SS 946 contribute to favorable conditions for the growth of microorgan- isms, especially Pseudomonas aeruginosa. Thus, frequent test- ing for disinfectant levels and pH, along with scheduled main- tenance, is necessary for safe whirlpool water quality." Studies have also shown that whirlpools can serve as a reservoir of Legionella pneumophila. This organism is often detected in water and filter samples during outbreaks; spray nozzles also should be checked because they can aerosolize the organism, Methods for P. aeruginosa are presented in 9213E and F. They clude a membrane filter procedure and a multiple-tube tech- nique. Methods for Legionella can be found in Section 9260). ¢ Microbiological indicators: The primary indicator of disin- fection efficacy is P. aeruginosa, with total coliforms, heterotrophic plate count, and staphylococci as supporting indicators of water ‘Quality. The standard index of water quality (ie, total coliforms) ‘may be insufficient ro judge the microbiological quality of whirl pool water. Pseudomonas aeruginosa is frequently isolated from ‘whirlpool water that is coliform-negative.® In the event of a whitl- pool-associated outbreak, collect samples as soon as possible. An- alyze for the suspected pathogen and P. aeruginosa 4. Sample preservation: Examine samples as soon as possible after collection. See Section 90608, MICROBIOLOGICAL EXAMINATION (aon 2, References 1. Govt ror Disease Cowes. 1981, Suggested Health and Satay Guidelines for Pubic Spas and Hor Tubs. DHHS-CDC 199.960 39 Government Printing Orie, Weshington, D.C. 2 Scuomion, SL. 1985. Host factors in whilpoo-associtedPseudan. ‘nas aeruginosa skin disease. infect, Control 6402. 3. Hicaswm, AK. PN. Les, RF. Kiaseaz & VP. Munn, 19, Characteristics of Pseudomonas aeruginosa isolated from whislon, and bathers. Infect. Control 6:407 4. Grooms, D.G., AH, HavELAnK & H.R. VESNENDAAL 18S, A note (on Legionella in whisipols. J. Appl. Bacteriol. 58479 5 Moussa, A.K. & M.S. Favero. 1985. Microbiological aspects of Public whirlpools. Clin. Microbiol. Newslewer 79. 6. Hatt, N. 1984, Whislpools and Pseudomonas aeruginose. UHL. Lab Hotline 219. 3. Bibliography Gareice, ELE, A.K. Hiceourn & WJ, Masrone. 1985, Public whit ools—the epidemiology and microbiology of disease. Infect. Con. trol 6:392. 9213 D. Natural Bathing Beaches 1. General Discussion @. Characteristics: A natural bathing beach ig any area of a stream, Take, ocean, impoundment, natural pool, or hot spring that is used for recreation. A wide variety of pathogens can be transmitted to humans through use of natural fresh and marine recreational waters contaminated by point sources, such as sew- age and industrial wastes, and non-point sources, such as streams, storm drains, and animals (e.g. birds and bathers)? Contaminating microorganisms may include enteric pathogens, such as Salmonella, Shigella, enteroviruses, protozoa, and “op. ortuniss,” such as P. aeruginosa, Klebsiella spp., and Aeromo- ‘nas hydrophila, which can multiply in recreational waters with sufficient nutrients. Other organisms of concer are those asso- ciated with the skin, mouth, or nose of bathers, such as Staph- ococcus aureus and other naturally occurring organisms (e., ontuberculous mycobacteria, Vibrio spp., and Naegleria).~? 1. Monitoring requirements: Historically, thermotolerant co- liforms have been recommended as the indicator of choice for evaluating the microbiological quality of recreational waters. ‘Many states use this indicator in their water quality standards, Studies havve demonstrated that E: coli and enterococci showed a Stronger correlation with swimming-associated gastroenteritis than do thermotolerant coliforms, and that both indicators were equally acceptable for monitoring fresh-water quality. For ma- ine waters, E. coli and other enteric bacteria have now been documented in numerous studies to enter the viable but non- culturable state in response to such stresses as elevated osmotic levels, so the inability to isolate such indicator organisms may ‘not prove @ lack of fecal pollution in such waters. In marine waters, enterococei concentration had the strongest relationship to the gastroenteritis incident rate. The recommended densities of these indicator organisms were calculated to approximate the degree of protection previously accepted for thermotolerantco- liforms. EPA-recommended water quality criteria are based on these findings." While the primary indicators of water quality are E. coli and enterococci, the enumeration of P. aeruginosa, Aeromonas hydrophila, Klebsiella spp., and Staphylococcus ax ‘reus in recreational waters may be useful in cases that involve discharge of pulp and paper wastes and effluents from textile finishing plants into receiving waters or in waters with higher bather densities, 2. Samples 4 Containers: Collect samples as directed in Section 9060 Select container size acconding tothe number and variety of ess to be performed. Adding Na;S,0, tothe botl is unecessary b. Sampling procedure: Collect samples 0.3 m below the water surface i the areas of greatest bather load. Samples m3) be taken ankle deep (at ~0.075 m below water surfice). deeper waters, if desired, take another sample approximately 0.075 m below the water surface, This area may be somewhet between the knees and the chest, depending on how deep the water is where the sample is taken, Take samples over the ange of environmental and climatic conditions, especially during times when maximal polation ean be expected (ie. periods ot tidal, current, and wind influences; stormwater runoff wastewater treatment bypasses). See 9213B.2b for methods of sample collection and Section 9222 for suggested sample volumes «Sample storage: See Section 30608.RECREATIONAL WATERS (9213)/Natural Bathing Beachos 3, Tests for Escherichia col «2 Media: Media deseribed in this section are available com- rnereially. Manufacturer's instructions should be followed for Torage and disposal after preparation. The formulation may be available commercially for use in the multipletube procedure, the mut-well procedure, or the presence-absence procedure ‘The need for good quality assurance and uniformity requires the se of a commercial substrate media. 1) mTEC agar:** Proteose peptone No. 3 50 8 Yeast extract. Saas 30 Lactose . 100 Sodium chloride, NaCl “13 B Dipotassium phosphate, K:HPO, on. 38 “Monopotassium phosphate KH,PO,, 10 g Sodium lauryl sulfate 02 8 Sodiuin desoxycholate Sn O1 g Bromeresol purple "008 Brompheno! red. eee 0.08 g Agar 150 8 Reagent grade water z 1 Sterlize via autoclaving; pH should be 7.3 + 0.2. Pour 4 t0 5 mL liquefied agar into culture dishes (50 X 100 mm). Store in refrigerator. 2) Urea substrate:** Urea : 208 Phenal red once coos 10 mg, Reagent-grade Water 100, mi [Adjust pH to 5.0 + 0.2. Store at 2 to 8°C. Use within 1 week. 3) Procedure Filter sample through a membrane filter (Gee Section 9222), place membrane on mTEC agar incubate at 35 = 05°C for 2 to rejuvenate injuced or stressed bacteria, and then incubate at 44.5 + 0.2°C for 22 h. Transfer filter toa filter pad saturated with urea substrate. After 15 min, count yellow or yellow-brown colonies using a fluorescent lamp and a magnify- ing lens. Verify a portion of these differentiated colonies via a commercial muli-test system (see Section 9222B.4/2)b)) +b. Modified mTEC method: 1) Medium—Modified mTEC agar: Prepare in same manner and with same ingredients as for mTEC agar, but omit brom- eresol purple and bromphenol red and add 005 g 5-bromo-6- chloro-3-indoxyl--b-glucuronide chromogen. Sterilize via av toclavng; final pH should be 7.3 * 0.2. Pour 4 to 5 mL liquefied agar into culture dishes (50 X 40 mm). Store in refrigerator. 