Growth Characteristics. Preliminary identification of mycobacterial isolates depends on the organisms’ rate of growth, colonial morphology (see Figure 1), colonial texture, pigmentation and, in some instances, the permissive incubation temperatures of mycobacteria. Despite the limitations of phenotypic tests, the mycobacterial growth characteristics are helpful for determining a preliminary identification (e.g., an isolate appears as rapidly growing mycobacteria). To perform identification procedures, quality control organisms should be tested along with unknowns (Table1). The commonly used quality control organisms can be maintained in broth at room temperature and transferred monthly. In this way they are always be available for inoculation to test media along with suspensions of the unknown mycobacteria being tested.

Fig1. Typical appearance of some mycobacteria on solid agar media. A, M. tuberculosis colonies on Löwenstein-Jensen agar after  8 weeks of incubation. B, A different colonial morphology is seen on culture of one strain of M. avium complex. C, M. kansasii colonies  exposed to light. D, Scotochromogen M. gordonae showing yellow colonies. E, Smooth, multilobate colonies of M. fortuitum on Löwenstein Jensen medium.

Table1. Controls and Media Used for Biochemical Identification of Mycobacteria

Growth Rate. The rate of growth is an important criterion for determining the initial category of an isolate. Rapid-growers usually produce colonies within 3 to 4 days after subculture. However, even a rapid-grower may take longer than 7 days to initially produce colonies because of inhibition by a harsh decontaminating procedure. Therefore, the growth rate (and pigment production) must be determined by subculture on the Evolve site). The dilution of the organism used to assess the growth rate is critical. Even slow-growing mycobacteria appear to produce colonies in less than 7 days if the inoculum is too heavy. One organism particularly likely to exhibit false-positive rapid growth is M. flavescens. This species therefore serves as an excellent quality control organism for this procedure.

Pigment Production. As previously discussed, mycobacteria may be categorized into three groups based on pigment production. To achieve optimum photochromogenicity, colonies should be young, actively metabolizing, isolated, and well aerated. Although some species (e.g., M. kansasii) turn yellow after a few hours of light expo sure, others (e.g., M. simiae) may take prolonged expo sure to light. Scotochromogens produce pigmented colonies even in the absence of light, and colonies often become darker with prolonged exposure to light (Figure 2). One member of this group, M. szulgai, is peculiar in that it is a scotochromogen at 35° C and nonpigmented when grown at 25° to 30° C. For this reason, all pigmented colonies should be subcultured to test for photoactivated pigment at both 35° C and 25° to 30° C. Nonchromogens are not affected by light.

Fig2. Initial grouping of mycobacteria based on pigment production before and after exposure to light. In one test system, sub cultures of each isolate are grown on two agar slants. One tube is wrapped in aluminum foil to prevent exposure of the organism to  light, and the other tube is allowed light exposure. After sufficient growth is present, the wrapped tube is unwrapped, and the tubes are  examined together. Photochromogens are nonpigmented when grown in the dark (tube A) and develop pigment after light exposure  (tube B). Scotochromogens are pigmented in the dark (tube C); the color does not intensify after exposure to light (tube D). Nonphotochromogens are nonpigmented when grown in the dark (tube E) and remain so even after light exposure (tube F). 

Biochemical Testing. Once categorized into a preliminary subgroup based on its growth characteristics, an organism must be definitively identified to species or complex level. Although conventional biochemical tests can be used for this purpose, new methods have replaced biochemical tests for identifying mycobacterial species because of the previously discussed limitations of phenotypic testing. Although key biochemical tests are still discussed in this edition, the reader must be aware that this approach to identification ultimately will be replaced by molecular methods. Table2 summarizes distinctive properties of the more commonly cultivable mycobacteria isolated from clinical specimens; key bio chemical tests for each of the major mycobacterial groupings, including M. tuberculosis complex, are listed in Table 3. The following sections address key biochemical tests.

Table2. Distinctive Properties of Commonly Cultivable Mycobacteria Encountered in Clinical Specimens

Table3.  Key Biochemical Reactions to Help Differentiate  Organisms Belonging to the Same Mycobacterial Group

Niacin. Niacin (nicotinic acid) plays an important role in the oxidation-reduction reactions that occur during mycobacterial metabolism. Although all species produce nicotinic acid, M. tuberculosis accumulates the largest amount. (M. simiae and some strains of M. chelonae also produce niacin.) Niacin therefore accumulates in the medium in which these organisms are growing. A positive niacin test is preliminary evidence that an organism that exhibits a buff-colored, slow-growing, rough colony may be M. tuberculosis (Figure 3). However, this test is not sufficient to confirm identification. If sufficient growth is present on an initial L-J slant (the egg-base medium enhances accumulation of free niacin), a niacin test can be performed immediately. If growth on the initial culture is scant, the subculture used for growth rate determination can be used. If this culture yields only rare colonies, the colonies should be spread around with a sterile cotton swab (after the growth rate has been determined) to distribute the inoculum over the entire slant.

Fig3. Niacin test performed with filter paper strips. With a  positive test result (A), the liquid turns yellow. With a negative result  (B), the liquid remains milky white or clear. 

The slant then is incubated until light growth over the surface of the medium is visible. For reliable results, the niacin test should be performed only from cultures on L-J medium that are at least 3 weeks old and show at least 50 colonies; otherwise, enough detectable niacin might not have been produced.

