9215 HETEROTROPHIG PLATE COUNT* 9215 A. Introduction 1. Applications ‘The heterotrophic plate count (HPC), formerly known as the standard plate count, isa procedure for estimating the number of live, culturuble heterotrophic bacteria in water and for measuring changes in swimming pools or during water treatment and dis: tribution, Colonies may arise from pairs, chains, clusters, or single cells—all of which are included in the term colons- Forming units (CFU). The final count also depends on interaction ‘among developing colonies. Choose the procedure and medium that best suit how the resulting information will be used. Only compare data generated using the same procedure and medium Four methods and five media are described. 2, Selection of Method «4 Pour plate method: ‘The pour plate method (92158) is simple to perform and can accommodate sample or diluted- sample volumes ranging from 0.1 to 2.0 mb. Kt praducessub- surface colonies that are relatively small compact, and less apt to eneroach on each other than surface colonies da, However. submerged colonies often are slower growing and difficult to transfer. A thermostatically controlled waterbath i essential for tempering the agar. by Spread plate meshod+ The spread plate method (9215C) causes no hea shock, All colonies are on the agar surface, wire Snalysts typically cam easily distinguish them from particles ae bubbles, and discer different colony morphologies. IF no, they can trina colonies quickly and easily discem colony mompbol- ‘ogy via comparisons to published descriptions. However, the "gat in this method can only absorb a small volume of sample or Ailted sample (0.1 0 05 mL, depeniing onthe degree to which prepoured plates have been dried). Alo, spe ould be interferences. To use this procedure, Of suitable pre-dried, absorbent agar plats (se 9215C:4), € Membrane fier method: The membrane filter method {@213D) can be ised to analyze large volumes of low-urbiiy water and isthe method of cholo for waters with low numbers of heterotraphie organisms (<1 10 10 CFUimL). Ic preduces no heat shock. However, there isthe expense ofthe membrane filter, the smaller area for colony development, possible damage 10 calls by excessive filtration pressures, posible variations in membranefilter quality (se Section 9020B.5i), and the nced to deteet colonies via reflected ight against a white background i colored filters or contrast stains are not used «Enzyme substrate method: The enzyme substrate method ‘SimPlate® (92158), canbe used 10 analyze drnking-and source water samples with a wide range of bacterial concentrations. It Approved hy Siandrd Methos Commitee, 2016 Joint Tas Gra Gi Dicer (arcu, Merk W. LeCheelier(-eha, produces no heat shock. Comparable to the pour plate method, this method? mses a medium in which substrates are hydrolyzed by multiple microbial enzymes, causing the release of 4-meth~ ylumbelliferone after 48 h of incubation at 35 = 5°C. The -methylumbelliferome Mluoresces when exposed to Tong-wane= length (365-366 nm) ultraviolet (UV) light, and the number of blue fuorescing wells corresponds to a most probable number (MPN) of bacteria inthe sample. Unfortunately, individual col- nies cannot be direetly recovered for subsequent analysis 3. Work Area. Provide a level table or bench top with ample area in a clean, Uraft-tee, well-lighted room or within a horizontal-low laminar hood. Use tables or bench tops with nonporous surfaces, and Aisinfous Ucfine any aualysin fy ak (Huctine 30200. ara e) 4, Samples Collect water as directed in Section 90G0A. Initiate analysis as soon as possible after enllection to minimize changes in bacterial populations, and do not exceed a holding time of 24 h (or, if the sample was collected for regulatory purposes, the maximum hholding time specified by the regulation). IF the sample cannot be tested within 30 min aftercollection, maintain it at “10°C burt do ot allow it to freeze during transit, 5. Sample Preparation Mark cach plate sith sample number, eition, date, and any