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    APHA 9222

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    © All Rights Reserved
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    400 views18 pages

    APHA 9222 TH 2017

    Uploaded by

    Umar Bahary Prijatna
    APHA 9222

    Copyright:

    © All Rights Reserved
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    MEMBRANE FILTER TECHNIQUE (2222Vintroduction oat 9222 MEMBRANE FILTER TECHNIQUE FOR MEMBERS OF THE COLIFORM GROUP* 9222 A. Introduction ‘The membrane filter (MF) technique is reproducible, can be sed to test relatively large sample volumes, and usually yields ‘umerical results more rapidly than the multiple-tube fermenta~ tion procedure, It is useful in monitoring drinking water and ‘sous natural waters, However, the MF technique has limita- ions, particularly when testing waters with high turbidity or large numbers of noncoliform (background) bacteria. If hetero- trophie bacteria interference occurs for example, sample results ‘may need to be invalidated and new samples collected Ta laboratory has not used the MF technique before, analysts should conduct parallel tests with the labs current method to demonstrate applicability and comparability. Many coliform per- formance studies have been reported in the literature; the rates of false-positive and -negative results can differ among various coliform media, so users should carefully select the medium and procedure that best fit their needs, 1. Terminology Inthe MF technique, the coliform group is defined as facul- tative anaerobic, Gram-negative, non-spore-forming, rod-shaped ‘nctria that develop colonies with distinctive characteristies on specific media, When purified cultures of coliform bacteria are resed, they produce negative cytochrome oxidase and positive egalactosidase test reactions. Details of these coliform charac- teristics are given below for the standard total coliform MF ) a below) and for two procedures for ‘otcting total coliforms and E. coli simultaneously (92225 and K) (ls band ¢ below — ine ff, Endo-type agar medium (Endo agar LES or Endo broth In this procedure, coliform bacteria ate defined as bacteria that develop red colonies with a metallic (golden-green) sheen within 24h at 35°C on an Endo-type medium containing lactose ‘Some members ofthe total coliform group also produce dark red, nnucoid, or nucleated colonies without a metallic sheen: when sesified, these are classified as atypical coliform colonies. Gen- taally, pink (non-mucoid), blue, white, or colorless colonies fucking sheen on Endo media are considered noncoliforms inthis ane Bic cbomogen m-Colibliedé madam: This sire! bane iter meso simultaneously det abd ene ea cafoms (TC) and Bote co on water ses DUh bad on hei specie enyie ates. Cal weM Alc ter thn cot) a dln ate seein cols win 24 ha 25°C on hs um, ih Tel eve and a nomelecine ye [23 Siphes tan fam lege CITC), Bala disingso om or eal 8m acre by Me to Dae clones fo the action of paoved by Standard Methads Commites, 2015 Jett Tonk Group: Neney H. Hall (har), Jenifer Best, Gil Dichter, Sandra M, ender Mark W. LaChevaliet, Mark Roope * feglucuronidese enzyme on S-bromo-tehloroindoy- ffdewronide(BCIG), aloo presen inthe medium, seoroge/ehromogen Mi medi: Ths differential mem: eee rnin inoenaly aceon ana sts ba tl cole TE) and Bahr a wa wpe ip 24 W faod on thelr ape enzyme” atv, ‘The metlum includesivo enzyme substates—the Auor cectylintellien:p-oyaactpyrnosie. OM0Gal) end the Chomogen indoryeeo-ghewonide BDG) to detect the sh Syne fgaanoidase an gucurniase produced by TC and Broth Sapostvely in tis procedure, colform bacerla are {dnod ee tetera at produce Suoreaett cones win 24h re eapooed to fongvave ultavilt (OV) ight (365-3651) WC nite Ecol enone appent ke oh ths median 2. Applications ‘The MF technique may be used to test drinking, surface ‘ground, swimming pool, and marine waters. Do not use il 1 (es primary wastewater-treatment effluent unless the sample is 24 b, For nonpotable water samples, funnels should be rinsed or sanitized with UV after iter removal or after each sample (as described above) because of the high number of coliform bac: ‘eri and background flora present in these samples. QC samples: Check for sterility and coliform contamination athe beginning and end of each filtration series, respectively, by iilering 20 to 30 mL. of dilution or rinse water through the filter ‘one funnel per sterilization batch). If controls indicate contam- ination, reject all data from affected samples and request new samples, Additionally, to check for possible cross-contamination or contaminated rinse water, insert a sterile rinse-water sample (00 mL) after fleation of 10 samples. Incubate these QC samples under the same conditions as the samples being an: lyzed. ¢, Alternative enrichment teclnique: Use this technique with mndo LES media only, Place a sterile absorbent pad in the lid ofa sterile culture dish, and pipet at least 2.0 ml. lauryl tryptose ‘oth (prepared as directed in Section 9221 B.3a) to saturate ped. Cuefully remove any excess liquid from absorbent pad) by decanting plate. Asepfically place flter—through which the sam- pleas been passed—on the pad. Incubate filter, without invert- ing dish, for 1.5 to 2.b at 35 * 0,5°C in an atmosphere of atleast 0% relative humicity Remove enrichment culture from incubator, lift filter from E exichment pad, and roll it onto the m-Bndo LBS agar surface, which has been allowed to equilibrate to room temperature Incomeet filter placement is instantly obvious because patches of | unstained membrane indicate entrapped air. If such patches ‘ovcur, carefully reseat filter on agar susface. If broth medium is wed, prepare final culture by removing enrichment culture from incubator and separating the dish halves. Place afresh sterile pad in dish bottom, saturate with atleast .0 mL. of m-Bndo medium, and carefully remove excess liquid from absorbent pad by de canting plate, Transfer filter, with same precautions as above, 1 new pad. Discard used enrichment pad. With either agar or broth medium and using this ¢wo-step, procedate, invert dish and incubate for 22 2h at 35 + 05°C. Proceed 10 below F. Counting: To count colony-forming units (CFU) on Endo: type membrane filters, use a low-power (10 to 15% magnlien tion) binocular wide-fild dissecting microscope or other optical device, with a cool white fluorescent light source directed to provide optimal viewing of sheen. The angle of light on she colony affects sheen detection for coliform colonies growing on indo plates. Rockisig and turing the Petri plate rflecis light at different angles and helps detect sheen on the colony. ‘The typical coliform €olony on Endo-type media has 2 pink 1 darkered colt with a metallic surface sheen. Count both types) and atypical coliform colonies promptly after incubation, ‘The sheen area may vary in size fron a small pinhead to compleic coverage of the colony surface. Alypical coliform colonies em be dark red, mucoid, or nucleated without sheen, Generally pink, blue, white, or colorless colonies lacking sheen are vou sidered noncoliforms. A high count of noncoliform coloni (©200 CEU) may interfere with the maximums developrn coliforms. Reftigerating cultures with high densities of noncol form colonies (after incubation) for 0.5 to 1 h before coun may aid sheen discemment. Samples of disinfected water or ‘wastewater effluent may include stressed organisms that grow relatively slowly and produce maximum sheen in 22 to 24h 8 Coliform verification: Occasionally on Endo-type medi, rnoncoliform organisms may produce typical sheen colonies. ane! atypical colonies (pink, dark red, or nucleated colonies witli sheen) may be coliforms; thus verification of all typica and atypical colony types is recommended, Verify all colonies on Endo media by swabbing the entire membrane or picking at les! five typical colonies and five atypical colonies from a rembrano filter culture, (See Section 9020B.9.) Based on need tind sample type, laboratories may incorporate more stringent QC measures (e.g. for nonpotable samples, verify at least one colony from each typical or atypical colony type from a gi membrane fiter culture, or verify 10% of positive sunpless ‘Adjust connts based on verification results, Verification tests si listed below. 