## 9213 RECREATIONAL WATERS*

9213 A. Introduction

1. Definition of Recreational Waters and Use of Appropriate Microbiological Indicators

Recreational waters are those in which primary contact occurs. Primary contact recreation activities are those that could be expected to result in the ingestion of, or immersion in, water (e.g., swimming, water skiing, and kayaking). There are two general types of recreational waters. The first type includes natural sources of water, such as lakes, streams, and coastal waters, where the water source cannot be disinfected and where these sources are at risk for contamination via point sources, such as sewage and industrial waste, and non-point sources, such as streams, storm drains, and animals (e.g., birds and bathers). This source of water is used primarily for swimming, wading, and surfing. The second type of recreational water is found infacilities using municipally treated water; it should be disinfected continuously. The designated uses of this type of water may be recreation, such as swimming pools, or therapy, such as whirlpools. The quality of recreational water should be suitable for its designated use and characteristic conditions. Historically, concentrations of selected microorganisms have been used to determine the suitability of recreational water for its designateduse. Ideally, recreational water quality indicators are microorganisms for which densities in the water can be related quantitatively to the degree of potential contamination and thus health hazards resulting from recreational use, particularly where upper body orifices are exposed to water. They are used to determine how well the water will protect human health. The ideal indicator is the one with the best correlation between density and the health hazards associated with a given type of pollution. It also is consistently and exclusively present when the contaminant is present, occurs in higher densities than the contaminant, cannotproliferate more than the contaminant, is more resistant to environmental stresses and more persistent than the contaminant, andhas characteristic, simple reactions for easy, simple identification. 1

Correlations of microbial density in water and predictable disease have been established for exposure to natural sources ofrecreational water but not for recreational waters that are routinely disinfected. Epidemiological studies have been conducted 1 to show a correlation between concentrations of E. coli and enterococci for natural fresh water sources used for swimming and for concentrations of enterococci for natural marine waters used for swimming. These studies used beaches contaminated by known point sources. A positive correlation was found between E. coli or enterococci and gastroenteritis in fresh waters and between enterococci and gastroenteritis in both fresh and marine waters. Water quality standards have been established using these indicators of fecal contamination. National Pollutant Discharge Elimination System (NPDES) permits and state permitsauthorized by the NPDES program require dischargers to monitor such waters for the parameters included in their permits. Fordischargers into coastal recreational waters, any permits including fecal indicators likely require monitoring for enterococci or E. coli. For continuously disinfected water, such as water in swimming pools and whirlpools, frequent monitoring is forconcentrations of disinfectant (e.g., free and total chlorine, bromine). In many U.S. communities, suitability of such waters is based primarily on hourly (pH, temperature, and disinfectant) or daily (hardness and alkalinity) water-chemistry measurementsrather than frequent monitoring for microorganisms or correlation with predictable health effects. Which parameters are measured and how often depends on the individual state, county, or municipal swimming pool codes. While most swimming pools use chlorine, they may also use bromine or a non-halogen-based system. If chlorine is used, free chlorine typically is monitored hourly (occasionally total chlorine also is monitored).

2. Transmission of Enteric and Non-enteric Pathogens by Recreational Use of Waters

In general, infections and disease associated with recreational water contact fall into two categories: those due to entericpathogens and those due to other (non-enteric) pathogens. Infections in the first category typically cause gastroenteritis symptoms and result from unintentional ingestion of water contaminated with fecal wastes. Enteric microorganisms that have been shown to cause gastroenteritis associated with recreational water contact include Giardia

, Cryptosporidium

, Shigella

, Salmonella

, E. coli O157:H7, Vibrio , Hepatitis A, Coxsackie A and B, andNorovirus. One exception is Adenovirus, which has been reported to cause conjunctivitis in those using swimming pools. 2