2) Procedure—Filter sample through a membrane filter (see Section 9222), place membrane on modified mTEC agar, incu- tate at 35 + 0.5°C for 2h to rejuvenate injured or stressed bacteria, and then incubate at 44.5 + 0.2°C for 22h. After incubation, count red or magenta colonies (E. coli) using & Suorescent lamp and a magnifying lens. Verify a portion ofthese differentiated colonies via a commercial multi-est system [see Section 9222B.472)6). © Enzyme substrate tes: See Section 92238, * Dito or equivalent 947 4, Tests for Enterococci Perform tests for enterococci using the multiple-ube tech nique (Section 9230B), membrane filter technique (Section 9230C), or fluorogenic substrate technique (Section 9230D), 5, Tests for Pseudomonas aeruginosa Perform tests for P. aeruginosa as directed in 9213E and F. ‘Use the multiple-tube test with samples but note that the proce dures may not be applicable to marine samples. 6. Tests for Aeromonas hydrophila, See Section 9260L. 7. Tests for Klebsiella spp. See Section 9222F. 8. Tests for Staphylococcus aureus Another method for enumerating total staphylococci and ‘S. aureus in marine waters and swimming pool water (but not natural freshwater) is a modification of the membrane filtration method using Baird-Parker medium (9213B.6al)].""_ This method adds 0.005% sodium azide to Tellurite Glycine Agar (TGA) or Vogel-Johnson Agar (VIA) as an additional inhibitor to increase the selectivity of these media to enumerate concen- trations of total staphylococci and S. aureus from natural coastal, ‘marine waters. TGA and VJA use the same basic ingredients (lithium chloride, glycine, and tellurite) as. selective agents against non-staphylococci bacteria, which form black target col- onies on these media. However, supplementing TGA or VIA ‘with 0.005% sodium azide was required so they could be used to enumerate total staphylococci and S, aureus from marine waters ‘but not from natural freshwater streams, Limited data show that this method also works in swimming pool water. Sodium azide inhibited the low percentages of non-staphylococci bacteria in ‘marine waters from forming black colonies, but not the much higher concentrations of non-staphylococci bacterin found in freshwater streams. All black colonies are presumptive counts of, total staphylococci colonies and must be verified as total staph- yylococei of S. aureus by further testing Gram staining (gram- positive cocci), catalase (positive), or lysostaphin (positive) to ‘confirm as total staphylococci and via coagulase (positive) to confirm as S. aureus. 9, References 1. Caneus, VJ. 1980, Health Effects Criteria for Marine Recreational ‘Waters. EPA-G0(/1-80-031, US. Environmental Protection Agency, Research Triangle Pat, N.C. 2, Durour, AP. 1984. Health Effects Criteria for Fresh Recreational ‘Waters. EPA-600/1 84-004, US. Environmental Protection Agency, Research Triangle Par, N.C. 3. Keswick, BH, CP, Grraa & SM. Gora. 1981. Occurrence of ‘enteroviraes in community swimming pool. Amer. J. Pub. Healt 71:1026. 4, Dutta, BJ. & KK. Kwan. 1978, Health indicator bacteria in water surface microlayers. Can. J. Microbiol, 24:187948 5, Capcuis, Vii, H. Kewweny & M.A. Levis. 1976. Pseidomonas ttemginoza and fresh recreational waters, J. Water Poll, Control Fed. 48361, 6, Sheer, 1B. SR. Kocwen & BJ. Dura. 1979. The occurence of Candida albicans in Lake Ontsio bathing beaches. Car. J. Micro Biol, 25:1036. 7, Smvens, AR, RL. TysDaLL, CC. Courar & B, Waar 1977 Teolation of the etiological agent of primary amoebic meningoen- ephaids from artcally heated waters. Appl, Environ. Microbiol 34701 8, Waunes, FM, P.T, Amuso, S.L, Cuno de AL. Lews. 1977, Isolation and identification of pathogenic Naegleria from Florida lakes. Appl. Environ. Microbiol. 34:661 9. N'Duate, A, P. Geonces, A. N'Go & B. Frsry. 1985. Soil amoebas tas biological markers 10 estimate the quality of swimming poo! waters. App. Environ. Microbiol 49:1072. MICROBIOLOGICAL EXAMINATION (20 10, US. Esvmonarextat Prorecon Acexcr. 1986. Ambient Wat Quality Criteria for Bacteria—1986. EPA-440/5-84.002, US. Ea. vironmental Protection Agency, Washington, D.C. 11, Chanoenea, N. & R, Furoxa, 1993. Assessment of Staphylococcas pactera in Hawaii's marine rereational waters. Water Sei. Tech 27:28. 10. Bibliography ‘Ourven, V.P, CW. Dause & K. Kawata. 1977. Micronganises in urban Stormwater. EPA-6002-77-087, US, Environmental Protec tion Agency, Cincinnati, Ohi. Roce, BW. LC. Covert, DK. Wap, D. Basso, S.A. Jroson & Ci ‘Jonson. 1993. Comparative resistance of Escherichia coli and Enterococci to chlorination. J. Environ, Health A28:89. 9213 E. Membrane Filter Technique for Pseudomonas aeruginosa 4. Laboratory Apparatus See Section 9222B.1 2, Culture Media a M-PA agar. This agar may not be available in dehydrated form and may require preparation from the basic ingredients. teLysive HCI... 50 8 Sodium chloride, NaCl 50 § ‘Yeast exit "20 8 Xylose Scie 25 8 Sucrose . 258 Lactose. 128 8 Phenol red 10.08 Ferric ammonivon citrate SOB B Sodium thiosulfate, N28;04.» 63 8 Agar See 150 g Reagent grade wer. eres ‘Adjust pH to 65 + 0.1 and sterilize via autoclaving. Coo! to 55 to 60°C; readjust pH to 7.1 + 0.2 and add the following dry ‘antibiotics per liter of agat base: sulfapyridine,* 176 mg; kana ‘mycin,® 85 mg; nalidixic aci.* 37.0 mg: and eyetoheximide.* 150 mg. After mixing, dispense in 3-ml- quantities in 50 X 12 ‘mm pets plates. Store poured plates at 2 to 8°C. Discard unused snedium after 1 month. b, Modified M-PA agart | & Milk agar (Brown and Scott Foster Modification Mixture A: Irstantsonfat milk: 100 = Reagentprade water. 500 inl. Sigma Chena! Co, St. Laity MO, ce equivalen. Sumas asile ns S¢PA-C spr. Contains magnesium, sulfite, Kans npn and naliineocd FCmaton or quale. Mixture B: Natrent broth 7 a 123g Sodium chloride, NaCl... 258 Agar. = ieee 150g Reagent grade water. os 500 ml Separately prepare and sterilize Mixtures A and B; cool rp. idly o 55°C; aseptically combine mixtures and dispense 20 025 mL per peti dish. 3, Procedure a. Presumptive tess: Filter 200 mL or less of natural wate o up to 500 mL of swimming poo! waters through sterile mem brane filters. Place each membrane on a poured plate of modified MPA agar so there is no ait space between the membrane and the agar surface. Tnvert plates end incubate at 41.5 # 0.5°C for 72h ‘Typically, P. aeruginosa colonies are 0.8 to 2.2 ram in din eter and appear flat with light outer rims and Grovwnish (0 frecnish-black centers. Count typical colonies, preferably fom filters containing 20 to 80 colonies. Use a 10- to 1S-power ‘magoifier as an aid in colony counting. ‘A two-plate method also may be used: this method will belP recover chlorine-stressed P aeruginosa (e.g. from swimming pools and whislpools), Use membrane fiter technique to prepa amples, Place membrane filters on R2A agar (Sesion 9215A.6c) and incubate at 37 + OSC for 24h. Asepically transfer the membranes to MLPA. ager and incubate at 35 ~ (05°C for another 24 h. Random and: typical colonies can be verified using confirmation tests. ’ Confirmation tests: Use milk agar to confirm a nuabet &f typical and atypical colonies. Make a single streak (2 1 4 6 Jong) from an isolated colony on a milk agar plate and incuba WOR LOPC for 24 h. P. aeruginosa hydrolyzes casein 24 produces 4 yellowish to grcen diffusible pigmentHETEROTROPHIC PLATE COUNT (9215)Intrcductisn 4, Interpretation and Calculation of Density Confirmation is not routinely required. In the absence of confirmation, report results as the number of presumptive P. aeruginosel\00 mL. 5, Bibliography Deans, CH, 1966, Evaluation of culture media for the isolation and Terumeration of Pseudomonas aeruginosa. Health Lab Sci. 3:10. eae Brown, MR.W. & J.H, Scorr Fostex. 1970. A simple diagnostic milk medium for Pseudomonas aeruginosa. J. Clin. Pathol. 23:172 Lev, MA. & VJ. CABRLLL 1972. Membrane filter technique for ‘enumeration of Pseudomonas aeruginosa. Appl Microbial. 24:34 Duka, BJ. & KK. Kwan. 1977, Coafirmation ofthe single-step mem- ‘brane filter procedure for estimating Pseudomonas aeruginosa den- Sites in wate. Appl. Bviron. Microbiol 33240, Broosey, MH. & B.W. Cin, 1978, Improved medium for recovery and enumeration of Pseudomonas aeruginosa from water using ‘membrane filles. Appl. Environ. Microbiol. 