caer necessary information before examination. For pour- oF Spread-plate methods use srl glase(65 cm" or pestered disposable plastic (57 en’) Pet dishes, For pourplate, spreal- plat, oc membrane-fllration methods, prepare at lost 0 1S)- Trae plates foreach volume of sample or iuion examined for compliance testing. Duplicates also are recomended for not- compliance testing ‘xual ik ll samples or tons by shuking vigor cusly for 7 + by hand (approximately 25 times wilh a I-A terest) orn ny cecal hala G15 9 ew apse ‘Analytics reauts depend on adoquie ssp: mixing: i the semcpeib oot aequely shaken before sliquot’ ore remade actual bacterial density could he underestimated. 6. Media ‘Users should ensure that the Formulations of purchased media ‘match those described below. Compare new lots of media with current lot in use according to Sections 90208.3f and 9p. 7 SimPinc for HNC, Mews Lora, West, ME.HETEROTROPHIC PLATE OUNT (@218)/ntroduetion 4, Plate count agar (also catfed wrypuone glucose yeast agar): ‘This medium can be used to examine leterotrophie bacteria in a wide variety of matrices. It can be used for both pour- and spread-plate methods. This high-nutrient agar, widely used in the past, may give lower counts than R2A or NWRI agar, Commerically available dehydrated medium should be used when available, Trptone sess 508 Yeast extract... se sovin OSB Giweose. erence LOY Agar Isae Reagent-rade water... : VL ‘The pH should be 7.0 = 0.2 after autoclaving at 121°C for 15 mit b. m-HPC agar (formerly called SPC agar): This ean be used to count heterotrophic bacteria in waters with Low levels of heteroirophs, such as treated potable water, This high-nutrient medium is used only with the membrune-fiter method. Com- merically available dehydrated medium should be used when available, Gelatin see re 2508 Giyeesot resin sectcenten ised SOMA, Agar Iso. Reagent-rae water TL For medium produced from individual ingredients, adjust to pH 7.2 with 1’ NaOH, Heat slowly to dissolve thoroughly, add ‘glycerol, and sterilize at 121°C for $ min, Commercially pec- pared medium should not requite post-sterilization pH adjust ment; see Section 9020B.5j1). Final pH is 7.1 = 02. & R2A agar: Use for pour-plate, spread-plate, and brane-filter methods. This low-nuttient agar was developed for use with potable treated water to monitor trends jn potable: ‘water production; this agar und a longer incubation period eun ‘improve the recovery of stressed and chlorine-tolerant bacte- ria. This medium may yield higher counts than high-nutrient formulations. Yeost exact os Protease peptone No. 3 oF polypeptone 05 8 Casamino acids 05 ¢ Glucose 05 ¢ Sole nc ase Dipoassium hydrogen phosphate (KHPO,) 03 9 “Magnesium sulfate heptahydrate (MgSO, 7H,0)..... 005 ¢ Sodium pyruvate . bones 03g Agar 150 2 Reagent-grade water... ro For medium produced from individual ingredients, adjust to pHl 7.2 with solid K,HPO, or KH,HPO, before adding: agar. Heat to dissolve agar and stetilize at 121°C for 15 mia, Com rmercislly-prepared medium should not require post-sterlization DH adjustment; see Section 9020851). Final pH is 7.2 = 0.2 d, NWKI agar (HPCA): Use for pour plate, spread-plate, and rmembrane-iller methods. Ths low-nutrient medium is likely to produce higher colony counts than high-nutrient media, This gar typically is not available in dehycrated form; it must be hipscidoi.op10.2109SWW 82.188 prepared from individual ingreuiems, making is use less desir- able Pepuone, deianmoemennn Ed Soluble case as g K.HPO, ‘ 5 se2 og Mes0, 905 Fecly se . . 001 g Asie Iso Reagentegrade water ak Final pH is 7.2 * 0.2 after amoclaving at 121°C for 15 min, 6, Encyine substrate medium: SimPlae® medium is available ‘commercially? in sterile vessels for 100-mL. multidose proce- dures or in test tubes for 10-mL unit-dose procedures. Store ‘medium between 2 and 25°C and use before expiration date, 7. Incubation ‘The media described in 92154.6 should be incubsted as follows; 4, Plate count agar (PCA): Incubate at 35°C for 48 © 3h b m-HPC agar: Incubate at 35°C for 48. 