Carbol-Fuchsin

Author: Kelsey Goldman, HTL (ASCP)

Last Update: 2 Sept 2024

Copyright: 2023, HistologyOutlines.com

Cite this Page! -> Goldman, Kelsey. “Carbol-Fuchsin.” HistologyOutlines.Com, 2 Sept 2024. Accessed (today) 

Special Stain

Purpose:

  • Carbol-Fushsin is a key reagent in several stains for acid fast bacteria including Kinyouns, Ziehl-Neelsen, and Fite Staining (Carson, 2001),(Disbrey & Rack, 1970).

Control Tissues:

  • Controls must include the subtype of Acid Fast Bacteria that each stain is meant to detect. Examples include-
    • Kinyoun’s- Any Acid Fast Bacteria (Carson, 2001)
    • Ziehl-Neelsen’s- Any Acid Fast Bacteria (Carson, 2001)
    • Fite- Mycobacterium leprae ( leprosy) (Carson, 2001)

Staining Pattern:

  • Acid Fast Bacteria- Red 
  • Other tissue elements- Blue (Carson, 2001)

Biochemistry Theory:

  • Carbol-fushsin is absorbed into many tissue elements. However, when absorbed into the lipoid capsule of acid fast bacteria, it resists decolorization when an acid is introduced. This staining mechanism is facilitated due to high solubility of the carbol-fushsin in the cell wall lipids of these bacteria (Carson, 2001) . 

  • Carbol-Fushsin containing stains are important  because these bacteria are not easily demonstrated by other methods such as the Gram stain (Carson, 2001).

  • Carbol-Fushsin is a stain dependent mixture of the following: Basic Fushsin, alcohol, and phenol (Morris et al., 2019),(cellpath, 2020). 

Additional References:

  • Carson, F. L. (2001). Histotechnology: a self-instructional text (2. ed., [Reprint]). ASCP Press.
  • cellpath. (2020, November 17). C is for Carbol Fuchsin. CellPath. https://www.cellpath.com/latest-news/c-is-for-carbol-fuchsin/
  • Disbrey, B. D., & Rack, J. H. (1970). Histological laboratory methods. Livingstone.
  • McCollough, C. (2008). Application of an aqueous acid-fast staining technique to detect pathogens of aquatic species. Biotechnic & Histochemistry, 83(3–4), 191–197. https://doi.org/10.1080/10520290802450780
  • Morris, G. B., Ridgway, E. J., & Suvarna, S. K. (2019). Traditional stains and modern techniques for demonstrating microorganisms in histology. In Bancroft’s Theory and Practice of Histological Techniques (pp. 254–279). Elsevier. https://doi.org/10.1016/B978-0-7020-6864-5.00016-5

Sample Protocol:

Kinyoun's Protocol- Adapted from Carson

Reagents-

  • Kinyoun Carbol- fushsin Solution

    • 28g of basic fushsin, 56mL pf phenol crystals, 140 mL of 95% alcohol, 700mL of filtered water) . Always filter this solution before use. This solution can be reused several times

  • Acid Alcohol 1%

    • Concentrated hydrochloric acid 5mL into 495 mL of 70% alcohol

  • Methylene Blue

 

Method-

  • Deparaffinize and bring to water. Make sure the water is filtered

  • Stain slides in the Kinyoun solution for 1 hour at room temperature or 30 minutes at 56C

  • Wash in running tap water

  • Differentiate in two changes of 1% Acid Alcohol. Color will run off of the slide, tissue will look pink after second solution

  • Wash in running tap water

  • Stain in Methylene blue. This is best done one slide at a time in one fast dip. Overstaining in this step will mask the acid fast signal

  • Rinse in running tap water

  • Dehydrate to xylene and mount in a xylene based mounting media

Adapted from Protocol found in (Carson, 2001)

Reagents

  • Ziehl- Neelsen Carbol Fushsin Solution

    • 0.5 g of basic fushsin, 2.5 mL pf phenol crystals, 5 mL of abs alcohol, 700mL of filtered water) . Always filter this solution before use. This solution can be reused several times

  • Acid Alcohol 1%

    • Concentrated hydrochloric acid 5mL into 495 mL of 70% alcohol

  • Methylene Blue

 

Method-

  • Deparaffinize  and bring to water. Make sure the water is filtered

  • Stain slides in the Ziehl-Neelsen solution  for 30 minutes

  • Wash in running tap water

  • Differentiate in two changes of 1% Acid Alcohol. Color will run off of the slide, tissue will look pink after second solution

  • Wash in running tap water for 8 minutes

  • Stain in Methylene blue. This is best done one slide at a time in one fast dip. Overstaining in this step will mask the acid fast signal

  • Rinse in running tap water. Then repeat the wash in deionized water

  • Dehydrate to xylene and mount in a xylene based mounting media

Adapted from protocol found in (Carson, 2001)

Reagents-

  • Xylene/ Peanut oil ( 2:1 ratio)

  • Ziehl- Neelsen Carbol- fushsin Solution

    • 0.5 g of basic fushsin, 2.5 mL pf phenol crystals, 5 mL of abs alcohol, 700mL of filtered water) . Always filter this solution before use. This solution can be reused several times

  • Acid Alcohol 1%

    • Concentrated hydrochloric acid 5mL into 495 mL of 70% alcohol

  • Methylene Blue

 

Method-

  • Use 2 changes of xylene/peanut oil for the deparaffinization instead of standard xylene, blot oil off in last change. After the xylene peanut oil solution continue with the standard deparaffinization to water. Make sure the water is filtered

  • Stain slides in the Ziehl Neelsen solution  for 30 minutes

  • Wash in running tap water

  • Differentiate in two changes of 1% Acid Alcohol. Color will run off of the slide, tissue will look pink after second solution.

  • Wash in running tap water for 8 minutes

  • Stain in Methylene blue. This is best done one slide at a time in one fast dip. Overstaining in this step will mask the acid fast signal

  • Rinse in running tap water then di water

  • Dehydrate to xylene and mount in a xylene based mounting media

Adapted from Protocol found in Carson, 2001