Gram, Brown-Brenn Modified Stain Kit

Used for differential staining of gram-positive and gram-negative bacteria in tissue sections, cultures and smears.

Product Options:

Part # 9123A 250ml kit $152.00 H

 

  

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GRAM, BROWN-BRENN MODIFIED STAIN KIT INCLUDES:

Part 9123A
Solution A: Crystal Violet-Oxalate Stain, Alcoholic 250 ml
Solution B: Iodine, Gram, Aqueous 250 ml
Solution C: Acetone-Alcohol 1:1 250 ml
Solution D: Basic Fuchsin Stain 0.25%, Aqueous 250 ml
Solution E: Picric Acid-Acetone 0.1% 250 ml
Solution F: Acetone-Xylene 1:1 250 ml

 

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:

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-Brenn Modified Stain Kit procedure is used for differential staining of gram-positive and gram-negative bacteria in tissue sections, cultures and smears.

 

METHOD:

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

Technique:  Paraffin sections cut at 4 microns and cultures/smears

      1. See Procedure Note #1.

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. Filter Solution A: Crystal Violet-Oxalate Stain, Alcoholic with high quality filter paper.

 

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 #2 and #3.
    2. Stain in freshly filtered Solution A: Crystal Violet-Oxalate Stain, Alcoholic (Step #2) for 30 seconds.
    3. Rinse well in distilled water, ensuring excess stain is removed.
    4. Mordant in Solution B: Iodine, Gram, Aqueous for 1 minute.
    5. Rinse well in distilled water, ensuring excess iodine is removed.
    6. Blot excess water from slide; decolorize one slide at a time in Solution C: Acetone-Alcohol 1:1 until blue stops running; approximately 1-3 dips.
    7. Place in Solution D: Basic Fuchsin Stain 0.25%, Aqueous for 3 minutes.
    8. Rinse in distilled water. Blot water from slide(s), but not to dryness.
        1. Proceed with Steps #11 to #14 one slide at a time.
    1. Dip once in Acetone, ACS (Part 10014).
    2. Dip in Solution E: Picric Acid-Acetone 0.1% until sections have a yellowish-pink color, 3-10 dips.
        1. Agitate slides until desired intensity is achieved.
    3. Dip in Solution F: Acetone-Xylene 1:1, 5-10 dips.
        1. Check control microscopically for proper differentiation.
        2. Repeat Step #12 if additional differentiation is needed.
    4. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

 

RESULTS:

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

 

PROCEDURE NOTES:

    1. For cultures/smears: Prepare within an accepted time frame a well-made culture/smear per your laboratories protocol with a focus on uniform cell distribution. Timings are based on paraffin sections and may need to be altered for optimal culture/smear staining.
    2. Drain slides after each step to prevent solution carry over.
    3. Do not allow sections to dry out at any point during procedure.
    4. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

REFERENCES:

    1. Bancroft, John D., and Marilyn Gamble. Theory and Practice of Histological Techniques. 6th ed. Oxford: Churchill Livingstone Elsevier, 2008. 312-313.
    2. Brown, J.H., and L. Brenn. “A Method for the Differential Staining of Gram Positive and Gram Negative Bacteria in Tissue Sections”.Bulletin of The Johns Hopkins2 (1931): 69-73.
    3. Luna, Lee G. Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. Gaitheresburg, MD: American Histolabs, 1992. 188-189.
    4. Modifications developed by Newcomer Supply Laboratory.