Infections in the second category are due to non-enteric pathogens that contaminate water and are transmitted by contact with water. These pathogens generally cause non-enteric diseases andmay be transmitted via inhalation, contact with mucosal membranes or abraded skin. The sources of the pathogens differ. For example, the bacteria that cause Leptospirosis have been found in the urine of infected animals. 3 The source may be from human bodies (e.g., Staphylococcus aureus ) or bacteria indigenous to that water (e.g., Pseudomonas aeruginosa, Legionella spp., Naegleria fowleri, Mycobacterium spp., and Vibrio spp). The illnesses or diseases caused by these non-enteric organisms include dermatitis or folliculitis, otitis externa (inflammation ofthe external ear), leptospirosis, Pontiac fever, granulomas (nodules of chronically inflamed tissue with granulations), primaryamebic meningoencephalitis (PAM), conjunctivitis, and potentially fatal wound infections ( Vibrio vulnificus ). * Approved by Standard Methods Committee, 2007. Joint Task Group: Robin K. Oshiro (chair), Roger S. Fujioka, James Oliver, Joan Shields.

13. Microbiological Monitoring Approaches and Limitations

The following sections describe recommended methods formeasuring pathogens or microbial indicators of recreational water quality. Several key monitoring factors should be considered (e.g., disinfectant residual, indicator organisms, and variousphysical and chemical characteristics). When selecting the microbiological method or indicator to be used, consider the type of water examined. No single procedure is adequate to isolate allmicroorganisms from contaminated water. While bacterial indicators may not adequately reflect risk of viral, fungal, or parasitic infection from recreational waters, lack of feasible methods limits monitoring for such organisms in routine laboratory operations.

Routine examination for pathogenic microorganisms is not recommended except for investigations of water-related illness and special studies. Certain pathogenic organisms, such as Giardia, Cryptosporidium, Mycobacterium, Naegleria, Hepatitis A, and Norovirus are more resistant to changes in environmental conditions than indicator bacteria; therefore, routine monitoringmay not always reflect the risk of infection from these organisms. In the case of water-related illness, focus microbiological analyses on the known or suspected pathogen and on the venue. For instance, if the venue is a disinfected swimming pool and symptoms include gastrointestinal distress, the initial focus should be on such organisms as Cryptosporidium, Giardia, and Norovirus. If the venue is heated whirlpools or spas where the issue is folliculitis or respiratory illness, the focus should be on P. aeruginosa and Legionella , which are more likely to be found. Methods for detecting several of these pathogens are given in Section 9260, Detection of Pathogenic Bacteria, Section 9510, Detection of Enteric Viruses, and Section 9711, Pathogenic Protozoa.

4. References

1. D UTKA , B.J. 1973. Coliforms are an index of water quality. J. Environ. Health 36:39. 2. AMERICAN WATER W ORKS A SSOCIATION . 1999. Waterborne Pathogens. AWWA Manual M48, American Water Works Assoc., Denver, Colo. 3. FAINE , S. 1994. Leptospira and Leptospirosis. CRC Press Inc., Boca Raton, Fla.

5. Bibliography

C ABELLI , V.J. 1977. Indicators of recreational water quality. In Bacterial Indicators/Health Hazards Associated with Waters. STP 635, American Soc. Testing & Materials, Philadelphia, Pa. D UFOUR , A.P. 1986. Diseases caused by water contact. In Waterborne Diseases in the United States. CRC Press Inc., Boca Raton, Fla. M OE , C.L. 1996. Waterborne transmission of infectious agents. In Manual of Environmental Microbiology. American Soc. Microbiology, ASM Press, Washington, D.C.



## 9213 B. Swimming Pools

1. General Discussion

a. Characteristics: A swimming pool is a body of water of limited size contained within a holding structure. 1 Most public swimming pools use potable fresh water and are designed to befiltered and disinfected continually to maintain high water quality. However, some public swimming pools use natural sources of water, including thermal springs and coastal marine waters, and rely on continuous circulation to maintain water quality. These natural water sources generally are not disinfected.

b. Monitoring requirements: 1) General—Monitor water quality in pools for changes in chemical and physical characteristics that may irritate a bather’s skin, eyes, and mucosal barriers or adversely affect disinfection. Microorganisms of concern typically are those from the bather’s body and its orifices and include those causing infections of theeye, ear, upper respiratory tract, skin, and intestinal or genitourinary tracts. Accidental release of feces into swimming pools occurs and is a serious source of contamination because the volume of water is restricted and exposure of other swimmers is highly likely. This kind of contamination in a swimming pool has been reported to be responsible for transmission of such microorganisms as Cryptosporidium

, Giardia , Norovirus, and E. coli O157:H7. 2 Under these conditions, all fecal-borne pathogens may be transmitted to other swimmers. Water quality depends on the disinfection efficacy, sanitary conditions, filtration efficacy, the number of bathers in the pool at any one time, and the total number of bathers per day. While the following describes various monitoring and sampling protocols, individual localities and states have their own pool codes that must be complied with.

