Acid-Fast Culture Health Article

Culture media and isolation methods

Several types of media are used for the cultivation of mycobacteria, and each facility determines which ones are most appropriate for use. A combination of culture media is often used to optimize recovery of mycobacteria as well as inhibit the growth of contaminants. Mycobacteria require a pH of 6.5-6.8 for growth and grow best at higher humidity. Commercially prepared solid culture media (in tubes with screw-top caps) consist of bovine serum albumin agar-based media (Middle-brook 7H10 and 7H11) and egg-based media (Lowenstein-Jensen). Liquid media (Middlebrook 7H9) is used to subculture stock strains or as part of a system (e.g., BACTEC 12B medium, Septi-Chek AFB) to cultivate and detect growth of acid-fast bacilli. Mycobacterium spp. grow more rapidly in liquid media; solid media takes approximately 17 days for the isolation of acid-fast bacilli whereas liquid media takes only about 10 days. The following are descriptions of three general types of media that are most often used.

• Lowenstein-Jensen media (L-J) is an egg-potato base solid media containing malachite green (an inhibitory agent). The use of L-J media is excellent for the recovery of M. tuberculosis from sterile-site specimens as well as decontaminated-digested sputum specimens.
• Petragnani media is an egg-milk-potato solid medium also containing malachite green. It is primarily used for specimens from highly contaminated areas (e.g., fecal material).
• Middlebrook 7H10 media is a liquid based media containing salts, vitamins, cofactors, oleic acid, albumin, catalase, glycerol, and glucose. This media enhances the recovery of MAC organisms (Mycobacteria avium complex).

Each culture medium described above represents a nonselective formulation, but selective formulations are also used which contain antibiotics to enhance the growth of mycobacteria and suppress the growth of contaminating bacteria. The enhanced formulas are used for specimens that are highly contaminated.

All culture tubes are incubated in an atmosphere of 5-10% CO₂ (for growth enhancement) even though mycobacteria are strict aerobes. The tubed media are kept in a high humidity incubator at 35°C in the dark in a slanted position with the caps loosened (in order for CO₂ to enter the tubes and excess fluid to evaporate). For specimens obtained from skin or superficial lesions, a lower temperature (25-30°C) is required for the recovery of M. marinum and M. ulcerans. A nutritional requirement of hemin and a temperature of 30°C are needed for the recovery of M. haemophilum (cultured from skin nodule specimens). If M. xenopi is suspected, a temperature of 42-45°C is required (cultured from hospital hot water tanks).

AFB cultures are held for six to eight weeks before reporting "No growth of AFB." Cultures are observed daily for the first two weeks, checking for any growth or colony formation. Rapid-growing mycobacteria usually appear on non-selective media in two to three days at temperatures between 20 to 40°C. The slow-growing mycobacteria associated with disease require four to six weeks of incubation on selective media. Since the use of liquid media allows mycobacteria to grow more rapidly and is considered the most sensitive primary isolation media, the Becton Dickinson Diagnostic Instrument Systems developed the BACTEC System. The BACTEC System utilizes Liquid Middlebrook 7H12 and 7H13 in an automated radiometric culture system. The broth is placed in commercially prepared vials containing a 14C-labeled substrate (palmitic acid) used by mycobacteria, liberating radioactive carbon dioxide (¹⁴C0₂) into the upper part of the vial. The ¹⁴C0₂ liberated is detected by the BACTEC 460 (instrument) and is recorded as a "growth index" denoting growth of mycobacteria in the vial of broth. This method of growth significantly improves the isolation rate of mycobacteria compared with conventional isolation using solid tubed media. The BACTEC vials must be checked within four days of inoculation. This method detects Mycobacteria spp. growth in clinical specimens in less than two weeks compared to four to six weeks for conventional methods.

Non-radiometric automated systems are also available for the detection of growth and recovery of mycobacteria from clinical specimens. An example is the Septi-Chek AFB system (BBL-Becton Dickinson Microbiology Systems) that detects, isolates, rapidly identifies, and performs antibiotic susceptibility testing. This is a biphasic media system (a bottle containing liquid media and solid media) that uses growth enhancing factors and antimicrobial agents in the liquid and three different solid media on a paddle inserted in the top of the vial. This system rapidly grows, isolates, and presumptively identifies M. tuberculosis (i.e., differentiates it from other mycobacteria).