Science of Water : Concepts and Applications

Environmental Biomonitoring, Sampling, and Testing 281
2. Sample procedure:
A. Keep the sample bottle unopened after sterilization until the sample is to be collected.
B. Remove the bottle stopper and hood or cap as one unit. Do not touch or contaminate
the cap or the neck of the bottle.
C. Submerge the sample bottle in the water to be sampled.
D. Fill the sample bottle approximately three quarters full, but not less than 100 mL.
E. Aseptically replace the stopper or cap on the bottle.
F. Record the date, time, and location of sampling, as well as the sampler’s name and any
other descriptive information pertaining to the sample.
3. Sample preservation and storage—examination of bacteriological water samples should
be performed immediately after collection. If testing cannot be started within one hour
of sampling, the sample should be iced or refrigerated at 4°C or less. The maximum recommended
holding time for fecal coliform samples from wastewater is 6 h. The storage
temperature and holding time should be recorded as part of the test data.
Multiple Tube Fermentation Technique
The multiple fermentation technique for fecal coliform testing is useful in determining the fecal
coliform density in most water, solid, or semisolid samples. Wastewater testing normally requires
use of the presumptive and confi rming test procedures. It is recognized as the method of choice for
any sample that may be controversial (enforcement related). The technique is based on the most
probable number of bacteria present in a sample that produces gas in a series of fermentation tubes
with various volumes of diluted sample. The MPN is obtained from charts based on statistical studies
of known concentrations of bacteria.
The technique utilizes a two-step incubation procedure. The sample dilutions are fi rst incubated
in lauryl (sulfonate) tryptose broth for 24–48 h (presumptive test). Positive samples are then
transferred to EC broth and incubated for an additional 24 h (confi rming test). Positive samples
from this second incubation are used to statistically determine the MPN from the appropriate
reference chart.
A single media, 24-h procedure is also acceptable. In this procedure, sample dilutions are inoculated
in A-1 media and are incubated for 3 h at 35°C, then incubated the remaining 20 h at 44.5°C.
Positive samples from these inoculations are then used to statistically determine the MPN value
from the appropriate chart.
Fecal Coliform MPN Presumptive Test Procedure
1. Prepare dilutions and inoculate fi ve fermentation tubes for each dilution.
2. Cap all tubes, and transfer to incubator.
3. Incubate 24 + 2 h at 35 ± 0.5°C.
4. Examine tubes for gas.
A. Gas present = Positive test—transfer
B. No gas = Continue incubation
5. Incubate total time 48 ± 3 h at 35 ± 0.5°C
6. Examine tubes for gas.
A. Gas present = Positive test—transfer
B. No gas = Negative test
√ Note: Keep in mind that the fecal coliform MPN confi rming procedure using A-1 broth test
is used to determine the MPN/100 mL. The MPN procedure for fecal coliform determinations
requires a minimum of three dilutions with fi ve tubes/dilution.
Calculation of Most Probable Number (MPN)/100 mL
The calculation of the MPN test results requires selection of a valid series of three consecutive
dilutions. The number of positive tubes in each of the three selected dilution inoculations is used
to determine the MPN/100 mL. In selecting the dilution inoculations to be used in the calculation,