1) Lactose fermentation—Transfer growth from each eolony ‘or swab the entire membrane with a sterile cotton swab {for presence-absence results) and place in single-strength lauryl tryptose broth; incubate broth at 35 + 0.5°C for up to 48 b, Gas formed in lauryl iryptose broth and confirmed in briliant green lgetose broth within 48 h verifies the colony as a coliform, (See Sections 9221B.3 and 4 for media preparation.) Simultaneous inoculation of both media is acceptable (if using same inocclat ing sterile loop, needle, oF wood stick, inoculate lauryl eyptose ‘broth first), Including EC broth inoculation incubated st 44.5 0.2°C for 24 + 2.1 will provide information on the presence of thermotolerant coliforms. Including EC-MUG inoculate: inew bated at 44.5 = 0.2°C for 24 h will provide information om presence of coli. The inoculation order should always be fea least to most inhibitory (1) EC or EC-MUG, 2) lauryl trypiose broth, 3) brilliant green lactose broth, (Seo 9222G and H for MI partition procedures.) p86 2) Alterative coliform verifications—Apply this alternative clifort verification procedure to isolated colonies on the Endo membrane filter media, IT a mixed culture is suspected or if colony separation is <2 mm, streak the growth to m-Endo ‘medium of MacConkey agar to ensure culture purity or submit the mixed growth to the fermentation tube method. a) Rapid est—A rapid verification of colonies uses test reac- sions For cytochrome oxidase (CO) and B-galactosidase. Coli- Form seactions are CO negative and -galactosidase positive ‘within 4 h inewbation of tube culture or micto (spot) test proce- lus. +») Commercial anultistest systems—Verify and/or identify the ‘coliform bacteria by selecting a well-isolated colony, streaking for isolation, and inoculating a pure colony into # multisest ‘dkniification system for Enferobacteriaceae that includes lac> tose fermentation and/or B-galactosidase and CO test reactions, 5, Calculation of Coliform Density Seleet the membrane(s) with acceptable number of colonies (Table 9222:11) and =200 colony-forming units (CFU) of all Iypes ner membrane, by the following equation: 1a eotifoams, NoZ100 mL coliform colonics connied 100 ai sample tered” NO CRU/GO me For drinking water samples, if no total coliform colonies are then report the ‘otal coliform colonies counted as <1 CFU/100 mi.” or report “total coliform bacteria absent per 1) eae sample.” For nonpotable water samples, if 10.0., 0.3-, and 0,01-mL portions age examined and all counts are-0, then calculate the hnunibet of coliforms per 100 mL that would have been reported if there had been 1 CFU om the filter representing the Inrgest filyation volume. For example, report <10 CFU/100 mL for # 10 ml. saraple volume with no coliform colonies, ie, LAO x 100 = <10 CFUFIO0 mL or verified coliform counts, adjust the initial count based on the positive verification percentage (both typical and atypical) sand report as follows: Verifies Gta cbifonms, No/100 md. . ‘ial number of coliform colonies iA subjected to verification 7 X ola wtber of presumptive colonies «4, Potable water: In its Total Coliform Rule, the U.S. Environ ‘mental Profeetion Agency (EPA) only requires a record of the presence or absence of total coliforms in 100-mL. samples of drink jing water; however, quantitative information can indicate the magnitude of a contaminating event and/or remediation progress, ‘specially when compating sample resulis from different locations (eg. repeat samples). Quantitative infomation only provides a MICROBIOLOGICAL EXAMINATION (8006 ross entimation of the actual coliform popolation at collection tin dhe to non-uniform digpbotion within the matrix, Tn good drinking water, coliform occurrence generally will ‘minim Therefore, count al coliform colonies (disregarding te lower limit of 20 cited above) and use the formula given above to obtain coliform density. if confluent growth ocears-—covering either the memirene's entire filtration area or a portion thereof with colonies that at pot discrote—reportcesults as “confluent growth with (or wit ut) coliforms.” If the total number of bactetial colonies~ coliforms plus noncoliforms—is >200 per membrane or if te colonies are not distinct enough t0 count accurately, then repot results as “too numerous 10 count” (INTC) or “confuent” respectively. Por drinking water samples using Endo-type mei, the presence of coliforms in such cultures may be confitmed (sg 9229B.4g). As an altemative, brosh entre iter surface witha sterile loop, applicator stick, or cotton swab and inoculate ti growth into 1) a tube of single-strenguh lauryl tryptose and 2) tube of brftiant green lactose bile broth, Ifthe brilliant gree bie ‘roth cube produces gas within 48 hat 35 2 0.5°C, coliforms a present, To comply with EPA'S Total Coliform Rule, rept Confhuent growth of TNT with atleast one detectable colin colony (vetfication only required with Endo-type medie) «2 “Total coliform positive sample.” Report confluent growth or ‘TNTC without detectable coliforms as “invalid.” a ‘or invalid. samples, xequest a new sample from the sat) location within 24h, Select more appropriate volumes to fee per membrane (kesping in mind that the standard drinking-wa portion is 100 mi, according tothe rule). So instead offing 100 mL through one membrane, filter SO-m. portions throu two membranes, 25-mL. portions through four separate mx branes, ete 0 teduce interference due to overcrowding. If a) membrane contains & ve entire sample as “otal coliform positive.” If a density dete nation is desired, total the coliform counts observed on membranes and report as “[number} per 100 mL.” (Ait heterotrophic bacterial interferences.) 'b, Other waters: As with potable water samples, if no filtec ‘coliform count within the ideal range, total the coliform cout fon all filers and report as "[oumber} per 100 mL.” For example if duplicate 50-mL portions were examined and the (wo m branes had five and three coliform colonies, respectively, tha report the total coliform count as 8 CBU per 100 mL: 15 43) % 100) +50) 8 CFU/IOO mL Altematively if 50+, 25, and 10-m portions were exami and the counts were 15, 6, and <1 coliform colonies, ep tively, ten calenlate based on the most nearly abceptable and report the total coliform count with a qualifying remark “estimated 30 CFU/100 ml." 1 (a5) x 100) q LO estas 30 CHLN00 mL On the other hand, if 10-, 1.04, and 0.1-mL. portions examined with counts of 40, 9, and <1 coliform cok MEMBRANE FILTER TECHNIQUE (9222)'Standard Total Colform Membrane Filer Procedure using Endo Media respectively, then calculate the coliform density based only on the 10-mL portion because this filter had a coliform count with the acceptable range (see Table 9222:T1) and report result as 400 CFU00 ml: 20100) 400 coliforns/100 mL, v9 In this tat example, ifthe membrane with 40 coliform coto- ties also had a total bacterial colony eount >200, then report the coliform count as 2400 CFU/I00 mL. or with a qualifying + rama as “estimated 400 CFU/I00 aL” 110.0 1.0- and 0.1-mL portions were examined with counts of TNTC, 150, and 92 coliform colonies, respectively, then calulate based on the most neatly acceptable value and report wih « qualifying remark as “estimated 92 000 CFU100 mL 2100) on estimated 92.000 CRU/100 mL. 1F1.0-, 0.3-, 0.1-,and 0.03-mL portions were examined with counts of TNTC, TNTC, 78, and 21 coliform cotonies, respec- lively, then sum the total coliform counts on the latter two {countable filters and divide by the sum of their volume to obtain the final reported value of 76 000 CFU/100 mL: (78+ 21) x 100 1+ 0.03) 16 000 CPLN00 mL. 1f1.0-,0.3-, and 0,01-mL portions were examined with counts of TNTC on all portions, then calculate using the maximum number of colonies acceptable for quantitative determination of that indicator with the smallest filtration volume and report result as >800 000 CFU/100 ml, (for total coliform): 50% 100 _- 800 000 CFU/100 ml. 6 Statistical reliability of membrane filter results: Although ME results are considered more precise than multiple-tube most probable number (MPN) results (S- and 10tube multiple-tube fermentation formats), membrane counts may underestimate the namiber of viable coliform bacteria and circumstances may affect this precision (background bacteria and dilution levels, sample Iypes, eic.). Table 9222411 illustrates some 95% confidence D limits for MF results, These values are based on the assumption that bacteria are distributed randomly and follow a Poisson distribution 4. Precision of MF results: Caloulate precision of replicate tnalyses for each type of sample examined and method used if there is enough sample avaiable for replicate analyses (drinking, wae, ambient water, ete.). (See Section 9020B.9e.) 6, Biography p Prev, CW. & CP, Scuawrus. 1958. hmproved membrane Mer me- ium forthe detection of coliform organisms. J- Amer. Water Works Assoc, 50:193, . our "Tans 9222sI1, Conripence Lavrs vor Mya Pace Caxonst Resuers Vaio 10D Sau Number of Coliform 25% Cont Colonies Counted Lower o op 1 ou 2 02 3 06 4 0 5 16 6 22 wa 28 8 ® 34 9 49 0 a7 ue 34 12 62, 1B 69) 14 1 5 a4 16 92 a 99 8 109 0 us 26 20 2 308 McCanniy, J.A. & JB, Datawey. 1958, Membrane filer mstia ste Water Sewage Works 105:282 Rioves, CE, & W.P, Cirvens. 1968. Decontamination of yeabrane filter holders by whraviolet light. J. Amer. Water Works stssos 57:500. Goprsten, BIE, HL. Jere & JA, Woven, 1967, Techaical conser ‘ations in applying the membrane filter procedure. Health ay Si 4113, WaTLNo, HLR, & RJ, Warne. 1975, Note on the trace metal conten ‘of membrane filters. Water Sa 1:28, Gaesicn, EE, 1976, Performance variability of membrane fer pro cxsdue. Pub, Heath Lab. 34: 100. Lin, S.D. 1976, Bvaluaton of Millipore HA and HC membrane ies For the enumeration of indicator bacteria. Appl Environ, Micubiol 32:300, Stanainge, HL. 1976, Comparison of surface pore mosplnlexy af veo brands of membrane filters. App. Environ. Microbiol. 31.316, US. Bavmonuevzas Prorecriox Aomcr. 1978, Microbiological Met ‘ods for Monitoring the Environment Water and Wastes; FPA 6 8-78-017, Off. Res, Develop, Cincinnati, Ohio Gratow, W.0.K. & M. Du Pxex2. 1979. Comparison of mine LBS, MacConkey and Teepol media for membrane filtration ‘counting of total coliform bacteria in water, Appl. Biniron Microbiol. 38351 Duvka, B.D, ed. 1981, Membrane Filation Appliations, Techniques ‘nd Problems. Marcel Dekker, Inc, New You, N.Y. Evans, TM, Rd. SapuaR & MAW. LECHEVALAIE FOS). Impact of verification media and resuscitation on accuracy ofthe ment fier tots coliform enumeration technique. Appl Bavivon, sire Biot 41-1144, Fravcniu, SG. B.. Hiwenesc, TIM. Kuuyey & NA, SINcLADi 19% Effect of noncolforms on coliform detection i poise grainy (er; improved recovery with an anacrobie membrane fier sigue. Appl. Environ. Microbiol 48:142 MeFinias, GA, JS. Kine & MW. LeCnevautut, 1986, Injures coliforms in drinking water. Appl. Erevan. Microbial. 51 pa Ioan KP, CC. Raweng, YR, Rovaat, GN, Sti, ty PL, Scarsino & A.P. Durour. 1993, New medium for the siultancous telecon of total coliforms and Escherichia colt in water. App. Eariron, Microbiol. $9°3534, Bronnre, KP, CC, Rao, M. Sivacanssan & PV. Seatono, 1996: Comparison of the recoveries of Excherichia coli and total col fonns fiom drinking water by the MI agar method andthe US. Bavinonmenal Protetion Agency-approved membrane filer msthod. Appl. Eavivon Microbiol 62/203. 9222 C. Delayed-Incubation Thix modification ofthe standard MF technique permits mem- ‘brane shipment or transport after filiradion toa distant laboratory for transfert another substrate, incubation, and completion of the test. The delayed-incubation test may be used when + conventional procedures sre impractical, + sesired sample temperature cannot be maintained dering transport, * the clapsed time between sample collection and analysis, would exceed the approved time limit; or + the sampling locaton is remote fioat laboratory services (see Section 906085), Independent studies using both fresh and marine water sam ples have shown consistent results betowen the delayed-ineuba- ‘ion and standard MF jesis. Determine the delayed-incubstion ‘ess applicability for a specific water souree by comparing with results of conventional MF methods Vo conduct the delayedincubation test iter stipe in the immediately after collection, place fiter on transport me- tlium, and ship laboratory. Complete coliform determination in the laboratory by transfering membrane to standard m-Endo ov Endo LES medium, inenbating at 35 = O.5°C for 20 10 22 h, ul counting the typical and atypical coliform colonies that slop. For drinking-water samples collected for compliance with EPA's Total Coliform Rule, zepot the presence or absence of verified coliforms in 100-mL. samples. Verify colonies as outlined previously in 9222B 4g casport media are designed to keep coliform organisms Viale and generally do not permit visible growth during transit ime, Bacteiostatic agents in holding/preservative media sup- press growth of microorganisms en route but allow normal coliform growth after transfer to a fresh medium,” The delayed-incubation test follows the methods outlined for the total coliform MF procedure, except as indicated below. Two alternative methods are ‘given: one using. the sv-Endo preservative medium and the other using te m-ST holding medium. If commercially preparéd medium is un available, prepate from individual components as described in 92928.24 and 9222¢.26. - 1. Laboratory Apparatus «Culture dishes: Use disposable, stele, plastic Petri dishes © 50 mm) with tighe-titing lids: Such containers ste light hv and less likely to break in transit. In an emergeney or MICROBIOLOGICAL, EXAMINATION 6000) US. EwenownetaL Pronscron AGENCY, 2002. Method 1604: Tol colifonms and Escherichia coll in water by membrane filtration ‘sing a simultaneous detection technique (ML medivin); BPA $2) R-02.024. Off. Water, Washington, D.C 4 U.S. Bnviorasenrat, PROTECYION AGENCY. 2013, National Primary § Drinking Water Regulations: Revisions to the Total Colin Rule; Final Rule. 49 CFR Parts [41 and 142; Fed. Reg ‘T8:10270, Total Coliform Procedure ‘when plastic dishes are unavailable, use sterile glass Petri dishes ‘wrapped in plastic film or similar material. (See 9222B.1e.) 6, Field filmation units: See 9222B.1f, Ulwaviolet light disie fection may be used in the field if an appropriate power source is available (115 V, 60 Hz). Glass or metal filtration units may be # steslized by immersing in boiling water for 2 min. Use reagent Water to avoid hard-water deposits. Use a hand aspirator obtain necessary vacuum, q €. Absorbent pads: See 92228.1h. 4. Foreeps: See 9222B.1i 2. Materials and Transport Media 4, m-Endo methods: 1) m-Endo preservaiive mediunt—Prepare m-Endo medium ss described in 9222B.2b, After cooling to <45°C, aseptically add 384 g sodium benzoate (U.S. Pharmacopein (USP) made or 32 mL 12% sodium benzoate solution te 100 mL medium. Mix ingredients and refiigerate poured plates. Discard unused medium after 96 h ' 2) Sodium benzoate solution—Dissolve 12 NaCyHs0. i sulicent reagent water to make 100 mt. Sterilize by a toclaving or by fering though 8 0.22-uan-poe-size mem bane filer, Discard after 6 months 3) Cyelohevimide™ Optionally, add “cycloheximide 1) 1n-Endo preservative medium. It may be used for samp that previously have shown overgrowth by fungi incluing yeasts. Prepare by aseptically adding 50 mg eycloheximie 100 miL to m-Endo preservative medium, Store cycle mide solution in reftigerator, and discard after 6 moat, Caumion: Cycloheximide fs a powerful skin iret Follow manufacturer's and SDS instructions for prope handling and storage of this chemfeal, b, ST method: ST holding median: ‘Sodium phosphate, monobasic (NaH,PO,* HO) ...... 