2) Disinfected indoor pools—Swimming pools should be disinfected continuously when in use. Test swimming pool water for total and free chlorine (to calculate combined) and pH when the pool is initially opened and at least 3 times/d. Collect samples from at least two locations for these determinations. Evaluate clarity of the swimming pool water before opening for the day and during periods of heavy use. 3 The heterotrophic plate count is the primary indicator of disinfection efficacy. Indicators of health risk include normal skin flora that are shed, such as Pseudomonas and Staphylococcus

. 4 –7 These organisms account for a large percentage of swimming-pool-associated illnesses. Take samples for microbiological examination while the pool isin use. The American Public Health Association (APHA) recommends that not more than 15% of samples collected for publicswimming pools during any 30-d period shall have a heterotrophic plate count of 200/mL or show a positive confirmed total coliform test in any of five 10-mL portions of sample examined via the multiple-tube fermentation test or more than 1 total coliform/50 mL when the membrane filter test is used. Whenever swimming pool samples are examined for total staphylococci or Staphylococcus aureus , not more than 50 organisms/100 mL should be present. 3

3) Disinfected outdoor pools—As well as the disinfectant levels (if chlorine, total and free) and pool chemistry, the level of RECREATIONAL WATERS (9213)/Swimming Pools

2cyanuric acid or stabilizer (if used and required by appropriatepool code) may be monitored. Thermotolerant coliforms (formerly known as fecal coliforms) and Pseudomonas species are the primary indicators of contamination from pets, rodents, stormwater runoff, and humans. Supporting indicators include coliform bacteria, heterotrophic plate count, and staphylococci.

4) Untreated pools—The primary indicator may be thermotolerant coliforms. Supporting indicators are those described for disinfected pools. Untreated pools are not recommended for recreational use because of increased health risks.

2. Samples

a. Containers: Collect samples for bacteriological examination of swimming pool water as directed in Section 9060A. Use containers with capacities of 120 to 480 mL, depending on analyses to be made. Add sufficient sodium thiosulfate (Na2 S 2 O 3 ) to the sample to provide a concentration of approxi

mately 100 mg/L in the sample. Do this by adding 0.1 mL of 10% solution of Na2 S 2 O 3 to a 120-mL bottle or 0.4 mL to a 480-mL bottle. After adding Na2 S 2 O 3 , stopper or cap and ster

ilize container.

b. Sampling procedure: Collect samples during periods of maximum bather load. Information on number of bathers may be helpful in subsequent interpretation of laboratory results. Usesampling frequency consistent with state and local health regulations.

Collect samples by carefully removing cap of a sterile sample bottle and holding bottle near the base at an angle of 45°. Do not rinse bottle (i.e., retain sodium thiosulfate). Fill in one slow sweep down through the water, with the mouth of the bottle always ahead of the hand. Avoid contamination of the sample by floating debris. Replace cap. For pools equipped with a filter, samples may be collected from sampling ports provided in the filter’s return and discharge lines.

Most bacteria shed by bathers are in body oils, saliva, and mucus discharges that occur near the surface; collect additional samples of the surface microlayer from the area in 1-m-deep water. Collect microlayer samples by plunging a sterile glass plate (approximately 20  20 cm) vertically through the water surface and withdrawing it upward at a rate of approximately 6 cm/s. Remove surface film and water layer adhering to both sides of plate with a sterile silicone rubber scraper and collect in a sterile glass bottle. Repeat until desired volume is obtained. To minimize microbial contamination, wrap glass plate and scraper in metal foil and sterilize via autoclaving before use. Wear sterile rubber or plastic gloves during sampling or hold glass plate with forceps, clips, or tongs.