3h. 4. NWRP agar: Incubate at 20°C for 7d ©, SimPlaze® medium: Incubate at 35 * 0.5°C for 48 h (range of 45 to 72 by. During incubation, maintain humidity so agar plates will not lose >15% moisture weight; this is especially important during [prolonged incubation. 4 pan of water placed inthe bottom of the incubator may be sufficient so long as the incubators interior walls and shelving are made of high-grade stainless steel or ‘anodized aluminum, which will not rust or oxidize, W the incu bbator is not humidtied, seal plates in plastic bags, removing as much air as possible before sealing hag, 8 Counting and Recording ‘a. Pour and spread plates: Count all plates promptly after incubation. If counting must be delayed emporarily, soe plates reriigetated for ~24 ly, but avoid this as routine practice. Record results of sterility controls on the report for eaeh lot of samples. Count colonies manually using # dark-field colony counter, such as a Quehee colony counter or equivalent. If sich equip- ‘ment is unavailable, then other counters can be used on ‘non-compliance samples so long as they provide equivalent ‘magnification. Automatic plate-counting instruments are avail- able: they generally use a computer program and scanner and sive computed results, Their use is aeceptable if, when evaluated by being run in parallel with « manual method, counting results are comparable. When preparing plates, between 30 and 300 colon ‘one dilution yield colony c provided below Ordinarily. do not pipet >2.0 mL. of sample; however, if this sample volume yields <30 colonies, record the result observed, pet sample volumes that will yield to have al least its, except as Fie Lakers, Wextrook, MEHETEROTROPHIC PLATE OUNT (@218)/ntroduetion ‘Otherwise, use only plates containing 30 10 300 colonies when determining plate count. Compute bacterial count per milliliter 8 follows: colonies counted FUL = 1f no plate contains 30 to 300 colonies, and one or more plates have >300 colonies, use the plates) whose count is nearest 30% colonies. Compute the count as above and report as e mated CFU/mL, If plates from all dilutions of @ sample have no colonies, report the count as 10 colonies/em*, count four representa five squares, take the average count per em’, and multiply by the appropriate factor (57 for disposable plastic plates and 65 for glass plates) to estimate colonies/plate, (Nore: The nom- inal diameter of both disposable plastic and nondisposable lass plates is 100 mm, However. the internal diameter of disposable plates is closer to 85 mm and that of nondisposable plates is closer 19 99 mm.) When bacterial counts on crowded plates are >100 coloniesfem’, report results as >6500 di Vide by the smallest sample volume plated for glass plates or 55700 divided by the smallest sample volume plated for plastic plates. Repent as estnnated CUAL. If spreading colonies (spreaders) are encountered on the plate(s) selected, count colonies on representative portions only \when colonies are well distributed in spreader-free areas and the area covered by the spreader(s) does not exceed one-half the pluce are. Phen spreading colonies mut be coumed,enunt each of the following types a one: « chain of eoloaies that appears to be caused by disintegration of a bacterial clump as agar and sample were mixed; a spreader that develops as a film or growth be ‘ween the agar and bottom of the Petri dish: and a colony that forms in film of water at the edge or over the ugar surTuee, The last evo types largely develop due to moisture accumulation st the point from whieh the spreader originates. They frequently cover more than half the plate and imterfere with obtaining a reliable plate count Count similar, adjacent colonies as individual colonies so long as they do not fouch and the distance between them is at least ‘equal to the diameter of the smallest colony. Count impinging colonies that fer in appearance (e.g, morphology ar color) as individual colonies, hipscidoi.op10.2109SWW 1 plates