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IS IT RAINWATER OR SEWAGE THAT ENTERED NCT?
As per the reports of the media and the city officials, it was rain water that drowned the
national college campus in the recent downpour. The department of biotechnology and
microbiology of NCT, undertook a scientific investigation and presents the facts here.
Key findings:
The fecal bacterial load in the water sample was greater than or equivalent to 1600, suggesting
poor microbiological quality.
A week after the sewage had been evacuated; the fecal coli form was still present in drainage
waters.
It is sewage water that has intruded, not rainwater.
On November 27, 2021, during a rainy time, Tiruchirapalli municipal authorities redirected rising
water outside the National college campus inside the
college premises (fig. 1).
Even though this activity caused significant disruption
inside the campus, the pungent odour and itching of
those exposed to the entering water enticed us to test
the water's microbiological quality. The objectives of
this work are to determine whether the entering
water is faecal polluted.
On the 01-12-2021, two water samples were taken
from the college's entry (sample 1) and flowing
points (sample 2), as shown in fig. 2. To detect faecal
contamination, we performed a 5-tube MPN (Most
Probable Number) test by inoculating varying
volumes of water samples (0.1ml, 1ml, 10ml) into
lactose broth. Both samples 1 and 2 were found to be
positive for coliforms, as demonstrated by gas
generation (fig 3) (an indicator for presence of fecal
bacteria).
Positive reactions were found in
all 5 tubes containing 0.1, 1, and 10
ml inoculums (5-5-5). By
comparing the results to the
standard MPN, we discovered that
the faecal bacterial load was
greater than or equal to 1600,
indicating that the water was of
poor microbiologic quality. We
Fig. 2: Water sampling points (1 & 2)
Fig. 1: Sewage entry ports were created to
allow sewage to flow into the campus.
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used presumptive test inoculums to corroborate the results. Retesting the inoculums for gas
generation in lactose broth, investigating the bacterial type using Gram's staining, and Indole
production are all part of the confirmatory test.
After a 24-hour incubation period, gas production was monitored again, and all tubes tested
positive for indole production (using Kovac's reagent) (fig. 4). Gram's reaction clearly showed the
presence of Gram negative rods, which were most likely E. coli (common faecal bacterium) (fig. 5).
Inoculums from the confirmatory test were streaked on EMB agar to confirm the presence of E. coli.
Following incubation, the presence of E. coli was verified (fig. 6). It is sewage water, not
rainwater that has intruded.
We analysed water samples from drainages for the presence of faecal coliforms after sewage water
had been discharged from the college site (on 08-12-2021). The results confirmed the presence of
Durham’s tube
with air trapped
within.
Fig. 3: Presumptive test for fecal coliforms. Presence of air bubble inside the durham’s tube
indicate the presence of fecal bacteria.
Fig. 4: All tubes from presumptive test were used to confirm the Indole production.
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faecal coliforms and indicated similar quantities in the college's drainage water. The faecal coli form
was still present in drainage waters a week after the sewage had been withdrawn (fig. 6).
The presence of fecal coliform in aquatic environments may indicate that the water has been
contaminated with the fecal material
of humans or other animals.
Escherichia coli are indicator bacteria
for other pathogens that may be
present in feces contaminated water.
We compared the culturable bacterial
load of drainage water to that of
campus tap water using the latest
samples. 5x10-6 CFU/ml was found in
drainage water, while 2.2x10-6
CFU/ml was found in tap water.
Drainage water has a bacterial load
2.3 times that of tap water.
What next?
For species confirmation, the E. coli culture was outsourced for 16S rRNA gene sequencing.
The study of faecal bacterial load in college soil samples is currently underway.
A scientific manuscript based on the case-study report is under preparation.
Presence of E. coli
confirmed by
nucleated metallic
sheen colonies
Fig. 5: Gram positive rods observed under 100X oil
immersion.
Fig. 6: Presence of metallic sheen colonies in EMB agar. The plate in left and right are from sampling
done on 01-12-2021 and 08-12-2021, respectively.
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Contributions
Mr. Vasanthakumar P., Mr. Kishore P., Mr. Raghuman F., Mr. Thangapandi R., Mr. Ranjith S.,
and Mr. Bernard D., students of II B. Sc. Microbiology, PG & Research Department of Biotechnology
and Microbiology, NCT, conducted the experiments under the supervision of their professor Dr.
Chinnamani Prasannakumar.