04 g Dipotassium hydrogen phosphate (KHPO,) 308 Sulfenitamide : 1g Etanol (954), 10 at ‘Tei (bydroxymethy!) aminomeshan, 308 Reagontsgrade water rk « Actidione®, ranufstured by the Upjoks Company, Kalannzc, Ml, or ese len Dissolve ingredients by rehydrating in water. Sterilize by iociaving at 121-124°C for 15 min, Final pH should be 8.6 (12. Dispense at Teast 2.0 to 3.0 mL. (depending on ped manu- By farurer) to tight-lidded plastic culture dishes containing. an bsorbent pad, and carefully remove excess liquid ftom pad by ‘decanting the plate. Store plats in the rftigerator for use within 6b & 9, Procedure 4. Sample preservation and shipment: Place absorbent pad in otto of sterile Petri dish and satorate with selected coliform holding medium (see 9222C.2). Remove membrane filter from filtration unit with sterile forceps and roll it, grid side up, onto surface of medium-saturated pad. Protect membrane from mois ture loss by tightly closing plastic Petri dish. Seal loose-fitting Fsishes with an appropriate sealing tapet to prevent membrane } cenydration during transit, Place culture dish containing mem- Fe brane in an appropriate shipping container and send to laboratory for test completion. The sample can be held without visible {gow for 2 maximum of 72 h at ambient temperatare on the Helinglpreservative fhedium. Visible growth occasionally be- ean, Sgma-Alish Co. LLC, ¢ euialan, ‘Thermotolerant (formerly fecal) coliform bacterial densities may be determined by either multiple-tube procedure or MF technique. (See Section 9225 for differentiation of Escherichia alt) The themmotolerant coliform MF procedure uses an en tiched laetose medium and incubation temperature of 44.5 (02°C for selectivity. Because incubation temperature is critical, F sobmerge waterproofed (plastic bag enclosures) MF culares in snnter bath for incubation at the elevated temperature or use a) | appropriate solid hext-sink or other incubator that is documented Bo hold the temperature at 44.5°C + 0.2°C throughout the ‘tamber over a 24-h period. The best type of incubator is a gable-covered circulating water bath. In general, this method is ‘applicable under the same circumstances as the multiple-tube ‘hermotolerant coliform procedures (see Section 9221F) ‘There ate limitations to the interpretation of a thermototerant F colform result from thermal waters (e.g, the topics) and pulp ed paper mill effluent samples where thermototerant Klebsiella >have predominated and not been indicative of a sewerage source, | As with all coliform results, a sanitary survey should be con- tucted 0 identify the most plausible Source and public lnealt «a, Sample bottles: See Section 9030B.19. », Diaion bottles: See Section 9030B.13, G), 6 Pipets and graduated cylinders: Sce Section 9030B.9. |. & Containers for culture medivn: See9222B. 1d | MEMBRANE FILTER TECHNIQUE (0222)/Thermotolorant (Focal) Colform Membrane Fiter Procedure 9.89 gins on transport medinm when high temperatures are eneou {ered during transit. b, Transfer and incubation: At the laboratory, transfer Hier from holding medium on which it was shipped (0 a second sterile Petri dish containing m-Endo or Endo LES medium and incubate at 35 0.S°C for 20 to 22 b. 4, Estimation of Coliform Density Procood a described 92228 8, Recor times of election, fhuation, and laboratory’ examination, nd calelats the apse dine, Report lapsed-tme with coliform resus. vt 5. Biography Gnomes, EXE, PAK Kamm, Hibs Jeor & WP. Cuare. 1955. A delayed incubation membrane filter test for coliform bacteris in watee, Amer J Pub. Health 45:1462 Panezal, AK Tl. Mackin & H,G. Cons 1965, Coft-aerogenes and ‘Bucherichia coli counts on water samples by means of wansporied membranes. Proc. Soe. Woter Treat. Exam. 14379, Baramssxs, PT. & 1A. Woeter, 1969. Use of the delayed inenbation rembrane filter tet for determining corm bactr Water Res. 1583. ‘Cun, M, & PJ. Hiexey, 1986, Blimination of overgrowth in deta incubation membrane filter test for iota coliouns by M-ST bol rmediuin, Appl Environ, Microbiol. 52:778, 9222 D. Thermotolerant (Fecal) Coliform Membrane Filter Procedure ¢, Culture dishes: Tight-fiting plastic dishes (see 9228.10) are preferred when MF culture plates will be submerged in 3 ‘water bath during incubation, Place thermovolerant cofifonn culture plates in plastic bags (remove as much air as possibie) ar seal individual dishes with waterproof (freezer) tape to proveut Teakage during submersion Filtration units: See 9222B.1f. '. Membrane filters: See 92228. fk. Absorbent pads: See 9228.1 i, Forceps: See 9222B.1i j. Water bath or incubator: The specificity of the thermotol rant coliform testis related disectly to incubation temperature To meet the need for greater temperature control, use a gable covered water bath, a heat-sink incubator, or any properly de signed and constructed incubator that can maintain a temperature tolerance of %0.2°C. Most circulating water baths equipped! with 1 gable top to reduce water and heat Toss ean maintain a en perature of 44.5 + 0.2°C, However, static air incubation may be ‘problem in some types of incubators because of potential heat layering in the chambes, slower heat transfer from air 10 the ‘medium, and slow temperature recovery each time the ineubster is opened during daily operations. 2. Materials and Culture Medium ‘mEC medium: The need for uniformity dictates the use of dehydrated media, Never prepare media from basic ingredients ‘when suitable debydrated media are available. Pollow the ns °-90 lufaotarer’s directions for rehydation. Commercial liquid media (sterile ampule, ete.) may be used if known to give equivalent results. See Section 9020 for QC specifications. If commercially prepared medium is unavailable, prepare from individual com- ponents as described below. mF C medi: Trypiose or biosate 1008 Proteose peptone No. 3 or polypoptone 508 Yeast extract 308 ium chloride (NaCI) 50g Lactose Dse Bile sats No, 3 or bile salts mixture, Isg Aailine bine Og ‘Ager (optional) 130g Resgontgrade water, re Rehydrate product or individual components in IL water ning, 10 mL. 1% rosolic avid in 0.2N’ NaQH.* Heat to near » promplly remove from heat, and cool 10 <50°C, Do not ize by autoclaving, Tf agar is used, dispense 4- to 6-mL. {quantities to 9- X $0-mm Petri plates (approximately 4 to 5 man «deop) and let solidify. Final pH should be 7.4 + 0.2. Refiigerate finished medium (preferably in sealed plastic bags of other containers to reduce moisture Joss) and discard unused broth after 96 h or unused agar after 2 weeks. Nore: For samples from soumes known to have minimal background growth (e.g. drink ing water), 1% rosolie acid addition ean be omitted from mFC ‘medium, but it should be used for all unknown sources, storm waters, and ambient water sources. Before use, fest each batch of laboratary-prepared MF medium for performance with positive and negative culture contrals (See Table 9020:V 1, Check for coliform contamination at the begin- ning and end of each filtration series by filtering 20 to 30 mL of cilution or rinse water through file. If controls indicate con- amination, reject all data from affected samples and request new samples. Test each new medium lot to confitm that its perfor~ ‘mance is satisfactory (see Section 9020B.5)), The use of control charts is helpful to identify tends and ensure long-term consis tency in media performance, 8. Procedure «, Select sample size: Select volume of water sample t0 be ‘examined in accordance with the information in Table 9222:1V, Use sampie volumes that will yield counts between 20 and 60 thermorolerant coliform colonies per membrane, ‘When the sample's bacterial density # unknown, filter severa) volumes or dilutions to