Efficacy of Centrifugation as a Method of Concentrating Tubercle Bacilli*

Klein, George C.; Maltz, Morris; Cummings, Martin M.; Fish, Charles H.

doi:10.1093/ajcp/22.6_ts.581

Cited by 8 publications

(6 citation statements)

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“…In general, buoyancy affects overall diagnostic sensitivity, independent of viability, a conclusion that is consistent with the results of Klein et al (8). These authors showed that (i) 88.8 and 82.4% of all specimens spun for 15 min at 2,000 rpm or 3,000 rpm, respectively, contained cultivatable material in the supernatant following centrifugation; (ii) a small percentage of specimens (11.1 and 17.5%, respectively) contained cultivatable material in the sediment only; and (iii) a small percentage of specimens (2.7 and 2.2%, respectively) contained cultivatable material in the supernatant only.…”

Section: Discussionsupporting

confidence: 90%

In Vitro Comparison of NALC-NaOH, Tween 80, and C ₁₈ -Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria

Thornton¹,

MacLellan²,

Brink³

et al. 1998

J Clin Microbiol

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A recent article (C. G. Thornton et al., J. Clin. Microbiol. 36:1996–2003, 1998) reported a new specimen-processing method for improved recovery of mycobacteria. This method used C18-carboxypropylbetaine (CB-18) and increased both smear and culture sensitivity. The companion article (C. G. Thornton et al., J. Clin. Microbiol. 36:2004–2013, 1998) described initial improvements to this method. Additional significant parameters of the CB-18 processing method are identified herein. First, eliminating the incubation step was shown to further improve culture sensitivity. Subsequently, recovery of several mycobacterial isolates by the CB-18 method was compared to a contemporary processing method that combines NALC and NaOH (NALC-NaOH) and a Tween 80-based method. Recovery of the tuberculous isolates following NALC-NaOH processing averaged 20% and ranged from 1.6 to 45%, whereas recovery of the nontuberculous isolates averaged 11% and ranged from 0.1 to 55%. Recovery of the tuberculous and nontuberculous isolates by the Tween 80-based method ranged from 22 to 92% and 27 to 93%, respectively, with averages of 58 and 65%, respectively. Recovery of the tuberculous and nontuberculous mycobacteria following CB-18 processing averaged 86 and 73%, respectively, with ranges from 61 to over 100% and from 43 to over 100%, respectively. Other parameters of the CB-18 method were also examined, including recovery versus CB-18 concentration and the relationship between CB-18 concentration and the tuberculocidal effect. The tuberculocidal effect was time dependent but independent of concentration, whereas recovery was directly proportional to concentration. Increasing the CB-18 concentration to 4 mM provided quantitative recovery on solid medium; however, higher concentrations of CB-18 were not compatible with liquid culture. Examination of the relationship between increasing CB-18 and lecithin concentrations suggested that lecithin could not overcome the deleterious effects of CB-18 in liquid culture at these higher concentrations.

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Section: Discussionsupporting

confidence: 90%

In Vitro Comparison of NALC-NaOH, Tween 80, and C ₁₈ -Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria

Thornton¹,

MacLellan²,

Brink³

et al. 1998

J Clin Microbiol

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“…For example, the solutions (e.g., NaOH) used to process mycobacterial specimens inhibit the PCR (14) and can also affect the sensitivity of the PCR (2). In addition, methods involving centrifugation that are used for preparing clinical specimens suspected of harboring mycobacteria are deficient because of the waxy cell wall (i.e., surface tension) and the buoyant nature of the mycobacteria (5,6,10,12,14). The difficulty associated with lysing these organisms further complicates detection.…”

mentioning

confidence: 99%

Comparison of C ₁₈ -Carboxypropylbetaine and Glass Bead DNA Extraction Methods for Detection of Mycobacterium bovis in Bovine Milk Samples and Analysis of Samples by PCR

