Published Online: August 27, 2018
Revised January 3, 2022
Abstract: 9221 A. Introduction

Coliform bacteria have long been used as water-quality indicators based on the premise that, because these organisms are present in the intestines of warm-blooded animals, their presence in water could indicate that recent fecal contamination has occurred. Historically, this group of organisms has been defined by their ability to ferment lactose, rather than through the tenets of systematic bacteriology, so the group consists of bacteria from several genera belonging to the family Enterobacteriaceae.

The methods described in this section use a lactose-based broth medium to detect the metabolic end products of lactose fermentation. The presence of coliforms must be confirmed in a lactose- and bile salt-containing medium [brilliant green lactose bile (BGLB) broth]. Thus, when the fermentation techniques in this section are used, coliforms are defined as all facultatively anaerobic, Gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose to produce acid, gas, or both in the presence of bile salts within 48 h at 35 °C.

The standard test for the coliform group may be carried out by the multiple-tube fermentation technique or presence-absence procedure (through the presumptive-confirmed phases or completed test) described herein, the membrane filter (MF) technique (Section 9222), or 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 (QC) procedures, which are outlined in Section 9020.

The fermentation technique can be used to detect coliforms in drinking water or quantitate coliforms in potable and nonpotable water. When multiple tubes are used, coliform density is estimated via a most probable number (MPN) table. This number, generated using specific probability formulas, is an estimate of the mean density of coliforms in the sample. Coliform testing results, together with other information obtained from engineering or sanitary surveys, provide the best assessment of water-treatment effectiveness and the sanitary quality of source water.

The fermentation test’s precision in estimating coliform density depends on the number of tubes used. The most satisfactory information is obtained when the largest sample inoculum examined shows production of acid or gas in some or all of the tubes; and the smallest sample inoculum shows no acid or gas in any or most 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 (9221 C.2). The number of sample portions selected is governed by the desired precision of the result. The MPN tables are based on the assumption of a Poisson distribution (random dispersion). However, if the sample is not adequately shaken before aliquots are removed or if bacterial cells clump, the MPN value will be an underestimate of actual bacterial density.


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Standard Methods Committee of the American Public Health Association, American Water Works Association, and Water Environment Federation. 9221 multiple-tube fermentation technique for members of the coliform group In: Standard Methods For the Examination of Water and Wastewater. Lipps WC, Baxter TE, Braun-Howland E, editors. Washington DC: APHA Press.

DOI: 10.2105/SMWW.2882.192