nave excessive spreader growth, report as “spreaders (Spr. When plates are uncountable due to missed dilution, accidental dropping, or contamination, or else the control plates indicate that the medium or other material was contaminated, report as “laboratory accident” (LA), 1h Menrane filter method: Comnt colonies on membrane filters using a stereoscopic microscope at 10 t0 15% magnifica- tion, Preferably slant Pets dish at @ 45° angle on a microscope stage and adjust light source vertical to the colonies. Optimal colony density per filter is 20 to 200, If colonies are small ancl uncrowded, a higher limit is acceptable ‘Count all colonies on the membrane when there are <2 col- fonies per square. For 3 10 10 colonies per square, count 10 squares and determine an average count per square. For 10 10 20 colonies per square, count 5 squares and determine an average ‘count per square, Multiply average count per square by 100 and divide by the sample volume to give colonies per mL, Mhere are >20 colonies per square, record the count as >2000 divided by the sample volume. Report averaged counts as estimated CFU ‘Make estimated counts only when there sre discrete, separated colonies without spreaders. , Enzyme substrate method: Count the number of blue fluo- rescent wells by holding a 6-W, 365-366 mm UV light about 5 in, ahove plate, preferahly with UV- filtering safety glasses or {in a UY cabinet, Altematively, count fluorescent wells through the back of the inverted plate following the procedure indicated above, Use the appropriate MPN Table (multi-dose or unit dose) provided by: the manufacturer to eoleulate MPNémL.. Adjust MPN to reflect sample volume and/or dilution made to yield a comected MPN value. Record as MPNink. 9. Computing and Reporting Counts The term colony-forming knits) (CFU) is descriptive of the ‘methods used; therefore, report all counts as CPUs, Also report the method used, the incubation temperature and time, and the ‘medium, For example: CFU/mL, pour plate method, 35°C/48 h, plate count agar. To compute the heterorophie plate count for pour plate, spread plate, and membrane filter methods (CFU/mL), divide either the total or average number of colonies per plate by the sample volume. (Use the average number of colonies if duplicate plates of the same dilution.) Record sample volumes used and ‘number oF colonies on each plate counted oF estimated, When colonies on duplicate plates andlor consecutive dilu- tions are counted and results are averaged before being recorded, ound off counts to two significant figures only when converting, wo CFUs Avoid creating fictitious precision and accuracy when come puting CFUs by recording only the first wo digits, Round up the second digit when the thd digit is $ through 9 and round down ‘when the third digit is 1 dough 4 (e.g, report a count of 142 as 140, 155 as 160, and 33 as 35) For the enzyme substrate method, record the MPN/m. ob- tained from the MPN ables provided by the manufacturer, adjusted for sample dilution if requiredHETEROTROPHIC PLATE COUNT (9215YPour Pate Method 40. Analytical Bas Avoid counting inaccuracies due to carelessness, damaged for dirty optics that impair vision, or failure to recognize colonies. Laboratory workers who cannot duplicate their ‘own counts on the same plate within 5% and other analysts ‘counts within 10%% should discover the cause and correct such disagreements 9215 B. Pour Plate Method 1. Samples and Sample Preparation Sce 921A and 5, 2. Sample Dilution Prepare water used for dilution blanks as directed in Section 9050C.1 4, Selecting dilttions: IF possible (eg based on historical information, select difutions s0 a least one plate in a series will contain 30 t0 300 CFU (Figure 9215:1). For example, if expe- rience indicate that heteroirophie plate counts ean be ws high as 300) CEU, prepare plates at 10"! and 10™ dilutions For most potable water samples plating |-mL. and 0.1 mk. undiluted sample and 1 mL. of the 10" dilution will produce plates suitable for counting . Measuring