nineteen isolates were multidrug - resistant ( mdr ) , eight 
 isolates were only resistant to inh and the remaining isolates were sensitive to both 
 inh and rif .
the bactec 460 tb system was used as the reference method for testing of the 
 mdr isolates and the bactec mgit 960 system was used as the reference method for testing 
 the remaining isolates .
the concentrations of inh and rif for the bactec 460 tb / bactec 
 mgit 960 systems were 0.1/0.1 and 2.0/1.0 g / ml , respectively .
the h37rv - atcc 25618 ( susceptible to all drugs ) , atcc 35822 ( resistant to inh ) , atcc 
 35838 ( resistant to rif ) and atcc 35820 ( resistant to str ) strains were used for quality 
 control .
preparation of antibiotics and malachite green ( mg ) - the inh and rif 
 antibiotics used in this study were purchased from sigma and the mg dye was from merck 
 ( germany ) .
inh and mg were dissolved in sterile distilled water and rif was prepared in 
 methanol .
stock solutions were prepared as 1,000 g / ml , 1,000 g / ml and 50 g / ml for 
 inh , rif and mg , respectively .
preparation of bacterial inocula - bacterial colonies were freshly 
 grown on lwenstein - jensen media , transferred into tubes containing physiologic saline 
 and 15 - 20 glass beads and vortexed for 30 sec .
the tubes were kept in a vertical 
 position for 30 min at room temperature to allow for the sedimentation of aerosols and 
 large particles .
the turbidity of the supernatant was adjusted to that of the mcfarland 
 1 standard . 

preparation of media - all m. tuberculosis isolates 
 were tested in middlebrook 7h9s broth ( containing 0.1% casitone , 0.5% glycerol and 10% 
 oadc ) .
inh and rif were tested at the critical concentrations of 0.25 g / ml and 0.5 
 g / ml , respectively ( martin et al . 2011 ) .
an 
 antibiotic - free growth control tube was also used for each bacterium . 
 performing the mgda test - three tubes containing inh , rif or 
 antibiotic - free control growth medium were used for each bacterium .
fifty microlitres of 
 inoculum adjusted to the mcfarland 1 standard was added to each tube and the tubes were 
 incubated at 37c . following a seven - day incubation period
, 150 l of mg ( 50 g / ml stock 
 solution ) was added into each test tube and the tubes were incubated for an additional 
 24 - 48 h. once the mg decolourised in the growth control tube , the test tubes containing 
 antibiotic were evaluated for colour change .
if the mg decolourised as a result of 
 bacterial growth in the tubes containing antibiotic , the bacterial sample was reported 
 as resistant ( figure ) .
the sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value 
 ( npv ) and agreement of the mgda test for inh were 92.5% , 91.3% , 92.5% , 91.3% and 92% , 
 respectively .
two isolates were sensitive to inh by the mgda test , but resistant by the 
 reference method .
two additional isolates were sensitive to inh by the reference method , 
 but resistant by the mgda test . for rif , the sensitivity , specificity , ppv , npv and 
 agreement of the mgda test were 94.7% , 100% , 100% , 96.8% and 98% , respectively .
one 
 isolate was resistant to rif by the reference method , but susceptible by the mgda test ( 
 table ) .
phenotypic methods that are inexpensive , rapid and reliable make susceptibility testing 
 of m. tuberculosis easier and more efficient .
studies 
 have shown that colourimetric tests are promising methods for drug susceptibility 
 testing , particularly the resazurin microplate method , the resazurin tube method , the 
 nitrate reductase test , the mtt and the xtt ( angeby et 
 al .
in contrast to most bacteria and fungi , mycobacteria 
 are resistant to mg . media for the cultivation of mycobacteria ( lwenstein - jensen ) 
 contain mg to reduce overgrowth by contaminating microorganisms .
mycobacterial resistance to mg may be due to dye reduction and 
 sequestration in the lipid fraction of the cells ( jones 
 & falkinham 2003 ) . 

