Twort's Gram Stain Set

(use: Hucker-Twort Gram Stain.)

  • Shelf Life 2 years from date of manufacture.

 

Sol’n A: Neutral Red Stain 1%, Alcoholic

Sol’n B: Fast Green Stain 1%, Alcoholic

Product Options:

Part # 14034A 250 ml set $57.80 H
Part # 14034B 500 ml set $96.20 H

 

  

SET INCLUDES:                                                                                       

Part 14034A Part 14034B
Solution A: Neutral Red Stain 1%, Alcoholic 250 ml 500 ml
Solution B: Fast Green Stain 1%, Alcoholic 100 ml 200 ml

 

Additionally Needed For Gram Stain, Hucker-Twort:                                                                                       

Gram, Multi-Tissue, Artificial Control Slides

                            OR

Gram+ & Gram- Bacteria, Artificial Control Slides

Part 4256

    OR

Part 4255

Crystal Violet-Oxalate Stain, Alcoholic, Hucker-Twort Part 10422
Iodine, Weigert & Lugol, Aqueous  Part 12092
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 Twort’s Gram Stain Set provides stain solutions for the Gram Stain, Hucker-Twort, a rapid and simple procedure that stains gram-positive and gram-negative bacteria without the use of picric acid.  The Neutral Red Stain combined with a Fast Green counterstain provides clear detection of any red gram-negative bacteria present against a green background.

 

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 Sets are designed to be used with Coplin jars filled to 40 ml following the provided staining procedure. Some solutions in the set may contain extra volumes.

 

PRESTAINING PREPARATION:

  1. If necessary, heat dry tissue sections/slides in oven.
  2. Filter Crystal Violet-Oxalate Stain, Alcoholic, Hucker-Twort (Part 10422) 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, Hucker-Twort (Step #2) for 30 seconds.
  3. Rinse quickly in distilled water.
  4. Mordant in Iodine, Weigert & Lugol, Aqueous (Part 12092) for 20 seconds.
  5. Rinse quickly in distilled water.
  6. Decolorize individually with Acetone, ACS (Part 10014); 2 quick dips.
    1. Or until majority of purple stain is removed and tissue remains light gray.
  7. Rinse quickly in distilled water.
  8. Prepare fresh Twort Stain; combine and mix well.
    1. Solution A: Neutral Red Stain 1%, Alcoholic      9 ml
    2. Solution B: 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.
  1. Rinse quickly in distilled water and carefully blot dry.
  2. Agitate slides quickly in clean Acetone, ACS to dehydrate (do not use any alcohols).
    1. See Procedure Notes #2 and #3.
  3. 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 needed, add extra dips in acetone to further differentiate and dehydrate sections.
    1. Check microscopically to avoid over-differentiation.
  3. The use of alcohol will remove Neutral Red staining.
  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. 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.