NEHCRI Makassar Laboratory: TB Unit

Prepared by

: Muh. Nasrum Massi

08
Date
: 04.09.2008
Revised by
: Sabai Phyu
Date
: 28.10.09
Approval by : Sabai Phyu
(
Effective date : 01.11.2009

Protocol:

Protocol 08
Identification of NTM by using PNB media
Principle
: Growth of the Mycobacterium tuberculosis complex (MTC) is
inhibited by p-nitrobenzoic acid (PNB), whereas non-tuberculous mycobacteria (NTM)
are resistant.
In this protocol, PNB added culture medium is used to evaluate its usefulness in the
screening of mycobacteria isolates.
Materials

1. PNB reagent (Dissolve 2.55 gr PNB in 100 ml DW. Filter through a 0.2 mm
membrane filter for decontamination. Add 100 microliter PNB into 4 ml MGIT liquid
medium + 0.5 ml OADC + 0.5 ml sample and the final concentration become 500
ug/ml in 5.1 total volume.
2. MGIT liquid medium
3. OADC
Equipment:
1. 37oC incubator
2. 1.5 ml appendorf tube with a screw cap
3. Microcentrifuge
4. Vortex
5. Waterbath
6. -20oC freezer
Microorganisms
1. M tuberculosis complex
2. M avium
3. M kansasii

Version : 3
Last Change : October 28, 2009

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NEHCRI Makassar Laboratory: TB Unit

Sample processing:
1. Inoculate a LJ medium with the mycobacterium strain of interest and incubate at 37 oC
until growth becomes clearly visible.
2. A few colonies were emulsified in flasks containing glass beads and 2 ml of sterile
distilled water.
3. Allowing the turbidity to be greater than McFarland 1 standard.
4. The suspension was left to stand for 15 min to allow larger clumps to settle
5. 1 ml of the supernatant was then transferred to another tube, where the turbidity level
was adjusted to McFarland scale no. 1.
6. This bacterial suspension was used as the working bacteria suspension
7. H37Rv strain is used as a negative control and M kansasi or M avium are used as
positive controls for this test..
8. Test were performed using the BBL-manual MGIT system (Becton Dickinson), which
consists of modified Middlebrook 7H9 broth medium enriched with OADC (albumin,
dextrose and catalase)
9. PNB was added 100 ul into a MGIT tube containing Middlebrok 7H9 media enriched
with OADC. A final concentration of the PNB becomes 500 ug/ml of medium.
10. Control media was BBL-MGIT medium with no addition of inhibitory substances.
11. For testing the culture sample, BBL-MGIT system was inoculated with 500 ul of 1:5
dilution of the work suspension and then incubated at 37oC.
12. Reading of the tubes in the BBL-MGIT system began on the third day after
inoculation, using BACTEC MicroMGIT (BectonDickinson) fluorescence reader
calibrated according to the manufacturers instructions.
13. Tubes containing the PNB were then read after the detection of fluorescence in the
control tube.
14. A strain was considered susceptible when the tube containing PNB did not show
fluorescence 2 days after positive results were observed in the control tube, whereas if
fluorescence occurred the strain was considered resistant.
REFERENCES:
1. C.M.S Giampaglia, M.C. Martins, V.T.G. inumaru, I.V. Butuem, M.A.S. Telles.
Evaluation of a rapid differentiation test for the Mycobacterium tuberculosis
complex by selective inhibition with p-nitrobenzoic acid and thiophene-2carboxylic acid hydrazide. Int J Tuberc Lung dis; 9(2);206-209;2005.
2. Mandira V.B, Sujeet K, Jitender Y, Nalim K, Mridula B. A simple method to
differentiate between Mycobacterium tuberculosis and non-tuberculosis
Mycobacteria directly on clinical specimen. Southeast Asian J Trop Med Public
Health 38(1) ;111-114;2007.

Version : 3
Last Change : October 28, 2009

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