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_ Jul-29-98 11:O1A ACZ Laboratories Inc <br />• <br />MEMBRANE FILTER TECHNIQUE (9222)Rotal Coldorms <br />consrstem rclatinnahlp m the total numher of bacteria present <br />m the original sample. However. a bleb count of noncoliform <br />colonies may interfere with the maximum de, rlopment of coli- <br />forms. RrlnCe raunC cultures latter ?? h mcuhauonl x~ilh hieh <br />den,ihes of noncnhlnrm mlonlrs for 0.5 to 1 h before counune <br />may deter spread of confluence while aldme shorn di,cemmem. <br />Anaerohtc incubatrnn at ?S°C for ?J h for some gruundwacer <br />samples may suppress nnncohlnrm colomo but must he earefulle <br />evaluated to insure nu loss of colifnrm rrern'en~. <br />Samples n( drain(ected x'ater or xa,lexak•r rf0ucm mac in- <br />clude stressed oreannm> that grow relatively slnwh and produce <br />maximum sheen m '_'_ to ?J h. Oreani,ms from undiv nfrctrd <br />wu«rs m:q produce sheen at Ili to IF h. and the sheen suhsr- <br />qucndv mss faJc alter sJ to 7U h. <br />f Colrlnnn rrnlu ruron: Typical ,horn colomes rosy hr pn•- <br />ducrJ oirtstonJlh he nnnmhBtnn nrcam,m,.:\l~ptcal culnnlr, <br />Nark red nr nuclratrd m!nmes wuhout +heenl occaaonalh ma. <br />be coti(orm>. Yerificauon of hush cnlunr types a adcuahlc \'er- <br />ifv be a It>l fur laclou lcrmcmaunn or he wing altrrnatrae <br />prMCdurn involving either a raped (~ h) test of two key hin- <br />chemtcal tractions or a mule-lest system (or sprcration. See Sec- <br />tion 9tg116 J[f. <br />II Laansc Icrmrmahnn-\'cri(c rill tvprcal and atypical cnl- <br />dorm cnlonle, incluJrd m the Jurct cnum or a minimum of fis'r <br />such colomes Grim drinking x•,nrr sample, by uansferring grox'th <br />Grim each colons to limn I trvptmc ninth: mcuhatn at i; x 0 9`C <br />(or J8 h Cias fnrmrJ m loon I trcpunc broth and amfirmcd m <br />brilliant ¢rrrn lei tear broth x~tlhtn JX h verifies the Mlnnv as a <br />mh(orm <br />?1 Ahernative rohfnrm vrnficannns-Applg this ahernauce <br />cohtorm eenficannn procedure to notated culnnin nn the mem- <br />brane filter culture. If a mixed culture is su,prcted or d colony <br />separauon a less than 1 mm, streak the cnrwth In M-Coda me- <br />dmm to assure culture puns' m >ut+mn the mucd uux'th to the <br />fermentation robe method. <br />a) Raped test-A rapt) venticaunn of colonies uulrzcs tt,t <br />reactions for ectnchntmr u.adase ICOI and (i-galachnidau <br />(ONPG).: cohfurm reactiom arc Cr7 nrgauve and ONpG pos- <br />itis•e within J h incuhatinn of tube calmer ur m¢ro Isputl test <br />procedure. <br />b) Commeraal multi-test s7stcm>-Vcnfc the culnm hr <br />streaking n for puri(icaUnn, ulccting a well-notated colony. and <br />moculaune into a mobs-rev rdenuficaunn system for Entero- <br />hattenaceae that mdude, tact n,e (ermentaWm and'nr O'iPG <br />and CO tot reuctmin. <br />6. Calculation of Coliform Density <br />Compute the count. ustne memhrme filters with ?U to FIt cnl- <br />dam colomes :rod nut more than '1m colomes of all types per <br />membrane. M• the Gtllox~ing equation. <br />cnlilnrm rolnmrs counted <br />(Total) culilorm culumr>'IINI ml. = mL Sample (ihereJ <br />Fnr verified Mli(orm counts. adurst the rmual cnum base) <br />upon the poan ivy .'rn fir:num percentage and report as "cenfied <br />colifnrm count per I(111 mL.