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Diff-Quick II Staining Protocol for Helicobacter pylori
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Diff-Quick II Staining Protocol for Helicobacter pylori

Principle: Helicobacter (formerly Campylobacter) pylori is a large curved gram negative bacillus that often colonizes the mucosal surface of the stomach. The Diff-Quik II stain, similar in action to the Giemsa stain, stains the bacillus fairly sensitively, though not at all specifically.

Specimen: The usual specimen is a gastric or duodenal biopsy specimen, fixed in 10% formalin or neutral buffered formalin, paraffin processed, and cut in the usual fashion. Diff-Quik II can also be used as a general bacterial stain for other tissues.

Reagents: Diff-Quik II®, a proprietary mixture of the thiazine dyes methylene blue and azure A in a water solution with a buffer, is manufactured by American Scientific Products, and the name is a registered trademark. Generic equivalents of this stain exist, such as Hema-Diff Stain Kit can be purchased from IHC World at IHC Store. Store at room temperature. Dispose of it down the drain, with plenty of water.


1. Deparaffinize sections and hydrate to water.

2. Stain 2 to 3 minutes in Diff-Quik II stain in a Coplin jar.

3. Rinse rapidly in water.

4. Dehydrate rapidly in 100% alcohol and pass to xylene or xylene substitute.

5. Mount in resin.


Run a control with every stain run. The control may be a gastric biopsy specimen known to be positive, or a specially prepared control (see reference below).


Bacteria and fungi are stained dark blue, with very distinct morphologic features. Cell nuclei and other histological structures are also distinctly blue. Helicobacter pylori is seen as large curved, helical, or gull-wing shaped bacilli. Gastrospirillum hominis is clearly stained and easily identified, in the author's experience of a single case.


1. Ray Skipper and Don B. DeStephano. A rapid stain for Campylobacter pylori in gastrointestinal sections using Diff-Quik. J. Histotechnol 12:303-304, Dec. 1989.

2. Billie L. Swisher and Mabel A. Nicholson (CDC, Atlanta). Development of staining controls for Campylobacter pylori. J. Histotechnol 12:299-301, Dec. 1989.

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