A. Typical Organisms

 In tissue, M. tuberculosis and other mycobacteria are thin, straight rods measuring about 0.4 × 3 μm (Figure 1). On artificial media, coccoid and filamentous forms are seen with variable morphology from one species to another. Because of their high lipid content in their cell walls, mycobacteria do not stain well with common aniline dyes, including the regular Gram-stain method. The organisms typically appear as “Gram-invisible” or appear as clear zones (“ghosts”). Some mycobacteria, specifically some of the rapid growers, may appear as beaded Gram-positive rods, but branching may not be observed. The extent of acid fastness depends on the integrity and amount of the mycolic acids within the organ isms’ cell wall. Aside from mycobacteria, some other bacteria also express the acid-fast staining characteristic; these bacterial organisms include the genus Nocardia, Rhodococcus, Gordonia, and Tsukamurella. The Ziehl-Neelsen technique of staining is used for identification of acid-fast bacteria. The method is detailed in Chapter 47. In smears of sputum or sections of tissue, mycobacteria can be demonstrated by yellow-orange fluorescence after staining with fluorochrome stains (eg, auramine and rhodamine). The ease with which acid-fast bacteria can be visualized with fluorochrome stains makes them the preferred stains for clinical specimens (Figure 1-B). The availability of ultrabright light-emitting diode microscopes, some of which do not require electricity, has advanced fluorescence microscopy in resource-limited countries.

Fig1. A: M. tuberculosis (arrows) in a processed sputum specimen stained by Ziehl-Neelsen stain. The M. tuberculosis is red against a blue background. B: The fluorescent dye Auramine O was used to stain a sputum sample. It shows two fluorescent M. tuberculosis. Original magnification ×1000. (Courtesy of G Cunningham.)

B. Culture

The media for primary culture of mycobacteria should include a nonselective medium and a selective medium. Selective media contain antibiotics to prevent the overgrowth of contaminating bacteria and fungi. There are three general formulations that can be used for both the nonselective and selective media. Agar-based (solid) media are useful for observing colony morphology, for detection of mixed cultures, for antimicrobial susceptibility testing, and can also provide some indication of the quantity of organisms in a particular specimen.

1. Semisynthetic agar media—These media (eg, Middle brook 7H10 and 7H11) contain defined salts, vitamins, cofactors, oleic acid, albumin, catalase, and glycerol; the 7H11 medium also contains casein hydrolysate. The albumin neutralizes the toxic and inhibitory effects of fatty acids in the specimen or medium. Large inocula yield growth on these media in several weeks. Because large inocula may be necessary, these media may be less sensitive than other media for primary isolation of mycobacteria.

2. Inspissated egg media—These media (eg, Löwenstein Jensen) contain defined salts, glycerol, and complex organic substances (eg, fresh eggs or egg yolks, potato flour, and other ingredients in various combinations). Malachite green is included to inhibit other bacteria. Small inocula in specimens from patients will grow on these media in 3–6 weeks.

 These media with added antibiotics (Gruft and Myco bactosel) are used as selective media.

3. Broth media—Broth media (eg, Middlebrook 7H9 and 7H12) support the proliferation of small inocula. Ordinarily, mycobacteria grow in clumps or masses because of the hydro phobic character of the cell surface. If tweens (water-soluble esters of fatty acids) are added, they wet the surface and thus permit dispersed growth in liquid media. Growth is often more rapid than on complex media. There are several commercial sources of these media that are used in many clinical and reference laboratories. These include the MGIT system (Becton Dickinson, Sparks, MD), VersaTREK® Culture Sys tem (ThermoFisher Scientific, Houston, TX), and MB Redox (Heipha Diagnostica Biotest, Eppelheim, Germany).

C. Growth Characteristics

 Mycobacteria are obligate aerobes and derive energy from the oxidation of many simple carbon compounds. Increased CO2 tension enhances growth. Biochemical activities are not characteristic, and the growth rate is much slower than that of most bacteria. The doubling time of tubercle bacilli is about 18 hours. Saprophytic forms tend to grow more rapidly, to proliferate well at 22–33°C, to produce more pigment, and to be less acid fast than pathogenic forms.

D. Reaction to Physical and Chemical Agents

Mycobacteria tend to be more resistant to chemical agents than other bacteria because of the hydrophobic nature of the cell surface and their clumped growth. Dyes (eg, malachite green) or antibacterial agents (eg, penicillin) that are bacteriostatic to other bacteria can be  incorporated into media without inhibiting the growth of tubercle bacilli. Acids and alkalies permit the survival of some exposed tubercle bacilli and are used to help eliminate contaminating organisms and for “concentration” of clinical specimens. Tubercle bacilli are resistant to drying and survive for long periods in dried sputum.

E. Variation

 Variation can occur in colony appearance, pigmentation, virulence, optimal growth temperature, and many other cellular or growth characteristics.

F. Pathogenicity of Mycobacteria

 There are marked differences in the ability of different mycobacteria to cause lesions in various host species. Humans and guinea pigs are highly susceptible to M. tuberculosis infection, but fowl and cattle are resistant. M. tuberculosis and Mycobacterium bovis are equally pathogenic for humans. The route of infection (respiratory vs intestinal) determines the pattern of lesions. In developed countries, M. bovis has become very rare. Some “atypical” mycobacteria, now designated as NTM (eg, Mycobacterium kansasii), produce human disease indistinguishable from tuberculosis; others (eg, Mycobacterium fortuitum) cause only surface lesions or act as opportunists.