Advanced Techniques in Diagnostic Microbiology

23. Diagnosis of Mycobacterium tuberculosis 393
Growth rate, colony morphology, and biochemical tests are other important differentiating
factors among mycobacteria. Mycobacteria that grow less than 14 days
are called rapid growers, and those that grow after 14 days are called slow growers.
Examination of the morphology of colonies is important especially in mixed cultures.
After 15 days growth, MTB produces thin, nonpigmented, rough colonies
on 7H11 agar. Biochemical tests also aid in the identification of MTB from NTM.
TB has the ability to reduce nitrate to nitrite. There are also other tests such as
production of catalase or urease, arylsulfatase test, iron uptake, tween hydrolysis,
tellurite reduction, and positive niacin test, which aid in the diagnosis of tuberculosis
(Ve’ronique Vincent, 2003).
Modern Methods for Identifying MTB
BACTEC 460TB
Automated or semiautomated liquid culture systems that detect growth of mycobacteria
species earlier than direct visualization have been developed. BACTEC
460TB system was commercially developed in the early 1980s by Becton
Dickinson Company Sparks, MD, USA (Watterson and Drobniewski, 2000;
Inderlied, 2004). This method is based on radiometric analysis of liquid growth
medium containing palmitic acid, labeled with radioactive carbon ( 14 C-palmitic
acid) as the substrate (Tenover et al., 1993). As the mycobacteria metabolize these
fatty acids, radioactive carbon dioxide ( 14 CO 2 ) is released and measured by the
system (Sharma and Mohan, 2004). This system has evolved to become the “gold
standard” (Scarparo et al., 2002). The advantage of this method is reduced detection
time of both smear-positive and -negative samples by nearly 50%; also, the
bacterial growth can be detected in 5–10 days (Brunello et al., 1999; Katoch, 2004).
Although this system considerably reduces the mycobacteria detection time, it is
labor intensive and has other limitations, including cumbersome manual loading
and unloading, potential risk of cross contamination, lack of computerized data
management, use of radioactive material, and accumulation of low-level radioactive
waste (Cruciani et al., 2004).
BACTEC 9000 MB
BACTEC 9000 MB is a fully automated, nonradiometric method developed by
Becton Dickinson Co. This fluorescence-based system uses an oxygen-specific
sensor to detect the mycobacterial growth (Flanagan, et al., 1999; Caws and
Drobniewski, 2001). In every vial, there is a silicon rubber disk impregnated with a
ruthenium metal complex as oxygen-specific sensor (van Griethuysen et al., 1996).
Although this system is more rapid and less labor-intensive than solid media, it requires
using a solid media, MYCO/F medium, a modified Middlebrook 7H9 broth
to suppress the growth of contaminating microorganisms and can be supplemented