Laboratory Manual andMicrobiology in Action Requiring Special Companies 2003 Workbook in Microbiology Laboratory Techniques 7e

ria must be handled with particular caution and strict asepsis. These organisms, with their thick waxy coats, can survive for long periods even under adverse environmental conditions. They can remain viable for long periods in dried sputum or other infectious discharges and they are also resistant to many disinfectants. Choosing a suitable disinfectant for chemical destruction of tubercle bacilli requires careful consideration.

Clinical samples such as sputum must be digested and concentrated before they are cultured for mycobacteria. During the digestion process, the thick, viscous sputum is liquefied so that any mycobacteria present, particularly if present in low number, are distributed evenly throughout the specimen. After digestion, the sample is centrifuged at high speed to concentrate the mycobacteria at the bottom of the centrifuge tube. The supernatant fluid is discarded into an appropriate disinfectant, and an acid-fast-stained smear of a portion of the centrifuged pellet is prepared and examined microscopically. Another portion is cultured on special mycobacterial culture media. The digestion and concentration steps greatly improve the microscopic detection of mycobac-teria in clinical specimens and their recovery in culture.

The Kinyoun stain (see Exercise 6) is commonly used to stain acid-fast bacilli. The organisms stain red against a blue background, whereas non-acid-fast organisms are blue (see color-plate 9). Tubercle bacilli are slender rods, often beaded in appearance. Another stain, using the fluorescent dyes auramine and rhodamine, permits rapid detection of the organisms as bright objects against a dark background (see colorplate 9). This technique is used in many laboratories today because the fluorescing organisms are easier to detect than bacilli stained with the acid-fast method. Thus, the preparation can be scanned at X400 magnification rather than X 1,000.

Most mycobacteria do not grow on conventional laboratory media such as chocolate or blood agar plates. Therefore, special solid media containing complex nutrients, such as eggs, potato, and serum, are used for culture. Lowenstein-Jensen medium is a solid egg medium prepared as a slant and is one of several in common use (see colorplate 39). Broth media are also available.

Tubercle bacilli and most other mycobacteria grow very slowly. At least several days, and up to 4 to 8 weeks for M. tuberculosis, are required for visible growth to appear. They are aerobic organisms, but their growth can be accelerated to some extent with increased atmospheric CO2. Automated instruments that detect the CO2 released by mycobacteria are used in many laboratories and permit detection before visible growth is apparent. The CO2 is released when mycobac-teria metabolize special substrates present in broth culture media.

M. leprae (also known as Hansen bacillus) cannot be cultivated on laboratory media. Laboratory diagnosis of Hansen disease is based only on direct microscopic examination of acid-fast smears of material from the lesions.

EXPERIMENT 29.1 Microscopic Morphology of Mycobacterium tuberculosis

Purpose To study M. tuberculosis in smears

Materials Prepared acid-fast stains

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