Publisher : Korean Society of Environmental Engineering
DOI : 10.4491/eer.2012.17.1.035
Title & Authors
Application of Multiparametric Flow Cytometry (FCM) to Enumerate the Diagnosis of Pseudomonas aeruginosa and Escherichia coli Hwang, Myoung-Goo; Oh, Jung-Woo; Katayama, Hiroyuki; Ohgaki, Shinichiro; Cho, Jin-Kyu;
In this study, multiparametric flow cytometry (FCM) was installed to enumerate the diagnosis of Pseudomonas aeruginosa ATCC 10145 and Escherichia coli K12 (IFO 3301). The nucleic acids (DNA/RNA) were double stained by a LIVE/DEAD bacLight viability kit, involving green SYTO 9 and red propidium iodide (PI), based on the permeability of two chemicals according to the integrity of plasma membrane. As the results showed, the gate for dead bacteria was defined as the range of to photo multiplier tube (PMT) 2 fluorescence (X-axis) and to PMT 4 fluorescence (Y-axis), and the gate for live bacteria was defined as the range of to PMT 2 fluorescence (X-axis) and to PMT 4 fluorescence (Y-axis). In the comparison of the number of the tested bacteria detected by FCM (viability assessment) and plate culture (cultivability assessment), the number of bacteria detected by FCM well represented the number of bacteria that was detected by the colony forming unit (CFU) counting method when bacteria were exposed to isopropyl alcohol and silver/copper cations. Consequently, it is concluded that the application of FCM to monitor the functional effect of disinfectants on the physiological status of target bacteria can offer more rapid and reliable data than the plate culture colony counting method.
Hwang MG, Katayama H, Ohgaki S. Effect of intracellular resuscitation of Legionella pneumophila in Acanthamoeba polyphage cells on the antimicrobial properties of silver and copper. Environ. Sci. Technol. 2006;40:7434-7439.
Lebaron P, Catala P, Parthuisot N. Effectiveness of SYTOX green stain for bacterial viability assessment. Appl. Environ. Microbiol. 1998;64:2697-2700.
Amor KB, Breeuwer P, Verbaarschot P, et al. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress. Appl. Environ. Microbiol. 2002;68:5209-5216.
Alvarez-barrientos A, Arroyo J, Canton R, Nombela C, Sanchez-Perez M. Applications of flow cytometry to clinical microbiology. Clin. Microbiol. Rev. 2000;13:167-195.
Oh JW. Evaluation of bacterial regrowth potential in drinking water system using cell cycle parameters [dissertation]. Tokyo: The University of Tokyo; 2004.
Bunthof CJ, Bloemen K, Breeuwer P, Rombouts FM, Abee T. Flow cytometric assessment of viability of lactic acid bacteria. Appl. Environ. Microbiol. 2001;67:2326-2335.
Haugen EM, Cucci TL, Yentsch CM, Shapiro LP. Effects of flow cytometric analysis on morphology and viability of fragile phytoplankton. Appl. Environ. Microbiol. 1987;53:2677-2679.
Gregori G, Citterio S, Ghiani A, et al. Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining. Appl. Environ. Microbiol. 2001;67:4662-4670.
Bowler PG, Duerden BI, Armstrong DG. Wound microbiology and associated approaches to wound management. Clin. Microbiol. Rev. 2001;14:244-269.
Davey HM, Kell DB. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single- cell analyses. Microbiol. Rev. 1996;60:641-696.
Hiraoka Y, Kimbara K. Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry. Appl. Environ. Microbiol. 2002;68:2031-2035.
Jepras RI, Carter J, Pearson SC, Paul FE, Wilkinson MJ. Development of a robust flow cytometric assay for determining numbers of viable bacteria. Appl. Environ. Microbiol. 1995;61:2696-2701.
Porter J, Edwards C, Pickup RW. Rapid assessment of physiological status in Escherichia coli using fluorescent probes. J. Appl. Bacteriol. 1995;79:399-408.
Veal DA, Deere D, Ferrari B, Piper J, Attfield PV. Fluorescence staining and flow cytometry for monitoring microbial cells. J. Immunol. Methods 2000;243:191-210.
Hoefel D, Grooby WL, Monis PT, Andrews S, Saint CP. A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity. J. Microbiol. Methods. 2003;52:379-388.
Szeto L, Shuman HA. The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. Infect. Immun. 1990;58:2585-2592.
Hwang MG, Katayama H, Ohgaki S. Accumulation of copper and silver onto cell body and its effect on the inactivation of Pseudomonas aeruginosa. Water Sci. Technol. 2006;54:29-34.
De Kievit TR, Parkins MD, Gillis RJ, et al. Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 2001;45:1761-1770.
Auty MA, Gardiner GE, McBrearty SJ, et al. Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining conjunction with confocal scanning laser microscopy. Appl. Environ. Microbiol. 2001;67:420-425.
Teitzel GM, Parsek MR. Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol. 2003;69:2313-2320.