36:26, @ 9213 F. Multiple-Tube Technique for Pseudomonas aeruginosa 1. Laboratory Apparatus See Section 9221 2, Culture Media 4. Asparagine broth: This medium may not be available in dehydrated form and may require preparation from the basic ingredient. Asparagine, 1. 308 “Anhydrous dipotassium hydrogen phosphate, K,HPO,...... 10 8 Magnesium sulfate, MgSO, 74,0... ose Reagent-grade water, aoe Ajst pH to between 6.9 and 7.2 before sterilization », Acetamide broth: This medium may not be available in dehydrated form and may requize preparation from the basic ingredients ‘Acetamide Sodium chloride, Nac. Anhydrous dipotassin aydrugen poosphate, K,HPO, 8 ‘Anhydrous potassium vihydrogen phosphate, KH,PO,..... 073 & Magnesium sulfate, MgSO, +740. as g Dissolve 1.2 g phenol red in 100 ml. 024 NaOH and add 1 ‘LIL of acetamide broth. Use phencl red stock solution within 9215 A. 1. Applications The heterotrophic plate count (HPC), formerly known as ® Standard plate sount, is a procedure for estimating the oo by Standard Methods Commits, 2004, Tuk Grp: Wayre choo (2a), Gi Dicer. Stephen C, Baber, Een Baga, Mark W. LeChoallr, Donald. Romer, gey A. Roce. 1 year. Adjust pH to between 7.1 and 7.3 before sterilization Final pHT should be 7.0 + 02. Prepare acetamide broth as described above. If agar slants are preferred, prepare as de- seribed above but add 15 g agar, heat to dissolve agar, and ispense 8-mL. quantities in 16-mm tubes. After autoclaving. incline tubes while cooling to provide a large slant surface 3. Procedure 4. Presumptive test: Perform a five-tube test. Use 10 mL single-strength asparagine broth for inocula of L mL or less and 10 mL double-stength asparagine broth for 10-mL. inocula. Higher dilutions may be necessary for swimming pools. Incubate inoculated tubes at 35 to 37°C. After 24h and again after 48h of incubation, examine tubes under long-wave ultraviolet tight (black light) in a darkened room. Production of a green fluores- cent pigment constitutes a positive presumptive test . Confirmed test: Confitm positive tubes by inoculating 0.1 mL of culture into acetamide broth or onto the surface of ‘acetamide agar slants. Development of purple color (alkaline pH) within 24 to 36 h of incubation at 35 to 37°C is a positive confirmed test for Pseudomonas aeruginosa. Computing and reporting results: Refer to Table 9221:V and Section 9221C. 9215 HETEROTROPHIC PLATE COUNT* Introduction number of live culturable heterotrophic bacteria in water and ‘measuring changes during water treatment and distribution, or in swimming pools. Colonies may arise from pairs, chains, clusters, or single cells, all of which are included in the term. “colony-forming units” (CFU). The final count also depends, ‘on interaction among the developing. colonies. Choose the procedure and medium that comply with the application of the information. To compare data, use the, same procedure and950 medium. Four different methods and five different media are described 2. Selection of Method 4. Pour plate method: The pour plate method (9215B) is simple to perform and can accommodate volumes of sample or diluted sample ranging from 0.1 t0 2.0 mL. The colonies pro- duced are relatively small and compact, showing less tendency to encroach on each other than those produced by surface growth, On the other hand, submerged colonies often are slower growing and are difficult to transfer. A thermostatically controlled water bath is essential for tempering the agar. Heat shock to bacteria, from the transient exposure of the sample to 45 t0 46°C agar may +. Spread plate method: The spread plate method (9215C) causes no heat shock and all colonies are on the agar surface. where they can be distinguished readily from particles and bubbles. Cotonies can be transferred quickly, and colony morphology easily can be discerned and compared to pub- lished descriptions. However, this method is limited by the small volume of sample or diluted sample that can be ab- sorbed by the agar: 0.1 100.5 mL, depending on the degree to which the prepoured plates have been dried. To use this procedure, maintain a supply of suitable predried, absorbent agar plates. € Membrane filter method: The membrane filter method (9215D) permits testing of large volumes of low-turbidity water and is the method of choice for low-count waters (<1 to 10 (CFUimL). This method produces no heat shock but adds the expense of the membrane filter. Further disadvantages include the smaller display area, the need to detect colonies by reflected light against a white background if colored filters or contrast stains are not used, possible damage to cells by excessive filtra- tion pressures, and possible variations in membrane filter quality (Gee Section $020B.4k). 4. Enzyme substrate method for heterotrophic bacteria: The enzyme substrate method (9215B) can be used with samples having a wide range of bacterial concentrations. The method* uses a substrate-based medium in which the substrates are hydrolyzed by microbial enzymes causing the release of 44-methylumbellferone maximally after 48 b of incubation at 35°C. 4-Methylumbelliferone fluoresces when exposed to long-wavelength (365-nm) ultraviolet light. The number of fuorescing wells corresponds to a most probable mimber (MPN) of bacteria in the original sample. The test may be used for the analysis of drinking water and source water samples. Individual colonies cannot be directly recovered for subsequent analysis. This method produces no heat shock to the bacteria in the sample. The enzyme substrate test is comparable to the pour plate method. 3. Work Area Provide a levei table or bench top with ample area in a clean, draft-fre, well-lighted room or within a horizontal-flow Jaminar * SimPiae® for HPC, sx Laboratories, Westrook, MB, MICROBIOLOGICAL EXAMINATION (G0g9 hhood, Use table and bench tops having nonporous surfaces an disinfect before any analysis is made. 4. Samples Collect water a directed in Section 9060A. Initiate analysis soon as possible after collection to minimize changes in bacter] population, The recommended maximum elapsed time between collection and analysis of sample is 8 h (maximum transit tine 66h, maximum processing time 2h). When analysis cannot begin within 8 h, maintain sample at a temperature below 4°C but dy not freeze, Maximum elapsed time between collection and ana. ysis must not exceed 24 h 5. Sample Preparation Mark each plate with sample number, dilution, date and any other necessary information before examination. Prepare a et two replicate plates for cach volume of sample or diuton examined. For the pour or spread plate methods use sterile ghss (65 cm?) or presterilized disposable plastic (57 cm’) peti dishes ‘Thoroughly mix all samples or dilutions by rapidly making bout 25 complete up-and-down (or back-and-forth) movements Optionally, use a mechanical shaker to shake samples or dit tions for 15 s. 6. Media Compare new lots of media with current lot in use according to Section 9020B.5j 4a. Plate count agar (trypione glucose yeast agar): Use fr pour and spread plate methods. This high-nutrient agar, widely used in the past, may gives lower counts than R2A or NWRI agar. ¢ ‘Tryptone . 508 Yeast exact Giucose . ohana serail Arar : 2.1808 Reagentgrade water a ee Ve pH should be 7.0 * 0.2 after autoclaving at 121°C for 15 mia. 2, mHPC agar:t Use this high-nutrient medium only for he membrane filter method Peptone 2006 Gelatin... 25.08 Glycerol... 100 mL ABBE sees eeeeees 1508 fed canst cigs aati with 1N NaOH. Heat slowly to dissolve thoroughly, add glycerol and sterilize at 121°C for 15 min, Commercially prepared m= dium should not require poststerlization pH adjustment; Section 9020B.5j1). Final pH is 7.1 * 02. €. R2A agar: Use for pour plat, spread plate and meribrtt fiker methods. This low-nutrent agar gives higher counts that Digh-nutient formulations 1 Format called -SPC aptHETEROTROPHIC PLATE COUNT (9218)/Introduction Yeast extract. 