Determine pH, temperature, total and free chlorine or other disinfectant levels at pool side at the time of sample collection (see Section 4500-Cl.G). Disinfectant levels and chemical and physical quality of pool water should be consistent with local, state, or APHA standards. The permissible bathing load should adhere to local or state pool codes, or APHA-recommendations.

c. Sample storage (holding time): Analyze microbiological samples as soon as possible after collection (see Section 9060B).

d. Sample storage temperature: Less than 10°C, but not frozen.

e. Sample volume: See Section 9222B.5. f. Sample dilution: If sample dilutions are required, use 0.1% peptone water or buffered dilution water as diluent to optimizerecovery of stressed organisms (see Section 9222B.5.a for suggested sample volume). Because peptone water has a tendency tofoam, avoid air bubbles when pipetting to ensure accurate measure.

3. Heterotrophic Plate Count

Determine the heterotrophic plate count as directed in Section 9215. Use at least two plates per dilution.

4. Tests for Total Coliforms

Determine total coliform bacteria as directed in Sections 9221, 9222, or 9223.

5. Tests for Thermotolerant Coliforms

Test for thermotolerant coliforms according to the multipletube fermentation technique (Section 9221), the membrane filter technique (Section 9222), or rapid methods (Section 9211).

6. Test for Staphylococci or Staphylococcus aureus

a. Single-plate procedure:

1) Baird-Parker agar base:
Tryptone................................................................................. 10.0 g Beef extract ........................................................................... 5.0 g Yeast extract.......................................................................... 1.0 g Glycine .................................................................................. 12.0 g Sodium pyruvate ................................................................... 10.0 g Lithium chloride.................................................................... 5.0 g Agar....................................................................................... 20.0 g Reagent-grade water ............................................................... 1 L Sterilize via autoclaving. Cool to 50°C and aseptically add 50 mL commercial egg yolk tellurite enrichment/L. Mix well. Final pH should be 7.0 0.2. 2) Procedure: Use membrane filter technique to prepare samples. Be certain to use 0.22-  m filters, because it has been reported that chlorine-stressed S. aureus will contract and can slip through 0.45-  m membranes. 8 Place membrane filter on Baird-Parker agar and incubate at 35 0.5°C for 48 4 h. Staphylococci typically form slate-gray to jet-black, smooth, entire colonies. If S. aureus is present, egg yolk clearing may be observed if the membrane filter is raised from the medium. Verify some differentiated colonies with a commercial multi-test system or on the basis of such key characteristics as catalasereaction, coagulase production, aerobic and anaerobic acid production from certain carbohydrates, and typical microscopic morphology.

b. Two-plate procedure : Alternatively, a two-plate method may be used, which will also help recover chlorine-stressed S. aureus . As above, use membrane filter technique to prepare samples. Place membrane filters on R2A agar (Section 9215A.6 c ) and incubate at 37 0.5°C for 24 h. Aseptically transfer the membranes to Baird-Parker agar and incubate at 35 0.5°C for another 24 h. Random and atypical colonies can be verified as described in ¶ 2) above. RECREATIONAL WATERS (9213)/Swimming Pools

3c. Modified multiple-tube procedure: 1) M-staphylococcus broth:
Tryptone............................................................................. 10.0 g Yeast extract ....................................................................... 2.0 g Lactose ................................................................................ 2.0 g Mannitol ............................................................................ 10.0 g Dipotassium hydrogen phosphate, K2HPO 4 ...................... 5.0 g Sodium chloride, NaCl...................................................... 75.0 g Sodium azide, NaN 3 ............................................................ 0.049 g Reagent-grade water ........................................................... 1 L Sterilize by boiling for 4 min; pH should be 7.0 0.2. For 10-mL inocula, prepare and use double-strength medium. 2) Lipovitellenin-salt-mannitol agar: This medium may not be available in dehydrated form and may require preparation from the basic ingredients or by adding egg yolk to a dehydrated base.
Beef extract......................................................................... 1.0 g Polypeptone ....................................................................... 10.0 g Sodium chloride, NaCl...................................................... 75.0 g

D -Mannitol ......................................................................... 10.0 g Agar ................................................................................... 15.0 g Phenol red............................................................................ 0.025 g Egg yolk ............................................................................ 20.0 g Reagent-grade water ........................................................... 1 L Sterilize via autoclaving; pH should be 7.4 0.2. 3) Procedure: Inoculate tubes of M-staphylococcus broth as directed in Section 9221. Incubate at 35 1°C for 24 h. Hold original enrichment sample but streak from positive (turbid) tubes on plates of lipovitellenin-salt-mannitol agar and incubate at 35 1°C for 24 h. Opaque (24 h), yellow (48 h) zones around the colonies are positive evidence of lipovitellenin-lipase activity (opaque) and mannitol fermentation (yellow).

