Diff-Quik II Stain 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 3 solution II, Curtin Matheson Scientific, and are satisfactory also. (The other two components of the full Diff-Quik stain are not needed for the present procedure.) Store at room temperature. Dispose of it down the drain, with plenty of water.
PROCEDURE:
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.
CONTROL: 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).
RESULT: 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.
REFERENCES:
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.
3. Package insert, Diff-Quik® Stain Set, American Scientific Products.
AUTHOR: Procedure written by Robert S. Richmond, M.D., F.C.A.P., December 1990.
This procedure is written in NCCLS format.
Davidson's fixative
PRINCIPLE: Davidson's (sometimes called Hartmann's) fixative is a rapid fixative which gives good nuclear detail with minimal formalin pigment. Specimens cut and stain as if fixed in ordinary formalin. Davidson's fixative contains no mercury or other metals.
SPECIMEN: Davidson's fixative is particularly useful for preparing tumors, bone marrow specimens, gynecologic material, fatty breast, and medical biopsies. It may be used for testicular biopsies and for overnight fixation of fat containing lymph nodes. Fixation of small specimens is rapid. Exposure to the fixative should be limited to 24 hours (tissue may be transferred to neutral buffered formalin or 70% alcohol for storage).
PROCEDURE: Mix
REAGENTS AND SUPPLIES: Any source of these chemicals should be satisfactory. Proportions are not very critical. Reagent alcohol, or waste alcohol from staining, may be used.
QUALITY CONTROL: Davidson's fixative is clear and has no color other than that imparted by eosin. Color should be sufficient to render identification easy. The odor changes with age as esterification produces a characteristic "airplane dope" odor of ethyl acetate. Tissues placed in Davidson's fixative rapidly turn white and opaque, while blood turns brown.
SAFETY AND ENVIRONMENTAL: Acetic acid is quite corrosive. Davidson's fixative may be stored at room temperature in plastic or glass containers for an indefinite time.
REFERENCES: Davidson's fixative is not mentioned in most standard reference works on histology. It is named after William McKay Davidson, a British hematologist, and was publicized by Moore and Barr in their well-known studies of sex chromatin. Davidson's fixative is often called Hartmann's fixative because of its introduction by Dr. William H. Hartmann (later at Vanderbilt) while he was at Johns Hopkins.