Environmental Engineering Reference
In-Depth Information
Glassware and disposable supplies —All glassware, equipment, and
supplies used in microbiological testing should meet the standards
specified in references cited in federal regulations.
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 detergent residue and finally rinsed
with distilled water. Laboratories should use a detergent certified to meet
bacteriological standards or at a minimum should rinse all glassware after
washing with two tapwater rinses followed by five distilled-water rinses.
For sterilization of equipment, a 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 mini-
mum of 60 minutes at 170°C. Sterilization of glassware in metal containers
requires a minimum of 2 hours. Hot-air sterilization cannot be used for liq-
uids. Sample bottles, dilution water, culture media, and glassware may also
be sterilized by autoclaving at 121°C for 15 minutes.
Dilution water used for making sample serial dilutions 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 references cited in fed-
eral regulations. The dilution water is then dispensed in sufficient quanti-
ties to produce 9 or 99 mL in each dilution bottle following sterilization. If
the membrane filter procedure is used, additional 60- to 100-mL portions of
dilution water should be prepared and sterilized to provide the rinse water
required by the procedure.
At times, the density of the organisms in a sample makes accurately deter-
mining the actual number of organisms in the sample difficult. When this
occurs, the sample size may need to be reduced to as little as 1 millionth of
a milliliter. To obtain such small volumes, a technique known as serial dilu-
tions has been developed.
DID YoU KNoW?
Like dinosaurs, bacteria left behind fossils. The big difference is
that it takes a microscope to see bacteria. Another big difference is
that bacteria are older … much older!
Multiple-Tube Fermentation Technique
The multiple-tube fermentation (MTF) technique for fecal coliform testing of
water, solid, or semisolid samples is based on the fact that coliform organ-
isms can use lactose (the sugar occurring in milk) as food and produce gas
 
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