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SOP10 – Toxicity Analysis
71
Enterococci
per 100 ml.
E
.
coli
and
Enterococci
densities are also estimated using the IDEXX
Colilert-18 and Enterolert kits (IDEXX Laboratories, Inc., Westbrook, ME), respectively.
When using IDEXX kits, the 100 ml of sample water is diluted ten folds with sterile deionized
water if the sample is from marine or estuarine waters (the MPN value has to be corrected to
include the dilution factor). The 100 ml of the diluted sample is then added to the provided
IDEXX resuspension bottle and mixed with the provided media in the kit. This inoculum is then
poured into a Quanti-tray and sealed. Trays are incubated at 41 ± 0.5ºC for 24 hours. For
Colilert-18 100 ml of sample water are mixed with the provided IDEXX resuspension bottles and
mixed with the provided media in the kit. This inoculum is then poured into a Quanti-tray and
sealed. Trays are incubated at 35 ± 0.5ºC for 18 hours.
Fluorescence with a 6-watt, 365 nm UV live held within 5 inches of the sample in a dark
environment determines a positive score for both
Enterococci
and
E
.
coli
. Indicator densities are
recorded as Most Probable Number (MPN) per 100 ml.
10.3.3. Total and Toxigenic
Vibrio cholerae
Total
V
.
cholerae
densities are determined by an RNA colony blot hybridization method. 1 ml of
sample water is passed through a sterile 0.22 µm nylon membrane that is then placed on TCBS
agar and incubated at 35 ± 0.5ºC for 18 to 24 hours. Membranes with 50 to 150 colonies are
removed and incubated on filter paper wetted with 10% SDS for 5 to 10 minutes at 65ºC, then
incubated on filter paper wetted with 3X SSC for 15 minutes at 65ºC. Membranes are then dried
at 37ºC for 10 minutes and baked at 70ºC for 15 minutes.
Toxigenic
V
.
cholerae
densities are determined by a DNA colony blot hybridization method that
detects
ctxA
gene. 1 ml of sample water is passed through a sterile 0.22 µm nylon membrane that
is then placed on TCBS agar and incubated at 35 ± 0.5ºC for 18 to 24 hours. Membranes with 50
to 150 colonies are removed and then placed on filter paper that is pre-wetted with 0.5N NaOH
at room temperature for 5 to 7 minutes. Membranes are then transferred to filter paper wetted
with 1M Tris-HCl/pH 8.0 for 5 minutes and then filter paper wetted with 1M Tris-HCl/pH 8.0,
1.5M NaCl for 5 minutes. Membranes are then washed in 2X SSC for 10 minutes, then dried at
37ºC for 10 minutes and baked at 70ºC for 15 to 30 minutes.
Total bacterial cells are fixed by filtering 100 mL of sample water through a 0.22 µm nucleopore
polycarbonate membrane that is then washed (vortexing) in 10 mL of 1X PBS to release cells. 1
ml of this is inoculated with 2.5% yeast extract and nalidixic acid and incubated at room
temperature and in the dark overnight. The inoculum is then fixed with formaldehyde to preserve
cells. Viable
V
.
cholerae
O1 cells are enumerated using a direct-fluorescent antibody
kit (DFA)
(New Horizons Diagnostics) for serogroup O1 using monoclonal antibodies tagged with
fluorescein isothiocyanate (FITC) under an epifluorescence microscope.
All data for all analyses will be recorded and stored in a laboratory notebook and electronically
on a spreadsheet. After each run water samples are kept at 4 ºC for one day in case any test need
to be re-run the following day.