9221 MULTIPLE-TUBE FERMENTATION TECHNIQUE FOR …

1 9221 MULTIPLE-TUBE FERMENTATION TECHNIQUE FOR MEMBERS OF THECOLIFORM GROUP*9221 A. IntroductionThe coliform group consists of several genera of bacteriabelonging to the family Enterobacteriaceae. The historical defi-nition of this group has been based on the method used fordetection, lactose FERMENTATION , rather than on the tenets ofsystematic bacteriology. Accordingly, when the fermentationtechnique is used, this group is defined as all facultative anaer-obic, gram-negative, non-spore-forming, rod-shaped bacteriathat ferment lactose with gas and acid formation within 48 h at35 standard test for the coliform group may be carried out bythe MULTIPLE-TUBE FERMENTATION TECHNIQUE or presence absenceprocedure (through the presumptive-confirmed phases or com-pleted test) described herein, the membrane filter (MF) TECHNIQUE (Section 9222) or the enzymatic substrate coliform test (Section9223).

2 Each TECHNIQUE is applicable within the limitations spec-ified and with due consideration of the purpose of the examina-tion. Production of valid results requires strict adherence toquality control procedures. Quality control guidelines are out-lined in Section multiple tubes are used in the FERMENTATION TECHNIQUE ,coliform density can be estimated by using a most probablenumber (MPN) table. This number, based on certain probabilityformulas, is an estimate of the mean density of coliforms in thesample. Results of coliform testing, together with other informa-tion obtained from engineering or sanitary surveys, provide thebest assessment of water treatment effectiveness and the sanitaryquality of source precision of the FERMENTATION test in estimating coliformdensity depends on the number of tubes used.

3 The most satis-factory information will be obtained when the largest sampleinoculum examined shows acid and/or gas in some or all of thetubes and the smallest sample inoculum shows no acid and/orgas in any or a majority of the tubes. Bacterial density can beestimated by the formula given or from the table using thenumber of positive tubes in the multiple dilutions ( ). Thenumber of sample portions selected will be governed by thedesired precision of the result. The MPN tables are based on theassumption of a Poisson distribution (random dispersion). How-ever, if the sample is not adequately shaken before the portionsare removed or if bacterial cells clump, the MPN value will bean underestimate of actual bacterial of Drinking Water QualityWhen analyzing drinking water to determine if the qualitymeets Environmental Protection Agency (EPA) standards,use the FERMENTATION TECHNIQUE with 10 replicate tubes eachcontaining 10 mL, 5 replicate tubes each containing 20 mL, or asingle bottle containing a 100-mL sample portion.

4 When exam-ining drinking water via the FERMENTATION TECHNIQUE , process alltubes or bottles demonstrating growth, with or without a positiveacid or gas reaction, to the confirmed phase ( ). Drinkingwater samples that are positive for total coliforms also must betested for thermotolerant (fecal) coliforms (9221E) orEsche-richia coli(9221F).For routine examinations of public water supplies, the objec-tive of the total coliform test is to determine the efficiency oftreatment plant operations and the integrity of the distributionsystem. It is also used as a screen for the presence of fecalcontamination. Some coliform occurrences in a distribution sys-tem may be attributed to growth or survival of coliforms inbacterial biofilms in the mains, rather than treatment failure atthe plant or well source, or outside contamination of the distri-bution system.

5 Because it is difficult to distinguish betweencoliforms entering the distribution system and coliforms alreadypresent in the pipe biofilm and sediments, assume that all coli-forms originate from a source outside the distribution of Other than Drinking Water QualityWhen examining nonpotable waters, inoculate a series oftubes with appropriate decimal dilutions of the water (multiplesof 10 mL), based on the probable coliform density. Use thepresumptive confirmed phases of the MULTIPLE-TUBE the more labor-intensive completed test ( ) as aquality control measure on at least 10% of coliform-positivenonpotable water samples on a seasonal basis. The objective ofthe examination of nonpotable water, generally, is to estimate thebacterial density, determine a source of pollution, enforce waterquality standards, or trace the survival of microorganisms.

