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 $35.30
Part # 10422B 500 ml $48.60

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
Acetone, ACS Part 10014
Picric Acid-Acetone 0.1% Part 1335
Acetone-Xylene 1:1 Part 10015

 

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

 

APPLICATION:

Newcomer Supply Gram Stain, Brown-Brenn is the traditional method 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 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 30 seconds.
  3. Rinse well in several changes of distilled water.
  4. Mordant in Iodine, Gram, Aqueous (Part 1140) for 1 minute.
  5. Rinse in distilled water; blot excess water from slide, but not from the tissue section.
  6. Decolorize one slide at a time by dipping in Acetone-Alcohol 1:1 (Part 10016) until blue color stops running. Approximately 1-3 dips.
  7. Counterstain in Basic Fuchsin Stain 0.25%, Aqueous (Part 1011) for 3 minutes.
  8. Rinse in distilled water; blot excess water from slide, but not from the tissue section.
    1. Proceed with Steps #11 to #14 one slide at a time.
  9. Dip once in Acetone (Part 10014).
  10. Dip in Picric Acid-Acetone 0.1% (Part 1335) until sections have a yellowish-pink color, 3-10 dips. Agitate slides until desired intensity is achieved.
  1. Dip in Acetone-Xylene 1:1 (Part 10015), 5-10 dips.
    1. Check control microscopically for proper differentiation. 
    2. Repeat Step #12 if additional differentiation is needed.
  2. 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 laboratory protocol with a focus on uniform cell distribution.  Timings offered in this protocol 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 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, Weigert & Lugol, 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 Gram Stain, Hucker-Twort is a rapid and simple procedure that stains gram-positive and gram-negative bacteria without the use of picric acid.  The Fast Green secondary counterstain provides the green background for clear detection of any red gram-negative bacteria present.

 

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, Aqueous (Part 12092); 20 seconds.
  5. Rinse quickly in distilled water.
  6. Decolorize one slide at a time with Acetone (Part 10014) until majority of the purple stain is removed, and tissue remains light gray. Approximately 2 quick dips.
  7. Rinse quickly in distilled water.
  8. Prepare fresh Twort Stain (Part 14034); combine and mix well. Use within 30 minutes of preparation:
    1. Neutral Red Stain 1%, Alcoholic      9 ml
    2. Fast Green Stain 1%, Alcoholic       3 ml
    3. Distilled Water                               30 ml
  9. Stain in fresh Twort Stain for 2 minutes.
  10. Rinse quickly in distilled water and carefully blot dry.
  1. Agitate slides quickly in clean Acetone to dehydrate; do not use any alcohols.
    1. See Procedure Notes #2 and #3.
  2. 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. To tone down excessive red staining, add extra dips in acetone to differentiate and dehydrate the section.
    1. Check microscopically to ensure that over-differentiation does not occur.
  3. Do not use alcohol in dehydration steps. The Neutral Red will be removed with alcohol exposure.
  4. If using a xylene substitute, closely follow the manufacturer’s recommendations 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.