‘After incubation, prompt! eile loop © transfer one eae e presumpive-postive bottle toa fern Sopfuls of fer GLB broth. Alternatively, insert a ten tube: East 25 eminto the clare, prompny ren orto he baton of@ fermentation tube ptt. Remove and discard applicator. Re empveosiive tudes andl ine Wood "Move, and. Containing 4 ulate a 35 an pl) ‘of culture from presumptive-posi Menseed into BC broth (for deter oc. also it thegmo- fee caifoms) andlor EC-MUG broth (for fy (em yate sae time, log asthe mone eka weg Peiinieelacdiite «Mitton, i ation: Gas prosuction in the BGLB broth eleamessh confirms the presence of coliform baer fest 8 FA test positive or negative for toad ctor MeL of sample. Drinking water samples that are positive nliforms also must be tested for thermotolerant (fecal (92216) or E coli (921F). weal 4, competed Prase rie sompleted phase, required for uonpotable water san fatied in 9221B.5 and Figure 9221.1. 9221 E. Thermotoi jnaly called fecal coliforms, thermotolerant coliforms ferment lactose to produce gas at 44.5°C) have been ‘organically rich waters or tropical climates inthe nt fecal contamination. So when looking for al contamination, testing for E, coli—a more ris recommended. Nevertheless, regutati hal thermotolerant (fecal) coliforms be identified ermotolerant coliformns, use one of the multiple s described here or the membrane-filter methods 9222D and E, In the multiple-tube fermen- motolerant coliforms are identified by their elose to produce gas at 44.5 0.2°C within doliform Test (EC Medium) ant coliform test using EC medium is appli S of drinking water, stream pollution, fees, wastewater treatment systems fers. and general water-quality moniton tes Flo directly igoace thermotoerant col fichment in presumptive medium overy of thermotalerant coliforms (To Heolonies growing on slid media EC medium following QC guidelines 8A Coliform on 5. Bibliography i Wars, Ten of Ck Her. 1939. Simp bctelgilexenina Chang ae Aer, Water Woks se 3.707 1 Seat: : ‘The detection of various bacteria indicative of water ute Presence-absence (P-A) procedure, Can. J. Micro- Late, indore, Wusor: 1973. Rlaontips among pllon Oy te et late om vate addin ems Cuan, mate tee PA) Heal Lab Sl 1016, ca rp iene of increcng numbers of ton ms upon the detection of indicator organisms bythe ‘nembrane filter and pres ee Wpresence-absence tesis. Can. J. Microbiol. Coax, ea CA. Benes & LE. Sauemos, 1982, Chutrizatin of a ace in mung aw wa, king water ad nw conn tat amps. Cor J Merb i Toes co baat. BC. Raz, TA. Sr, & EW, Ree Tone, Cimrson of manbrane tr, mkipl-fementaton- cont resecethece cies for dein wil ‘oliforms in-small community water systems. vi Microbiol, $1:1001, : ses Rice, EM. EE Gator & B Reso. 199, The pesece aber coliform test for monitoring drinking water qual sn ig drinking water quality. Pub. Heal rant (Fecal) Coliform Procedure Teyptose or tryplicase .. Lacioxe i alieeaee. le Bile sass misture or bile salts No. 3 Dipotassium hydrogen phosphate (K,HPO.).. Potassium ditydrogen phosphate (KH,PO,) Sodium chloride (NaC), Reagent-grade water. ‘Add dehydrated ingredients to water, mix thoroughly, and heat to dissolve. Before sterilization, dispense sufficient medium in tation tubes with an inverted vial to cover the inverted ferment ter sterilization. Close tubes ‘vial at least one-half to two-thirds at ‘with metal of heat-resistant plastic caps. Autoclave medium at 121°C for 12 to 15 mi, Ensure that inverted vials are ree of ait bubbles. Medium pHT should be 6.9 * (1.2 after sterilization 1b. Procedure: 1) After incubation, gently shake or rotate fermentation tubes or bottles showing gas, growth, or acidity (0 resuspend the Prompily