If the plate is negative, streak another plate from the original enrichment tube before discarding. Lipovitellenin-lipase activity has a 95% positive correlation with coagulase production. Ifnecessary, confirm positive isolates as catalase-positive, coagulase-positive, fermenting mannitol, fermenting glucose anaerobically, yielding typical microscopic morphology, and grampositive.

7. Tests for Pseudomonas aeruginosa

Tests for P. aeruginosa are presented in Sections 9213E and F and include a membrane filter procedure and a multiple-tube technique. 8. References

1. C ENTERS FOR D ISEASE C ONTROL . 1983. Swimming Pools—Safety and

Disease Control through Proper Design and Operation. DHHS— CDC No. 83-8319, Centers for Disease Control, Atlanta, Ga. 2. C RAUN , G.F., R.L. C ALDERON & M.F. C RAUN . 2005. Outbreaks associated with recreational water in the United States. Internat. J. Environ. Health. Res. 15:243. 3. AMERICAN PUBLIC HEALTH A SSOCIATION . 1981. Public SwimmingPools. Recommended Regulations for Design and Construction, Operation and Maintenance. American Public Health Assoc., Washington, D.C. 4. S EYFRIED , P.L., R.S. T OBIN , N.E. B ROWN & P.F. NESS . 1985. A prospective study of swimming-related illness. II. Morbidity and the microbiological quality of water. Amer. J. Pub. Health 75:1071. 5. KLAPES , N.A. & D. V ESLEY . 1988. Rapid assay for in situ identification of coagulase-positive staphylococci recovered by membrane filtration from swimming pool water. Appl. Environ. Microbiol. 52: 589. 6. C OVERT , T.C. & P.V. S CARPINO . 1987. Comparison of Baird-Parkeragar, Vogel-Johnson agar, and M-Staphylococcus broth for the isolation and enumeration of Staphylococcus aureus in swimming pool waters. Abstr. Annu. Meeting American Soc. Microbiology, Atlanta, Ga., American Soc. Microbiology, Washington, D.C. 7. C HAROENCA , N. & R.S. FUJIOKA . 1995. Association of staphylococcal skin infections and swimming. Water Sci. Technol. 32:11. 8. ABID , N., S. M AALEJ & S. R OUIS . 2004. Morphological and physiological changes of Staphylococcus aureus exposed to hypochlorous acid. Lett. Appl. Microbiol . 38:245-25.

9. Bibliography

W ORKING PARTY OF THE PUBLIC HEALTH LABORATORY S ERVICE . 1965. A bacteriological survey of swimming baths in primary schools. Monthly Bull. Min. Health & Pub. Health Lab. Serv. 24:116. GUNN , B.A., W.E. D UNKELBERG , J R . & J.R. C RUTZ . 1972. Clinical evaluation of 2% LSM medium for primary isolation and identification of staphylococci. Amer. J. Clin. Pathol . 57:236. HATCHER , R.F. & B.C. PARKER . 1974. Investigations of Freshwater Surface Microlayers. VPI-SRRC-BULL 64. Virginia Polytechnic Inst. and State Univ., Blacksburg. U.S. ENVIRONMENTAL PROTECTION A GENCY . 1985. Test Methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure. EPA-600/4-85/076. HURST , C.J. 1991. Disinfection of drinking water, swimming-pool-water and treated sewage effluent. In S.S. Block. Disinfection, Sterilization and Preservation, 4th ed. Lea & Febiger, Philadelphia, Pa.



