Hixon 1 Concentrations and identities of fecal coliform bacteria from ...

Hixon 7
Testing began with using the membrane filtration technique: triplicates of 50mL of each sample
was filtered on 0.45µm filters and then placed onto M-FC agar (Fisher Scientific Catalog
Number DF0677173), which is selectable for fecal coliform bacterial growth, in 50x9mm Petri
dishes. The incubation period for the 1/29/11 collection was 24 hours and 37 minutes and 18
hours and 35 minutes for 3/12/11 collection at 46 ºC. Fecal coliform CFUs were visually counted
using a Leica Quebec Darkfield Colony Counter and these numbers were multiplied by two to
calculate fecal coliform CFUs per 100mL (table 1). Only blue colonies were counted, all brown
or off white colonies were considered not to be fecal coliform bacteria CFUs. Water pH was
measured using a Corning pH meter 125, which was calibrated prior to use. To calculate total
bacterial counts, triplicates of serial dilutions using 0.85% NaCl in 100mL dilution containers
were completed in tenfold increments from tenfold to one-thousand fold, followed by plating a
0.1mL sample onto TSA and incubating for 42 hours and 5 minutes at 37 ºC prior to counting.
IDENTIFICATION OF FECAL COLIFORM BACTERIA
Morphological, physiological, and metabolic tests were used to identify and differentiate
fecal coliform bacteria from each of the sampling locations. Three random and separate fecal
coliform bacteria colonies, one from each of the three triplicate plates per sample site (totaling 18
isolates), were chosen and successively quadrant streaked six times on m-endo agar (Fisher
Scientific Catalog Number DF0677173), in order to promote only fecal coliform bacteria growth.
Single colonies were used for testing and each sample was quadrant streaked on a new m-endo
plate weekly in order to have living bacteria available for all tests. Before individual fecal
coliform bacteria testing for differentiation among fecal coliform bacteria began, m-endo slants
were streaked, incubated for 11 hours and 20 minutes, sealed tight, and store in a refrigerator
with a temperature ranging from 0 to 5ºC for preservation. The methods for identification were