sample portions: Do not prepare dilutions and pour plate in direct sunlight. Use sterile pipet for al transfers ‘rom each container; one pipet per dilution I any pipet becomes contaminated before transfers are completed, replace it with @ sterile one. Use caution when removing sterile pipets from the Ceontainer; to avoid contamination, do not drag pipet tip across ‘exposed ends of pipets in the pipet container or across lips and necks of dilution otles. When removing sample, do not insert, pipet 2 to 3 em below the surface of sample or dilution. «. Measuring diluions: Add sample 1 a serile Per ish before ‘adding melted, tempered culture medium, When discharging sam sample Delivery volume amt od mt Ccuture dishes ; med Sncndeh tm om. pile portions, hold pipet or miropipet tip at an angle of about 45°, wih ip touching bottom of Pett dish or inside neck of dilution bolic. Lilt Petr-dish cover just high enough to inser pipet or Imicropipet tip. Allow 2 to 4 for liquid to drain from IemL graduation mark to pipet tip. When measuring .1-ml. quantities, et diluted sample drain from chosen referenes graduation until 0.1 mL. has been delivered. Use decimal dilutions when preparing sample ‘yolumes <0.1 mL. When examining sewage or turbid water, do not ‘measure «0. 1-ml. inoculum of original sample: insted, prepare an ‘appropriate dilution (9215.5), If pipet is nota blowout type, touch tip of pipet ance against aa dey spot on Petri dish bottom. If using # cotion-plugged blow-out type pipet and a pipet bulb (less preferable), gently blow out remaining volume of sample dilution, Remove pipet without further touching it to dish ‘Aller depositing test portions for each series of plates, pour culture medium and mix carefully. Do not let >20 min elapse between starting pipeting and pouring plates. 3. Media for Plating 4, Melting medium: Melt sterile solid agar medium in being ‘water, via exposure to flowing steam in a parially elesed container fr via microwave [Section S020B.3)3)]. Avoid prolonged exposure ‘tounnacessriy high temperatures during and after melting, Do not resterilize plating medium. If medium is melted in to or more 99m. ‘ane amt oa mt 01m. 0.001 mt Figure 92152, Preparation of dilaton, hapscdoorp 0.2105HETEROTROPHIC PLATE COUNT (9215)/Spread Plate Method batches, use al of each butch in order of melting, so longs contents ‘remain fully melted. Discard melted agar tht contains precipitate. “Maintain melted medium in a waterbath between 44 and 46°C until used, preferably ao longer than 3 b. To tack the tempera- ture, place a thermometer in a separate container of medium that has been exposed to the same heating and cooling as the plating mediuin; do not depend on the sense of touch to indicate proper medium temperature when pouring agar. Prepare media [plate count agar (PCA), R2A agar, or NWRI agar] as described in S215A.6, », Pouring plates: Limit number of samples to be plated in any nie Series suo mvoxe than 20 in (gueterably 10 i elapse between dilution of frst sample and pouring of the lat plate in the series. After adding sample to Petri dish, gently lift cover just high enough to pour at least 10 to 12 mL liquefied medium {maintained at 44 t0 46°C) in each dish. Carefully avoid spilling medium on outside of dish lid when pouring. When ponring agar fiom flasks or tubes that have been held in a water bath, wipe ‘outside of bottle with a clean towel and avoid drigping water from water bath onto work surface. As each plate is poured, mix melted medium thoroughly with test portions in Petri dish— taking care not fo splash mixture over the edge—by rotating the dish slockwise and counterclockwise, oF by rotating ond tng Let plates solidify Gvithin 10 min) on a level surface. After medium solidifies, invert plates and place in incubator. For plates incubated at 35°C, stack plates no more than four high. Sterility controls: Check sterility of medium and dilution ‘water blanks by pouring contol plates for each seties of samples. Prepare additional controls to determine eontarninaion of plates, Pipets, and room ai 4, Incubation See 9215.7 for incubation temperatures andl times. ‘5. Counting, Recording, Computing, and Reporting Seo VEISA.S and 9. 