Products

Crystal Violet-Oxalate Stain, Alcoholic

Crystal Violet-Oxalate Stain, Alcoholic

(use: Brown-Brenn & Hucker-Twort Gram Stain.)

      • Shelf Life is 2 years from date of manufacture.

Product Options:

Part # 10422A 250 ml $38.00
Part # 10422B 500 ml $52.60

Tech Memo 1: Crystal Violet-Oxalate Stain, Alcoholic for Brown-Brenn Gram Stain

 

SOLUTION:

250 ml 500 ml
Crystal Violet-Oxalate Stain, Alcoholic Part 10422A Part 10422B

 

Additionally Needed:

Gram, Multi-Tissue, Artificial Control Slides
OR
Gram+ & Gram- Bacteria, Artificial Control Slides		 Part 4256
OR
Part 4255
Xylene, ACS Part 1445
Alcohol, Ethyl Denatured, 100% Part 10841
Alcohol, Ethyl Denatured, 95% Part 10842
Iodine, Gram, Aqueous Part 1140
Acetone-Alcohol 1:1 Part 10016
Basic Fuchsin Stain 0.25%, Aqueous Part 1011
Tartrazine Stain 0.25%, Acetic Aqueous Part 14016
Acetone-Xylene 1:1 Part 10015

 

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

 

APPLICATION:

Newcomer Supply Crystal Violet-Oxalate Stain, Alcoholic, a component of the Brown-Brenn Gram Stain, is used for differential staining of gram-positive and gram-negative bacteria in tissue sections, cultures and smears.  Tartrazine provides the yellow background in this procedure, replacing picric acid-acetone counterstain.

 

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 procedures are designed to be used with Coplin jars filled to 40 ml following the provided staining procedure.

 

PRESTAINING PREPARATION:

    1. If necessary, heat dry tissue sections/slides in oven.
    2. Filter 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 Crystal Violet-Oxalate Stain, Alcoholic for 1 minute.
    3. Rinse in distilled water.
    4. Mordant in Iodine, Gram, Aqueous (Part 1140) for 1 minute.
    5. Rinse well in distilled water; removing excess iodine.
    6. Decolorize one slide at a time in Acetone-Alcohol 1:1 (Part 10016) until blue stops running. Approximately 7-10 dips.
    7. Rinse well in distilled water.
    8. Place in Basic Fuchsin Stain 0.25%, Aqueous (Part 1011); 90 seconds.
    9. Rinse well in distilled water.
    10. Dip once in Acetone-Alcohol 1:1.
    11. Counterstain in Tartrazine Stain 0.25%, Acetic Aqueous (Part 14016) 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
Gram-negative bacteria Red
Nuclei Red
Background tissue Yellow

 

PROCEDURE NOTES:

    1. For cultures/smears: Prepare a well-made culture/smear with a focus on uniform cell distribution.
        1. Timings in this protocol are based on paraffin sections and may need to be altered for 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, follow manufacturer’s recommendation 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 Hopkins 48.2 (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.

 

Tech Memo 2: Crystal Violet-Oxalate Stain, Alcoholic for Hucker-Twort Gram Stain

 

SOLUTION:

250 ml 500 ml
Crystal Violet-Oxalate Stain, Alcoholic Part 10422A Part 10422B

 

Additionally Needed:

Gram, Multi-Tissue, Artificial Control Slides
                         OR
Gram+ & Gram- Bacteria, Artificial Control Slides		 Part 4256
     OR
Part 4255
Xylene, ACS Part 1445
Alcohol, Ethyl Denatured, 100% Part 10841
Alcohol, Ethyl Denatured, 95% Part 10842
Iodine, Lugol’s, Aqueous Part 12092
Acetone, ACS Part 10014
Twort’s Gram Stain Set
    Solution A: Neutral Red Stain 1%, Alcoholic
    Solution B: Fast Green Stain 1%, Alcoholic		 Part 14034

 

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

 

APPLICATION:

Newcomer Supply Crystal Violet-Oxalate Stain, Alcoholic, a component of the Hucker-Twort Gram Stain that stains gram-positive bacteria. The Twort Stain combines Neutral Red and Fast Green for clear detection of red gram-negative bacteria with a green counterstain.

 

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 procedures are designed to be used with Coplin jars filled to 40 ml following the provided staining procedure.

 

PRESTAINING PREPARATION:

    1. If necessary, heat dry tissue sections/slides in oven.
    2. Filter 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 Note #1.
    2. Stain in freshly filtered Crystal Violet-Oxalate Stain, Alcoholic for 30 seconds.
    3. Rinse quickly in distilled water.
    4. Mordant in Iodine, Weigert, Lugol’s, Aqueous (Part 12092); 20 seconds.
    5. Rinse quickly in distilled water.
    6. Decolorize individually with Acetone, ACS (Part 10014); 2 quick dips.
    7. Rinse quickly in distilled water.
    8. Prepare fresh Twort Stain (Part 14034); combine and mix well.
        1. Neutral Red Stain 1%, Alcoholic 9 ml
        2. Fast Green Stain 1%, Alcoholic      3 ml
        3. Distilled Water               30 ml
        4. Use within 30 minutes.
    9. Stain in fresh Twort Stain for 2 minutes.
    10. Rinse quickly in distilled water; carefully blot dry.
    11. Agitate slides quickly in clean Acetone, ACS to remove excess stain and dehydrate; do not use any alcohols.
        1. The use of alcohols will remove Neutral Red Stain.
    1. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

 

RESULTS:

Gram-positive bacteria Dark blue
Gram-negative bacteria Red
Cytoplasm and red blood cells Shades of green
Nuclei Red

 

PROCEDURE NOTES:

    1. Drain slides after each step to prevent solution carry over.
    2. If using a xylene substitute, follow manufacturer’s recommendation for deparaffinization and clearing steps.

 

REFERENCES:

    1. Bancroft, John D. and Alan Stevens. Theory and Practice of Histological Techniques. 3rd ed. Edinburgh: Churchill Livingstone, 1990. 290-292.
    2. Culling, C.F.A. Handbook of Histopathological and Histochemical Techniques (including museum techniques). 3rd ed. London: Butterworth, 1974. 393-395.
    3. Twort, F.W. “An Improved Neutral Red, Light Green Double Staining for Animal Parasites, Microorganisms and Tissues.” Journal of State Medicine 32 (1924). 351.
    4. Modifications developed by Newcomer Supply Laboratory.