achieve a countable plate. Estimate the volume and/or dilution expected to'7eld a countable membrane, and select two additional quantities representing one-tenth ag ‘en times (or one third and tree times) this volume, respectively », Filrer sample: Potlow the sane procedure snd precautions given in 9222B 4c : ©. Prepare culsure dish: Using aseptic technique, place a sterile absorbent pad in each culture dish and pipet at Teast «Rosle aid eagen wit decompose if sterilized by autataving, Refigroe suck ofsion in he dak and serdar 2 weeks, o Sooner fs colo henge From died 0 muy owe Water Soe 100 S010 1 inking water x Lakes, reservoirs x Wells, springs x Water supply intake Natural bathing waters ‘Sewage teaenent plant Farm ponds rivers Stormwater runoft Raw municipal sewage Feedlot runoff Sewage slodge see etn MMe oe Dot pe ee Pe ee pare Anchor dishes below water surface; if anchor devices (e8, tings, bricks, ot water bottles) will also be submerged, make si does not allow the plates to reach temperature for many hou . Counting; Colonies produced by thermotolerant coliform bi ‘medium are various shages of blue, Non-thet rant coliform colonies are gray to cream-colored, Normally, ratory. Verify typical blue colonies and any atypical grey i} green colonies (see Section 9020B.10) for thermotolerant cold {orm analysis, Simultaneous inoculation into single-strength ry] tryptose and EC broth (or EC-MUG broth) incubated at 9 and 44.5°C, respectively, is acceptable during verification, 4. Calculation of Thermotolerant Coliform Density @ General: Compute the density from the sample quanti that produced MF counts within the desired range of 20 to 6 thermotolerant coliform colonies. This colony-density range i ‘more restrictive than the 20 to 80 total coliform range because [MEMBRANE FILTER TECHNIQUE (2222)/Delayed-ncubation Thermotolerant (Fecal) Coliorm Procedure 291 coliform density as directed in 9222B,5, Record thermotolerant coliform densities as CPU per 100 mL. +, Sediment and biosolids samples: For tora solids (dry weight bss), see Section 2540G, Calculate thermotolerant coliforms E per gram dry weight for biosolids analysis as follows: Themotolerant coliform, CFUIg dry weight 7 colonies counted ~ Gllation chosen) (% dry solids) ‘where dilution and % dey solids are expressed in decimal form, Example 1: Aualyst observed 22 colonies on the 1:10 000 lution plate of a biosolids with 4% dry solids. 2 Woon 0.045 = 5.5 10° CFUlg dey weight Jf no filter has a thermotolerant coliform count falling in the ileal range (20 to 60), total the thermotolerant coliform counts cn all countable filters, and report as thermotolerant coliforms per gram dry weight: Bxample 2; Analyst observed 18 colonies on the 1:10 000 Aileion plate and 2 colonies on the 1:100 000 dilution plate of a 1 biosolids sample with 4% dry solids. Wooar + onowary was” *6* 1° ‘To compute # geometric mean of samples, convert the ther- ofolerant coliform densities of each sample to logio values. Determine the geometric mean for the given number of sémplest ty averaging the logy, values of the thermototerant coliform densities and taking the antilog of that value Wooly seve if collecting for EPA's Patopen Reduction Ru, 40 CTR Pat By 5, Roforence 1, US, Fivwmnnral, PROTECTION AGENCY. 1993, Stands for te Use of Disposal of Sewage Sludge: Final Role. 40 CPR Pare 50s Fed, Reg, 58:9248, 6. Bibliography Gapnsicn, BE, HF. Cane, CB. Huse & LC. Bas. 1965. Fecal coliformzorganiem medium for the membrane filter wehnigue J. Amer. Water Works Assoc. $7:208. Rose, RLE., EE. Granarict & W. Lrrsky, 1975, Improved membrane filtgrymethod for fecal coliform analysis. Appl Microbiol 29532. ® Ly, SD. 1976, Membrane filter method for seeove forms in chloctiated sowage effuents. Appl. Bi 32:587.* Presswoon, W.G. & D.K. Stmoxa. 1978. Modif diam by eliminating rosolic acid. Appl. 36:90. Ganun, BL, W. Lrsky & KJ. Stapak, 1980. Evaluation of memb fier methods for enumeration of faecal coliforms from maine waters. Mar, Environ. Res. 67:261. Saxrony, D.P. 1980. Membrane filtration Faecal cofiform determin tions with wamodified and modified MPC 6113, Grawow, WOK. CA, Hosur & P. Cousnouen, 1981. Evaluation wf standard and modified MFC, MacConkey, and Teepol modi ‘membrane tiles counting of fecal eolifoom in water. Microbiel. 42:192. Ryouenr, RC & GR. Sruienson. 1981. Atypical Escherichie co in streams, Appl. Environ. Microbiol. 41:1276. Probl, 1, A.A. Qunesit, BLM. Youn & L:P, Viassore. 1982. Cou parison of four membrane Miter methods for fecal coliform enum ‘ion, Appl. Environ. Microbiol. 43787, US. Exvinonweat ProvecrioN AGENCY. 1992, Environmental Reg ‘lations and Technology, Control of Pathogens and Vector A traction in Sewage Sivdge, EPA.626/R-92-013. Washington De, of fecal coi iron. Microbiol on of MFC ave viron. Microbiol wi, Water 83 tase 9222 E, Delayed-Incubation Thermotolerant (Fecal) Coliform Procedure ‘This delayed-incubation procedure is similar to the delayed- innbation total coliform procedure (92220). Use this test only wen the standard immediate thermotolerant coliform test can nat be performed (e.g., if the appropriate field incubator is unevalable or circumstances indicate that a specialized labora tery service is advisable to examine, confirm, ot speciate the ‘uspect colonies) | Results obtained by this delayed method have been consistent ith results from the standard thermotolerant (fecal) coliform Uc test under vacious laboratory and field use conditions. How- # ver, determine test applicability for a specific water source by jeomparison with the standard MF test, especially for saline U \wers, chlorinated wastewaters, end waters containing toxic substances. H) To conduct the delayed-incubation tes, filter sample in the Sie immediately after collection, plage filter on m-ST holding medium (see 9222C.2b), and ship 10 the laboratory. Complete thermotolerant coliform test by transferring filter (0 mifC me. liu, incubating at 44,5°C for 24 + 2h, and counting therm. tolerant coliform colonies, The m-ST medium keeps thermotolerant coliform orgasms viable but prevents visible growth during transit. Membrane filters can be held for up t0 3 d on m-ST holding medium with litle effect on thermotolerant coliform cout. 1. Laboratory Apparatus 4, Cultwre dishes: See 9222B. le. 1, Field filtration units: See 9222B.1f. 6. Absorbent pads: See 9222B.1h 4. Forceps: See 9222B.1i. 2, Materials and Transport Medium a. eST mecliuna: Prepare as desevibed in 9222C.2b, b. mC medium: Prepare as described in 9222D.2. 3. Procedure «6, Membrane filter transport: Using aseptic technique, place an slosorbent pad in a plastic Petri dish with a tight lid and saturate with mST holding medium, After filtering sample, remove membrane filer from the filtration nit and place it on medium-saturited pad, Use only tightly lidded dishes to prevent nioistute loss but avoid excess liquid in the dish, Place culeure ‘lish containing the filter in an appropriate shipping container and send 10 laboratory. Membranes can be held on the transport meclivin at ambient temperature for a maximum of 72h with Tinie effect on thermotolerant coliform counts ‘Transfers AC the laboratory, aseptically remove membrane Irom holding medium and place it in another dish containing nik svediun Incubation: After transferring filter 0 mFC medium, place Lidded dishes in waterproof plastic bags, invert, and sub- MICROBIOLOGICAL EXAMINATION (9000) merge in a water bath at 44.5 02°C for 24 2h, or use a solié hreat-sink oF equivalent iRcubator . Counting: Colonies produced by thermotolerant coliform bacteria are various shades of blue, Non-thermotoferant coliform colonies ate gray to cream-colored. Count colonies with a bin ‘ocular wide-field dissecting microscope at 10 to 15 magaif- cation. , Verification: Verity colonies at a frequency established by the laboratory. Verify typical blue colonies and any atypical (grey to green) colonies as described! in Section 9020B.10 for thermotolerant coliform analysis. 