05 § Proteose peptone No, 3 or polypeptone 05 8 Ccasamino acids 05 £ Ghcose. - 05 g Soluble starch 05 ¢ Dipotassium hydrogen phosphate, K3HPO,. 03 ¢ Magnesium sulfate bepiahydrate, MgSO, - 7H,0. 005 g Sodium pyruvate.» 03 2 Asie = . 130 Reagentgrade water poner PL For medium produced from basic ingredients, adjust ro pH 7.2 with solid K,HPO, or KHHPO, before adding agar. Heat to dissolve agar and sterilize at 121°C for 15 min. Commercially @ prepared met should not require pot sterilization pl adjust Trent; see Section 9020B.5j1). Final pH is 7.2 + 0.2 id. NWRI agar (HPCA): Use for pour plate, spread plate, and ‘membrane filter methods, This low-nutrient mediuin is likely to jroduce higher colony counts than high-nutrient media. Ie is not Pirreatly available in dehydrated form and requires preparation from the basic ingredients; this makes its usage less desirable. Peptone : 3 30 8 Soluble casein os K,HPO, 02 M80. 005 FeCl, 0.001 g Ar - i 150 8 Reagent-grade water es row Final pH is 7.2 + 0.2 after autoclaving at 121°C for 15 min. e Enzyme substrate medium: See 9215E3. 7. Incubation For compliance monitoring purposes under U.S. EPA's Sur- face Water Treatment Rule (40 CFR 141.74) provision on het- erotophie bacteria, incubate pour plates at 35°C for 48 h, Oth- erwise, select from among recommended times and temperatures for monitoring. changes in water quality. The highest counts ‘ypically wili be obtained from 5- to 7-d incubation ata temper ature of 20 to 28°C. During incubation, maintain humidity within the incubator $0 that the agar plates will have no moisture weight loss eater than 15%. This is especially important if prolonged incubation is used. A pan of water placed at the bottom of the incubator may be sufficient, but note that to prevent rusting or Okidstion of the incubator, the inside walls and shelving Should be of high-grade stainless steel or anodized aluminum. For long incubation in nonhumidifed incubators, seal plates in plastic bags. 8 Counting and Recording 4 Pour and spread plates: Count all colonies on selected Plates prompily after incubation. If counting must be delayed lemporarily, store plates at 5 to 10°C for no more than 24 h, but 20d this as routine practice. Record resus of sterility controls 0n the report for each lot of samples. For compliance samples, count colonies manually wsing a field colony counter, such as a Quebec eolony couster. IF ost such equipment is not available, for samples not for compliance purposes, use other equipment, provided such equipment gives ‘equivalent magnification. Automatic plate counting instruments are available, These generally use a television scanner coupled to ‘a magnifying Tens and an electronics package. ‘Their use is acceptable if evaluation in parallel with manval counting gives comparable results. In preparing plates, pipet sample volumes that will yield from 30 1 300 colonies/plate. The aim isto have a least one dilution giving colony counts between these limits, except as provided below. Ordinarily, do not pipet more than 2.0 mL of sample; however, ‘when the total number of colonies developing from 2.0 mL. is less than 30, disregard this rule and record result observed. With this exception, consider only plates having 30 to 300 colonies in ‘determining the plate count, Compute bacterial count per mil liter by the following equation: pastes colonies counted ‘eval volume of sample plated, i If there is no plate with 20 to 200 colonies, and one or more plates have more than 300 colonies, use the plate(s) having a count neazest 300 colonies. Compute the count as above and report as estimated CFU per milliliter. If plates from all dilutions of any sample have no.colonies, report the count as less than one () 6500 divided by the smallest sample volume plated for glass plates or (©) 5700 divided by the smallest sample volume plated for plastic plates. Report as estimated colony-forming-units per milliliter. If spreading colonies (spreaders) are encountered on the plate(s) selected, count colonies on representative portions only when col nies are well distributed in spreader free areas and the area covered by the spreader(s) does not exceed one-half the plate area. ‘When spreading colonies must be counted, count each of the following types as one: a chain of colonies 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- tween the agar and bottom of the petri dish; and a colony thog forms in a film of water atthe edge or over the agar surface. The last two types largely develop because of an accumulation of ‘moisture atthe point trom which the spreader originates. They frequently cover more than half the plate and interfere with ‘obtaining a reliable plate count. Count as individual colonies similar-appearing colonies grow- ing in close proximity bat not ovching, provided that the dis- tance between them is at least equal to the diameter of the smallest colony. Count impinging colonies that differ in appear- ance, such as morphology or color, as individual colonies. If plates have excessive spreader growth, report as “spreaders” (Spr.). When plates are uncountable because of missed dilution, accidental dropping, and contamination, or the control plates indicate that the medium or other material was contaminated, report as “laboratory accident” (LA), » Membrane iter method: Count colonies on membrane filter using a stereoscopic microscope at 10 to 15%. magnification. Preferably slant peri dish ata 45° angle on microscope stage and adjust light source vertical to the colonies. Optimal colony density per filter is 20 t0 200. If colonies are small and there is no crowding, a higher limit is acceptable. Count all colonies on the membrane when there are =2 colonies per square. For 3 to 10 colonies per square count 10 squares and obtain an average count per square. For 10 to 20 colonies per square count 5 squares and obtain an average count per square. Multiply average count per square by 100 and divide by the sample volume to give colonies per 100 mL. If there ate more than 20 colonies per square, record the count as >2000 divided by the sample volume. Report averaged counts as estimated colony-forming units. Make estimated counts only when there are discrete, separated colonies with ot spreaders . Enayme substrate method: See 9215E.6. MICROBIOLOGICAL EXAMINATION ace, 9. Computing and Reporting Counts ‘The term “colony-forming unit(s)” (CFU) i descriptive of te methods used; therefore, report all counts a5 colony-forming units. Include in the report the method used, the incubation temperature and time, and the medium, For example: CFU/m, pour plate method, 35°C/48 h, plate count agar. To compute the heterotrophic plate count for pour pl spread plate, and membrane filter methods, CFU/ml, divide the total number of colonies or average number (if duplicate plates ‘of the same dilution) per plate by the sample volume. For the ‘enzyme substrate method, use the MPN obtained from the MPN tables, adjusted for sample dilution. Record sample volumes used and number of colonies on each plate counted or estimated. When colonies on duplicate plates and/or consecutive di tions are counted and results are averaged before being recorded, round off counts to two significant figures only when converting to colony-forming unis. ‘Avoid creating fictitious precision and accuracy when com- puting colony-forming units by recording only the fist two left-hand digits. Raise the second digit to the next higher number when the third digit from the left is 5,6, 7, 8, or 9; use zeros for cach successive digit toward the right from the second digit, For example, reporta count of 142 as 140 and. count of 155 as 160, but report a count of 35 as 35. 1. Analytical Bias Avoid inaccuracies in counting due to carelessness, dam- aged or ditty optics that impair vision, or failure to recognize colonies. Laboratory workers who cannot duplicate their own counts on the same plate within 54% and the counts of other analysts within 10% should discover the cause and correct such disagreements, 9215 B. Pour Plate Method 1. Samples and Sample Proparation See 9215A.4 and 5. 