Flow cytometric detection and quantification of mycobacteria in metalworking fluids

Chang, Shou-Yi; Rihana, A.; Bahrman, S.; Gruden, Cyndee; Khijniak, A.I.; Skerlos, Steven J.; Adriaens, Peter

doi:10.1016/j.ibiod.2004.03.019

Cited by 11 publications

(17 citation statements)

References 34 publications

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“…Since aggregation of mycobacterial cells interferes with accurate optical quantification using both EFM and FCM, the cells were dispersed by a 45-s exposure of probe sonication at amplitude level 2 (Virsonic 50, Virtis Company Inc., Gardiner, NY). Sonication after formalin fixation has been suggested to reduce the damage due to strengthened cellular layer (Tso and Taghon, 1997) and the specific results of mycobacterial cell disaggregation in MWFs were reported previously (Chang et al, 2004). The microcentrifuge tubes were put into an ice bath while applying probe sonication to prevent sample heating and cavitation (Harrison, 1991).…”

Section: Sample Preparationmentioning

confidence: 98%

“…Probe sonication resulted in effective disaggregation of M. parafortuitum and M. immunogenum based on EFM and FCM quantification, giving a good linear correlation between both enumeration methods (Chang et al, 2004). It is plausible that the components in the semisynthetic MWF had prevented the 62 CHANG AND ADRIAENS mycobacterial cells from reaggregating (Calleja, 1984;Ortalo-Magné et al, 1996).…”

Section: Pretreatment and Amendment Of M Immunogenum In Mwfsmentioning

confidence: 99%

“…It is plausible that the components in the semisynthetic MWF had prevented the 62 CHANG AND ADRIAENS mycobacterial cells from reaggregating (Calleja, 1984;Ortalo-Magné et al, 1996). Optical interference due to MWF matrices has been successfully overcome by adding low-polarity solvents (Chang et al, 2004(Chang et al, , 2005. Recently, other researchers (Skerlos et al, 2003;Veillette et al, 2004) have also reported their enumeration results of mycobacteria.…”

Section: Pretreatment and Amendment Of M Immunogenum In Mwfsmentioning

confidence: 99%

“…This calibration curve is good over five orders of magnitude based on VA. At the lowest cell concentrations (10 3 cells/mL), the FCM counts showed slight deviation from the curve. It is probably due to background noise that came from incomplete destabilization of surfactant-dye complex by the resolving agent (Chang et al, 2004). The same data files were reanalyzed by using CAAVA and the calibration curve is Y ϭ 0.0061 ϫ X with R 2 ϭ 0.9998, and it is good over four orders of magnitude.…”

Section: Calibration Curves and Detection Limit For M Immunogenum Inmentioning

confidence: 99%

“…Traditional enumeration methods for bacteria in MWFs include direct and indirect detection technologies (briefly reviewed in Chang et al, 2004). Due to the slow growth rate and aggregate-forming characteristics of mycobacteria, these approaches may either underestimate their cell density or are too time-consuming to be effectively used for process control.…”

Section: Introductionmentioning

confidence: 99%

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Nano-Immunodetection and Quantification of Mycobacteria in Metalworking Fluids

Chang

Adriaens

2007

Environmental Engineering Science

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The accurate detection and enumeration of mycobacteria in metalworking fluids (MWFs) is imperative from an environmental protection and occupational health perspective. We report here on a comparison of the labeling efficiency of nano-immunomagnetic particles (NIMP) and free antibody (FAb) to detect mycobacteria in semisynthetic MWF by using both traditional visualization analysis and cluster analysisaided visualization analysis (CAAVA). The NIMP labeling method involved coating nanometer-scale magnetic particles with Protein A, and oriented conjugation of polyclonal antibodies specific to Mycobacterium spp. The FAb labeling method is modified from the traditional immunofluorescence (IF) method for more efficient detection of mycobacteria in a model MWF. The labeling efficiency of NIMP and FAb were 7.2 Ϯ 4.6 and 16.3 Ϯ 5.5%, and the specificity 85.0 Ϯ 6.1 and 88.1 Ϯ 10.5%, respectively, based on traditional visualization analysis. Based on CAAVA analysis, the labeling efficiency of NIMP and FAb increased to 12.4 Ϯ 1.6 and 20.5 Ϯ 3.9%, and the specificity to 97.8 Ϯ 3.2 and 98.5 Ϯ 2.5%, respectively. A linear relationship of FCM counts and seeded concentrations was observed over four orders of magnitude (R 2 Ͼ 0.99) in pure and ternary cultures. The results strongly support the applicability of either FAb or NIMP coupled with CAAVA and flow cytometry for rapid detection and enumeration of mycobacteria in complex matrices.

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Section: Sample Preparationmentioning

confidence: 98%

Section: Pretreatment and Amendment Of M Immunogenum In Mwfsmentioning

confidence: 99%

Section: Pretreatment and Amendment Of M Immunogenum In Mwfsmentioning

confidence: 99%

Section: Calibration Curves and Detection Limit For M Immunogenum Inmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Nano-Immunodetection and Quantification of Mycobacteria in Metalworking Fluids

Chang

Adriaens

2007

Environmental Engineering Science

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The microbiology of metalworking fluids

Saha

Donofrio

2012

Appl Microbiol Biotechnol

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Metalworking fluids (MWFs) are complex mixtures of chemicals and are indispensable materials in industry. They are used as cooling and lubricating agents in different machining process such as grinding, milling, and cutting. The quality of MWFs is affected by physical, chemical, and microbial contaminates. In particular, MWFs are highly vulnerable to microbial contamination, which may act both as potential pathogens and deteriorgens. Microbial contamination is of major concern due to potential health hazards such as skin dermatitis and hypersensitivity pneumonitis. The contaminated MWFs can exhibit high degrees of microbial loading, ranging from 10(4) to 10(10) colony-forming units (CFU)/ml. Wide varieties of microorganisms are reported to colonize MWFs. Traditional culturing techniques are not only laborious and time consuming but also underestimate the actual distribution of the microorganisms present in the contaminated MWFs. Therefore, rapid molecular methods such as real-time PCR and fluorescent in situ hybridization are implemented to monitor the microbial load. In industry, biocides are presently used to control microbial contamination. However, it has its own disadvantages and therefore, in recent years, alternative methods such as UV irradiation were evaluated to reduce microbial contamination in MWFs. Microbes inhabiting the MWF are also capable of forming biofilm which is detrimental to the MWF system. Biofilm is resistant to common disinfectant methods, and thus further research and development is required to effectively control its formation within MWF systems. This review is intended to discuss the overall microbiological aspects of MWF.

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Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

Chang

Anderson

Bahrman

et al. 2005

J IND MICROBIOL BIOTECHNOL

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The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.

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Flow cytometric detection and quantification of mycobacteria in metalworking fluids

Cited by 11 publications

References 34 publications

Nano-Immunodetection and Quantification of Mycobacteria in Metalworking Fluids

Nano-Immunodetection and Quantification of Mycobacteria in Metalworking Fluids

The microbiology of metalworking fluids

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

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