6 Themultiple-tube FERMENTATION TECHNIQUE may be used to obtainstatistically valid MPN estimates of coliform density. Examine asufficient number of water samples to yield representative resultsfor the sampling station. Generally, the geometric mean ormedian value of the results of a number of samples will yield avalue in which the effect of sample-to-sample variation is SamplesThe MULTIPLE-TUBE FERMENTATION TECHNIQUE is applicable to theanalysis of salt or brackish waters as well as muds, sediments,and sludges. Collect samples as directed in Sections 9060A andB, using sample containers specified in Section Fol-low the precautions given above on portion sizes and numbers oftubes per dilution.* Approved by Standard Methods Committee, Task Group: Ellen B. Braun-Howland (chair), Paul S.

7 Berger, Robert , Clifford H. Johnson, Shundar Lin, Mark C. Meckes, Eugene W. prepare solid or semisolid samples, weigh the sample and adddiluent to make a 10 1dilution. For example, place 30 g sample insterile blender jar, add 270 mL sterile phosphate buffered or dilution water, and blend for 1 to 2 min at high speed(8000 rpm). Prepare the appropriate decimal dilutions of the ho-mogenized slurry as quickly as possible to minimize B. Standard Total Coliform FERMENTATION samples as directed in Sections 9060A and B, usingsample containers specified in Section PhaseUse lauryl tryptose broth in the presumptive portion of themultiple-tube test. If the medium has been refrigerated aftersterilization, incubate overnight at room temperature (20 C)before use. Discard tubes showing growth and/or Reagents and culture medium:If possible, use a commer-cially available tryptose broth:Tryptose.

8 GDipotassium hydrogen phosphate, gPotassium dihydrogen phosphate, gSodium chloride, gSodium lauryl sulfate .. gReagent-grade 1 LAdd dehydrated ingredients to water, mix thoroughly, and heatto dissolve. Before sterilization, dispense in FERMENTATION tubeswith an inverted vial (Durham tube) sufficient medium tocover the inverted vial at least one-half to two-thirds aftersterilization. Alternatively, omit the inverted vial and g/L bromcresol purple to lauryl tryptose broth to determineacid production, an indicator of a positive result in this part of thecoliform test. Close tubes with metal or heat-resistant lauryl tryptose broth of such strength that adding100-mL, 20-mL, or 10-mL portions of sample to the mediumwill not reduce ingredient concentrations below those of thestandard medium.

9 Prepare in accordance with Table 9221 medium at 121oC for 12 to 15 min. Ensure thatinverted vials, if used, are free of air bubbles. Medium pH shouldbe after Procedure:1) Arrange FERMENTATION tubes in rows of five or ten tubes eachin a test tube rack. The number of rows and the sample volumesselected depend on the quality and character of the water to beexamined. For potable water, use five 20-mL portions, ten10-mL portions, or a single bottle of 100-mL portion; for non-potable water, use five tubes per dilution (of 10, 1, mL, etc.).When making dilutions and measuring diluted sample vol-umes, follow the precautions given in Section UseFigure 9215:1 as a guide to preparing dilutions. Shake sampleand dilutions vigorously about 25 times. Inoculate each tube ina set of five with replicate sample volumes in increasing decimaldilutions, if decimal quantities of the sample are used.

10 Mix testportions in the medium by gentle ) Incubate inoculated tubes or bottles at 35 C. After 24 2 h swirl each tube or bottle gently and examine it for growth,gas, and/or acidic reaction (shades of yellow color) and, if no gasor acidic reaction is evident, re-incubate and re-examine at theend of 48 3 h. Record presence or absence of growth, gas,and/or acid production. If the inner vial is omitted, growth withacidity (yellow color) signifies a positive presumptive Interpretation:Production of an acidic reaction and/or gasin the tubes or bottles within 48 3 h constitutes a positivepresumptive reaction. Submit tubes or bottles with a positivepresumptive reaction to the confirmed phase ( ).The absence of acidic reaction and/or gas formation at the endof 48 3 h of incubation constitutes a negative test.