Nitrate Reduction. This test is valuable for identifying M. tuberculosis, M. kansasii, M. szulgai, and M. fortuitum. The ability of acid-fast bacilli to reduce nitrate is influenced by the age of the colonies, temperature, pH, and enzyme inhibitors. Although rapid-growers can be tested within 2 weeks, slow-growers should be tested after 3 to 4 weeks of luxuriant growth. Commercially available nitrate strips yield acceptable results only with strongly nitrate-positive organisms, such as M. tuberculosis. This test may be tried first because of its ease of performance. The M. tuberculosis–positive control must be strongly positive in the strip test, or the test results are unreliable. If the paper strip test is negative or if the control test result is not strongly positive, the chemical procedure must be carried out using strong and weakly positive controls.

Catalase. Most species of mycobacteria, except for certain strains of M. tuberculosis complex (some isoniazid resistant strains) and M. gastri, produce the intracellular enzyme catalase, which splits hydrogen peroxide into water and oxygen. Catalase can be assessed by using the semiquantitative catalase test or the heat-stable catalase test.

• The semiquantitative catalase test is based on the relative activity of the enzyme, as determined by the height of a column of bubbles of oxygen (Figure 4) formed by the action of untreated enzyme produced by the organism. Based on the semiquantitative catalase test, mycobacteria are divided into two groups: those that produce less than 45 mm of bubbles and those that produce more than 45 mm of bubbles.

• The heat-stable catalase test is based on the ability of the catalase enzyme to remain active after heating (i.e., it is a measure of the enzyme’s heat stability). When heated to 68° C for 20 minutes, the catalase of M. tuberculosis, M. bovis, M. gastri, and M. haemophilum becomes inactivated.

Fig4. Semiquantitative catalase test. The tube on the left  contains a column of bubbles that has risen past the line (arrow),  indicating 45-mm height (a positive test result). The tube on the right  is the negative control. 

Tween 80 Hydrolysis. The commonly nonpathogenic, slow-growing scotochromogens and nonphotochromo gens produce a lipase that can hydrolyze Tween 80 (the detergent polyoxyethylene sorbitan monooleate) into oleic acid and polyoxyethylated sorbitol, whereas pathogenic species do not. Tween 80 hydrolysis is useful for differentiating species of photochromogens, nonchromogens, and scotochromogens. Because laboratory prepared media have a very short shelf life, the CDC recommends use of a commercial Tween 80 hydrolysis substrate (Becton-Dickinson, Franklin Lakes, N.J. or Remel Laboratories, Lenexa, Kansas) that is stable for up to 1 year.

Tellurite Reduction. Some species of mycobacteria reduce potassium tellurite at variable rates. The ability to reduce tellurite in 3 to 4 days distinguishes members of MAC from most other nonchromogenic species. All rapid-growers reduce tellurite in 3 days.

Arylsulfatase. The enzyme arylsulfatase is present in most mycobacteria. Test conditions can be varied to differentiate different forms of the enzyme. The rate at which this enzyme breaks down phenolphthalein disulfate into phenolphthalein (which forms a red color in the presence of sodium bicarbonate) and other salts helps to differentiate certain strains of mycobacteria. The 3-day test is particularly useful for identifying the potentially pathogenic rapid-growers M. fortuitum and M. chelonae. Slow-growing M. marinum and M. szulgai are positive in the 14-day test (Figure5).

Fig5. A positive arylsulfatase test result is shown on the left;  the tube containing the negative control is on the right. 

Growth Inhibition by Thiophene-2-Carboxylic Acid Hydrazide (TCH). This test is used to distinguish M. bovis from M. tuberculosis, because only M. bovis is unable to grow in the presence of 10 mg/mL of TCH.

Other Tests. Other tests are often performed to make more subtle distinctions between species (see Table 3). However, performing all the procedures necessary for definitive identification of mycobacteria is not cost-effective for routine clinical microbiology laboratories; therefore, specimens that require further testing can be forwarded to regional laboratories.

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم شؤون المعارف ينظم دورة متقدمة في تحقيق المخطوطات لملاكاته

قسم الشؤون الفكرية يصدر العدد العشرين من مجلة دراسات إفريقية

المجمَع العلمي يكرم الأساتذة المشاركين في مشروع الدورات القرآنية الصيفية في كربلاء

العتبة العباسية المقدسة تختتم فعاليات البرنامج المركزي الأسبوعي لمنتسبيها

المزيد

الآخبار الصحية

هل من الآمن شرب القهوة على معدة فارغة؟

تحذير: مرض السكري يسرق طاقة قلبك

حقيقة صادمة.. لماذا يجب أن تتوقف عن شراء الجزر الصغير؟

بعد عمر الستين.. توقيت وجبة الإفطار مؤشر على صحتك

المزيد

الآخبار التكنلوجية

تركيب مقعد الطفل بأمان في سيارتك: دليل كامل وسهل

مليار مليار عملية في الثانية.. ألمانيا تدشن الحاسوب الفائق

أخيرا.. إنستغرام يصل رسميا إلى آيباد

حل يثير جدلا طبيا.. حقنة قادرة على إخفاء الشيب من رأسك ؟!

المزيد

مواضيع ذات صلة

Genetic Sequencing and Nucleic Acid Amplification for Mycobacterial Infections

Acid-Fast Stains for Mycobacterial Infections

Vibrio Parahaemolyticus and Vibrio vulnificus

Vibrio Cholerae

Burkholderia cepacia Complex

Burkholderia pseudomallei and Burkholderia mallei

Rapidly Growing Nontuberculous mycobacteria (RGM)

Slow-growing Nontuberculous Mycobacteria

Mycobacterium Tuberculosis Complex

Anaerobic Gram-Positive and Gram-Negative Cocci

The Salmonellae

The shigellae