Staining Characteristics of Mycobacteria

Tuberculosis and Nontuberculous Mycobacterial Disease

Mycobacteria

Gaby E. Pfyffer, Clark B. Inderlied, in Infectious Diseases (Third Edition), 2010

Acid-fast stain and smear microscopy

An acid-fast stain remains the most rapid and least expensive method for directly detecting mycobacteria in clinical specimens, and is highly specific. Nevertheless, the laboratory must be aware that there are organisms other than mycobacteria with various degrees of acid-fastness such as Rhodococcus spp., Nocardia spp. and Legionella micdadei, as well as cysts from Cryptosporidium, Isospora, Cyclospora and Microsporidium spores. The presence of AFB in a specimen from a patient with signs and symptoms of or another mycobacterial infection is an important guide to effective treatment and the initiation of public health measures. Furthermore, an acid-fast stain can quickly assess patient infectiousness, and is also used to confirm that a culture is acid fast. The detection limit of an acid-fast stain has been estimated to be 5000–10 000 bacilli/ml of sputum; overall sensitivity is between 22% and 78%.55 The importance of the acid-fast stain and the need for a prompt turnaround time cannot be overemphasized. In laboratories that do not perform cultures, an acid-fast stain can be performed using a specimen treated for a short time with 5% sodium hypochlorite. Above all, the predictive value of a positive smear for M. tuberculosis in expectorated sputum is very high (~90%).

The three stains that are commonly used to detect AFB are Ziehl–Neelsen, Kinyoun and auramine–rhodamine fluorochrome stains.51 With each of these procedures, acid- fastness is defined as resistance to decolorizing with acid–alcohol (e.g. 3% hydrochloric acid in 95% ethanol). When mycobacteria are stained with Gram's and they often appear as beaded Gram-positive bacilli or fail to stain at all. It is important to note, however, that culture media, incubation conditions and the age of the culture influence the acid fastness of mycobacteria. The critical role of the mycobacterial cell wall in acid fastness is underscored by the observation that INH causes a loss of ‘acid fastness’ in susceptible strains of M. tuberculosis.

The preparation of the smear is the critical first step in performing an acid-fast stain. Using a glass slide cleaned with ethanol, make a smear of approximately 1 × 2 cm of a single specimen or isolate. For CSF specimens previously concentrated by centrifugation, three drops of the concentrate are placed on a clean glass slide, one drop at a time. The drop is allowed to air dry before the next drop is added. The slide is air dried or alternatively dried at 176°F (80°C) for 15 minutes in a biosafety cabinet class II – it is important to note that heat fixing may not kill all the mycobacteria on the slide. Mycobacteria appear brightly fluorescent against a dark background in the auramine–rhodamine stain and the increased sensitivity of the fluorescence stain compared with the other stains is used to rapidly screen slides at a lower (250–450×) magnification. However, fluorescent objects must be examined at 800–1000× magnification to confirm morphology. Positive fluorescent smears should be confirmed with either the Ziehl–Neelsen or Kinyoun stains. The Ziehl–Neelsen stain requires that the carbolfuchsin stain be heated for the stain to penetrate the mycobacterial cell. In contrast, heating is unnecessary with the Kinyoun stain because the concentration of basic fuchsin and phenol have been increased to ensure that the basic fuchsin penetrates the mycobacterial cell wall. Using a 100× oil immersion lens, 100–300 fields of a properly prepared smear and stain should be examined. It should be remembered that in examining even 100–300 fields, only 1– 4% of a 1 × 2 cm smear would be examined. Mycobacteria usually appear as pink, slender rod-shaped bacilli; however, pleomorphic shapes are common and range from coccoid to long rods with curves or bends (Fig. 174.11).

Sign in to download full-size image Fig. 174.11. Ziehl–Neelsen acid-fast stain of sputum containing 4+ tubercle bacilli. Courtesy of S Froman and A Gaytan. Read full chapter

Purchase book