In this analysis, 150 mL samples were passed through a filter
with a diameter of 0.42 micron by the help of a vacuum pump.
To avoid possible contamination, it was conducted as class 2
laminar flow.
Separation of microbes from the filter paper was carried out in
two ways:
A - Washing with distilled water.
B - Washing with diethylpyrocarbonate (DEPC).
Filter paper was placed in 50 cc Iso glass and about 10 cc of
0.1% solution of autoclaved DEPC was added to the Iso glass.
Filter was properly washed three times with the help of sampler
in the Iso glass wall through immersion and extreme turbulence.
Then the solution resulted from washing were transferred into
centrifuge Falcon tubes. Then, the solution was centrifuged for 10 min with 14 000 r/min.
Supernatant was removed carefully with a sampler and about
10 cc of autoclaved saline was added to the deposits. Then it
was centrifuged after application of vortex and turbulence.
Thus, DEPC was washed from the deposits. About 300 micro liters
of the final deposits were transferred to a microtube 1.5 and
finally about 50 micro liters of the final deposit was transferred
as a bacterial mass to a microtube 1.5, after it was centrifuged
at 14 000 r/min for 2 min and at the next stage, it was analyzed
by PCR test. Moreover, a number of samples were comparatively
tested with or without DEPC to evaluate the necessity of its
application.
From 250 mL sample in the sample container, 100 cc was used
for doing MPN test, about 30 cc was used for doing turbidity test
and the rest was used for doing molecular filtrations.
In this study, three types of samples were studied:
1 - Wells and water distribution system samples: MPN culture
test were first performed as the gold standard tests, then the
samples were filtered and analyzed by PCR test.
2 - Standard bacteria: E. coli and CRM.
3 - Synthetic samples: In order to evaluate PCR, synthetic
samples were used as positive examples. These samples were
positive BGB (their microbial contamination with coliform was
confirmed) and they are kept and expanded for setting up PCR.
After isolation of bacteria, purified bacteria were added to the
negative MPN water sample. In this study, all water samples sent
to the laboratory culture were kept in a refrigerator for 24 h after
being cultured in a lauryl tryptose broth for doing MPN.