Gram Positive & Gram Negative Bacteria, Animal

Validation Stain: Gram, Brown-Brenn
Other Applicable Stains: Gram, Brown-Hopps and Gram-Twort

 

Gram (+) and Gram (-) In animal tissue with specific infection or disease.

Tissue: 
(+) Animal Tissue / (-) Lung
Adhesive or Charged Slides: 
Superfrost +

Product Options:

Part # 4254A 10/Set $53.00
Part # 4254B 98/Set $372.30

 

PRODUCT SPECIFICATIONS:

Tissue:  Positive staining animal tissue and negative staining human lung.

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

Section/Glass: Paraffin sections cut at 4 microns on Superfrost™ Plus slides.

Quality Control Stain:  Brown-Brenn quality control stained slide(s) included.

Reactivity: Guaranteed product specific reactivity for one year from date of receipt. Revalidate after one year to verify continued reactivity. 

Storage: 15-30°C in a light deprived and humidity controlled environment.

Intended Use: To verify histological techniques and reagent reactivity.

Before using unstained control slides, review the enclosed stained slide(s) to ensure that this tissue source is acceptable for testing needs.

CONTROL SLIDE VALIDATION:

With Gram, Brown-Brenn Stain Kit: Part 9123A Individual Stain Solution
Solution A: Crystal Violet-Oxalate Stain, Alcoholic 250 ml Part 10422
Solution B: Iodine, Gram, Aqueous 250 ml Part 1140
Solution C: Acetone-Alcohol 1:1 250 ml Part 10016
Solution D: Basic Fuchsin Stain 0.25%, Aqueous 250 ml Part 1011
Solution E: Tartrazine Stain 0.25%. Acetic Aqueous 250 ml Part 14016

 

APPLICATION:

Newcomer Supply Gram Positive & Gram Negative Bacteria, Animal Control Slides are for the positive histochemical staining of gram positive and gram negative bacteria in a naturally occurring infection. 

PRESTAINING PREPARATION:      

    1. Heat dry sections in oven according to your laboratory protocol.
    2. Filter Solution A: Crystal Violet-Oxalate Stain, Alcoholic.

 

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 in freshly filtered Solution A: Crystal Violet-Oxalate Stain, Alcoholic (Step #2) for 1 minute.
    3. Rinse well in distilled water.
    4. Mordant in Solution B: Iodine, Gram, Aqueous for 1 minute.
    5. Rinse well in distilled water, removing excess iodine.
    6. Decolorize in Solution C: Acetone-Alcohol 1:1 until blue stops running; 7-10 dips.
    7. Rinse well in distilled water.
    8. Place in Solution D: Basic Fuchsin Stain 0.25%, Aqueous for 90 seconds.
    9. Rinse well in distilled water.
    10. Dip once in Solution C: Acetone-Alcohol 1:1.
    11. Counterstain in Solution E: Tartrazine Stain 0.25%, Acetic Aqueous for 5-15 seconds.
    12. Rinse well in distilled water.
    13. Dehydrate in two changes of 100% ethyl alcohol, 5 dips each. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.
      1. Do not use 95% alcohol in the dehydration step.

 

RESULTS:

Gram positive bacteria Blue/violet
Gram negative bacteria Red
Nonreactive lung Negative for gram positive/negative bacteria

 

PROCEDURE NOTES: 

    1. Drain slides after each step to prevent solution carry over.
    2. Do not allow sections to dry out at any point during procedure.
    3. 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.