2. Sample Dilution Prepare water used for dilution blanks as directed in Section 0s0c. 4a, Selecting dilutions: If possible, e.g, on the basis of histor- ‘cat information, select the dilutions so tat at Teast one plate in «series will contain 30 to 300 CFU (Figure 9215:1). For exam- ple, where a heterotrophic plate count as high as 3000 is sus- pected, prepare plates with 10"? dilution. For most potable water samples, plates suitable for counting will be obtained by plating 1 mL and 0.1 mL. undiluted sample and 1 mL of the 10 dilution. b, Measuring sample portions: Use a sterile pipet for initial and subsequent transfers from each container. If pipet becomes contaminated before transfers are completed, replace with sterile pipet. Use a separate pipet for transfers from each ent dation, Do not prepare dilutions and pour plates i Use caution when removing sterile pipets ftom the container; to avoid contamination, do not drag pipet tip across lexposéd ends of pipets in the pipet container or across lips and necks of dilution bottles. When removing sample, do not insert pipets more than 2 to 3 em below the surface of the same of dilution ¢. Measuring dilutions: When discharging sample portions, hold pipet at an angle of about 45° with tip touching bottom of petri dish or inside neck of dilution bottle. Lift cover of petr dish {ust high enough to insest pipet. Allow 2 to4 s for liquid to drain. from I-mL, graduation mark to tip of pipet. If pipet is not # blow-out type, touch tip of pipet once against a dry spot on pet dish bottom. Less preferably, use a cotton-plugged blow-ovt type pipet and a pipet bulb and gently blow out remaining volume of sample dilution, When 0.t-mL quantities are measured, let luted sample drain from chosen reference graduation until 0.) mL. has been delivered. Remove pipet without touching it ©eTeROTROPHIC PLATE COUNT (9216/Pour Plate Method Water sample Dotivery volume oa mt. |}— 1m. 99 mt. blank “A560 Aetual volume of sample in ish ost mt. 0.01 mt 0.008 mt ea Figure 9215:1. Preparation of ditions ish, pet 1 mL, 0.1 mL, or other suitable volume into a sterile ets dish before adding melted, tempered culture medium, Use decimal dilutions in preparing sample volumes of less than 0.1 mL. In examining sewage or turbid water, do not measure a (0.L-mL inoculum of original sample, but prepare an appropriate Jlution. Prepare at least two replicate plates for each volume of sample or dilution examined. After depositing test portions for ‘each series of plates, pour culture medium and mix carefully. Do sat let more than 20 min elapse between starting pipetting and poring plates. 8. Plating 4. Melting medium: Melt sterile solid agar m boiling water or by exposure to flowing steam in a partially closed Container, but avoid prolonged exposure to unnecessarily high temperatures during and after melting. Do not resterilize plating snedium. Ifthe medium is melted in to or more batches, use all, of each batch in order of melting, provided that the contents remain fully melted. Discard nielted agar that contains precipi- tte ‘Maintain melted medium in a water bath between 44 and 46%C ‘nil used, preferably no longer than 3 h. In a separate container ace a thermometer in water or medium that has been exposed ‘0 the same heating and cooling as the plating medium. Do not ‘depend on the sense of touch to indicate proper medium tem- Perature when pouring agar. Use plate count agar, R2A agar, or NWRI agar as specified in ISAS. +. Pouring plats: Limit the number of samples tobe plated in ‘ny one series so that no more than 20 min (preferably 10 min) ‘lapse between dilution ofthe fist sample and péuring of the last Pate inthe series. Pour at least 10 to 12 mi. liquefied medium ‘aintained at 44 to 46°C in each dish by gently lifting cover just igh enough to pour. Carefully avoid spilling medium on outside dish id when pouring. When pouring agar from flasks or tubes ‘hac have been held in a water bath, wipe with clean paper towel and flame before pouring. As each plate is poured mix melted ‘medium thoroughly with test portions in petri dish, taking care not to splash mixture over the edge, by rotating the dish first in ‘one direction and then in the opposite direction, or by rotating and tilking. Let plates solidify (within 10 min) on level surface. After medium solidifies, invert plates and place in incubator. . Sterility controls: Check sterility of medium and dilution ‘water blanks by pouring control plates for each series of samples. Prepare additional controls to determine contamination of plates, pipets, and room air. 44, Incubation See 92157. 5. Counting, Recording, Computing, and Reporting See 9215A.8 and 9, m 6. Bibliography Broxo, RS. & W.D. Dorrensx. 1916. he numberof colonies allowable ‘on satisfactory agar plates. Tech, Bul. 53, New York Agricultural Experimental Sta Burrexsiio, C.T, 1933. The selection of a dilution water for bacterio- logical examinations, J. Bacteriol. 23:355; Pub. Health Rep. 4: 81 Ancuasanuit, J, J. Curor & MH, MeCrapr. 1937, The need of ‘uniformity of conditions for counting plates (with suggestions fora standard colony counter). Amer. J. Pu Health 27:809, Ricuasps, OW. & P.C. Heim. 1945. An improved dark-feld Quebec colony counter. J. Milk Technol. 8253, Bexny, IM, DA. MeNene & LD, Wires, 1969. Effect of delays in pour plating on bacterial counts. J. Dairy Sci. $2:1456. Garonne, EE, HD. Nash, DJ. Reasonen & RH. Tavion. 1972. The necessity of controling bacterial populations in potable waters: Community water supply. J. Amer. Water Works Assoc, 64:596,———————_— 954 ‘Gaesicn, EE. 1973: Is the total count necessary? Proc. Ist Annu. "AWWA Water Quality Technol. Conf, Dec. 34, 1973, Cincinnati, Obio, p. VIEL. American Water Works Assoc., Denver, Colo. nsec, W. 1973. Improved total count techniques. Proc. Ist Ann. "AWWA Water Quality Technol, Conf, Dee. 3-4, 1973, Cincinnati, Ohio, p. VI-1. American Water Works Assoc, Denver, Colo. umes, BJ., AS.Y. Cru & J. Cosuns. 1974. Relationship of hetero- ‘wophic bacterial indicators of water polfution and fecal sterols. Water Res, 8:1087 Kun, DA. & S. Wu, 1974. Suess: a factor to be considered in heterotrophie microorganism enumeration from aquatic environ- iments, Appl. Microbiol. 37:429. Guneerc, EE, HD. Nasu, DJ. Reasower & RH. Tartor. 1975. The necessity for controlling bacterial populations in potable waters: MIGROBIOLOGICAL EXAMINATION (ag) Botled water and Assoc. 67:117 Bar, CR, MA. Hotogt-Fraweun & M. FRveLn. 1980, Hetero. Trophic bacteria in yo Canadian rivers. —I. Seasonal variation in the predominant bafterial populations. Water Res. 14:44 ‘Maass, E.G, L. Havagn, GF. Riooeway & BH, OLsox. 1981. Ev ating mediums ahd plating techniques for enumerating bacteria jg ‘water distribution systems. J. Amer. Water Works Assoc. 73:58, Reasower, DJ. & BEE. Gipnscu. 1985. A new medium forthe en. ‘eration and subculture of bacteria from potable water. App Environ. Microbiol. 49:1, [Awenican Punic Heat AssocuTion. 1993. Standard Methods forthe Examination of Dairy Products, 16th ed. American Public Heald ‘Assoe., Washington, D.C. water supplies. J. Amer. Water Wor 9215 C. Spread Plate Method 1, Samples and Sample Preparations See 9215.4 and 5. 2. Laboratory Apparatus 1a. Glass rods: Bend 4-mm-diam, fie-polished glass rods, 200 rm in length, 45° about 40 mm from one end, Sterilize before using. . Pipet: 1.0-mL glass or plastic pipets. «Turntable (optiol).