Critical Parameters and Troubleshooting

A plausible explanation for minor discrepancies is that the majority of the population of M. tuberculosis was not in the exponential growth phase when tested by flow cytometry. Hydrolysis of FDA is affected by the metabolic state or activity of the M. tuberculosis cells. This may be especially true when an inoculum is scraped from solid medium for susceptibility testing (the most common case). Generally, the central portion of the colony has the lowest viability. Therefore, efforts should be made to use M. tuberculosis cells grown in broth for <14 days to perform susceptibility testing. Even M. tuberculosis cells grown in broth can vary in their viability. Maximum viability can be obtained by inoculating 10 ml of 7H9 broth with M. tuberculosis cells contained in 70-ml tissue culture flasks with canted 0.2-|m vented blue-plug seal caps (Falcon 3109). Cultures should be gently shaken daily to disperse the tubercle bacilli and to maintain exposure to atmospheric air and 5% CO2. Generally, 5 to 9 days of incubation is required to obtain sufficient M. tuberculosis organisms (1 x 107/ml) to perform susceptibility testing.

Biosafety is a serious concern. Viable my-cobacterial cells with or without exposure to antituberculosis agents are being processed by the flow cytometer, which can generate droplet nuclei of <5 |m containing a single cell of M. tuberculosis. It takes only one tubercle bacillus to establish infection in humans. Therefore, safety is primary. This procedure can be safely performed only by public health laboratories or large reference laboratories having a Biosafety Level 3 tuberculosis laboratory and experience with Biosafety Level 3 precautions. To address this issue, the authors have developed a procedure that kills the mycobacterial cells after they have been stained and yet will not compromise the differential effect of FDA to distinguish between viable and nonviable cells (Moore et al., 1999).

The high cost of the flow cytometer has also been a critical issue for universal implementation and acceptance of this rapid susceptibility test. However, considering the high costs of supplies for performing the radiometric proportion tests, including the cost of the instrument, the flow cytometer is less expensive, especially with a refurbished instrument. The reagents used for flow cytometry are also relatively inexpensive. Cost are restricted to the purchase of 7H9 broth, microtubes, FDA, and the antituberculosis agents. Technician times for performing the radiometric proportion and flow cytometric methods are similar.

In conclusion, flow cytometry with FDA staining of M. tuberculosis cells is a simple and accurate method for obtaining susceptibility test results in 24 hr, and should greatly assist public health personnel in the control of tuberculosis.

Anticipated Results

Isolates of M. tuberculosis susceptible to ethambutol, isoniazid, or rifampin will yield a susceptibility index of <0.75. Resistant isolates will yield a susceptibility index of >0.76; generally, resistant isolates have a susceptibility index of >0.95.

Time Considerations

Results of the flow cytometric susceptibility tests are available after 24 hr of initiation (setup) of the testing procedure. Generally, 98% of the susceptibility tests can be processed by the flow cytometer 24 hr after incubating the antimycobacterial agent with an isolate of M. tuberculosis. The remaining tests (2%) may require an additional 24 hr of incubation before results are obtained by flow cytometry. Other methods of susceptibility testing require 4 to 21 days of incubation before susceptibility results are available.

Literature Cited

Bownds, S.E., Kurzynski, T.A., Norden, M.A., Dufek, J.L., and Schell, R.F. 1996. Rapid susceptibility testing for non-tuberculosis mycobacteria using flow cytometry. J. Clin. Microbiol. 34:1386-1390.

Centers for Disease Control and Prevention (CDC). 1998. Tuberculosis morbidity—United States, 1997. Morbid. Mortal. Weekly Rep. 47:253-257.

CDC. 1996. Tuberculosis morbidity—United States, 1995. Morbid. Mortal. Weekly Rep. 45:365-370.

Kirk, S.M., Schell, R.F., Moore, A.V., Callister, S.M., and Mazurek, G.H. 1998. Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid and rifampin in 24 hours. J. Clin. Microbiol. 36:1568-1573.

Moore, A.V., Kirk, S.M., Callister, S.M., Mazurek, G.H., and Schell, R.F. 1999. Safe determination of susceptibility of Mycobacterium tuberculosis to antimicrobial agents by flow cytometry. J. Clin. Microbial. 37:479-483.

National Committee for Clinical Laboratory Standards (NCCLS). 1995. Antimycobacterial Susceptibility Testing for Mycobacterium tuberculosis. Proposed Standard M24-T. NCCLS, Villanova, Pa.

Norden, M.A., Kurzynski, T.A., Bownds, S.E., Callister, S.M., and Schell, R.F. 1995. Rapid susceptibility testing of Mycobacterium tuberculosis (H37Ra) by flow cytometry. J. Clin. Microbiol. 33:1231-1237.

Siddiqi, S.H., Hawkins, J.E., and Laszio, A. 1985. Interlaboratory drug susceptibility testing of Mycobacterium tuberculosis by a radiometric procedure and two conventional methods. J. Clin. Microbiol. 22:919-923.

Vena, R.M., Munson, E.L., DeCoster, D.J., Feh, D.B., Callister, S.M., and Schell, R.F. 2000. Flow cytometric testing of Mycobacterium avium to amikacin, ciproflaxin, clarithromycin and ri-fabutin in 24 hours. Clin. Microbiol. Infect. 6:365-375.

Microbiological Applications

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