Antimicrobial Susceptibility Testing (AST)

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The page below is a sample from the LabCE course Tracking Antibiotic-Resistant Tuberculosis. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

Learn more about Tracking Antibiotic-Resistant Tuberculosis (online CE course)
Antimicrobial Susceptibility Testing (AST)

The FDA-approved automated systems, including the BACTEC MIGIT 960 (Becton-Dickinson) and the ESP® Culture System II (Trek Diagnostic Systems) for first-line antibiotics, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA) are state-of-the-art AFB susceptibility testing systems. These antibiotics have been recommended by the Clinical and Laboratory Standards Institute (CLSI) for testing.
Automated systems
  • Preferences for automated systems include decreased time for the indirect method (utilizes a pure culture of the test isolate) and the time from media inoculation to reporting is two to four weeks.
  • The standard agar proportion method is more labor-intensive and time-consuming than the commercial broth-automated methods.
  • Greater laboratory safety is maintained when bottles growing AFB are contained inside the automated system cabinet.
  • A recent study demonstrates that first-line and second-line drug testing on the BACTEC MGIT 960 also provides acceptable results in a distinctly shorter time. Second-line drugs usually include four or more of the following: Increased dosages of INH and EMB, capreomycin, ethionamide, amikacin, p-aminosalicylic acid (PAS), rifabutin, streptomycin, and levofloxacin.
Manual methods: Agar proportion
  • The agar proportion method for susceptibility testing of M. tuberculosis complex (MTBC) became the gold standard for testing in the U.S. and Europe after its development in the 1960s.
  • Susceptibility testing of MTBC includes M. tuberculosis, M. bovis, and M. africanum.
  • Either Middlebrook 7H10 medium, to which drugs made from reference powders are added, or agar diffusion, with drug-impregnated discs placed after the agar solidifies, may be employed.
  • The 7H10 medium is supplemented with oleic acid-albumin-dextrose-catalase (OADC) as standard procedure. However, when drug-resistant strains are present, growth may not be adequate unless 7H11 agar, with higher concentrations of INH, RIF, PZA, and EMB, is used.
  • Quality control strains from the American Type Culture Collection (ATCC) are recommended by the CLSI as essential to the successful performance of the method. Resistant strains of MTB (INH and RIF or other drugs) are available from ATCC.
Using the indirect method...
  • A pure culture of MTBC is suspended in 7H9 broth and incubated at 35⁰ to 37⁰C until the turbidity matches a 1.0 McFarland standard.
  • The number of CFU/mL varies from 10-2 - 10-4.
Using the direct method...
  • The inoculum from a processed specimen or an untreated sterile specimen is diluted (according to number of AFB observed on a smear) to the correct concentration.
  • Because the concentration of tubercle bacilli is presumably greater, the direct method has the advantage of a shorter time to reporting results.
  • The contamination rate (in non-sterile specimens) with this method may be as high as ≥ 15%, rendering the test a failure. The contamination must be ≤ 5%, which is more easily obtained by the indirect method.
Agar proportion testing is often performed on quadrant drug plates with 0.1 mL of diluted inoculum added to each quadrant.
  • The test specimen is added to one quadrant and control to the next.
  • After drying, the plates are enclosed in CO2 permeable polyethylene bags or individually sealed and incubated at 35⁰ to 37⁰ C in 5-10% CO2 in air.
  • Plates are checked for contamination after one week when the procedure may be repeated by the indirect method, if necessary.
  • Interpretation of the test requires that the proportion of mutant colonies resistant to a drug is greater than 1% (the concentration of drug in the test quadrant).
  • The percent resistance is derived by dividing the colony count on the drug-containing quadrant by the count on the control quadrant, which must grow ≥ 50 colonies for a valid test.
  • Three weeks are required to determine susceptibility, though resistance may be determined earlier.
11. CDC/ Dr. UP Kokko. 1963. #12128 Caption: "This historic 1963 photograph depicted a laboratorian performing an examination of an agar Petri dish culture plate, known as a Felsen quadrant culture plate, as part of a Mycobacterium tuberculosis drug susceptibility test." PHIL image, public domain. Accessed May 11, 2022.

TB susceptibility agar proportion test. (11)