4, Estimation of Thermotolerant Coliform Density Count as directed in 9222D.3e and compute thermotolerant coliform density as described in 9222D.4. Record time of col Feotion, filtration, and laboratory examination, and calculate and report elapsed time, 5. Bibliography Chen, M. & P.J. Hickey. 1983. Modification of delayed-incubation procedure for deretion of fecal coliforms in water. App. Envion. Microbiol. 46:889. 9222 F. Klebsiella Membrane Filter Procedure Klebsiella bacteria belong tothe fareily Enteobaeteriaceae and sue included in the total coliform group. Klebsiella spp. are exerted in she feces of many healthy humans and animals, and are readily ‘cicote in sewagepoluted waters. Approximately 60 to 80% ofall Kiebsietl fom feees and from clinical specinens are positive inthe shermotolerant coliform test and are Klebsiella pneumoniae Klebsiella bern also ave wisely dstibated in nae, occatng in soil, water, grain, vegetation, ete. Wood pulp, paper mil, texte Iinsing plants, and sugee-cane processing operations contin large ininbers of Klebsiella spp in Hr efiuets (10 to 10° per 100m), and Klebsiella spp. are often tho predominant coifom in such eyes. Rapid quantitation may be achieved in the MF procedure by sneditying mPC agar ase through substitution of inesitol For lac voso and adding earbencilin or by using mKleb aga. These meth- ‘ads roduce the necessity for biochemical testing of pure strains. Prctiminary vetfication of cifferentated colonies is recommended 1. Laboratory Apparatus ‘ vr 0. Sample bottles: See Section 9030B.19.* >. Dilurion bottles: See Section 9030B.13.—- &, Pipets and graduated eylinders: Ses- 9222B. le. 4. Containers for culture medium: See 9222B. 1d. , Cultze dishes: See 9222B. 16. f. Filwation units: See 9222B.1f sg: Membrane filters: See 9222B.1g. fh Absorbent pads: See 9222B.1h. Forceps: See 9222B.1i. j. Incubators: See 92228. 1. 2. Materials and Culture Media ‘4. mECIC agar: This medium may not be available in dehy. rated form and may require preparation from the basic ingre- dients Tiyptase or bist, 1008 Proteose peptone No, 3 ot polypeptone. 308 Yeast exact 308 Sodium chloride (NaCI) 508 Iositol 7 2 1008 Bile salts No. 3 or bile salts mintre 15e Aailine blue o1g ‘Agar 1508 Reagent-grale water : rk Heat medium to boiling, and add 10 mL. 19% rosolic acid dissolved in 0.2 N NaOH, Cool to <45°C, and add 50 mg carbenieillia.f Dispense aseptically in 4 to mL. quantities ito 5. $0-mm plastic Peti dishes (approximate depth of 4 to 5 mm), Refrigerate until needed. Discard unused agar mediuin after 2 weeks. Do not sterilize by autoclaving. Final pH should be 74 + 02. b. miKleb agar Phenol red agar 310e Adonitel 508 Aniline bloe ole TRovlieaid reaguat will devompoce fserifized by astoclaing. Roger "Hook soution in he dank nd sate ater 2 weeks, o Sooner is Coke Cm $n dark eo maddy bow FAvaslebe tom Geopen, Reig Phe, Ine, New York, NY. og an nat 121-124°C. After au- ig; cool fo SO°C in a water bath; add 20 mb. 95% ethyl (ot denatured) and 0.05 g filter-stetlized carbenicil- B) psc cate plats. The fiat pt should bo 7.4 © 02. Bf poche trad owte ve er membrane, Place membrane filter on agar surface: i for 24 2h ot 35 = 05°C, Klebsiella colonies on ‘gee are be or buish-gray, Most atypical colonies are {gx brownish, Occasional false-positive occurrences are Bl by Buerobacter species. Klebsiella colonies on mKleb deep blue 16 bive gray: other colonies most often are 3 occasionally pale yellow. Count colonies with a low- FP U0t0 15% muigsitcation) bincculer wie Feld dissecting 8 or other optical device. Venfiaton: Verify Klebsiella colonies from the first set of from ambient waters and effluents, ad when Kiebsielia in weter-supply distribution systems. Verily # mini= ‘of five typeal colonies by teansfersing growth from a Total coliform verification: Verity total coliforms before using By tesmctolerant coliform partition method. Swab surface growth Eo fre ttal-coliform-positive filter or, if quantification is desired, Eger small portions of each target colony on the filter to the Reloriate total coliform verification medium using a sterile nee- 09 (positive), motility (negative), lysine decarboxylase positive) ornithine decarboxylase (negative), and urease (positive). A Klebsiella strain that is indole-positive, liquefies pectin, and demonstrates a negative thermotolerant coliform respowse. is ‘most likely of nonfecal origin, 4. Bibliography Duncan, DW, & W.B, Reza. 1972, Klebsiella biotypes among coli foums isolated ftom fprest envitoamonts and fatm produce. Aj! Microbiol 24933, Stan, SL. 1976” Presumptive identification of Kebs ‘manig® on M-FC mesinm, Cun, J. Mcrabiot, 22/1774, Bastsy, 8.7. & RL Sufbime, 1977. Signifieance of fecal colt posiive Klebsiella. Appl. Environ. Microbe. 33:1 141 Kowrret, M.D., RJ, S0um, C. Ebr & LM. Cant, 1977, Colonization ‘of the botatical environment by Klebseta isolates of pathogen tigin. Appl Brviron. Microbiol. 34557, Bowouson, A.S., EM. Coos, A.P-D. Witcoct & R, Suivnaun, 1980. A ‘comparison of the propestios of Kledsiela isolated from differen sources, J. Med. Microbiol, (UK) 13:S41 Sum, RIB. 1981. A Critical Evaluation of Media for the Selective Identification and Enumeration of Klebsiella. M.S. thesis, Der Cuil & Favironmental Engineering, Univ, Cincinnati, Ohio. Nise. $1. & P. VaataNen. 1982. Survival in foe water of Kiehsills neuoniae discharged by a paper mill, Appl. Environ, Mievel 44268. Gaioneicn, EE. & EW. Rice, 1987, Occurrence, significance, snd detection of Klebrietta in water systems. J. Amer. Water Works Astoc, 79:14, Donean, LBLR. 1988, Waterbome Klebsiella and human disease. Tass icity Assess. 3581 pe ¢ Partition method for thermotolerant coliform devermines tion: Using aseptic technique, transfer total-coliform-positive colonies from the membrane fiker (0 @ tube containing EC medium by one of the following method + remove membrane containing total coliform colonies from the substrate with sterile forceps and carefully curl and insert membrane into wbe of EC medium (do not vortex tbe to avoid introducing air bubbfes to inverted vial, swab the entize membrane filter surface with a sterile cotton swab and transfer the inoculum to EC medium (do not leave cotton swab in the medium), or if quantification is desired, inoculate individual toual coli form-positive colonies into separate EC tubes. Simuliancors inoculation of both total-coliforn: verification tests and IEC broth is acceptable (order of inoculation should always be EC broth first and then other more inhibitory media), Incubate tubes in 44.5 + 0.2°C water-bath incubator within 30 min after inoculation, Maintain a sufficient water depth in incubator to immerse tubes to upper level of medium. Gas production in an EC broth (92218.1a) culture in =24 his considered a positive response for thermotolerant coliform bacteria. Mawis, A.&M, Sinmun. 1989. Menfyane filttion difentiton af Ecol ‘om corms inthe examination of wate. J. App, Bacterial 67:23, U.S. Exvomasrat Prorecnon Acaxey. 1989. Drinking Water; Na- ‘ion! Primary Drinking Water Regulations; Totet Coliforms (in ‘hiding Fecal Coliforms and eo): Binal Rule. 40.CFR Pats 141 142; Fed, Reg, 34227544, US, Cvmonmenta, Prorucon AGENCY. 1991, National Primary ‘Drinking Water Regulations; Analytical Techniques; Coliform Bae: teria, 40 CFR Part 141; Fed. Reg. 36:636 9222 H. Partitioning E. coli from MF Total Coliform using EC-MUG Broth Excherichia coli is @ member of the thermotolerant coliform roup of bacteria; its presence is indicative of fecal contamins- lion. Rapid quantitation and verification for E, coli may be nchieved for a total-coliform- or thermotolerant-coliforn= positive MP sample by using media containing 4-methylumbel- lifery1-B-D-glucuronide (MUG). In this method, £ coli is defined «s any coliform that procuces the enzyme f-D-glucuronidase and hyolyzes the MUG substrate to produce a blue fluorescence, When examining drinking-water samples, use one of the two Partition methods to determine the presence of E. coli from a ‘otal-cotiform-positive MF sample on Endo-type media: nutrient containing MUG (92221) or EC containing MUG. When examining wastewater and other nonpotable water samples, use ‘one of the partition methods to defermine the presence of E- coli from thermotolerant (feeal)-coliform-positive MF samples on mPC media 1. Laboratory Apparatus 4, See 9222B,14-k 4, Ultraviolet lamp, long-wave (365-366 nm), 6 W. See Seetion 9030B,23, 2. Materials and Culture Medium EC broth with MUG (EC-MUG): See Section 9201F.1. 