* Incubator or drying oven, set at 42°C, of laminar-flow hood. 3. Media See 9215A.6a, ¢, and d. If R2A agar is used, best results are obtained at 28°C with 5 to 7 d incubation; if NWRI agar is used, incubate at 20°C for 7 d. 4, Preparation of Plates Pour 15 mL of the desired medium into sterile 100- x 15-mm or 90- X 15-mm petri dishes; let agar solidify. Predry ‘Faher Seienif hand operate, No, (8-758 or Lab-Line, motor driven, No 1580, er equtalent plates inverted so that there is a 2- to 3-g water loss overnight with lids on. See Figure 9215:2, Table 9215:1, or Figure 9215:3. Use predried plates immediately after drying or store for 2 weeks in sealed plastic bags at 4°C. For predrying and using plates the same day, pour 25 mL. agar into petri dish and dry in a laminar-flow hood at room temperature (24 to 26°C) ‘with the lid off to obtain the desired 2- to 3-g weightloss. See Figure 92153, 5. Procedure Prepare sample dilutions as directed in 9215B.2. a. Glass rod: Pipet 0.1 or.0.5 mL sample onto surface of 2 predied agar plate. Using a sterile bent glass rod, distribute Jnoculum over surface of the medium by rotating plate by hand ‘or on a turntable, Let inoculum be absorbed completely into the ‘medium before incubating. >. Pipet: Pipet desired sample volume (0.1, 05 mL) onto te surface ofthe predried agar plate while plate is being rotated 02 a turtable, Slowly release sample from pipet while making om to-and-fro motion, starting at center of plate and stopping 0.5 cm from plate edge before reruming to center. Lightly touch the pipet to plate surface. Let inoculum absorb completely ilo ‘medium before inverting and incubating. trance 9215:1, Berecr oF Tewrexarine oF Davos on Wetcer Loss oF 1S-ul. Acar PLares Sronep SErARATELY® “Time for Pats to Lose 1 0 4 g of Water (Avg. for 5 Plats) h “emp. Plates Inverted with Lids on ates Inverted with Lids Removed °c ig 28 38 4e 1g 28 35 a 2 2 4 95 ns 3a 10 105 37 1 38 st o 7 35 53 50 6 2 18 4 07 3 19 @ 4 : 2 16 : = zi ‘Referenced in Canada Cenze for Inland Waters Manual, Burlington, Om.HETEROTROPHIC PLATE COUNT (8215)Spread Plate Method 9-55 5 4 sore w eis & 7 are = 8 S2 a a ° 5 1) 15S Time, h ie] Pure 9215:2. Drying weightloss of SL agar plates stored separately Inverted with ids on, SOLACE: Unpalished data, Wat Paiaton Lab, Chicago Dep, Wate. a 8 = ° s Near screen, lids off A Near screen, 1/2 lids oft Near outside edge, lids off Near outside edge, 1/2 lids off Random placement, lids off ° 20 40 60 20 100 120 ; Time, h mi, “9215: Weight loss of 25-ml. agar plates (100 x 15 mm) dried seporaely ina laminar‘ow hood at room temperature (24 to 36°C), relative ‘humidity (30 t0 33%), and air velocity 0.6 mis. SouRCE: Unpublished data, Alberta Environmental Cente, Vegreville, At,9568 6. Incubation See 9215A.7. 7, Counting, Recording, Computing, and Reporting See 921548 and 9. 8. Bibliography Bucs LD. & RC. Chsvenwon, 1960. The spread pate asa metbod for *2 eaumeration of marine bacteria. Limaol Oceanogr. 578. cunn’ DS, 167, Comparison of pour and surface plate menos for ve jexminaton of bacterial counts. Car. J. Microbiol. 131409, ‘yu Sossraetcan, AA. & CH. Lez. 1971. Poor plates or teak rates, ‘Appl Microbiol. 18:1092. cua 7DS. 1971. Studies on the surface plate method of counting ‘bacteria: Can. J. Microbiol 17:943. Gucwusn IE, JE. Cowen, CB. Dower, LT. Pars & ‘purrs. 1973, Spiral plate method for bacterial determination Appl. Microbiol. 25:24. : MICROBIOLOGICAL EXAMINATION (cop, ran, DM, & W, Gnsoura 1976. Pour ple steak ae ret so Aan, AWWA Water Quality Techslogy Cont, De, re a san Diego, Cal p 285. American Wats Works As sec. Denver. Coo. Dunn hed. 1978, Mehods for Microbioloreal Anais ot Ty Bo acewaters and Sediments. Inland Water Direc, Ware pention Div, Canada Cente fr Inland Wate, Burlington, Ont AT Naas &RR.Canwa. 178 anf ey mn of emetang aoe eos in We ST ye Maem Appl Eni. Mraiok 35:76. one M919, A road spread plate techie fr the det Me po concenations of viable heterotopic baci, STP aaaaays American Soc. Testing & Motrin Pildeph, Pa. Gamer. EB. 1981. Curent stats of mirobioogieal water gay criteria, ASM News 47:23. “Taveoe, REL, MJ. Auto BE. Gaon 1981, Standard pt count A -crparisnlo pou plate and spread pate metbods Pre. 9 Am EW WA Water Quality Techno. Con,.Des.6-9, 1981, Seale, Wash, 223, American Water Works Assce., Denver, Colo, 9215 D. Membrane Filter Method 4. Samples and Sample Preparation See 9215A.4 and 5. 2. Laboratory Apparatus See Section 9222B.1. 3, Media See 9215A.6. Use m-HPC agar or, alternatively, R2A or NWRI ager. 4, Preparation of Plates Dispense 5-ml. portions of sterile medium® into 5O- > 9 mm pet dikes Let soiify at oom temperature, Prepared pes Peay te stored inverted ina plastic box of tight container in 2 refrigerator for no longer than 2 weeks. 5, Sample Size “The volume to be filtered will vary with the sample. Select & smaximam sample size to give 20 to 200 CFU per filter 6. Procedure Filter appropriate volume through a stele 47-mm, 045-pen-pore diam, gilded membrane iter, under partial vacuim, Rinse funne! ‘asHPC agar may not be tere vith hee 20-to30-mi portions of sterile ution wate. Place ern ‘aga in pets ish 7. Incubation Place dishes in lose-fting box o plas bag containing moistened eke Incubate a 35 = O.5°C for 48h ifwsing m- HPC est Meese using R2A medium, o at 29 1 28°C for Sto 7 di wis ANWR ce ROA agar. Duplicate ples may be ince for ter ie ane temperature conitions as desired. 8, Counting, Recording, Computing, and Reporting See 9215A.8 and 9, Report as CFU/mL, membrane Fier ‘method, time, and medium, 9. Bibliography Cuan, HF. BE, Grupenon, FLL. Jermx & PW. Hanes 1951. The Mt Tabane fier in sanitary bacteriology. Pub. Health Rep. 66551. Srorent, EM., W'T. Soxosc & 37. Nowra. 1962: The actor of Temperature in the beter recovery of bacteria from wate by fli tion. Can. J. Microbiol. 8809. Tana RH, & EE Gasnsct, 1979. A new membrane A proce sree for bacterial counts in potable water and swimming samples. J. Amer. Water Works Assoe. 71:402, Cuan, JA 1980. “Ths fnfhuenct of nereasing numbers of non-indcaee O, Mo dann inter pss by be mera fe presenceabsenor tests. Car. J. Microbiol 20827 Dyn BJ, ed 1981, Membrane Filaion, Applications, Tbe a ‘Pleas. Marcel Dekker, Inc, New York, NY and Basel, wisn oa. A\W. 1981. Eifet of injury on the recovery of ‘acters oF eerbrane fiters. In B.. Dutka, ed. 1981. Membrane irsi®- ‘Applications, Technigues, and Problems, p. 413 ‘Marcel Del Tne, New York, N-Y. and Basel, Switzerland. |DIRECT TOTAL MICROBIAL COUNT (@216y/ntroduction 957 9215 E. Enzyme Substrate Method 4, Samples and Sample Preparation See 9215A.4 and 5. 2, Laboratory Apparatus 4, Pipets, 0.1-, 1.0-, and 10-mL, sterile, graduated, glass or plastic. », Incubator set at 35 + 05°C. ¢, Ultraviolet light source, long wavelength, 6-W, 365 nm. 4. Sample plates.* ‘B Medium ‘Te formulation is available commercially in sterile medium ves- «els for 100-mL. multidose procedures os avalable in test tubes for 10m unit dose procedures. Store medium between 2 and 25°C. The hus a shel life of up wo 12 months from date of manufacture. Test cach lot for erty by following the inoculation procedure (5 tulow), using 10 mL. rehydrated medium without sample. incubate for 48h at 35°C. No wells should fluoresce afte incubation, 4, Sample Diluent Use cither sterile deionized water, distilled water, buffered water, or 0.1% peptone. 