6. Bibliography arto, RS. & W.D, Doriesex, 1916, The numberof colonics allowable ‘on satisfactory agar ptes Tech, Bull. $3. New Yorke Agricultural Experimental Ss, ertrarein, CT. 1053. The sletion faction wate for hacteriokagal xO, J. Bacteral 23385; Pub, Heald Rep 8081. Ancianiatr, Ly. Conor & MAL MeCkaoy. 1937. The need of uniformity of eomdions for counting pte (with suggestions for Standard colony counter) Amer. J. Pub. Heath 27-800. Recianns, OW. & P.C. Hous. 1945. An improved darkfeld Quebec colony counter. J. Milk Technol, 8253, Danas, 1M, DA. MeNet & LD. Wirret. 1969, lex of Uelays in ‘ou plating on bacterial counts. J. Datry Sei. 2:1456. Gaoneicn, EE, HD. Nasi, DJ. Reasonex & RH, Tayi. 1972. The necessity of eonirolling bacterial populations in potable water Community water supply. J Amer. Water Works Assoc. 64:56 Gitnasicn, EE, 1973. she total count necessary? fn Poe. Ist Annva AWWA Water Quality Techrol. Conf, Dee 31973, Cinsinna, Ohio, p. VIEL. American Water Works Assoc., Denver, Colo ‘Grune, W. 1973 raved otal count tesingus, ln roe, Ist Annu ‘AWWA Wat Quilty Toshnol. Cont, Doe 3-4, 1973, Cincina, Ohio, p Vill-1, American Water Works Asso, Denver, Cola Dorks, BJ, AS. Chav & J. Cows, 1974, Relationship of hetero Teicade nereke teaevs af este patel ant Boal eee Water Res 81047 Kus, DA, & 8. Wu, 1974, Suess: « facta to be comidered in heterotophie mieroorganism enumeration fom aquatic ensiton- ents. App. Microbiol. 37:29, cH, EE, HD. Nast, DJ, Reason & RM, Tavion, 1978. The necessity for eanolling hateial papaations in potable waters: ‘Bot water and emergency water supplis. J Aner. Wier Works ‘Assoo, 67:17. Ba, CR, MA. Hower Buankun & M, BRawxun, 1980 Hetero {wophichactsi in two Canadian rivers. Seasonal waitin in the predominant bacterial populations. Waver Res. 14:44. Means, EG, L. Havant, GF Riparway & BH, O1S0%, 1981. Esai: ing mediums and plating techniques for enumerating bacteria in water distribution systems. J Amer. Water Works Assoc. 73:58. Reason, DJ. & EE, Gunkdes. 1985, A new medium forthe ‘eration and subculture of bacteria from potable water. Appl Environ. Microbiol, 49:1 Awomcan Puauic Hentrw Association, 1993, Standard Methods forthe Examination of Dairy Products, 1th ed. Washington, D.C. Gu 9915 C. Spread Plate Method 1, Samplas and Sample Preparations Seo 9215A4 and 5, 2. Laboratory Apparatus 4 Glass rods: Bend 4-mm-tiam, 200-mm-long,fire-polished ils rods, 45° about 40 mm from one end. Sterilize before use. ‘Traditionally, the glass rod is dipped into ethanol and flamed. It should not be overheated, as 118 not felt in the flame. Stenle disposable plastic spreaders may also be used. », Piper: .O-ml. sterile glass or plastic disposable pipers or micropipets with sterile disposable tips hapscidoiorp 10.2108 Turntable (optional. 4. Incubator or drying oven, sett 42°C, oF laminar flow hood. 3. Media See 9215A.6a, ¢, and d. I R2A agar is used, best resulis ane ‘obtained when incubated at 28°C for $ 10 7 d; if NWRI agar is used, incubate at 20°C for 7 d. Faker Scene, No 758 qn epi), No ISK (Lain, ete diem of equivalentHETEROTROPHIC PLATE COUNT (8218)/Membrane Fiter Method 4. Preparation of Plates Pour 15 mL. of desired medium into sterile 100- 15-mm or ‘90+ 15mm Petri dishes: let agar solidify. Use pre-dried plates immediately after drying or store for up to 2 weeks refrigerated ‘at -8°C in sealed plastic bays, If pre-drying and using plats the ‘same day, pour 25 mL, agar into Petri dish and dry in laminar- How hood at room temperature (24-26°C) with the lid off to ‘obtain the desired 2- to 3-g weight loss 5. Procedure Prepare sample dilutions as directed in 9215852. 