SC25

CARBOL FUCHSIN STAIN
(ZIEHL-NEELSEN)
- For in vitro use only - Catalogue No. SC24K
Our Carbol Fuchsin (Ziehl-Neelsen) Stain is Formulation per 100 mL

used in the microscopic detection of acid-fast
microorganisms such as Mycobacterium. SC25 Carbol Fuchsin Stain (Ziehl-Zeelsen)
Acid-fast organisms such as Mycobacterium Basic Fuchsin ..................................................... 0.3 g
have cell walls that are resistant to conventional Phenol ................................................................ 5.0 g
staining by aniline dyes such as the Gram stain. Ethanol ............................................................ 10 mL
However methods that promote the uptake of dyes De-ionized Water ............................................. 90 mL
are available; once stained these organisms are not
easily decolorized even with acid-alcohol or acid- SC26 Carbol Fuchsin Decolorizer
acetone solutions therefore they are described as Hydrochloric Acid .......................................... 3.0 mL
acid-fast. Their resistance to destaining is a useful Ethanol .......................................................... 97.0 mL
characteristic in differentiating these organisms
from contaminating organisms and host cells. SC27 Carbol Fuchsin Counterstain (Methylene Blue)
The Ziehl-Neelsen staining procedure is often Methylene Blue ................................................. 0.3 g
referred to as hot carbolfuchsin because of the need De-ionized Water ............................................100 mL
to apply heat during the staining process. The
primary stain for the Ziehl-Zeelsen procedure is the
aniline dye, basic fuchsin, that stains all the cells Recommended Procedure
present red. The unique ability of mycobacteria to
resist decolorization by acid-alcohol is why they are 1. Prepare slides by applying sample in a thin smear
termed acid-fast, and will keep their red coloration and heat fixing the sample to the slide.
throughout the staining process. The decolorizer 2. Flood the entire slide with Carbol Fuchsin Stain
used is a hydrochloric acid-ethanol mixture that will (Ziehl-Zeelsen).
decolorize non-acid-fast material present. The last 3. Heat the slide using a bunsen burner or electric
step in the staining procedure is the application of staining rack until it is steaming.
the counterstain, methylene blue, which colors other 4. Maintain steaming for 5 minutes by using low or
cells and background material present on the slide intermittent heat.
blue. 5. Allow the slide to cool briefly and rinse the slide
The acid-fast smear plays an important role in with water.
early diagnosis of mycobacterial infection because 6. Flood the slide with Carbol Fuchsin Decolorizer
of the lengthy incubation times required to culture and allow smear to decolorize for 2 minutes;
mycobacteria. Nonmycobacterial organisms with flood until no more color drains from the slide.
various degrees of acid-fastness include 7. Rinse the slide thoroughly with distilled water
Rhodococcus species, Nocardia species, Legionella and shake off any excess moisture.
micdadei, and the cysts of Cryptosporidium, 8. Flood the slide with Carbol Fuchsin Counterstain
Isospora, Cyclospora and microsporidia. Detection (Methylene Blue) and allow the slides to stain for
of these organisms is possible using some of the 30 to 45 seconds.
staining reagents provided but in most instances 9. Rinse thoroughly with water and allow to air dry.
requires a modified staining procedure and Do not blot.
additional reagents not provided. 10. Examine the smear microscopically under a 100x
oil immersion objective.
Interpretation of Results Storage and Shelf life
Acid-fast mycobacteria will appear as dark- Our Carbol Fuchsin Stain, Decolorizer and
pink to red bacilli against a blue background when Counterstain should be stored at room temperature and
examined microscopically. Mycobacteria are protected from light. Under these conditions they have
typically slender, 1 to 10-µm long rods that may a shelf life of 52 weeks from the date of manufacture.
appear curved or bent. Individual bacilli may
display heavily stained areas and area of alternating
stain, producing a beaded appearance. Some Ordering Information
nontuberculous mycobacteria may appear
pleomorphic, appearing as long filaments or coccoid Cat# Description Format
forms, with uniform staining. Mycobacterium SC24K-250 Carbol Fuchsin Stain Kit 3 x 250mL
kansaii are often recognized by their large size and (Ziehl-Neelson)
cross-banding appearance. [Includes stain, decolorizer & counterstain]
When a carbol fuchsin smear is read a SC25-250 Carbol Fuchsin Stain 250-mL
(Ziehl-Neelson)
minimum of 300 fields should be examined before
the smear is reported as negative. To verify the SC26-250 Carbol Fuchsin Decolorizer 250-mL
(Ziehl-Neelson & Kinyoun)
staining procedure and staining intensity of the
acid-fast organisms it is recommended that a SC27-250 Carbol Fuchsin Counterstain 250-mL
positive and negative control slide be included (Ziehl-Neelson)
with each run of stains.
Non-acid-fast organisms and background
material will stain blue. References
• Rapidly growing mycobacteria may vary in 1. Baron EJ, Finegold SM. Bailey and Scott's
their ability to retain acid-fast dyes and may diagnostic microbiology. 8th ed. St. Louis:
fail to stain Mosby, 1990.
2. Isenberg HD, Ed. Clinical microbiology
• Be aware of adequate safety precautions and procedures handbook, Vol 1. Washington, DC:
procedures required when handling specimens ASM, 1992.
that are submitted for mycobacterial evaluation 3. Bloom BR, Ed. Tuberculosis: pathogenesis,
protection, and control. Washington, DC: ASM
• Mycobacterial staining should always be used 1994.
as a adjunct to culture methods since culture 4. Murray PR, Baron E, Pfaller M, Tenover F,
techniques are much more sensitive than all Yolken. Manual of clinical microbiology. 7th
acid-fast staining procedures ed. Washington, DC: ASM, 1999.
Original: December 2004
Revised / Revisited: October 2014
Quality Control
Organism Expected Result
Mycobacterium tuberculosis Dark pink to red

ATCC 25177 (H37Ra) bacilli

SC25

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CARBOL FUCHSIN STAIN

Our Carbol Fuchsin (Ziehl-Neelsen) Stain is Formulation per 100 mL

Organism Expected Result

Mycobacterium tuberculosis Dark pink to red

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