[Histonet] Stain for H.Pylori

Sun Dec 7 06:41:18 CST 2003

Alcian yellow is not being made anymore, anywhere.

ANATECH, Ltd. has a substitute, called HP Yellow, if I remember correctly. The usual TB/AY staining procedure has to be modified slightly, in order for this yellow dye to bind, but according to the procedure I read, it's just one extra solution. 1-800-ANATECH. (No, I don't work for them. Just passing the information along.)

We don't do the Tol. Bl/Al Y stain. We do the Diff-Qwik - the same one that is done in cytology. It's fast, reusable (we use it about 1 week, then throw it out, but then we're staining 20-30 slides/day).

The directions say to run down to abs. reagent alcohol, then go into the DQ fixative (methanol). If you run your slides down to water, it's OK. Just put them directly into the DQ fix before staining them. I have tried to go from abs. directly into the stain, skipping the methanol. (I figured, alcohol is alcohol.) Well, it doesn't stain right if you skip the methanol fixative. Don't know why. Just thought I'd pass this on.

Our procedure is below.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI

MICRO-ORGANISM - DIFF-QUIK 

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

ADOPTED BY: _______________________________ DATE: 

     DATE
     INITIAL
     DATE
     INITIAL
     DATE
     INITIAL
     DATE
     INITIAL

      REVIEWED

      REVIEWED

      REVIEWED

      REVIEWED

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:

1. Do not use this stain with Hollandes fixed tissues. Steiner and Steiner will demonstrate these.

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

EQUIPMENT:

Erlenmeyer flask, graduated cylinders, forceps.

CAUTION: FOLLOW STANDARD SAFETY PROCEDURES WHEN PREPARING STAINS.

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 FIXATIVE

DIFF-QUIK SOLUTION I 

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

ACID-ALCOHOL SOLUTION

95% alcohol 368.0 mL

Distilled water 132.0 mL

Acetic acid (HCOOH) 1.25 mL

Mix together alcohol and water. Slowly add, drop by drop, acetic acid to solution. Store at room temperature in a plastic or glass container. Good for several months.

PROCEDURE - Diff-Quik:

1. Deparaffinize and place in several changes of absolute alcohol. 

NOTE: If possible, proceed to Step 2 after the absolute alcohol. However, if slides have already been run in water, go to Step 2 and extend the time in Step 2 to 2 minutes.

NOTE: If a frozen section or cytology specimen, follow directions under "PROCEDURAL NOTES."

2. Place slides directly into Diff-Quik Fixative Solution 1 minute

3. Place slides directly into Diff-Quik Solution I 2 minutes

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

  ----- Original Message ----- 
  From: Jill Cox 
  To: histonet <@t> lists.utsouthwestern.edu 
  Sent: Wednesday, December 03, 2003 11:58 AM
  Subject: [Histonet] Stain for H.Pylori

  Hello everyone,
   Has anyone done a Toluidine blue/Alcian yellow stain for H. Pylori? If so how do the Doctors like it?   Or, what other stains are you doing for that. I am trying to get away from the Genta!! Thanks in advance

  Jill Cox HT (ASCP) 
  Histology Supervisor
  Seattle Histology Lab

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