3. Procedure Selection of sample size and filtration procedure: See 2228.4. ? ». Total coliform verification: Verity total coliforms before ting the £ coli partition method. Swib surface groyyth on the ‘otal-coliform-positive filter or, if quantification is desired, tans- Fer small portions of cach target colony on the filter to the appropriate total coliform verification*medium using a sterile noodle, See 9222B.4g for total coliformverification procedures, Partition method for E. coli determination: Use aseptic technique to transfer total coliform-positive colonies on the membrane filter to @ tube containing EC-MUG medium by one of the following methods: + remove membrane containing total coliform colonies from the substrate with sterile forceps and carefully curl and ingert membrane into tube of EC-MUG medium, Mares, A. & M. Suanpex. 1992, Quantitative determination of fh chia col from ecliforms and fecal coliforms in sea water. Mt ios 11:27 Santony, D, & L. Howaeo. 1992. A mediuun detecting betn-glucu dase for the simultaneous membrane ftraion enumeration of crichia coli and coliforms from deiaking water. Le Amp. Meng Diol. 15.273, vonMENTAL Paorscsion Aguncy, 2013. National Prins Drinking Water Regulaions; Revisions to the Total Colif Role; Final Rule, 40 CPR Parts 141 and 142: Fed Reg. 76:10) us. + swab entre membrane fier surface with 2 sterile cota swab and transfer the inoculum to EC-MUG medium (dq not leave cotton swab in EC-MUG medium), o + if quantification is desired, inoculate individual tot cl form-postve colonies into separate EC-MUG tubes. Incubate EC-MUG media at 44.5 = 0.2°C for 24 + 2h Pe all EC-MUG tubes in water-bath incubator within 30 min at inoculation, Maintain a sufficient water depth in incubator immerse tnbes to upper level of medium q Observe ECMUG tubes using long-wavelength G6 366-nm) UV light soures, preferably containing a 6-W bal Cauriox: UV lamp should never be viewed directly. Pre ably view tes ina viewing box or hold UY light a few in in front of tubes (eg Aso, using a UV lamp equipped with a speci filter toe nate most of the visible ight interference is desirable an vil facilitate Muorescence determination. The presence of a bight tue fluorescence in the tube isa postive response for Ect Recont presence of absence of fluorescence, For nonpouble water samples, this partition method can be used to determin E.coli from the thermotoleren coliform MF procedure wig ‘mFC medium for initial isolation before wansfer to EC-MUG: medium. The procedar isthe same athe above, except for total coliform verification process A positive contol consisting of «known E.coli (MUG poste) culture, a negative contol consising of a temmotoerant Keil prewroniae (MUG-negatve) cuts, end an uninoculated medi control may be necessary wo interpret sample resus and ai risidettyng the medium's week yellow-biteauoflaoresoeace at 8 positive response, (See Section 9221) 4. Bibllography ‘Mates, A. & M. Sussman. 1989. Membrane filtration differentiation of . coli from coliforms in the examination of water J. App Bac teriol. 67:343, USS. Bwmonenra, Provecrion Acrscy. 1989. Drinking Wate: Ne ‘ional Primary Drinking Water Regulations; Total Coliforms (i cluding Fecal Coliforms and E. col); Fina! Rule, 40 CFR Pars 41 and 142; Ped, Reg, S4:27844, US. EwononmentaL Provecon AcENcY, 1991. National Pimry Drinking Water Regolatios; Analytical Techuigues; Coliform Bee- teria. 40 CFR Part Idl; Fed. Reg, 56:636, Manes, A. & M. Snarren. 1992. Quantitative determination of scke richia coli from coliforms and fecal coliforms in sea water. Miro ios 71:2. sony, D, & L. Howano. 1992, A medium detecting beta-uevroni ‘se for the simoltancous membrane fkiaon enumeration of Escherichia coli isa member of the thermototerant coliform ou of bac its presence is indiative of fecal contamina- Rapid quantitation and verification for E. colt may be ved for a. total-oliform- or thermotolerant-coliform- [| When examining drinking water samples, use one ofthe to Iiction metnods to determine the presence of E. cli from a Esivcoliforn-positive MF sample on Endo-.ype media, use Micieat agar containing MUG or EC broth containing MUG HisaH). When examining wastewater and other nonpotable ecsamples, use one of the pastion methods to determine the sence of E. coli from thermotolerant (feval)-coliform-positive Forceps: See 9222B.1i Incubator: See 92228. | Uiravioler lamp, long-wave (365-366 nm), 6 W: See Sec- 508 308 5 1508 Pdetiynmbetiteryt ols Reagen-grade water tL [hs ceryratot ingens wo reagentgzade water, six toe Aish, and heat to dissolve. Sterilize by autoclaving for 15 min Hipi“124°C. Dispense 4. to G-sL. quantities aseptically into [am pose cultare plates (approximate depth of 4 to 5 mm) BU sow to solidify, Final pH should be 6 8 0.2, Refrigerated Hpared medium may be held for 2 weeks. Procedure Sdection of sample size and fitration procedure: See ops, Fs Total coliform verification: For drinking water samples WF: Eodo-type medium, jotal coliform verification procedures Gi performed before or after the partition method. Swab j= growth on the filter or, if quantification is desied PNEVORANE ALTER TECHNIQUE (B222VParttioning Ecol rom MF Total Coliforms using NA-MUG Agar Bscherichia coli and coliforms from deinkiny Lett, Appl Microbiol, 15273, 9222 |. Partitioning E. coi from MF Total Coliforms using NA-MUG Agar appropriate total coliform verification medium using a stele acedle. Alternatively, ater uansferting filter (o NA-MUG media, incubating it, and reading the results on this media, either trans fer individual colonies, swab surface growth on filter, or plave whole filter into appropriate fotal coliform veriBeation medi. (See 9222B.4g foF total coliform verification procedures.) c. Patiition method for B. coli determination: Aseplically transfor membrane filter with at least ene coliform-positive cul ony to NA-MUG plate. If quantification is desired, mul eich sheen colony (eu, use a One-tip pen to mark sheen colony's, Tocation on lid and fiker/lid orientation) and uaaster lid 10 NA-MUG plate, or use a sterile needle to make a hole in ‘membrane filter next to sheen colony after transferring, men brane to NA-MUG medium. Incubate NA-MUG immediately after trnsfer at 35 + 0.5°C for 4h. Observe individval colonies (on NA-MUG plates) using a long-wavelength (365~366-nm) UY light source, preferably von: (aining a 6-W bulb. Caution: UV lamp should never he viewed directly, Preferably view plates in a viewing box or hol the UV light a few inches above the plates (e.2., 3 t0 4 in.) facing away from the viewer. The presence of a bright blue fiuorescence on the periphery (outer edge) of a colony, oF ob served from the back of the plate is a positive response for E, coli, Record presence or absence of fluorescence, oF if gu ation is desired, count and record the number of invget colonies. For nonpotable water samples, this partition methuxl can be used 10 determine E. coli from the thermotolerant col form MF procedure using mBC medium for initial isolation before transfer to NA-MUG medium. The procedure is the same as the above, except for the total coliform verification process. 4, Bibliography Manes, A. & M, Siiara. 1989. Membrane Filtration diffeenaton of eo from coliforms in the examination of water, J. App. Ha terial 67:35, US. Envikonuvtat PaorscHon AcaNey. 