Determination of MICs for Mycobacterium avium-M. intracellulare complex in liquid medium by a colorimetric method

Gómez-Flores, Ricardo; Gupta, Subash C.; Taméz-Guerra, Reyes; Mehta, R.

doi:10.1128/jcm.33.7.1842-1846.1995

Cited by 81 publications

(40 citation statements)

References 18 publications

(20 reference statements)

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“…As first described by Mosmann (1983), the tetrazolium salt MTT has been used to assess cell viability in various bioassays (Peck 1985; Denizot & Lang 1986; Hansen, Nielsen & Berg 1989; Stevens & Olsen 1993; Gomez‐Flores, Gupta, Tamez‐Guerra & Mehta 1995; Botsford 1997; Mshana et al . 1998; Schaller, Sun, Yang, Somoskovi & Zhang 2002).…”

Section: Discussionmentioning

confidence: 99%

“…The process involves the conversion of the yellow dye MTT by mitochondrial dehydrogenase enzymes to produce insoluble, purple formazan crystals which are then solubilized and measured spectrophotometrically. Although the procedure described by Mosmann (1983) utilized eukaryotic cells, Gomez‐Flores et al . (1995) and Mshana et al .…”

Section: Discussionmentioning

confidence: 99%

“…The inoculum level of 10 8 CFU mL −1 for each bacterial species was chosen for setting up the bioassay based upon the CFU used in previous studies and to help provide sufficient CFU to help produce measurable MTT reduction as previous studies showed that the degree of MTT reduction correlates with the number of bacteria (Gomez‐Flores et al . 1995).…”

Section: Discussionmentioning

confidence: 99%

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A rapid bioassay for bactericides against the catfish pathogens Edwardsiella ictaluri and Flavobacterium columnare

Schrader¹,

Harries²

2006

Aquaculture Res

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The most common bacterial diseases in pond-raised channel cat¢sh Ictalurus punctatus (Ra¢nesque) are enteric septicemia of cat¢sh and columnaris, caused by Edwardsiella ictaluri and Flavobacterium columnare respectively. Medicated feed containing antibiotics is one management approach that cat¢sh producers use in the treatment of bacterial diseases. However, the future use of all types of medicated feed in cat¢sh aquaculture is uncertain. To discover e¡ective alternatives to antibiotics, a rapid 96-well microplate bioassay utilizing E. ictaluri and F. columnare to evaluate natural compounds and extracts was developed. In this bioassay, bacterial growth is determined by absorbance measurements of microplate wells after 24 h incubation and then con¢rmed by detecting cell viability after the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide with additional incubation for 24 h. The minimum inhibitory concentration , minimum bactericidal concentration and 50% inhibition concentration (IC50) are determined by graphing the absorbance data. The 24 h IC50 results of test compounds are compared with the 24 h IC50 results of the drug controls oxytetracycline and £orfenicol. Among the antibiotics evaluated, doxycycline and tetracycline appear more e¡ective against E. ictaluri and F. columnare than either drug control. This bioassay is rapid, reproducible and economical for evaluating a large number of compounds and extracts.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A rapid bioassay for bactericides against the catfish pathogens Edwardsiella ictaluri and Flavobacterium columnare

Schrader¹,

Harries²

2006

Aquaculture Res

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“…Several molecular methods for the rapid detection of drug resistance have been described [6][7][8], but cost implications have precluded their routine implementation in tuberculosis-endemic countries, creating an urgent need for simple, rapid and inexpensive methods. Two promising alternative techniques are colourimetric assays, which use oxidation-reduction indicators (redox methods), and the mycobacteriophage assay [9][10][11][12][13][14][15]. In the present study, two redox indicators, Alamar Blue and 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT), were tested against an in-house D29 phage assay for their ability to detect resistance to isoniazid and rifampicin in M. tuberculosis.…”

Section: Introductionmentioning

confidence: 99%

Comparison of redox and D29 phage methods for detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis

Silva

Boffo

Mattos

et al. 2006

Clinical Microbiology and Infection

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Rapid, accurate and inexpensive methods are essential to detect drug-resistant Mycobacterium tuberculosis and allow timely application of effective treatment and precautions to prevent transmission. The proportion method, the MTT and Alamar Blue redox methods, and the D29 mycobacteriophage assay, were compared for their ability to detect resistance to isoniazid and rifampicin. When tested against a panel of known M. tuberculosis strains, the redox methods and the D29 assay showed good sensitivity and specificity compared to the proportion method, suggesting that they could be useful alternatives for identifying multidrug resistance in M. tuberculosis.

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“…Broth macro-and microdilution methods are widely accepted as standards for evaluation of activities of compounds against aerobic and anaerobic organisms. Such methods, employing both turbidimetric and colorimetric endpoints, have been described for rapid-and slow-growing mycobacteria, including M. tuberculosis (4,9,14,(20)(21)(22)(23). Strategies for susceptibility testing of M. tuberculosis by a bioluminescence assay based on mycobacterial ATP have been successfully applied (16).…”

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confidence: 99%

Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare

Shawar

Humble

Dalfsen

et al. 1997

Antimicrob Agents Chemother

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The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts. Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well minitube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M. intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values. As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually. A total of 480 extracts were tested. Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M. intracellulare. With activity defined as a relative change in luminescence of <1% (i.e., >99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods. Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro. With this method, it is possible to screen a large number of samples in a short period of time. MATERIALS AND METHODSMycobacterial strains. The strains used in this study were the M. bovis BCG substrain Connaught (ATCC 35745) and M. intracellulare ATCC 35761. The integrating shuttle vector pMV361 was constructed and electroporated into rBCG and M. intracellulare as described previously (10).Culture and growth conditions. Stock strains of mycobacteria were maintained in 7H9 broth with 15% glycerol at Ϫ80ЊC. Subcultures of the microorganisms were made in Middlebrook 7H9 broth (Difco, Detroit, Mich.) containing 10% ADC (albumin-dextrose-catalase) enrichment (BBL, Cockeysville, Md.), 0.05% Tween 80 (BBL), and 20 g of kanamycin (Sigma, St. Louis, Mo.)/ml. Cultures of rBCG and recombinant M. intracellulare were incubated in an ambient atmosphere for 48 and 24 h, respectively. Following incubation, the culture suspension was sonicated for 10 s with a VibraCell Sonicator (Sonics & Materials, Inc., Danbury, Conn.). To prepare the inoculum, the sonicated culture was diluted in Middlebrook 7H9 broth without kanamycin to an optical density at 540 nm of 0.05. This procedure yielded a suspension containing approximately 10 7 CFU/ml, as confirmed by a plate count on 7H11 agar (Remel, Lenexa, Kans.). This diluted suspension was used to inoculate test trays as described below, resulting in a final inoculum of 5 ϫ 10 5 CFU/ml in the reaction tube.

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Determination of MICs for Mycobacterium avium-M. intracellulare complex in liquid medium by a colorimetric method

Cited by 81 publications

References 18 publications

A rapid bioassay for bactericides against the catfish pathogens Edwardsiella ictaluri and Flavobacterium columnare

A rapid bioassay for bactericides against the catfish pathogens Edwardsiella ictaluri and Flavobacterium columnare

Comparison of redox and D29 phage methods for detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis

Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare

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