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

doi:2012.17.1.35

Environmental Engineering Research 2012;17(1): 35-39. DOI: https://doi.org/10.4491/eer.2012.17.1.035

Application of Multiparametric Flow Cytometry (FCM) to Enumerate the Diagnosis of Pseudomonas aeruginosa and Escherichia coli

Myoung Goo Hwang¹^†, Jung Woo Oh², Hiroyuki Katayama³, Shinichiro Ohgaki⁴, and Jin Kyu Cho⁵

¹Kumho Engineering & Construction, Seoul 110-857, Korea
²Korea Coast Guard Research Institute, Incheon 406-741, Korea
³The University of Tokyo, Tokyo 113-8656, Japan
⁴National Institute for Environmental Studies, Ibaraki 305-8506, Japan
⁵Kimpo College, Gimpo 415-761, Korea

Corresponding Author: Myoung Goo Hwang ,Tel: +82-2-6303-0509, Fax: +82-2-6303-0745, Email: Mghwang1@kumhoenc.com

Received: June 7, 2011; Accepted: February 23, 2012.

ABSTRACT

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 0.2 × 10⁰ to 6.0 × 10¹ photo multiplier tube (PMT) 2 fluorescence (X-axis) and 2.0 × 10⁰ to 2.0 × 10² PMT 4 fluorescence (Y-axis), and the gate for live bacteria was defined as the range of 6.0 × 10⁰ to 6.0 × 10² PMT 2 fluorescence (X-axis) and 2.0 × 10⁰ to 4.0 × 10² 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.

Keywords: Bacterial diagnosis | Escherichia coli | Flow cytometry | Pseudomonas aeruginosa