Acid-Fast Culture Health Article

Acid fast and fluorescent staining

The smears made after the process of decontamination and digestion of sputum are stained using either an acid-fast staining procedure or a fluorochrome stain. Mycobacteria do not stain well with the Gram staining procedure used routinely in the microbiology laboratory. Specimens obtained from sterile sites (bone marrow, tissue, etc.) do not need processing and smears are made directly from the specimen onto glass microscope slides. Mycobacteria are slightly curved or straight bacilli, about 0.2 to 0.6 by 1.0 to 10 micrometers in size. The cell wall of mycobacteria contains a high lipid content, and is made up of long-chain, multiply cross-linked fatty acids (mycolic acids). In the acid-fast staining procedure, a basic dye, carbolfuchsin stain, is used to stain the cell wall. The long-chain mycolic acids and waxes in the mycbacteria cell wall serve to complex the carbolfuchsin. The Ziehl-Neelsen acid fast stain for mycobacteria uses heat to fix the dye in the cell wall, while the Kinyoun staining method uses an increased concentration of basic fuchsin and phenol eliminating the heat requirement. In the Ziehl-Neelsen procedure, the carbolfuchsin stain is left on the smear for five minutes while heat is applied under the slide by a bunsen burner or a hot plate. The carbolfuchsin dye penetrates the cell wall and the excess stain is washed off with a 3% acid-alcohol mixture (95% ethanol and 3% hydrochloric acid). The mycobacteria cell wall retains the dye (a red-purple color) and will not be decolorized (washed out) by the acid-alcohol, thus the term acid-fast. A second dye, methylene blue, is used to stain any background material including any other bacteria that may be present. This dye results in a light background providing good contrast to the red-purple stain of the carbolfuchsin dye, thus aiding in the detection of acid-fast bacilli. If mycobacteria are present in the smear, the appearance of red-purple short or long bacilli are observed at 1000 X magnification. Some species of mycobacteria appear "beaded" while others may appear pleomorphic (a mixture of coccoid and rod shapes), or filamentous (branching of the bacillus).

Another staining method used for the detection of mycobacteria is the auramine-rhodamine fluorochrome stain. This method requires a fluorescent microscope. Smears are scanned at a lower magnification (250 X to 400 X). The fluorochrome dyes used in this procedure complex to the mycolic acids in acid-fast cell walls. The fluorescing mycobacteria are seen as bright yellow-orange bacilli against a dark background. Fluorescent stained smears can be read more rapidly than acid-fast stains, but there are drawbacks. Mycobacteria spp. that are rapid-growers may not appear fluorescent with these stains; artifacts may fluoresce; material on the oil objective may have floated off a previous positive smear causing a false-positive reading for the next smear examined. All positive smears from the auramine-rhodamine fluorochrome method should be confirmed using the Ziehl-Neelsen method for acid-fast bacilli.

Acid-fast bacillus (AFB) smear report

Laboratories performing staining procedures and reporting smear results must adhere to guidelines from the U.S. Department of Health and Human Services (Public Health Service, Centers for Disease Control, Atlanta). The rule for reporting acid-fast smears for mycobacteria requires scanning the smear for a minimum of 15 minutes (at least 300 oil immersion fields) before calling the slide negative for acid-fast bacilli or "No AFB seen." The following are recommended interpretations and ways to report smear results:

• A request for another specimen or a doubtful report is the result of seeing AFB of 1-2/300 fields for the Ziehl-Neelsen (Z-N) stain and AFB of 1-2/70 fields for the auramine-rhodamine (fluorochrome) stain.
• A "1+" report for AFB seen = 1-9/100 fields for the ZN method and 2-18/50 fields for the fluorochrome stain.
• A "2+" report for AFB seen = 1-9/10 fields for the Z-N method and 4-36/10 fields for the fluorochrome stain.
• A "3+" report for AFB seen = 1-9/field for the Z-N method and 4-36/field for the fluorochrome stain.
• A "4+" report for AFB seen = less than 9/field for the Z-N method and less than 36/field for the fluorochrome stain.