- Catalog order # K7164 - 4oz kit - $61.00
- K7168 - 8oz kit - $101.00
APPLICATION
Mammalian Tissue
DESIGNED TO SHOW
Helicobacter Pylori, Rickettsias, Cytoplasm, Collagen, Muscle, Erythrocytes
SPECIMEN REQUIREMENTS
Fixation: Tissue: Any well fixed tissue, however, it is suggested to use formalin fixed tissue.
Smears: Fix air dried blood or marrow films for 10 minutes in a 70% alcohol or by a 30-minute formaldehyde vapor fixation.
Sections: Cut Paraffin sections at 5 microns.
COMPONENTS AND REAGENTS
1. Wolbach's Giemsa Stain Solution - 4 oz / 120 ml
2. May Grunwald Stain Solution - 4 oz / 120 ml
3. Phosphate Buffered Stain Solution - 4 oz / 120 ml
4. 1 Stained bone marrow and three unstained controls
* Working May Grunwald Stain
Combine equal parts of May Grunwald Stain with Distilled Water. Mix. Make fresh before use.
**Working Giemsa Stain
Add 2.5 ml of Wolbach's Giemsa Stain to 50 ml of distilled water. Mix. Make fresh before use.
***Diluted Phosphate buffered rinse
Combine one part Phosphate buffered rinse solution with 6 parts distilled water. Mix.
Make fresh before use.
1% Acetic Acid
Add 1 ml of Acetic Acid, Glacial to 99 ml of distilled water.
Mix thoroughly.
PROCEDURE
1. Deparaffinize sections and hydrate to deionized water.
2. Treat sections in two changes of Methanol for 2 minutes in each change.
3. Stain sections in **Working May Grunwald stain for 6 minutes.
4. Transfer sections directly to **Working Giemsa Stain and stain for 45 min.
5. Quickly rinse sections in ***Phosphate Buffered Rinse.
6. Differentiate sections in 1% acetic acid until nuclei are well differentiated.
7. Quickly rinse sections in distilled water.
8. Dehydrate sections through alcohols. Clear in Xylene or Xylene substitute. (Increase clearing time in Xylene Substitutes)
RESULTS
Cytoplasm - Varying shades of light blue.
Nuclei-Blue to Violet
Collagen and Muscle-Pale pink
Erythrocytes-Gray to Yellow
Rickettsias-Reddish Purple
Helicobacter Pylori-Deep blue
NOTES
1. Change to fresh stains when intensity of stain decreases.
2. Staining time for the demonstration of Helicobacter Pylori can be reduced to 5 minutes in **Working Giemsa Stain.
Do not differentiate section following short staining time.
Eliminate the use of May Grunwald and stain only in **Working Giemsa Stain.
Definition of surrounding tissue may not be as intense using May Grunwald and increased staining times, but target organisms will stain a dark blue to violet.
REFERENCES
1. Laboratory Medicine: Vol. 25, No. 6 June 1994: pg. 389
2. Theory and Practice of Histotechnology; Second Edition, 1960,
Dezna C. Sheehan, pp. 156-157
3. Laboratory Manual of Histochemistry; Linda L. Vacca, Raven Press, 1985, pp. 352-353
Dev. 8/97
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