (CAR) bacillus infection in conventionally reared rabbits. Zentralbl Veterinarmed B. 1998 Aug;45(6):363-71.

Pravettoni D, Toccacieli S, Monestiroli S, Crippa L, Canali E, Salvi S, Scanziani E. Cilia-Associated Respiratory (CAR) bacillus infection in veal calves and adult cattle. Dtsch Tierarztl Wochenschr. 2001 Sep;108(9):386-9.

Huerta B, Arenas A, Carrasco L, Maldonado A, Tarradas C, Carbonero A, Perea A. Comparison of diagnostic techniques for porcine proliferative enteropathy (Lawsonia intracellularis infection). J Comp Pathol. 2003 Aug-Oct;129(2-3):179-85.

Van Der Heijden HM, Bakker J, Elbers AR, Vos JH, Weyns A, De Smet M, McOrist S. Prevalence of exposure and infection of Lawsonia intracellularis among slaughter-age pigs. Res Vet Sci. 2004 Dec;77(3):197-202.