44, Glass rod: Pipet 0.1 oF 0.5 ml. sample onto surface of pre-dtied agar plate. Using a sterile bent glass or disposable plastic md, distribute inoculum over surface of medium by rotating plate manually or on a wmntable. Let inoculum absor’ completely into medium before inverting and incubating, b, Pipet: Pipet desired sample volume (0.1, 0.5 mL) omto surface of pro-dried agar plate, preferably while plat is being rotated on a turntable. Slowly release sample from pipet while making one to-and-fro motion, starting at center of plate and Stopping 0.5 em from plate edge before returning (© center, [tiny uct piper plate suave, Lec incu abs pletely into medium before inverting and incubating. 6. Incubation See 921547 7. Counting, Recording, Computing, and Reporting See 9215A.8 and 9. 8. Bibliography Bock, LD, & RC. CuRvERDON, 1960. The spread plate as & method for the enumeration of marine bacteria Lranol. Oceanogr. 5.78, (Cuan, D.S, 1967. Comparison of pour and surface plate methods for determination of bacterial counts. Can. J Microbiol. 131408. CCuane, DS. 1971, Studies on the surface plate method of conning bacteria, Cin J Microbiol. 17983, ‘Vow Socstutxuan, A.A. & CH, Lut. 1971. Pour plates or steak pla ‘Appl. Microbiol. 18:1002 Gucuast, LE, LE. Cova, CB. Dour, 1. Paine & JM, Daaney. 1973, Spiral plate method for bacterial determination. Appl. Microbiol, 25:248 Prax, DAM, & W, Gras. 1976, Pour plate 9. streak pate method. Proc, ath Annual AWWA Water Quality Technology. Cont Dee. 6-7, 1976, San Diego, Calif. p.2B-S. American Water Works Assoc, Dente. Ca, eres, BJ ye. 1978 Mths for Microbiological Analysis of Waters, ‘Westewaters and Sediments, Inland Water Directorate, Scientific Operation Dis, Canada Cente for Taiand Waters, Burlington, Ont Kerok, LB, AL, Mus & RR Conve, 1978, Evaluation of the ‘accuracy amd precision of enumerating serobic eterotrophs in ‘water samples by the spread plale method. App. Environ. Miero- biol. 35:75, ‘Yount, M1979: A mould spread plate technique for the determina ‘ion of concentrations of viable heterotrophic bates: STP 673: ALS American Soc. Testing & Materials, Philadelphia, Pa. Gupnzicn, EE, 1981, Current status of microbiological water quality criteria, ASM News 47:23, Tomtom, RH. MJ Auss & EE, Geionncn. 1981, Standond plate unt: A eomparison of pour plate and spread pate methods. Jn Proc. th Anaual AWWA Water Quality Teehiol, Con, Dec. 6-3 1981, Seattle, Wash, .223, American Water Works Asse, Dene ver, Coto, 9215 D. Membrane Filter Method. 1. Samples and Sample Preparation See 9215.4 and 5 2, Laboratory Apparatus See Section 92228. 3, Media See 9215.6, Use mHHPC agar or else R2A oF NWRI aga 4, Preparation of Plates Dispense S-mL. portions of sterile medium into 50- * 9mm Pett dishes, Let solidify at room temperature. Prepared plates may be stored inverted in a plastic box of tight container in « refrigerator at 2-8°C for =52 weeks. 5, Sample Size “The volume to be filtered will vary with the sample, Select maximum sample size ta give 20 to 200 CFU per fier. hipscidoiorp0.2105 6. Procedure Filter appropriate volume through a sterile 47-mm, 0.45-pan- ppore-diam, gridled membrane filter under partial vacuum. Rinse funnel with three 20- to 30-mL. portions of serle dilution water. Using flame-sterilized forceps, aseptically place filter on agar in Peti dish, Make sure to position the filer extefully, rolling from ‘one side 10 the vier So air bubbles are not tapped ser i 7. Incubation Place dishes in close-fiting box or plastic bag containing ‘moistened paper towels. See 9215A.7 for incubation tempera- tres and times, 8. Counting, Recording, Computing, and Reporting See 9215A.8 and 9, Report ax CFL/mL, membrane filter ‘method, time, and medium, 9. Bibliography Cuike, HAP, BE. Guuonach, HL Jone & PW. Kaun, 1981. The menorane filer in sanitary bacteriology. Pub Health Rep. 66:381HETEROTROPHIC PLATE COUNT @215)Enzyme Substrate Metho ‘temperature inthe better recovery of bacteria from water by fle tion. Can, J. Micrabio. 880. Taviok, RH. & EE, Graperica. 1979, A new membrane fier proce dure for bacterial counis i potable ater and svimmiag, pool samples, J. Amer. Water Works Assoc, 1402, (Chang. LA. 1980. The ntuence of mereasing numbers of non-indeator “organisms upon the detection of indicator organisms by the mem ume NE und presence-absence sts. Cum J. Seeroto. 