Steiner-Chapman Modified Silver Stain Kit

 

 

No Uranyl Nitrate!

Stains spirochetes, Helicobacter pylori, Legionella pneumophila, other nonfilamentous bacteria and fungus. Eliminates the use of Uranyl Nitrate.

Product Options:

Part # 9172A 250ml kit $224.40 H
Part # 9172B 500ml kit $394.70 H

 

                                                                                                 

STEINER-CHAPMAN MODIFIED SILVER STAIN KIT INCLUDES:

Part 9172A Part 9172B
Solution A: Zinc Formalin Sensitizer 250 ml 500 ml
Solution B: Silver Nitrate 1%, Aqueous 250 ml 500 ml
Solution C: Gum Mastic 2.5%, Alcoholic 175 ml x 2 350 ml x 2
Ingredient D: Hydroquinone, Powder 5 Grams 10 Grams
Mini Sampling Spoon

 

COMPLIMENTARY POSITIVE CONTROL SLIDES: Enclosed are two complimentary unstained positive control slides for the initial verification of staining techniques and reagents.  Verification must be documented by running one Newcomer Supply complimentary positive control slide along with your current positive control slide for the first run. Retain the second complimentary control slide for further troubleshooting, if needed.

Individual stain solutions and additional control slides may be available for purchase under separate part numbers.

 

Additionally Needed:

Hydrochloric Acid 5%, Aqueous Part 12086 (for acid cleaning glassware)
Xylene, ACS Part 1445
Alcohol, Ethyl Denatured, 100% Part 10841
Alcohol, Ethyl Denatured, 95% Part 10842
Acidulated Water pH 4.0-4.1 Part 10013
Coplin Jar, Plastic Part 5184 (for microwave modification)

 

For storage requirements and expiration date refer to individual bottle labels.

 

APPLICATION:

Newcomer Supply Steiner-Chapman Modified Silver Stain Kit procedure, with included microwave modification, is used for staining spirochetes, Helicobacter pylori, Legionella pneumophila, other nonfilamentous bacteria and fungus. This modified method eliminates the use of Uranyl Nitrate and its regulatory and disposal requirements.

 

METHOD:

Fixation: Formalin 10%, Phosphate Buffered (Part 1090)

Technique:  Paraffin sections cut at 4 microns

Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

All Newcomer Supply Stain Kits are designed to be used with Coplin jars filled to 40 ml following the staining procedure provided below.  Some solutions in the kit may contain extra volumes.

 

PRESTAINING PREPARATION:

    1. If necessary, heat dry tissue sections/slides in oven.
    2. All glassware/plasticware must be acid cleaned prior to use.
        1. See Procedure Notes #1 and #2.
    3. Preheat Solution A: Zinc Formalin Sensitizer to 60°
    4. Preheat Solution B: Silver Nitrate 1%, Aqueous to 60°
    5. Prepare Hydroquinone Working Solution; combine and mix well.
        1. Ingredient D: Hydroquinone, Powder 5 gm (or one rounded scoop with reusable mini sampling spoon)
        2. Distilled water     25 ml
        3. Save for use in Step #15.
    1. Do not preheat solutions if using Microwave Modification.

 

STAINING PROCEDURE:

    1. Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each. Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each.  Wash well with Acidulated Water pH 4.0-4.1 (Part 10013); two changes, 5 minutes each.
        1. See Procedure Notes #3 and #4.
    2. Sensitize slides in preheated Solution A: Zinc Formalin Sensitizer (Step #3) in a 60°C water bath or oven for 15 minutes.

        Microwave Modification:  See Procedure Note #5.

        1. Place slides in a plastic Coplin jar containing Solution A: Zinc Formalin Sensitizer and microwave to 60°C.
        2. Remove from microwave; incubate and agitate in warm solution under fume hood for an additional 5 minutes.
        3. Heating Zinc Formalin Sensitizer will release fumes. Use appropriate protective actions.
    1. Rinse in several changes of Acidulated Water pH 4.0-4.1 (Part 10013).
    2. Place slides in preheated Solution B: Silver Nitrate 1%, Aqueous (Step #4) and incubate in 60°C water bath or oven for 1 hour.

                Microwave Modification: 

        1. Place sides in a plastic Coplin jar containing Solution B: Silver Nitrate 1%, Aqueous and microwave to 60°C.
        2. Remove from microwave and let stand an additional 90 seconds; agitate occasionally for even heat distribution.
    1. Rinse well in several changes of distilled water.
        1. Excessive rinsing may cause nuclei to pick up silver.
    2. Dip briefly in two changes each of 95% and 100% ethyl alcohols.
    3. Place in Solution C: Gum Mastic 2.5%, Alcoholic for 5 minutes.
    4. Air-dry slides for 1-5 minutes until slides are milky white.
    5. Prepare fresh Reducing Solution by combining:
        1. Solution C: Gum Mastic 2.5 %, Alcoholic 15 ml
        2. Hydroquinone Working Solution (Step #5) 25 ml
        3. Filter, then add and mix well:
        4. Solution B: Silver Nitrate 1%, Aqueous            3 ml
    6. Rinse air-dried slides quickly in distilled water.
    7. Place in fresh Reducing Solution. Incubate in 45°C water bath for 10-30 minutes with frequent agitation; examine microscopically at 10 minutes.
        1. Check staining progress at timed intervals.
        2. Tissue will turn tan in color; bacteria will be black when the tissue reaches a golden brown color.
        3. Dip in warm distilled water before and after examination.

        Microwave Modification:  See Procedure Note #6.

        1. Heat slides in a plastic Coplin jar containing fresh Reducing Solution and microwave at 45°C for 30 seconds.
        2. Remove from microwave; continue to incubate in warm solution for an additional 2 minutes.
    1. Wash for 3 minutes in running tap water; rinse in distilled water.
    2. Dehydrate in two changes each of 95% and 100% ethyl alcohol. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

 

RESULTS:

Spirochetes Dark brown to black
Helicobacter pylori Dark brown to black
Legionella pneumophila Dark brown to black
Nonfilamentous bacteria and fungus Dark brown to black
Background Golden brown

 

PROCEDURE NOTES:

    1. Acid clean all glassware/plasticware (Part 12086) and rinse thoroughly in several changes of distilled water.
    2. Plastic (Part 5500), plastic-tipped or paraffin coated metal forceps must be used with any silver solution to prevent precipitation of silver salts. No metals of any kind should be in contact with any silver solution. Only glass thermometers should be used.
    3. Drain slides after each step to prevent solution carry over.
    4. The use of Acidulated Water pH 4.0-4.1 rinses (Steps #7 and #9) is recommended for proper tissue pH and enhanced staining.
    5. The suggested microwave procedure has been tested at Newcomer Supply. This procedure is a guideline and techniques should be developed for use in your laboratory.
    6. The Reducing Solution contains alcohol and will reduce its boiling point. Do not boil solution. Adjust microwave times and power levels accordingly.
    7. Some xylene substitutes may diminish spirochete staining. If using a xylene substitute closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

 

REFERENCES:

    1. Carson, Freida L., and Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press, 2015. 238-239.
    2. Chapman, Clifford, and Lorelei Margeson. “Use of Zinc Formalin as a Sensitizer in Silver Stains for Spirochetes.” The Journal of Histotechnology 2 (1996): 135-138.
    3. Steiner, Gabriel, and Grete Steiner. “New Simple Silver Stain for Demonstration of Bacteria, Spirochetes and Fungi in Sections of Paraffin Embedded Tissue Blocks.” Journal of Laboratory Clinical Medicine 29 (1944). 868-871.
    4. Modifications developed by Newcomer Supply Laboratory.