1989. Drinking Water: No Regolaiions; Tol Colfomis tn cluding Fecal Coliforms and E cli); Final Rule. 40 CER Pants (1 and 142; Fed, Reg. 5427544, US. Ewvnosmenrat, Prorctios AoENcy, 1991. National Primary Drinking Water Regulations; Analytical Techniques: Calon Bae teria. 40 CER Past 141; Fed. Reg, 56:636 Mares, A. & M, Shura. 1992. Quaniative determination of fe ‘la eo from coliforms and Feal elif in 304 water: dies 11:27 Sawrory, B. & 1: Howarn, 1992. A medion detecting betrghica aso for the simultaneous membrane estion enamoratin f Escherichia coli and coliforms from dinking Water. Lett. yp Microbiol. 15273 Suabbe, LC., MLE. Dnoucan & EW. Ries, 1993, Detetion of Bet Tichia coli by the nutrient agar plus 4smethylumbellifry ‘slucuronide (MUG) membrane fier method. Cen. J. Microbiol 39:1068. 9222 J, Simultaneous Detection of Total Coliform and E: coli by Dual-Chromogen Membrane Filter Procedure 1. Laboratory Apparatus For MF analyses, use glassware and other apparatus composed rial free from agents that may affect bacterial growth. 1. Sample botles: See Section 9030B.19. '. Dilution bores: See Section 9030B. 13. 6 Pipets, sample containers, and graduated cylinders: See Section 9030B.9. d, Culture dishes: See 9222B.1e. ¢, Filteation units: See 9222B.1f [) Membrane filters: See 92228. 1g. 1. Absorbent pads: See 9228.1, 1, Forceps: See 9222B.1i. 4. Incubators: See 9222B.1. 2, Materials and Culture Medium Prchase thie medium from a comamercial vendor it cannot Be prepared from basic ingredients, Seo Section 9020B.5j for media ‘QC specifications Belowe use, lest each lor with postive and negative eultere 0.2 Place absorbent pads in 9 50-mm Petri dishes and saturate with 2.0 to 3.0 ml. MI broth containing 5 ygitol, final concentration of cefsslodin, Store plates in reftigerator, and discard after 96 bh, Pour off excess broth before using plates. 8. Procedure 4, Selection of sample size: Soe 92228 4a »b. Sterile filtration units: Seo 9222B.4b, ¢, Filtration of sample: See 9222B.4c . Counting: To count colonies on membrane fillers, use a low-powered (10 to 15x magnification) binocwlar wide-field dissecting mictoscope or other optical device with a cool white Muorescent light source directed to provide optimal viewis ‘Count all blue colonies on each MI plate under nornval/ambient light and record as £: coli results. Positive results that occur in <24.h are valid, but results cannot be recorded as negative wil the 24-h incubation period is complete. Expose eal Ml plate (0 Jong-wave UV fight (366 nm), and count all fluorescent caloies {blue/green fluorescent F: coli, blue/white Muorescent TC exer than E, coli, and bluesgreen with fluorescent edges (also F.coti}} to obiain the TC count. Record the data. If any blue, ron ‘uorescent colonies are found on the same plate, ald their taal to the TC coun, Caloulate the final values using the following formula: umber of blue colonies , colt!100 mt. = Nes OES colonies 100 m= Shame of sample fitered i) x 100 Detyated Difeo™ pM Broth (No, 214882), o ntvaen 998 number ef uorescent colonies iver hu, renome ~ 4 ©. Coliform verification: For drinking water, total coliform ‘colony verification is not requited, For waters other than drink ing water, verify a a frequeney established by the laboratory (see Section 9020B.10), Laboratories may incorporate more sttingent QC measires (e.g, verify atleast one colony from each typical or alypical eolony type from a given membrane filter culture, verify 10% of positive saraples) based on need and sample type (soe Section 90208,10). Adjust counts based on verification results, Verification tes are listed in 92228.4g. 4. Caloulation of Coliform Density Soe 9222.5. 9223 ENZYME SUBSTRATE COLIFORM TEST* 9223 A. Introduction wzyme substrate tests use hydrolyzable chromogenic and ‘loorogenie substrates to simultaneously detect enzymes pro: ‘duced by total coliforms and Escherichia cali (E, coll. In this ‘method, total coliform bacteria produce the enzyme f-D-palac ‘osidase, which cleaves the chromogenic substrate in the medium {o release chromogen. Most E. coli strains produce the enzyme P-glucuronidase, which cleaves a fluorogenic substrate in the ‘medium to release fluorogen. The release of chromogen indicates ‘hat coliform bacteria are present, and the release of fluorogen indicates that F, coli ave present, Muluple-tbe, multi-well, or presence-absence (Single 100-mL, sample) formats are available for use with these enzyme sub- swale tests, 1. Principle 4. Total coliform bacteria: Coliler®, Coliler-18®, and Colisire® media use the chromogenic substrates ortho-nitrophe= nyl-B-b-galactopyranoside (ONPG) and chlorophenol 1ed--b- gslactopyranoside (CPRQ), respectively, 10 detect the enzyme B-p:ealactosidase, which is produced by total coliform bacteria. "The -b-galactosidase enzyme hydrolyzes the chromogenic sub- rate that produces a color change, therebyindicating the pres ccace of total coliforms without additional procedures Although non-coliform bacteria (e.g Aeromonas, flavobac Jerium, abd Pseudomonas species) may produce smal amounts ol the enzyme -b-galactosidase, the growth of these organisms 's suppressed so they generally will no¥ produce false-positive resal unless >10° CFU/100 mL are present |. Bxchevichia coli: The Muorogenic substrate 4-methyl-umbel- lifery!-B-o-glacuronide (MUG) is used to detect the enzyme f-D- curonidase, which is produced by most stains of E, coli: The Appvd by Standard Methods Commitee, 2016. Join’ Tsk Grog: Demifer Best (has, Bete L. Cocker, iy Git Dich, ancy Mi Ua Wiliam W. Nordine, Viel Reyold, Helena Slo Gale MICROBIOLOGICAL EXAMINATION pul 5. Bibliography Busan, KP. CC. Rew, Y.R. Roveal, GN, Sttus, In, PE Scawwano d A.P, Durour. 1993. New medivn for ihe stmulneoed Aetection of woul coliforms and Escherichia coli in water, Enviren. Microbiol. 393534, Basser, KP, CC. Raver, N. Sivaganasass & P.V. Scanmno, 15g ‘Comparison of the recoveries of Hsckerichia col! and totale US. Environmental Protection Agency-approved membre fi method. App Environ Microbiol, 62:204, 821-R.02-024, OFF. Water, Washington, D.C. £-P glucuronidase enzyme hydrolyzes the fluorogenic subse produces blush forescence when viewed under ong-wavleng (265-366 nm) ultraviolet (UY) light. Together, the color (due to B-o-galactosidase) and the fuorescence (due to B>-ghcay rontdase) indicate that a sample contains B. coli. { Large numbers of some bacteria or sirains of bacteria (elf some strains of Shigella and Salmonella spp.) may case sample to fluoresce but will not change its color because the lack B-v- galactosidase. Such samples would be considered ative for E coli 2. Applications ‘Those enzyme substrate coliform tests are recommended fr analysis of drinking wate, source water, groundwater, and wast water samples. If laboratory has not used this method befor. il desirable to conduct parallel testing (including seasonal variations ‘withthe existing method to assess site-specific effectiveness and Qi compare results. The results of many method-performance ste ae available in the literature and the rates of false-positive -negative results differ among various media. Users should cae fully select the medium and procedure that best fs their neds. Section 9020B.11 for guidance on validating new methods. Water samples containing humic or other material may tf colored. If there isa natural background color, note what tis. the water is yellow enough to be misinterpreted as a wed positive after incubation, se amedam tht docs nature (e.g Colisure). Some waters’ high calciunn-salt content cause precipitation, but this should not affect the reaction. Tf samples with excessive chlorine, a blue flash may be seen whl adding Coliler or Colilen-18 media. If this occurs, consid sample invalid and discontinue testing, Do not use the enzyme substiate test 10 verify presunp coliform cultures or membrane-filter colonies, because the subst tay be overloaded by the heavy inoculum of weak (-D-gelacos

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