MICROBIOLOGICAL EXAMINATION (9000)
9221 MULTIPLE-TUBE FERMENTATION TECHNIQUE FOR MEMBERS OF THE COLIFORM GROUP*
9221 A. Introduction
The coliform group consists of several genera of bacteria belonging to the family Enterobacteriaceae. The historical deﬁnition of this group has been based on the method used for detection (lactose fermentation) rather than on the tenets of systematic bacteriology. Accordingly, when the fermentation technique is used, this group is deﬁned as all facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas and acid formation within 48 h at 35°C. The standard test for the coliform group may be carried out either by the multiple-tube fermentation technique or presenceabsence procedure (through the presumptive-conﬁrmed phases or completed test) described herein, by the membrane ﬁlter (MF) technique (Section 9222) or by the enzymatic substrate coliform test (Section 9223). Each technique is applicable within the limitations speciﬁed and with due consideration of the purpose of the examination. Production of valid results requires strict adherence to quality control procedures. Quality control guidelines are outlined in Section 9020. When multiple tubes are used in the fermentation technique, results of the examination of replicate tubes and dilutions are
* Approved by Standard Methods Committee, A–E, 1999; F, 2001. Joint Task Group: (9221C): Eugene W. Rice (chair), Paul S. Berger, James A. Clark, Stephen C. Edberg, Wallace E. Garthright, Nancy H. Hall, Shundar Lin; (9221F): Mark C. Meckes (chair), Paul S. Berger, James A. Clark, Wallace E. Garthright, Nancy H. Hall, Shundar Lin.
MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Standard Total Coliform Fermentation Technique
reported in terms of the Most Probable Number (MPN) of organisms present. This number, based on certain probability formulas, is an estimate of the mean density of coliforms in the sample. Coliform density, together with other information obtained by engineering or sanitary surveys, provides the best assessment of water treatment effectiveness and the sanitary quality of source water. The precision of each test depends on the number of tubes used. The most satisfactory information will be obtained when the largest sample inoculum examined shows gas in some or all of the tubes and the smallest sample inoculum shows no gas in all or a majority of the tubes. Bacterial density can be estimated by the formula given or from the table using the number of positive tubes in the multiple dilutions (9221C.2). The number of sample portions selected will be governed by the desired precision of the result. MPN tables are based on the assumption of a Poisson distribution (random dispersion). However, if the sample is not adequately shaken before the portions are removed or if clumping of bacterial cells occurs, the MPN value will be an underestimate of the actual bacterial density. 1. Water of Drinking Water Quality
A high proportion of coliform occurrences in a distribution system may be attributed not to treatment failure at the plant or the well source, but to bacterial regrowth in the mains. Because it is difﬁcult to distinguish between coliform regrowth and new contamination, assume all coliform occurrences to be new contamination unless otherwise demonstrated. 2. Water of Other than Drinking Water Quality
When drinking water is analyzed to determine if the quality meets the standards of the U.S. Environmental Protection Agency (EPA), use the fermentation technique with 10 replicate tubes each containing 10 mL, 5 replicate tubes each containing 20 mL, or a single bottle containing a 100-mL sample portion. When examining drinking water by the fermentation technique, process all tubes or bottles demonstrating growth with or without a positive acid or gas reaction to the conﬁrmed phase (9221B.2). Apply the completed test (9221B.3) to not less than 10% of all...