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Frontiers of Environmental Science & Engineering

ISSN 2095-2201

ISSN 2095-221X(Online)

CN 10-1013/X

Postal Subscription Code 80-973

2018 Impact Factor: 3.883

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Front. Environ. Sci. Eng.    2016, Vol. 10 Issue (6) : 12    https://doi.org/10.1007/s11783-016-0884-4
RESEARCH ARTICLE
Flow cytometric assessment of the effects of chlorine, chloramine, and UV on bacteria by using nucleic acid stains and 5-cyano-2,3-ditolyltetrazolium chloride
Xuebiao Nie1,Wenjun Liu1(),Mo Chen2,Minmin Liu1,Lu Ao3
1. School of Environment, Tsinghua University, Beijing 100084, China
2. China Gezhouba Group Real Estate Corporation, Beijing 100020, China
3. Department of Architecture and Environmental Engineering, Logistic Engineering University of PLA, Chongqing 401331, China
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Abstract

Flow cytometry based on nucleic acid stains and CTC was established and optimized.

Membrane of S. aureus is more resistant to chlorine/chloramine than E. coli.

The metabolic activity of bacteria persisted after the cytomembranewas damaged.

S. aureus showed more resistance to UV irradiation than E. coli by FCM.

MP-UV was a stronger inhibitor of metabolic activity than LP-UV.

Flow cytometry (FCM) has been widely used in multi-parametric assessment of cells in various research fields, especially in environmental sciences. This study detected the metabolic activity of Escherichia coli and Staphylococcus aureus by using an FCM method based on 5-cyano-2,3-ditolyltetrazolium chloride (CTC); the accuracy of this method was enhanced by adding SYTO 9 and 10% R2A broth. The disinfection effects of chlorine, chloramine, and UV were subsequently evaluated by FCM methods. Chlorine demonstrated stronger and faster destructive effects on cytomembrane than chloramine, and nucleic acids decomposed afterwards. The metabolic activity of the bacteria persisted after the cytomembranewas damaged as detected using CTC. Low-pressure (LP) UV or medium-pressure (MP) UV treatments exerted no significant effects on membrane permeability. The metabolic activity of the bacteria decreased with increasing UV dosage, and MP-UV was a stronger inhibitor of metabolic activity than LP-UV. Furthermore, the membrane of Gram-positive S. aureus was more resistant to chlorine/chloramine than that of Gram-negative E. coli. In addition, S. aureus showed higher resistance to UV irradiation than E. coli.
Keywords Flow cytometry      Escherichia coli      Staphylococcusaureus      UV      CTC      SYTO 9     
PACS:     
Fund: 
Corresponding Author(s): Wenjun Liu   
Issue Date: 14 November 2016
 Cite this article:   
Xuebiao Nie,Wenjun Liu,Mo Chen, et al. Flow cytometric assessment of the effects of chlorine, chloramine, and UV on bacteria by using nucleic acid stains and 5-cyano-2,3-ditolyltetrazolium chloride[J]. Front. Environ. Sci. Eng., 2016, 10(6): 12.
 URL:  
https://academic.hep.com.cn/fese/EN/10.1007/s11783-016-0884-4
https://academic.hep.com.cn/fese/EN/Y2016/V10/I6/12
Fig.1  Effects of R2A concentration on Staphylococcus aureus count:(a) comparison between noise and bacterial points; (b) comparison between CTC+ and SYTO 9+ results; and (c) CTC+ results (R2A 0%–25%)
Fig.2  FL1-FL2 plots of E. coli solutions stained with SYTO 9 plus PI after treatment with 1 mg·L-1 chlorine for (a) 10 s, (b) 30 s, and(c) 60 s
time density of live cells
/(103·mL-1)		 density of dead cells
/(103·mL-1)		 density of total cells
/(103·mL-1)		 percentage
of live cells/%		 percentage
of dead cells/%
0 s 988.4 151.8 1140.2 86.7 13.3
5 s 1146.0 190.3 1336.3 85.8 14.2
10 s 997.7 147.7 1145.3 87.1 12.9
30 s 190.8 945.0 1135.8 16.8 83.2
45 s 219.9 947.6 1167.5 18.8 81.2
60 s 123.2 1362.0 1485.2 8.3 91.7
2 min 75.1 1200.0 1275.1 5.9 94.1
5 min 61.5 1010.4 1071.9 5.7 94.3
10 min 64.8 1313.0 1377.8 4.7 95.3
15 min 62.6 1135.8 1198.4 5.2 94.8
30 min 51.8 1266.2 1318.0 3.9 96.1
60 min 51.4 1222.8 1274.2 4.0 96.0
Tab.1  FCM counting results for E. coli solutions stained with SYTO 9 plus PI after treatment with 1 mg·L-1 chlorine for different reaction times
Fig.3  FL1-FL2 plots of S. aureus solutions stained with SYTO 9 plus PI after treatment with 1 mg·L-1 chlorine for (a )30 s and (b) 10 min
Fig.4  FL1-FL2 plots of S. aureus solutions stained with SYTO 9 plus PI after treatment with 2 mg·L-1 chloramine at (a) 0 s, (b) 10 min, and (c) 20 min
action time density of E. coli
/(103·mL-1)		 density of S. aureus
/(103·mL-1)
0 s 893.2 729.4
30 s 844.6 444.8
60 s 1122.2 430.4
2 min 877.1 412.4
5 min 912.4 381.6
10 min 765.6 322.4
20 min 303.1 130.9
30 min 236.2 117.5
45 min 244.9 118.5
60 min 215.8 99.1
Tab.2  FCM counting results for E. coli and S. aureus solutions stained with SYTO 9 plus PI after treatment with 2.0 mg·L-1 chloramine for different reaction times
Fig.5  Fluence-response curve of E. coli and S. aureus to LP-UV
Fig.6  Ratio of CTC-positive to SYTO 9-positive E. coli decreased with increasing UV dosage: (a) LP-UV and (b) MP-UV
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