~~ <br />OM1PG Imrbnmuapb. and uLa n~wJ.l n iM1. •ub.uam Iw wr U c.rL.ao.Na.. <br />,n~ <br />Iql <br />970 879-2216 P.03 <br />• <br />9-57 <br />Percentage s'r nLrd a,hlurms <br />numher of ,cnhcJ [vunmrs <br />= IINI <br />pool numbs of n+hlurm culmm~ .ubl: nrJ I+~ ,: nh:abon <br />a. R'arrr n(drmLrng Horn quuho. \\'hlla• Ihr EPA Total Cul- <br />dorm Rulr.[or public water wpply samples rryuu is only a record <br />of cohfurm presence or ah,ence in inU-mL santplrs. a may he <br />advisable to drcermmc athlnrm dcn,nlr, m rrpuut sampbng sn- <br />uauons. Thn a of parucular Impon.mcc x'hrn a cohfurm hioGlm <br />problem rs suspected m [he dntnhmion as stem Qu:mmanse <br />mformahnn rosy provide an mdieulon n( the maemmde nl a <br />mmaminaune evcm that cannrn hr JrlrrmmrJ m Ihr presence. <br />absence concept <br />N'tth rater of Mod qualit v. Ihr n.currencr tit cubforms ecn- <br />erally x•tll he nunimal. Therrlnre. i.+um all cohtorm rnlomra <br />(disregardine the lower limit u( _'U inrJ olio+rl and ust thr <br />formula gn'rn abuse to nMmn cohlonn drnsits <br />If mnflurm erowth occur,. that Is crnx'th co, trine either the <br />enure filtrauun area nl the mrmhranr or a pnn a+n thereat. and <br />colonies are not dncrcte. report remits as -'n,n0uem ernxth <br />with for without) coliforms"~ and rcqursl a Hex ,ample Grim the <br />same location. If the total numher n(hactrri:J mlome,. cnhtnrms <br />plus noncoli(nrmx. exceeds '_lln per mrmhnmr. nr d the cnlnme, <br />are not dnunn enoueh for accuram rounun? repon re,uin a, <br />"too numerous to counY~ f pVTCI. Thr prc,rn:r of colifnrm m <br />such cultures ,hnwtnc nn shun mss hr mdrmtrJ by plaan~ the <br />entire memhrane filter cultnrr into a •trnlr tube of hrilhant green <br />lactose bile broth. As an ahernarut. brush the enure filter wr <br />face with a sterile loop. applicator stick. or covon swab and <br />inoculate this growth ro the wbe of brilliant Green lannse hJr <br />broth. if gas is produced from this iuhure robin JF h al ? 9 n <br />0.5°C. conclude that nuifnr m, arc prraem Fur compliance wuh <br />the EPA Total Cnli(orm Rule. report con0uem crox'th nr TVTC <br />wuh at Icas1 unr deteaahlr colifnrm cnlom Ixhich is seriGrdl <br />as a total colifnrm posnisr sample. Rrpun mn0umt gear th nr <br />TNTC without deteaahle rnhfnrm, a, invalid. Reyurn a nix <br />sample from the same locauun and +elrn more appropriate enl- <br />umes to ht filtered per mrmhranc. nbsers mg the regwrrmcm <br />that the standard drinking water ponlon n IUU mL. Thus. to <br />reduce mlerference from overcrowdmr. msteaJ of filtering 1(111 <br />mL per membrane. fiber u)-mL pnruons Ihmu~h each of two <br />membranes _'S-mL poruona through rash of four memhrane. <br />etc. Total the cnldorm counn uhse•rs rd on all mtmhranes and <br />repon as numher per ItNI mL. <br />h. l1'aMCOf other than drreknre +. ~nrr gaoler' As xilh potable <br />water samples. if nn filter has a udrtorm uwm tolling m the Ideal <br />ranee. total the cub(nrm counts nn ;JI (ihcr, and rrpurt as numher <br />per IW mL. For example. d dupuiatr ~U-mL portions x~ere <br />examined and the rx'o memhrane haJ for and Ihrcc aihlorm <br />colonies. respecuvrly, repon the cnum as right adilorm cnlome, <br />per 100 mL. i.e.. <br />Ala }{ IINI~ <br />l ill - il4 <br />Similarly. d SU .'_S-. and 10-mL pornoro rote examined and <br />the counts were 15 6. and '<1 cohtorm column. rrsprctis elr. <br />report the count as '_57uU mL. I e . <br />tits - q - U) + IWl <br />I ill ~a IDI <br />