Cornejo

Sahagún-Ruíz

Suárez-Güemes

et al. 1998

Appl Environ Microbiol

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The purpose of this prospective study was to compare two different milk preparation methods to assay for the presence ofMycobacterium bovis by PCR. Detection by a C18-carboxypropylbetaine (CB-18)-based sample processing method was compared to extraction of DNA from milk with glass beads. Samples from 17 skin test-positive cattle were analyzed. Following CB-18 processing and glass bead extraction, the sensitivity of IS6110-based PCR was 94.1 and 58.8%, respectively (P < 0.025). Because CB-18 processing will permit the proficient use of PCR for diagnosis and surveillance of bovine tuberculosis, it will contribute to the more efficient detection and control of tuberculosis.

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“…Mycobacteria have a low specific gravity and may remain buoyant during centrifugation (7). The importance of examining the supernatant fluid for mycobacteria has been stressed in the past (1,7). However, the advantage of this procedure in routine practice has not been established (7).…”

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Effect of relative centrifugal force and centrifugation time on sedimentation of mycobacteria in clinical specimens

Ratnam¹,

March²

1986

J Clin Microbiol

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Optimum relative centrifugal force (RCF) and centrifugation time to concentrate mycobacteria in clinical specimens were determined by processing split samples of sputa and urines containing mycobacteria with combinations of different RCFs and centrifugation times. Although individual test results showed considerable variation in the recovery rates of mycobacteria in the sediment, the data indicated that higher recovery rates occurred as centrifugation speed and time were increased. With a 15to 20-min centrifugation time, on the average, 67 to 71 % of mycobacteria were recovered at an RCF of 2,074 x g, and 76 to 80% were recovered at 3,005 or 3,895 x g at maximum radius. The remainder of mycobacteria was mostly recovered from the supernatant, but culturing of supernatant was not profitable. Increasing RCF had a negligible effect on acid-fast bacillus smear sensitivity. The smear sensitivity for about 25,000 clinical specimens processed with an RCF of 3,800 x g for 20 min was 71% compared with 69% as determined for over 30,000 specimens processed in a similar manner but with an RCF of 2,000 x g. An RCF of 3,000 x g applied for 15 min, or an RCF of about 2,000 to 2,500 x g applied for 20 min, is considered adequate to concentrate mycobacteria in clinical specimens.

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Efficacy of Centrifugation as a Method of Concentrating Tubercle Bacilli*

Cited by 8 publications

References 0 publications

In Vitro Comparison of NALC-NaOH, Tween 80, and C ₁₈ -Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria

In Vitro Comparison of NALC-NaOH, Tween 80, and C ₁₈ -Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria

Comparison of C ₁₈ -Carboxypropylbetaine and Glass Bead DNA Extraction Methods for Detection of Mycobacterium bovis in Bovine Milk Samples and Analysis of Samples by PCR

Effect of relative centrifugal force and centrifugation time on sedimentation of mycobacteria in clinical specimens

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Efficacy of Centrifugation as a Method of Concentrating Tubercle Bacilli*

Cited by 8 publications

References 0 publications

In Vitro Comparison of NALC-NaOH, Tween 80, and C 18 -Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria

In Vitro Comparison of NALC-NaOH, Tween 80, and C 18 -Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria

Comparison of C 18 -Carboxypropylbetaine and Glass Bead DNA Extraction Methods for Detection of Mycobacterium bovis in Bovine Milk Samples and Analysis of Samples by PCR

Effect of relative centrifugal force and centrifugation time on sedimentation of mycobacteria in clinical specimens

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Product

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In Vitro Comparison of NALC-NaOH, Tween 80, and C ₁₈ -Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria

In Vitro Comparison of NALC-NaOH, Tween 80, and C ₁₈ -Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria

Comparison of C ₁₈ -Carboxypropylbetaine and Glass Bead DNA Extraction Methods for Detection of Mycobacterium bovis in Bovine Milk Samples and Analysis of Samples by PCR