Water & Wastewater Asia March/April 2022

ADVERTORIAL
Live bacteria cells with intact
membrane staining with
green fluorescent dye
both dead and live bacterial cells. The
assay reports the total cell count (TCC) in a
sample.
The BacCount Viable assay contains an
additional red fluorescent dye that stains
DNA and is used as an indicator of dead
cells. The combination of both dyes in this
assay allows the discrimination between
the live and dead cells, thus provide the
enumeration of viable cell count (VCC) in a
sample.
Rinse Washout
Figure 1 demonstrates that the flow
cytometry method provides greater
sensitivity compared to the plate count
method. The higher BacCount Total and
Viable cell counts indicated more bacterial
cells were detected with flow cytometry.
The discrepancy in flow cytometry vs
Dead bacteria
cells with
membrane
defects staining
with both red
fluorescent
and green,
fluorescent dye
plate count can be attributed to “viable
but nonculturable (VBNC)” bacteria that
are physiologically active but cannot be
cultured under laboratory conditions.
CASE STUDY #2: ESTABLISH
THE BACTERIA FINGERPRINT OF
A WATER SOURCE WITH CYSTAIN
BACCOUNT VIABLE
Using the immense resolution of a flow
cytometer, detectable bacteria can be
further differentiated into low nucleic acid
(LNA) and high nucleic acid (HNA) bacteria
populations. LNA and HNA ratios can
differ across water sources, presenting
a signature “fingerprint” of the water
source.
HNA bacteria are thought to be larger and
more active organisms, whereas the LNA
bacteria primarily consist of relatively
small, less active bacteria in environmental
water samples. However, growing evidence
suggest that LNA bacteria are just as active
and represent a different physiological
state of bacterial cells 3 .
Types of water sample
Figure 1: Measured
microbial population of
rinse water over seven
days using BacCount
Total and BacCount
Viable compared to the
plate count method.
Rinse washout here
refers to the samplings
of a machinery washout
solution collected from
a single manufacturing
site in Singapore.
Measurements were
performed with
technical triplicates
over a period of seven
days.
A deep well water source in Germany
presents similar bacterial fingerprint to that
of river water (Table 2). At a waterworks
facility in Germany, the same deep well
water source undergoes chlorination as
part of the water treatment process. Post
treatment analysis showed a shift in its
LNA-to-HNA ratio 4 .
CONCLUSION
Flow cytometric analyses facilitate
expedient monitoring of the quality of water
treatment processes and allow intervention
within a timely manner. Furthermore,
the establishment of “fingerprints”
may increase efficiency of treatment
process and for automated 24/7 process
monitoring.
Total Cell Count
per mL
River water 58,022
LNA: 76%
HNA: 24%
Deep well water 34,889
LNA: 69%
HNA: 31%
Chlorination 27,333
LNA: 43%
HNA: 57%
Table 2: Bacterial count of water samples from a waterworks facility in Germany 4
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REFERENCES
1 Allen et al. (2004) Heterotrophic plate count
bacteria—what is their significance in drinking
water? Int. J. Food Microbiol. 1;92(3):265-74.
2 Eawag. Drinking water is unexpectedly rich in
microbial life. Retrieved Jan 2022, from https://
www.admin.ch/gov/en/start/documentation/
media-releases.msg-id-47549.html
3 Cranfield Water Science Institute. Understanding
the Use of Flow Cytometry for
Monitoring of Drinking Water. Retrieved Feb
2022, from https://cdn.dwi.gov.uk/wp-content/
uploads/2020/10/27111348/DWI70-2-321.pdf
4 Sysmex Partec GmbH, 2021. Bacterial count of
disinfected drinking water. Application Note.
Total Viable Count
per mL
52,205
LNA: 72%
HNA: 28%
33,127
LNA: 70%
HNA: 30%
4,601
LNA: 85%
HNA: 15%
WATER & WASTEWATER ASIA | MARCH/APRIL 2022 13