River water samples (10 ml) were aseptically pipetted into a
sterile Erlenmeyer flask and diluted tenfold by adding 90 ml of
quarter-strength Ringers solution followed by subsequent decimal
dilution (up to 10−5) using the same diluent. Produce samples were
prepared for subsequent analyses by adding 90 ml quarter-strength
Ringers solution to 10 g of uncut leaf material (or florets in the case of
cauliflower) in a sterile Erlenmeyer flask followed by a 10 min
treatment on an orbital shaker at 200 rpm at ambient temperature
prior to decimal dilution (up to 10−5) using the same diluent as
above. Aerobic plate counts for water and produce were conducted in
triplicate according to the South African National Standards (SANS,
2007) procedure 4833 using plate count agar as specified therein.
Results are expressed as the weighted mean (log10) with the
calculated standard error indicated. Total and fecal coliforms were
enumerated by using a MPN (most probable number) method
according to the method MFHPB-19 suggested by Health Canada
(2002). This involved an initial presumptive test in Lauryl Sulphate
Tryptose broth (LST) (Oxoid) and a confirmation test by inoculating
Brilliant-green lactose bile broth (BGLB) (Merck) with one loopful
from the gas-positive LST tubes. Escherichia coli was quantified by
inoculating gas-positive LST tubes into E. coli (EC) broth (Merck)
containing 4-methylumbelliferyl-β-D-glucuronide (MUG). Fluorescence
under ultra violet (UV) light, visible growth and the production
of gas indicated a positive test, and E. coli presence was confirmed
using Levine-Eosin Methylene Blue (L-EMB) agar (Conda) and the
prescribed biochemical tests (i.e. gas production at 45 °C (G), indole
formation from tryptophane (I), Methyl-Red (M), Voges-Proskauer
(Vi) and Simmon's citrate assimilation (C)=GIMViC). The results are
expressed as log10 MPN counts per 100 ml of river water or gram of
produce sample with a 95% confidence interval established according
to Garthright and Blodgett (2003).