Carbol-Fuchsin Stain, Ziehl-Neelsen

This product has applications for both the Ziehl-Neelsen and Fite AFB staining procedures.

Product Options:

Part # 1030A250 ml $36.70H
Part # 1030B500 ml $56.20H
Part # 1030C1 L $96.40H

Technical Memo 1: For AFB Stain, Ziehl-Neelsen

 

SOLUTION:                                                        

  250 ml 500 ml 1 Liter
Carbol Fuchsin Stain, Ziehl-Neelsen Part 1030A Part 1030B Part 1030C

 

Additionally Needed:

Acid Fast Bacteria (AFB) Control Slides Part 4011
Acid Alcohol 1% Part 10011

Methylene Blue Stain 1.4%, Alcoholic

                          OR

Methylene Blue Stain 0.14%, Working Alcoholic 

Part 1240

     OR

Part 12401

Xylene, ACS Part 1445
Alcohol, Ethyl Denatured, 100% Part 10841
Alcohol, Ethyl Denatured, 95% Part 10842

 

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

 

APPLICATION:

Newcomer Supply Carbol Fuchsin Stain, Ziehl-Neelsen, a crucial element in the AFB Stain, Ziehl-Neelsen is used to demonstrate the presence of acid-fast mycobacteria in tissue sections. Phenol is employed in this solution to render the cell walls of bacteria permeable to the fuchsin stain.  The use of weak acid for differentiation allows excess stain to be removed from tissues, but will not remove stain from the acid-fast organisms.

 

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

 

STAINING PROCEDURE:

  1. Filter Carbol Fuchsin Stain, Ziehl-Neelsen before use.
  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 Notes #1 and #2.
  3. Stain in freshly filtered Carbol Fuchsin Stain, Ziehl-Neelsen for 60 minutes at room temperature. Keep solution covered.
  4. Rinse in running tap water for 2 to 3 minutes.
  5. Differentiate in Acid Alcohol 1% until color no longer runs off the slide and sections are pale pink; 3 to 10 rapid dips.
  6. Wash in running tap water 3 to 5 minutes; rinse in distilled water.
  7. Counterstain in Methylene Blue Stain 0.14%, Alcoholic (Part 12401). Or dilute 5 ml Methylene Blue Stain 1.4%, Alcoholic (Part 1240) with 45 ml tap water to a 0.14% solution.
    1. Dip slides a few times in counterstain; rinse in tap water, followed by a distilled water rinse and check microscopically.  Sections should be pale blue.
    2. See Procedure Notes #3 and #4.  
  8. Wash in running tap water for 1 minute; rinse in distilled water.
  9. Dehydrate in two changes each of 95% and 100% ethyl alcohol. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

 

RESULTS:

Acid-fast bacilli Bright red
Background Pale blue

 

PROCEDURE NOTES:

  1. Drain staining rack/slides after each step to prevent solution carry over.
  2. Do not allow sections to dry out at any point during staining procedure.
  3. It is important not to over-counterstain, as the organisms may be masked.  If section is over-stained, remove methylene blue with Acid Alcohol 1% (Part 10011), rinse thoroughly, and repeat methylene blue step (Step #7).      
  4. If laboratory tap water is generally acidic, the methylene blue stain may be pale. Adjust staining time accordingly.
  5. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

 

REFERENCES:

  1. Carson, Freida L., and Christa Hladik. Histotechnology: A Self-Instructional Text. 3rd ed. Chicago, Ill.: American Society of Clinical Pathologists, 2009. 226-227.
  2. Sheehan, Dezna C., and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 236-237.
  3. Modifications developed by Newcomer Supply Laboratory.

 

 

Technical Memo 2: For AFB Stain, Fite

 

SOLUTION:                                                                                                                    

  250 ml 500 ml 1 Liter
Carbol Fuchsin Stain, Ziehl-Neelsen Part 1030A Part 1030B Part 1030C

 

Additionally Needed:

Fite Stain, Nocardia Sp. Control Slides Part 4215
Xylene/Peanut Oil, 2:1  Part 1449
Sulfuric Acid 1%, Aqueous Part 14012
Methylene Blue Stain 0.5%, Aqueous Part 12402
Acid Alcohol 1%  Part 10011 (If staining for Mycobacterium leprae sp.)
Xylene, ACS Part 1445

 

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

 

APPLICATION:

Newcomer Supply Carbol Fuchsin Stain, Ziehl-Neelsen, a crucial element in the AFB Stain, Fite is used to detect the presence of either Nocardia sp. or Mycobacterium leprae sp. (causative agent of leprosy) in tissue sections with minor variations in the procedure.

 

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

 

STAINING PROCEDURE:

  1. Filter Carbol Fuchsin Stain, Ziehl-Neelsen.              
  2. Deparaffinize slides in Xylene/Peanut Oil, 2:1, two changes, 12 minutes each.
    1. See Procedure Note #1
  3. Drain slides, wipe off excess oil, and blot to opacity taking care to remove residual oil.
    1. See Procedure Note #2. 
  4. Stain slides in freshly filtered Carbol Fuchsin Stain, Ziehl-Neelsen for 30 minutes at room temperature.
  5. Wash in running tap water for 3 minutes.
  6. Differentiation:
    1. If staining for Nocardia sp., differentiate slides in Sulfuric Acid 1%, Aqueous (Part 14012) for 3 minutes. 
    2. If staining for Mycobacterium leprae sp., differentiate slides individually in Acid Alcohol 1% (Part 10011) until sections are light pink; approximately 1 minute.
  7. Wash in running tap water for 3 minutes.
  8. Counterstain lightly with Methylene Blue Stain 0.5%, Aqueous, for 5-10 seconds.
    1. See Procedure Notes #3 and #4.
  9. Rinse off excess Methylene Blue Stain 0.5%, Aqueous in running tap water. Background should be a light sky blue.
  10. Blot excess water from slide and air-dry completely.
  11. Dip dried slides in xylene and coverslip with a compatible mounting medium.

 

RESULTS:

Acid-fast bacilli and Mycobacterium leprae sp. Red
Nocardia sp. Red
Red blood cells Yellow-orange
Other tissue elements Pale blue

 

PROCEDURE NOTES:

  1. Acid-fastness of the leprosy organisms is enhanced when the waxy capsule is protected by the mixture of xylene-peanut oil and the avoidance of dehydrating solutions.
  2. It is important to blot well, residual oil may produce staining artifact.
  3. If over-stained with methylene blue, organisms may be masked. Check microscopically before air drying. If over-stained, remove methylene blue with Acid Alcohol 1% (Part 10011); rinse thoroughly; repeat Step #8 with a shorter timing.   
  4. If laboratory tap water is generally acidic, the methylene blue stain may be pale. Adjust staining time accordingly.
  5. A small percentage of Nocardia sp. organisms may resist taking the red stain and remain blue due to the growth phase of the individual organism.
  6. If using a xylene substitute, closely follow the manufacturer’s recommendations for coverslipping step.

 

REFERENCES:

  1. Carson, Freida L., and Christa Hladik. Histotechnology: A Self-Instructional Text. 3rd ed. Chicago, Ill.: American Society of Clinical Pathologists, 2009. 228-229.
  2. Fite, George, P.J. Cambre and M.H. Turner. “Procedure for Demonstrating Lepra Bacilli in Paraffin Sections”. Archives of Pathology 43 (1947). 624-625.
  3. Luna, Lee G. Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. Gaitheresburg, MD: American Histolabs, 1992. 180-181
  4. Sheehan, Dezna C., and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 237.
  5. Modifications developed by Newcomer Supply Laboratory.