200827 Derk, BJ, ed, 981, Membrane Filtration, Applications, Technigues, tnd Problems. Marcel Dekker, Inc, New Yor, N.Y. and Base Switzerland, Hoapisy, A.W. 1981. Beet of injury on the recovery of bacteria on ‘eibrane filers, Ja BL. Dutka, ed, 1981. Membrane Filtration, ‘Applications, Techniques, and Problems, p. 415. Maree! Dekker. Inca Now York, NY, and Basel, Swivzeriand, 9215 E. Enzyme Substrate Method 1. Samples and Sample Preparation See 9215A.4 and 5 2. Laboratory Apparatus 4 Pipets, O.l-, 10, and 10-mL, sterile, graduated, glass or disposable plasticor micropipsters with sterile disposable tips. bs bmn wh at 35 OFC. © Ultraviolet tight source, tong wavelength, 6-W, 365~ 366 nm. dd. Sample plates.* 3. Medium SimPlate® medium is available commercially? in sterile ves- sels for 100-m multi-dose procedures or in test tubes for 10-m unit-dose procedures, Store medium between 2 and 25°C, and use before expiration dat, “Test each lot for sterility by following the inoculation procedure (9215E.5), using 10 ml rehydrated medium without sample. Inew- bate at 35 O.°C for 48 h. No wells should fluoresce afte incubation when viewed under a 6W 365-364 nm UY lamp. 4, Sample Diluent Use either sterile deionized water, distilled water, buffered Water, of 0.1% peptone (see Section 9050C. 1), 5. Procedure «4, Muli dose: Rehyérate medium by filling medium vessel to 10%>-mL mark with sterile diluent, re-capping, and shaking until medium has dissolved. Aseptically pipet 1.0 mL sample and then 9 mL. rehydrated medium onto center of the sample plate. Ale tematively, aseptically pipet 0.1 mL sample and thea 9.9 ml ‘ehiyiate! median nk cemter of the sauple plate. (Nove, Final volume of sample plus medium must be 10 0.2 mL.) 4 Mexs Laborstres, Westbrook, ME: hapscAdoiorp0.2105 Cover plate with lid and gently swisl wo distribute mixture into wells. Tip plate 90° (0 120° to drain excess mixture into the absorbent pad. Invert plate and incubate at 35 + 0.5°C for 48 b (range of 45 to 72 h). LF a sample is suspected t have a count >738 MPN, dilute by adding 1 ml. sample toa sterile vessel containing 99 ml. sterile diluent. Make additional dilutions as required to keep counts <738 MPN/nl. in final dilution. +, Unit-dase: Rehydrate medium by either aeding 10 mL. sample sas if seh ia vagal We ie > 72.0 MPNAnLy pips 9 als a sterile diluent and I ml of sample, e-cap, and shake until medium has dissolved, Add entre eontsns to center of plate, (Nove: Final volume of sample plas medium must be 10 0:2 ml.) Cover plate with id and gently swirl wo distribute mixture into ‘wells. Tip plate 90° to 120° to drain excess mixture into absor- bent pad. Invert plate and incubate at 35 + 0.5°C for 48 h (range of 45 to 72 by. 6. Counting, Recording, Computing, and Reporting Aflet incubation, coun the munuber of blue Muoresceut wells by holding a 6-W, in. above plate preferably with UV-fitering safety glasses or in a UV cabinet, Alternatively, count fluorescent wells through the back of the inverted plate following the procedute as indicated. Use the appropriate MPN Table (multidose or unit dose) provided by the manntacturer to calculate MPN/mL., Adjust MPN to reflect sample volume and/or difution made to yield a comected MPN value, Record as MPNimL. 7. Bibliography Stuincs, A.D. Heezo0 & B. Rott. 1998. Comparative assessment of the newiy developed SimPlate® method with the existing EPA. approved pou plate method for the detection of heterotrophic Pate fount bacteria in ozone-teated drinking water. Presented! at Inter- ‘ational Ozone Assoc. Cont, Ostober 19-23, 1998, Vancouver, BC, Canada, {scxson, R.W.,K. Osmo, G. Baas, C_ Jota, D, Zana B. Rot ‘A. Sriuines, D. Hinze, 8. Casnon & S, Lovntasts 2000, Muli regional evaluation of the SimPlate® heterotrophic plate count ‘method compared wo the standard plsto count agar pour plate method in water. Appl, Environ. Microbiol. 66:483,