5, Procedure Retydrate medium by filing medium vessel to 100-miL mark with stile diluent, re-capping, and shaking until medium has dissolved. * SinPlaes®, IDEXX Laboratories, Westhrook, ME o equivalent, 1 IDEXX Laboraries, Westbrook, ME. -Aseptically pipet LO mL sample and 9 ml. of reydrated medium onto the center ofthe sample plate. Altematvely, aseptically pipet 0.1 ml. sample and then 9.9 mi rehydrated medium onto center ofthe sample plate, Nore: Final volume of sample plus medium must be 10 = 0:2 mL. Cover plate with lid and genly swirl to distribute mixture into wells. Tip plate 90° to 120° to drain excess mixture into the absorbent pad. Invert plate and incubate for 48h (range of 45 to 72) at 35°C. If 8 sample i suspected of having a count greater than 738 CFU/mL, due by adding 1 mL. sample to a sterile vessel containing 99 mL sterile diluent. Make addtional dations as required to keep counts ‘below 738 CFUmL in final cilton, 6. Counting, Recording, Computing, and Reporting Aer incubation, examine plate for uoresent wells. Count number of fluorescent wells by olding a 6-W, 365-nm UV light about 13 cm above plat. Preferably use UVfitering laboratory safety glasses dur ing counting, Count numberof wel exhibiting blue fuorescence. Use MPN chart (provided by the medium manufacturer) to cal culate MPNimL. Adjust MPN to reflect sample volume andor Auton made to yield a corrected MPN value, Record as MPN. 7. Bibliography ‘Smuxss, A, D. Hiswoa & B. Rou. 1998, Comparative asessment of the newly developed SimPtie® method with the existing EPA approved pour plate method for tie detection of heterotopic pate coum bacteria in ‘ovone treated drinking water. Prseriad at Intemational Ozone Assoc. Cont, Ober 19-23, 1998, Vancouver, BC, Canada, Incson, R.W., K. Osnoene, G. Barns, C. Jour. D. Zaman, B. Rott, ‘A. STILLNGs, D. Hexz06, S. Cannon & S. LoveLan, 2000. Multi regional evaluation of the SimPlate® heterotrophic plate count ‘method compared f0 the standard plate count agar pour plate ‘method in water. Appl. Environ. Microbiol. 66:453, 9216 DIRECT TOTAL MICROBIAL COUNT* 9216 A. {Staining using deoxyribonucleic acid (DNA)-Specific fuoro- ‘Firomes, such as 3,6-bis (dimethylamino) acridinium chloride } (tdi orange) or 4", 6-diamidino-2-phenylindole (DAPI), fol- |; loved by epifluorescence microscopy, has been used to count ‘}al microbial cells in aquatic samples. Other uorochromes, APoroved by Standard Methods Committee, 2008. Herrero Cnn Ve ht Cate Dak se 9. dau land, Chcistinn P, Chouret, Archie J. Degoan, Isabel Escobar, Marion G. Sis ce tees he De tee ile. Sommer, and Margaret M. Willams. Introduction such as S-cyano-2,3-ditoly! tetrazolium (CTC) or SYTO 9/pro- pidium iodide, can be used for viable or total bacteria cell counts Direct total cell counts of bacteria in water or wastewater usually exceed counts obtained via heterotrophic plate counts (HPC) and most probable number (MPN) methods because they preclude the errors caused by viability-related phenomena, such as selectivity of growth media, cell clumping, and slow growth Fates or errors inherent to the methods. Some water samples might contain large amounts of dead or nonculturable cells. ‘The method described in this section uses acridine orange as the staining agent258 igRoRIOLOGICAL EXAMINATION (2009, 9216 B. Epifluorescence Microscopic Method Using Acridine Orange ‘Te epiurescence microscopy mehog produces direct total calc with relative speed 20 10,3) Me from time of Sfmpling) and senitivy, Te does 0° ‘permit differentiation of tutral cells on the basis of txon0"y, metabolic activity, OF es iy, and it cannot be used 1 eRe ‘jerobial biomass canis the volume of individual ces YAN ‘considerably. The Toque an experienced technician 91 ‘phosphate buffer ‘Prepare fresh daily. =P Fuorochrome, 0.1% (w!¥) acridine corangedt in phosphate buffer. i Immersion oil ow fuorescing ref the potential heal ffxi of XPS to gluta ence and acridine orange, handle ‘chemicals in ventilated alse fne ood), Keep sluions in GENY sealed containers, are gear appropiate persons protective equipment 4, Procedure collect water samples as directed in Seco 19060, Add 9. ini snmple to a est tube containing, 1 ml. fixative. Fixed mpc ean be stored in ily CAPPED containers at 4°C for wp sar pecks without a significant Jerease © ‘ell number isperse and iu samples from smesotrophi sources to obtain reprogucible Fess ‘Mix sample using & tender or vortex mixer, then make tenfold iutions in pba Phat buffer as necesary. Clean 891 samples may not require tn, but anger sample volumes 1) im) may be required espn reliable counts (higher vole ‘of water might Be fcred to concentrate samples With OY ‘bacteria concentrations). Samples containing clumped bate (uch as biofilm sample) Sard be subjected 1 witrasoni eaten disrupt aggre Stage 1 ra. of sample or aiution 09 @ nonfluorescent oly carbonate fier supported by 2 cellulose membrane filter th carbetlder. Using dspossbe senile SY filters, add 1 ml fer Moe and incubate or 15 rain te ark, then add abot Fa tered phosphate buffer to Promo ‘oiform distibaion ‘Of calls across the membrane. ‘Kitermatively, combine Meer oF epeand sample ina sia clean vill ‘allow to react, Coe Uo the ier bolder ter One ‘vacuum (about 13 the ach membranes with 2 mi DDO ‘bier and op) ra. Remove polyesbonste tet wih ‘lean forceps a8 “ty for ito 2 min, The filter can Ue quarter sections aoe if neoded. Place dried filter oe ¢ drop of immersion saver ean gase mieoscope slide, Ad small drop of imine oe to ler surface. Gently cove. EE ‘vith a clean BB “coc heey named stack 107 om cia Geigy Com 1" Jen. F attire Com. of eit. nena Co, Bal EAE Te Sig ortnes fe Type By oF eCASSIMILABLE ORGANIC CARBON (9217)/Introduction coverslip. Samples can be stored in the dark for several months without significant loss of fluorescence. ‘Examine a least 1O randomly selected fields on the filter using. the oil immersion lens to establish that distribution of microbial cells is uniform and that individual cells can be enumerated (i rol, dilute sample and repeat) Preferably count 10 to 50 cells per field Ifthe distribution and number of cells is adequate, count umber of cells in atleast 20 squares of the counting graticule in the ocular lens. When processing samples free of debris, an image analysis system can be used for microscopic counts and calculations of cell concentration, Include negative and positive controls to confirm sterility and staining efficacy. For the negative control, perform the staining on an aliquot (Same volume as sample to be tested) of filtered phosphate buffer. AS a positive control, use a suspension of laboratory cultivated cells (eg., E.coli) that has been fixed and Givied in filer-sterilized phosphate buffer to a predetermined cell concentration. To test for interference of the sample matrix with the proce- ure, add an aliquot of the positive control cell suspension 0 | mL of sample and perform acridine orange staining. Enumerate total cells, subtract sample cell number from the sample + positive control cell number, and perform the calculation in ‘§216B.5, accounting for the dilution of the positive control aliquot in the dilution factor to obtain the number of cells per nilliter. Compare the positive control enumeration with the predetermined positive control cell concentration to determine staining efficacy for the sample, 259 5. Calculations Calculate the average number of cells per filter. Obtain effee- tive filter area from filtration unit specifications. Extrapolate to determine number of cells per milliliter of sample: ‘Total cells. = (avg cells/square) x (squaresfiter) > (diluion factor/sample volume, ml. 6. Bil lography Homi, JE., RJ. DALry & S. Jason, 1977. Use of nclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Mi- crobiol. 33:1225, Stexack, M.E, P.W. Jowson & J. McH. Siepurni. 1985. Detection, ‘enumeration, and sizing of planktonic bacteria by image-analyzed epiftuarescence microscopy. Appl. Environ. Microbiol. 49:79. ‘Awenea Socery for TesreG and Mareuats. 1987, Standard test ‘method for enumeration of aquatic bacteria by epifluorescence ‘microscopy counting procedure. ASTM D4455-85, Annual Book of ASTM Standards, Vol. 11.02, Water. American Soc. Testing & ‘Materials, Philadelphia, Pa, Keren, RL Jt. & JR, Peart. 1994 Use of fluorochromes for direct, ‘enumeration of total bacteria in environmental samples: past and present. Microbiol. Rev. 58:63. Bou.os LM. Prevost, B. Bane, J. Coausam & R, Desiaxoms. 1999 Liveldead Bac.ight: application of a new rapid staining method for iret enumeration of viable and total bacteria in drinking water. ‘Microbiol. Methods. 32.77. 