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SOP10 – Toxicity Analysis
70
10.0 SOP 9 – Microbial Analyses
Version 2.0
Date of Issuance: July 6, 2012
Authors: Anwar Huq, Fred Dobbs, Daniela Ceccarelli, Sadaf Yahyai
10.1. Summary
The University of Maryland (Baltimore, MD) and Old Dominion University (Norfolk, VA)
laboratories examines the pre and post treated water samples for bacterial abundances. The
bacterial density includes pathogens as well as fecal indicator densities as a proxy for human
fecal-oral pathogen densities.
10.2. Field Protocols
Bacteria samples are collected via an integrated flow system into a whole water reservoir as
described above and in section 4.5, 7.2.4 and 9.2. Immediately after filling of each reservoir, 2L
of raw water are collected in clean, labeled 2L bottles. Each bottle is sample rinsed 3 times, the
sample collected, and then the bottles placed in a cooler to keep dark and cool for immediate
transport back to the lab to be tested for total heterotrophic bacteria,
Escherichia coli
,
Enterococci
, and
Vibrio cholerae
.
10.3. Laboratory Protocols
10.3.1. Total Heterotrophic Bacteria
To determine total bacterial densities, 1ml of water sample is appropriately diluted in a 10-fold
dilution series in sterile Phosphate Buffered Saline (PBS), or sterile ambient water, to obtain a
countable number of colonies on the plate (between 30 and 300 colonies). Preliminary screen
may be done to asses the load of bacteria in the sample. A 100 µl aliquot of each appropriate
dilution are spread in triplicate onto Marine Agar
(MA), for marine water samples, or R2A agar,
for fresh water samples, and incubated at 25ºC or room temperature for 5 days. Colony forming
units will be counted at day 5, or sooner (day 3 or day 4) if the plate is overloaded, and recorded
as colony forming units (CFU) per 100 mL of sample water.
10.3.2.
E
.
coli
and
Enterococci
Two methods will be employed to determine both
E
.
coli
and
Enterococci
densities in collected
water samples. Following the USEPA Method 1603 10 mL and 100 mL of water are passed
through a 0.45 µm membrane which is then placed on modified thermo tolerant
E
.
coli
agar
(mTEC) (Becton Dickson, Sparks, MD) and incubated for 2 hours at 35 ± 0.5ºC to allow for cell
wall repair and then further incubated at 44.5ºC in a waterbath for 22-24 hours. After incubation,
total red and magenta colonies are be scored as
E
.
coli
colonies and reported as
E
.
coli
colonies
per 100 mL of sample water.
To determine
Enterococci
densities, 10 mL and 100 mL of water are passed through a 0.45 µm
membrane which is then transferred onto mEnterococcus agar (mEA) and incubated at 35 ±
0.5ºC for 24 hours. Membranes with light and dark red colonies are then transferred to Esculin
Iron Agar (EIA) and incubated for 30 min at 35 ± 2ºC (EPA method 1106.1). After this
incubation period colonies with a black halo are scored as
Enterococci
and reported as