PREPARED BY: Peggy A. Wenk, BS, HTL(ASCP)SLS

ADOPTED BY: _______________________________ DATE:

SUPERSEDES:

PURPOSE:

This stain demonstrates Helicobacter pylori, which is present in many gastric biopsy specimens with active gastritis. It can also be used to non-differentially stain bacteria and for the demonstration of some parasites.

PRINCIPLE:

Diff-Quik is a modified Giemsa/ Romanowsky stain. Diff-Quik solution I contains eosin, which gives a pink color to cytoplasm. Diff-Quik solution II contains Azure A and Methylene Blue. The methylene blue is an impure dye, and will oxidize into azure A, azure B, and methylene violet, thereby giving a wider range of colors. Both solutions contain buffers, which establish the best pH for staining.

FIXATION:

Formalin fixed tissues preferred.

Hollandes fixed tissues may stain false negative, due to the Hollandes solution creating holes in the walls of the bacteria. Steiner and Steiner is recommended for Hollandes-fixed tissues.

TECHNIQUE:

Cut routine paraffin sections at 5 um.

CONTROL:

Gastric tissue with H. pylori preferred.

Any tissue with small, gram negative bacteria may also be used, such as E. coli.

QUALITY CONTROL:

2. Use a microscope to determine the assess quality of staining.

EQUIPMENT:

Erlenmeyer flask, graduated cylinders, forceps.

DIFF-QUIK Fixative contains methanol which is poisonous. May be fatal or cause blindness

if swallowed. Cannot be made nonpoisonous. Liquid and vapor are flammable. Harmful if

inhaled or absorbed through skin. May cause skin and eye irritation.

DIFF-QUIK Solution I - Contains Eosin, Buffer and 0.001% sodium azide.

DIFF-QUIK Solution II - Contains Azure A, Methylene Blue, and a buffer.

ACETIC ACID is an acid. Add drop by drop to solution. May cause skin/eye burns.

REAGENTS:

DIFF-QUIK SOLUTION I

DIFF-QUIK SOLUTION II (Baxter catalog #B4132-12)

95% alcohol 368.0 mL

Distilled water 132.0 mL

Acetic acid (HCOOH) 1.25 mL

PROCEDURE - Diff-Quik:

4. Place slides directly into Diff-Quik Solution II 4 minutes

5. Rinse quickly in 2-3 changes of distilled water 1-2 seconds each

6. Pour on, pour off quickly Acid-Alcohol Solution 1-2 seconds

7. Rinse in distilled water, 2-4 changes 5 seconds each

8. Allow slides to air dry completely 2 minutes or longer

9. Coverslip with synthetic mounting media

RESULTS:

Bacteria, including Helicobacter pylori deep-blue

Fungus deep-blue

Nuclei blue

Cytoplasm, collagen, muscle, cytoplasm varying shades blue and pink

PROCEDURAL NOTES:

1. Check slides after water rinse (Step #6). Slides may be restained, if necessary.

2. If tissues have been fixed in Hollandes, use the Steiner and Steiner procedure, as the bacteria may be false negative is stained with the Diff-Quick.

3. Slides may be left in Diff-Quik Fixative solution (blue) longer than 1 minute, without harm to the tissue.

4. IF SPECIMEN IS A FROZEN SECTION: Use the following procedure:

     Do NOT deparaffinize and dehydrate (Step #1)

     Place in Diff-Quik Fixative solution (blue) 1-5 minutes

     Go directly to Step 2.

5. IF SPECIMEN IS A CYTOLOGY SPECIMEN: Use the following procedure:

     Deparaffinize and hydrate through absolute alcohol.

     Place in Diff-Quik Fixative solution (blue) 1 minute

     Go directly to Step 2.

6. If specimen must go through the Diff-Quik Fixative solution, tissue may be left in solution longer than 1 minute, without harm to the tissue.

REFERENCES:

Manufacturer's notes that come with Diff-Quik Solutions

Ray Skipper and Don B. DeStephano: A Rapid Stain for Campylbacter pylori in gastrointestinal sections using Diff-Quik. J. Histotechnol 12:303-304, Dec. 1989