( 2012 ) reported that mg interfered 
 with the recovery of mycobacteria on solid culture media following exposure to certain 
 antibiotics , including inh and ethionamide ( etm ) , that target cell wall biogenesis .
this 
 interference did not affect the test results for inh sensitivity because mg was used to 
 determine the viability of bacteria in this test . 

( 2008 ) reported that mgda could be 
 used for the rapid detection of drug susceptibilities of m. 
 tuberculosis clinical isolates ; the sensitivity , specificity and agreement 
 of the mgda test were found to be 100% for both rif and inh and the results were 
 obtained within six-17 days ( average of 12 days ) . in our study , the inh - rif 
 sensitivities , specificities , ppvs , npvs and agreements of the mgda test were 
 92.5 - 94.7% , 91.3 - 100% , 92.5 - 100% , 91.3 - 96.8% and 92 - 98% , respectively and the results 
 were obtained within eight - nine days .
there was a major discrepancy in the tests of two 
 isolates that were sensitive to inh by the mgda test , but resistant by the reference 
 method .
in addition , there was a minor discrepancy in the tests of two additional 
 isolates that were sensitive to inh by the reference method , but resistant by the mgda 
 test .
the group reported that 50 l of a 0.02 g / ml mg stock 
 solution was used for the direct susceptibility test , whereas 50 l of a 0.02 mg / ml mg 
 stock solution was used for the indirect susceptibility test .
therefore , the 
 concentration of the stock solution used in the study is not clear . our preliminary 
 studies indicated that using a 0.02 g / ml stock solution was not suitable for the mgda 
 test ; an mg titration showed that 150 l of a 150 g / ml stock solution was suitable for 
 the susceptibility testing using 1 ml test tubes ( figure ) .
2010 ) 
 reported that mgda could be used for the rapid detection of pyrazinamide resistance . 

they used 50 l of a 50 g / ml mg solution in their study .
no additional studies using 
 mgda were found in searches of medline , pubmed , isi web , web of science and google 
 academic databases . in conclusion ,
the mgda test for the rapid detection of inh and rif resistance is an 
 inexpensive , easy to perform and interpretable method that can be used in laboratories 
 for routine drug susceptibility testing .
inh : isoniazid ; npv : negative predictive value ; ppv : positive predictive value ; r : 
 resistant ; rif : rifampicin ; s : susceptible .
 early detection of drug resistance in mycobacterium tuberculosis 
 isolates allows for earlier and more effective treatment of patients . 
 the aim of this 
 study was to investigate the performance of the malachite green decolourisation assay 
 ( mgda ) in detecting isoniazid ( inh ) and rifampicin ( rif ) resistance in m. 
 tuberculosis clinical isolates . 
 fifty m. tuberculosis 
 isolates , including 19 multidrug - resistant , eight inh - resistant and 23 inh and 
 rif - susceptible samples , were tested . the sensitivity , specificity , positive 
 predictive value ( ppv ) , negative predictive value ( npv ) and agreement of the assay 
 for inh were 92.5% , 91.3% , 92.5% , 91.3% and 92% , respectively . 
 similarly , the 
 sensitivity , specificity , ppv , npv and agreement of the assay for rif were 94.7% , 
 100% , 100% , 96.8% and 98% , respectively . 
 there was a major discrepancy in the tests 
 of two isolates , as they were sensitive to inh by the mgda test , but resistant by the 
 reference method . 
 there was a minor discrepancy in the tests of two additional 
 isolates , as they were sensitive to inh by the reference method , but resistant by the 
 mgda test . 
 the drug susceptibility test results were obtained within eight - nine days . 
 in conclusion , 
 the mgda test is a reliable and accurate method for the rapid 
 detection of inh and rif resistance compared with the reference method and the mgda 
 test additionally requires less time to obtain results . 