use a sterile 3 to 3.5-mam-diam loop to or more loopfuls of culture from bottles or tubes Shoring growth with aid anor gas production a fennens tion tube containing EC broth, Alternatively, insert a sterile at least 2.5 cm into the culture, promptly wooden applicator @ : veove, and plunge applica the otto ofa fermeniaoon robe coftsining EC broth, Remove and discard apvca Re- Ail other presurptive-posiive tubes and incubate al organisms, transfer one peat for 44,5 + 02—- 9-78 Simultaneous inoculation into EC broth and/or EC-MUG broth along with BGLB broth is acceptable, if the most inhibi- tory medium (BGLB broth) is inoculated last. 2) Place all EC tubes into circulating water bath (preferably with a gabled cover) within 30 min after inoculation. Incubate inoculated EC broth tubes at 44.5 + 0.2°C for 24 + 2h. Maintain a sufficient water depth in the water bath incubator to immerse tubes to the upper level of the medium. ¢, Interpretation: Gas prodvetion with growth in an EC broth culture within 24 + 2h or less is considered a positive thermmo- tolerant (fecal) coliform reaction, Failure to produce gas (with litte or no growth) constitutes a negative reaction. If multiple tubes are used, calculate the MPN of thermotolerant coliforms from tie number of positive EC broth tubes, as described in 9221¢. When using only one tube for subculturing from a single presumptive botle, report asthe presence or absence of thermo- tolerant coliforms. If heavy growth occurs with no gas produ tion, subject the culture to a thermotolerant coliform or E. coli test using a different medium. 2. Thermotolerant (Fecal) Coliform Direct Test (A-1 Medium) 4a. A-1 medium: This medium may be used to directly isolate thermotolerant coliforms from unfiltered source water, treated tewater, and seawater, but not drinking water. Follow guide- 9221B.1 for sample collection. Unlike EC medium, A-L ‘medium does not require prior enrichment in presumptive medium for optimum recovery of thermotoleraat coliforms. Use QC guidelines cited in 9221B.2. Lactose . Bez, A eae 50g Tryptone E : 200g Sodium ciloride (NaCl). 502 ic OSg .... LOmL Pal 2 Heat to dissolve solid ingredients, add polyethylene glycol -isooctylphenyl ether, and adjust to pH 69 + O.1. For 10-mL MICROBOLOg Gy samples, prepare double-tren, of ingredions afer sample adios So dispense sufficient medium in fern vial © cover the inverted viel ar sterilization, Close with mello by autoclaving a 121°C for 10 man 6 fre of air bubbles. Sorin the day longer than 7d. Ignore precipitate b, Procedure: inoculate tubes oy 8 9221B.3b. Incubate for 3 h at 3§ a water bath at 44.5 + 0.2°C and inaye 6. Inlerpretation: Gas " within 24 h or less is a ae oh (Fecal) coliforms are present) Caley 8 erant (fecal) coliforms froin the gunk ® Ne tubes, as described in 9221C, "iy least eal-resi 8, Bibliography Peney, CA. & AA. Hawa 7 Bacteriol 26419, Eng Perry, CA. & AA. Hama, 1944, Funter ey the isolation of coliform baceria and Pub, Health 24:735. Gatoreicn, EE. HF. CLaRe, P.W. Kanes, 1958. The coliform group. I. Reaction in Appl. Microbiol. 6347, Geinesicn, FE, RH. Boronce, CB. Hur, HF Guy Kanter, 1962. Type distribution of fart of warm-blooded animais. J. Weser Fly 34.295, Getpkcicn, EB, 1956. Sanitary Significance ik Environment; FWPCA Pub, WP-202. US, De ington, D.C ‘ANDREWS, W.HL, & M.W. PeesveL., 1972. Ropid soe coll from estuarine water. Appl. Miroil 235 Otson, BAH. 1978. Enhanced accuracy of coliforms 4 modification of the most-probable-nunix nn bio. 36:438 Stanbatooe, JH. & JJ, Detewo. 1981, 4-1 meu Al nique for fecal coliform organism enunertin wastewaters, Appl. Environ. Microbiol. #2918 alin Eicher Hor ang EC neg