Gram, Hucker-Twort Stain Kit

gram stain kit hucker twort

Product Options:

Part # 9125A250ml kit $149.90H

 

  

GRAM STAIN KIT INCLUDES:

    Part 9125A
Solution A: Crystal Violet-Oxalate Stain, Alcoholic 250 ml
Solution B: Iodine, Weigert & Lugol, Aqueous 250 ml
Solution C: Neutral Red Stain 1%, Alcoholic  125 ml
Solution D: Fast Green Stain 1%, Alcoholic  50 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, Hucker-Twort Stain Kit 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 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. Filter Solution A: Crystal Violet-Oxalate Stain, Alcoholic with high quality filter paper.
  2. 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.
  3. Stain in freshly filtered Solution A: Crystal Violet-Oxalate Stain, Alcoholic for 30 seconds.
  4. Rinse quickly in distilled water.
  5. Mordant in Solution B: Iodine, Weigert & Lugol, Aqueous for 20 seconds.
  6. Rinse quickly in distilled water.
  7. 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.
  8. Rinse quickly in distilled water.
  9. Prepare fresh Twort Stain; combine and mix well. Use within 30 minutes of preparation:
    1. Solution C: Neutral Red Stain 1%, Alcoholic   9 ml
    2. Solution D: Fast Green Stain 1%, Alcoholic  3 ml
    3. Distilled Water                                               30 ml
  10. Stain in fresh Twort Stain for 2 minutes.
  11. 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.

 

GRAM STAIN KIT RESULTS:

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

 

PROCEDURE NOTES:

  1. Drain staining rack/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.  Check microscopically to ensure that over-differentiation does not occur.
  3. Do not use any alcohol 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.