Gram, Brown-Hopps Stain Kit

gram stain kit brown and hopps

Product Options:

Part # 9124A250ml kit $134.00H
 
  

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GRAM STAIN KIT INCLUDES:

    Part 9124A
Solution A: Crystal Violet Stain 1%, Aqueous, Brown-Hopps 250 ml
Solution B: Iodine, Gram, Aqueous 250 ml
Solution C: Basic Fuchsin Stain 0.25%, Aqueous 250 ml
Solution D: Gallego Solution 250 ml
Solution E: Picric Acid-Acetone 0.05% 250 ml
Solution F: Acetone-Xylene 1:1 250 ml

 

COMPLIMENTARY POSITIVE CONTROL SLIDES: Enclosed with this kit are two complimentary unstained positive control slides to be used 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:

Xylene, ACS Part 1445
Alcohol, Ethyl Denatured, 100% Part 10841
Alcohol, Ethyl Denatured, 95% Part 10842
Acetone, ACS Part 10014

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

 

APPLICATION:

Newcomer Supply Gram, Brown-Hopps Stain Kit procedure, is a superior and consistently reliable Gram stain that uses Gallego Solution to differentiate and fix staining of gram-negative and gram-positive bacteria in tissue sections. 

 

METHOD:

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

Technique:  Paraffin sections cut at 5 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.

                                                                               

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 distilled water.
    1. See Procedure Notes #1 and #2.
  2. Stain slides in Solution A: Crystal Violet Stain 1%, Aqueous, Brown-Hopps for 2 minutes.
  3. Rinse well in distilled water, ensuring excess stain is removed.
  4. Mordant in Solution B: Iodine, Gram, Aqueous for 5 minutes.
    1. Sections should turn black.
  5. Rinse well in running tap water, ensuring excess iodine is removed. 
  6. Blot excess water from slide; decolorize one slide at a time in Acetone, ACS (Part 10014) until blue color stops running; 1-2 dips.
    1. Sections should be very light gray in color.
  7. Rinse quickly in running tap water to remove excess Acetone.
  8. Stain in Solution C: Basic Fuchsin Stain 0.25%, Aqueous for 5 minutes.
  9. Rinse well in running tap water.
  10. Differentiate sections in Solution D: Gallego Solution for 5 minutes.
  11. Rinse thoroughly in running tap water.  Blot excess water off slide, but not to dryness.
    1. Proceed with Steps #12 to #15 one slide at a time. 
  1. Dip quickly in Acetone, ACS (Part 10014); 1-2 dips.
  2. Dip directly in Solution E: Picric Acid-Acetone 0.05%; 3-10 dips.
  3. Dip quickly in Solution F: Acetone-Xylene 1:1; 5 dips.
  4. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium. 

RESULTS:

Gram-positive bacteria Blue/Violet
Gram-negative bacteria Red
Nuclei Red
Background tissue Yellow

          

PROCEDURE NOTES:

  1. Drain staining rack/slides after each step to prevent solution carry over.
  2. Do not allow sections to dry out at any point during staining procedure.
  3. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

       

REFERENCES:

  1. Brown, Robert C., and Howard C. Hopps. “Staining of Bacteria in Tissue Sections: A Reliable Gram Stain Method.” American Journal of Clinical Pathology 60.2 (1973): 234-240.
  2. Carson, Freida L., and Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press, 2015. 222-224.
  3. Luna, Lee G. Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. Gaitheresburg, MD: American Histolabs, 1992. 194-195.
  4. Modifications developed by Newcomer Supply Laboratory.