Science of Water : Concepts and Applications

280 The Science of Water: Concepts and Applications
1. Reagents and Media—All reagents and media utilized in performing microbiological
tests on water samples must meet the standards specifi ed in the reference cited in Federal
Regulations.
2. Reagent Grade Water—Deionized water that is tested annually and found to be free of dissolved
metals and bactericidal or inhibitory compounds is preferred for use in preparing
culture media and test reagents, although distilled water may be used.
3. Chemicals—All chemicals used in fecal coliform monitoring must be ACS reagent grade
or equivalent.
4. Media—To ensure uniformity in the test procedures, the use of dehydrated media is recommended.
Sterilized, prepared media in sealed test tubes, ampoules, or dehydrated media
pads are also acceptable for use in this test.
5. Glassware and Disposable Supplies—All glassware, equipment, and supplies used
in microbiological testing should meet the standards specifi ed in the references cited in
Federal Regulations.
Preparation of Equipment and Chemicals
All glassware used for bacteriological testing must be thoroughly cleaned using a suitable detergent
and hot water. The glassware should be rinsed with hot water to remove all traces of residue from
the detergent and, fi nally, should be rinsed with distilled water. Laboratories should use a detergent
certifi ed to meet bacteriological standards or, at a minimum, rinse all glassware after washing with
two tap water rinses followed by fi ve distilled water rinses.
For sterilization of equipment, the hot air sterilizer or autoclave can be used. When using the
hot air sterilizer, all equipment should be wrapped in high-quality (Kraft) paper or placed in containers
prior to hot air sterilization. All glassware, except those in metal containers, should be
sterilized for a minimum of 60 min at 170°C. Sterilization of glassware in metal containers should
require a minimum of 2 h. Hot air sterilization cannot be used for liquids.
When using an autoclave, sample bottles, dilution water, culture media, and glassware may be
sterilized by autoclaving at 121°C for 15 min.
Sterile Dilution Water Preparation
The dilution water used for making serial dilutions of the sample is prepared by adding 1.25 mL of
stock buffer solution and 5.0 mL of magnesium chloride solution to 1000 mL of distilled or deionized
water. The stock solutions of each chemical should be prepared as outlined in the reference
cited by the Federal Regulations. The dilution water is then dispensed in suffi cient quantities to
produce 9 or 99 mL in each dilution bottle following sterilization. If the membrane fi lter procedure
is used, additional 60- to 100-mL portions of dilution water should be prepared and sterilized to
provide rinse water required by the procedure.
Serial Dilution Procedure
At times, the density of organisms in a sample makes it diffi cult to accurately determine the actual
number of organisms in the sample. When this occurs, the sample size may need to be reduced to
as one millionth of a milliliter. To obtain such small volumes, a technique known as serial dilutions
has been developed.
Bacteriological Sampling
Proper technique, equipment, and sample preservation are critical to obtain valid test results that
can be utilized in the evaluation of process effi ciency of water quality. These factors are especially
critical in bacteriological sampling.
1. Sample dechlorination—When samples of chlorinated, effl uents are to be collected and
tested and the sample must be dechlorinated. Prior to sterilization, place enough sodium
thiosulfate solution (10%) in a clean sample container to produce a concentration of 100
mg/L in the sample (for a 120-mL sample bottle, 0.1 mL is usually suffi cient). Sterilize the
sample container as previously described.