Delft University of Technology
Section Sanitary Engineering
Department Water Management
Faculty of Civil Engineering and Geosciences
Section Environmental Biotechnology Department Biotechnology
Faculty of Applied Sciences
Chlorine cell disinfection determination
with flow cell cytometry and plate count
Marjolein Peters, Maarten Keuten, Merle de Kreuk, Mark van Loosdrecht, Luuk Rietveld
INTRODUCTION
Chlorine is used for disinfection in different water systems. This research focuses on chlorine disinfection in swimming pool water. In the Netherlands, free available chlorine concentrations in swimming pools are limited between 0.5-1.5 mg/L, which is based on a 4-log removal of
Pseudomonas aeruginosa
within 30 seconds. Another indicator organism (Escherichia coli
) is used to validate the swimming pool water quality based on plate count. Plate count is used for many years and has proven its worthy in viability tests. However, this technique is time consuming, since colonies have to grow overnight. With new techniques like flow cell cytometry a faster result can be obtained and if needed actions can be made directly to protect humans health.OBJECTIVE
In this research the use of flow cell cytometry for chlorine disinfected microorganisms is compared to the regular plate count technique. Different populations are used to determine the disinfection of indicator organisms and a more realistic anthropgenic population.
METHODOLOGY
Lab experiments were performed with the indicator organisms
Pseudomonas fluorescens
(P17) andEscherichia coli
(WR1). For the more realistic population a anthoropogenic (shower) population was used assuming the release of similar microorganisms during a 1 minute shower compared to a jump in a swimming pool. Non-chlorinated shower water of 10 different persons was collected of which 60L was filtered (0.2μm). The microorganisms were concentrated on the filter and rinsed off with a mineral medium.All three populations were exposed to three chlorine concentrations (0.5 – 1.0 – 5.0 mg/L free chlorine) at different contact times and directly dechlorinated afterwards with an excess of sodiumthiosulfate. The samples were analyzed with flow cell cytometry, cell count based on live/dead DNA staining figure 1, and plate count, figure 5.
RESULTS & DISCUSSION
Figure 2, 3 and 4 show that the results of flow cell cytometry are not comparable with plate count. The reason why this number is not equal can be found in the chlorine disinfection reaction. Hypochlorous acid (HOCl) is a neutral compound that can easily diffuse over the cell membrane. However, this doesn’t mean that it will disrupt the cell membrane. When the cell membrane is still intact PropiumIodide fluorescence cannot enter the cell and stain the DNA. Intact dead cells from chlorine disinfection disturb the flow cell cytometry results because they appear as intact (living) cells. This results in a high number of intact cells (flow cell cytometry) but a low number of viable cells (plate count).
Based on the decrease of viable cells it can be concluded that a higher chlorine concentration results in a faster disinfection of all three populations. Only the anthropogenic population shows a decrease of intact cells as well, figure 4.
M.C.F.M.Peters@TUDelft.nl
+31 (0)15 27 87 894
CONCLUSION
Figure 1: Flow cell cytometry: Live/dead staining. Total cell count
based on fluoresence of all DNA with SyberGreen. Indication of intact (living) cells based on the difference between total cells and broken cells which are determined with PropiumIodide fluoresence.
Figure 2: Disinfection of P.fluorescence with the free chlorine
concentrations 0.5 - 1.0 - 5.0 mg/L. Full markers represent the number
of intact cells per μL during flow cell cytometry (FC) and the open markers represent the number of viable cells per μL during plate count (PC).
Figure 3: Disinfection of E.coli with the free chlorine concentrations 0.5 - 1.0 - 5.0 mg/L. Full markers represent the number of intact cells per
μL during flow cell cytometry (FC) and the open markers represent the number of viable cells per μL during plate count (PC).
Figure 5: A LLA-plate from the anthropogenic population.
Figure 4: Disinfection of an anthropogenic population with the free chlorine concentrations 0.5 - 1.0 - 5.0 mg/L. Full markers represent the
number of intact cells per μL during flow cell cytometry (FC) and the open markers represent the number of viable cells per μL during plate count (PC).
• Flow cell cytometry live/dead staining is unsuitable for determining chlorine disinfection of cells.
• A higher chlorine concentration results in a faster decrease of viable cells.
• Disinfection of an anthropogenic population is faster than disinfection of
Escherichia coli
.• Disinfection of an anthropogenic population is
slower than disinfection of
Pseudomonas
fluorescens
, meaning thatP.aeruginosa
might not be a good indicator organism*.• Further study will define the anthropogenic population.
*) It was assumed that disinfection of Pseudomonas fluorescens is similar to disinfection of
Pseudomonas aeruginosa, which is the official used indicator organism in swimming
pools. Number of intact (FC) and viable (PC) E.coli cells after
exposure to chlorine 0 5 10 15 20 25 30 0 2 4 6 8 10 12 14 16 18 20 22 Time (min) N u m b e r o f c e lls ( x 1 0 0 ) p e r u L 0,5 - FC 0,5 - PC 1,0 - FC 1,0 - PC 5,0 - FC 5,0 - PC