9217 ASSIMILABLE ORGANIC CARBON* 9217 A. 1. ‘Significance Growth of bacteria in drinking water distibution and storage ‘ems can lead to the deterioration of water quality, violation of Water quality standards, and increased operating costs. Growth or rowth resulis from viable bacteria surviving the disinfection Frcess and utilizing nutrients in the water and biofilm to sustain ‘Bomth. Factors ther than nutrients tha influence regrowth include ‘emperature,* residence time in mains and storage units,” and the ‘icacy of disinfection Tests to determine the potential for bacte- “id regrowth focus on the concentration of nutrients." Not all organic compounds are equally susceptible to micro- bial decomposition; the fraction that provides energy and carbon €tacterial growth has been called labile dissolved organic ‘kina! biodegradable organic carbon (BDOC), or asiile "© Organic carbon (AOC).* Easily measured chemical surro- files for AOC are not available now." As alternatives to al methods, bioassays have been proposed?" Pe ‘Ateroved by Standard Metods Commitee, 1997. Introduction Ina bioassay, the growth of a bacterial inoculum to maximum density can be used to estimate the concentra nutrients; the underlying assumptions of the AOC bioassay are that nitrogen ‘and phosphorus are present in excess, ie, that organic carbon is limiting, and that the bioassay organisms) represent the physiological capabilities ofthe distribution system ‘microflora. Various bioassay suse an inoculum of one to four species of bacteria” growing in log phase or present in late stationary phase, or may use undefined bacteria attached to a sand substratum,’ suspended in the sample,° or filtered from the sample and then resuspended."* Incubation vessels vary as to material,” size," closure," and cleaning procedure.*"* Water to be tested for nutrient concentrations hhas been variously prepared.*""* The AOC bioassay is an indi- rector surrogate method, wherein nutrient concentrations are not measured directly, but colony-forming units (CFU) of the bio- assay organism(s) are the test variable. Nutrient concentrations have been estimated directly from changes in dissolved organic carbon concentrations within the test vessel” or indirectly from «epifluorescence microscopic counts of the maximum number of bacterial cells grown," turbidity," or incorporation of tritiated Ba960 thymidine ino bacterial DIVA.‘2° CFU densities, total cell den ara bacterial production are converted (0 nutrient concen: tration by the growth yield | ‘of bacteria, defined as either the ratio: ‘between CFU or cells ‘produced and organic carbon used, oF tiomass produced and organic carbon used.” 2. Selection of Method -The method described below is a two-species bioassay using Pseudomonas fuorescens stain P-I7 and Spirillum stain NOX {oan der Kei)” tat has Deen modified o reduce robles of tucteial and carbon contamination." It uses a defined inoe- Tum and miniaturized incubation vessels, requires no special jaed equipment, and hasbeen rated to the presence of coliforms ina drinking water distribution system.” The two-species ino ham probably underestimates the, total quantity of AOC. is vpnsistenty lower than BDOC estimates, and docs not provide . Preparation of stock inoculum: Prepare individual rabid sus- pensions of P-17 and NOX by transferring growth from a slant culture on R2A agar into 2 to 3 ml. filtered (0:2 pm), autoclaved ‘sample. Use slant not older than 6 months. The autoclaved sample ccan be any water that supports growth of P-17 and NOX and is arbor-limited, Neutralize chlorinated samples with s0- dium thiosulfate (42 1/50 mL). Transfer 100 pL of suspension to ‘50 ml filtered, autoclaved sample ina sterile 125-mL. ground-glass- stoppered erlenmeyer flask. Add 125 pL. sodium acetate solution (Gaspension contains 1 mg acetate-C/L). Incubate at room temper- ature (25°C) until the viable cell count reaches the stationary phase. Organic-carbon Limitation will insure complete uilzation of ‘acetateC so n0 AOC is transferred with the inoculum. The station- ary phase is reached when the viable cell count, as measured by spread plates, reaches maximum value, Store stock cultures for not ‘more than 6 months at $°C. Before inoculating a bioassay vessel, rake a viable count of the culture (spread plate) to determine the appropriate volume of inoculum to be added to each bioassay vessel ‘Preparation of incubation water: Collect samples directly {nto ten 45-ml. vials. Use 9 vials for AOC measurement and 1 for growth control. Fill each vial to the neck (40 mL) within as short atime as possible. Place septa on the vials, TFE side down, and secure with open-topped screw caps. Altematively, collect 500 mL. sample in an organic-carbon-free vessel and pour into ‘each vial. Neutralize samples containing disinfectant residuals ‘with 33 LL sodium thiosulfate soltion added to each vial or 0.5 rn per 500-mL sample. Preferably, collect an extra val to check for residual chlorine after neutralization. In the laboratory, cap ‘vials tightly and pasteurize in 70°C water bath for 30 min. ‘d:Inoculation and incubation: Cool, inoculate with 500 col- ony-forming units (CFUYimL each of P-17 and NOX, either by injecting through the septum or by removing cap and using 2 catbon-free pipet. Plastic, sterile tips for continuously adjustable Pipets are suitable. Use the following equation to calewate ‘volume of inoculum: (500 CFU/ml.) X (40 mLivial) ‘Vohune of inoculum = f AL stock inoculum Hold vials at 15°C in the dark for 1 week. Ifa 15°C incubator is unavailable, incubate at room temperature not to exceed 25°C. ‘Because incubation temperature influences growth yield, record {Avail rom the American Type Culture Collection, + Perce Varian MICROBIOLOGICAL EXAMINATION tog and report temperature-Determine yields as directed below if ay alternative temperature is used ¢e. Enumeration of test bacterium: On incubation days 7, 8, and, remove three vials from the incubator. Sample an individ via cq only 1 d. Shake vials vigorously for 1 min, remove 1 mL witha sterile pipet, and prepare a dilution series (ee Section 9215B), Phe three dilations (102, 10°, and 10 in duplicate. Incubate plates 125°C for 3 to 5 d and score the number of colonies ofeach stain, -17 colonies appear on plates frst; they are 3 to 4 mm in diameter with diffuse yellow pigmentation. NOX colonies are smal (1-g 2mm diam) white dots. It may be necessary to count P17 and NOX colonies at different dilutions. Sample vals on three separate days to check whether maximum density has been reached. Day. today variations of between 11 and 16% of the mean for batch cultures of P-I7 in staionary phase are typical! A’ consisext increase in cell densities of 20% or more over the 3-d petnd indicates thatthe cultures are notin stationary phase; repeat assy with longer incubation period. Altematively, colleet more samples (three foreach additional sampling day) and prepare asin above 0 extended incubation can be used. A sharp population decrease ct approximately 05 log over the 3 dis unstal, but may occur. his happens repeat the assay. if Determination of yield of P-I7 and NOX: The yields of P-17 and NOX on model carbon compounds should be constant if organic carbon is limiting andthe incubation temperature is kept Constant, Itis acceptable to use the previously derived erp Yield values of 4.1 x 10® CFU P-17/ug acetate-C, 1.2 X 10° CFU-NOX/pe acctate-C, and 2.9 X 10° CFU-NOX/yg. ox- alate-C at 15°C However, the determination ofa yield contol provides an important check on both the bioassay (see also 9217B.6) and carbon limitation in the sample. 5. Calculation ‘a. AOC conceniration: Average viable count results for the 34 ‘and calculate concentration of AOC as the product of the mean of the viable counts and the inverse of the yield: ug AOCIL = {(mean P-17 CFU/mL\yielé) ++ (mean NOX CFUhmL)H/yield)(1000 mLIL) When the empirical yield factors? are used, the equation becomes: ‘ug AOCIL = [(onean P-17 CFUMmL(Uyield) -+{anean NOX CFU/ml (yield) (1000 mA.) When the empirical yield factors® are used, the equation becomes: ug AOCIL = ((mean P-17 CFU/mLXug actate-CH.1 X 10° CFU) -+(mean NOX CFUMiL ng oxaate-C72.9 x 10° CFU (1000 mL)