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Journal of Clinical Microbiology, June 2007, p. 1941-1948, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02616-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid and Reliable Method for Quantification of Mycobacterium paratuberculosis by Use of the BACTEC MGIT 960 System

Sung Jae Shin,^1, Jun Hee Han,² Elizabeth J. B. Manning,¹ and Michael T. Collins^1*

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, 2015 Linden Drive, Madison, Wisconsin 53706-1102,¹ Department of Statistics, College of Letters and Sciences, University of Wisconsin—Madison, 1300 University Avenue, Madison, Wisconsin 53705²

Received 30 December 2006/ Returned for modification 6 March 2007/ Accepted 28 March 2007

A simple method for the enumeration of viable Mycobacterium paratuberculosis cells was developed and evaluated using the MGIT 960 culture system. For each of 12 M. paratuberculosis strains isolated from either cattle or humans, single-cell suspensions of M. paratuberculosis cells were adjusted to an optical density at 600 nm of 1.00 (10^7.6 to 10^8.2 cells/ml), and serial dilutions were prepared. Standard curves were established by relating the MGIT time-to-detection data to the log₁₀ CFU for these suspensions using standard plate counting and BACTEC 460 results as reference methods. Universal and strain-specific standard quantification curves were generated. A one-phase exponential decay equation best fit the universal standard curve and strain-specific curves (R² of 0.96 and >0.99, respectively). Two subgroups within the universal curves were distinguished: one for laboratory-adapted strains and the other for recently isolated low-passage bovine strains. The predictive errors for log₁₀ estimations using the universal standard curve, each subgroup's standard curve, and strain-specific curves were ±0.87, ±0.45, and ±0.31 log₁₀ units, respectively. CFU estimations by all three standard curves were highly reproducible, regardless of the M. paratuberculosis strain or inoculum volume. In comparison with the previously described BACTEC 460 M. paratuberculosis counting method, quantification with MGIT 960 was less expensive, more rapid, more accurate, and more sensitive (<10 CFU). This MGIT counting method has broad applications for studies requiring the quantification of viable M. paratuberculosis cells, such as drug susceptibility testing or environmental survival studies.

Published ahead of print on 11 April 2007.

Current address: Department of Microbiology, College of Medicine, Chungnam National University, Daejon 301-747, South Korea.

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