## 9213 C. Whirlpools

1. General Discussion

a. Characteristics: A whirlpool is a shallow pool with a maximum water depth of 1.2 m; it has a closed-cycle watersystem, a heated water supply, and usually a hydrojet recirculation system. It may be constructed of plastic, fiberglass, redwood, or epoxy-lined surfaces. Whirlpools are designed for recreational as well as therapeutic use and may accommodate one or more bathers. These pools usually are not cleaned, drained, and refilled after each use. They are located in homes, apartments, hotels, athletic facilities, rehabilitation centers, and hospitals.

b. Monitoring requirements : Whirlpool-associated infections are common because of the whirlpool’s inherent design andcharacteristics, which include high temperature, reduced disinfection efficacy, and increased organic material. All these factors RECREATIONAL WATERS (9213)/Whirlpools

4contribute to favorable conditions for the growth of microorganisms, especially Pseudomonas aeruginosa . Thus, frequent testing for disinfectant levels and pH, along with scheduled maintenance, is necessary for safe whirlpool water quality. 1–5 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. Theyinclude a membrane filter procedure and a multiple-tube technique. Methods for Legionella can be found in Section 9260J.

c. Microbiological indicators: The primary indicator of disinfection efficacy is P. aeruginosa , with total coliforms, heterotrophic plate count, and staphylococci as supporting indicators of water quality. The standard index of water quality (i.e., totalcoliforms) may be insufficient to judge the microbiological quality of whirlpool water. Pseudomonas aeruginosa is frequently isolated from whirlpool water that is coliform-negative. 6 In the event of a whirlpool-associated outbreak, collect samples as soon as possible. Analyze for the suspected pathogen and P. aeruginosa

.

d. Sample preservation: Examine samples as soon as possible after collection. See Section 9060B. 2. References

1. C ENTERS FOR D ISEASE C ONTROL . 1981. Suggested Health and Safety

Guidelines for Public Spas and Hot Tubs. DHHS-CDC #99-960. U.S. Government Printing Office, Washington, D.C. 2. S OLOMON , S.L. 1985. Host factors in whirlpool-associated Pseudomonas aeruginosa skin disease. Infect. Control 6:402. 3. H IGHSMITH , A.K., P.N. LEE , R.F. KHABBAZ & V.P. M UNN . 1985. Characteristics of Pseudomonas aeruginosa isolated from whirlpools and bathers. Infect. Control 6:407. 4. GROOTHUIS , D.G., A.H. HAVELAAR & H.R. V EENENDAAL . 1985. A note

on Legionella in whirlpools. J. Appl. Bacteriol . 58:479. 5. H IGHSMITH , A.K. & M.S. FAVERO . 1985. Microbiological aspects of public whirlpools. Clin. Microbiol. Newsletter 7:9. 6. HALL , N. 1984. Whirlpools and Pseudomonas aeruginosa. UHL Lab Hotline 21:9.

3. Bibliography

GELDREICH , E.E., A.K. H IGHSMITH & W.J. M ARTONE . 1985. Public whirlpools—the epidemiology and microbiology of disease. Infect. Control 6:392.



## 9213 D. Natural Bathing Beaches

1. General Discussion

a. Characteristics: A natural bathing beach is any area of a stream, lake, 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 marinerecreational waters contaminated by point sources, such as sewage and industrial wastes, and non-point sources, such as streams, storm drains, and animals (e.g., birds and bathers). 1,2 Contaminating microorganisms may include enteric pathogens, such as Salmonella, Shigella, enteroviruses, protozoa, and “opportunists,” such as P. aeruginosa
, Klebsiella spp., and Aeromonas hydrophila, which can multiply in recreational waters withsufficient nutrients. Other organisms of concern are those associated with the skin, mouth, or nose of bathers, such as Staphylococcus aureus and other naturally occurring organisms (e.g., nontuberculous mycobacteria, Vibrio spp., and Naegleria ). 3–9

b. Monitoring requirements: Historically, thermotolerant coliforms 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 have demonstrated that E. coli and enterococci showed a stronger correlation with swimming-associated gastroenteritis than do thermotolerant coliforms, and that both indicators wereequally acceptable for monitoring fresh-water quality. For marine waters, E. coli and other enteric bacteria have now beendocumented in numerous studies to enter the viable but nonculturable state in response to such stresses as elevated osmotic levels, so the inability to isolate such indicator organisms may not prove a lack of fecal pollution in such waters. In marine waters, enterococci concentration had the strongest relationship to the gastroenteritis incident rate. The recommended densities of these indicator organisms were calculated to approximate thedegree of protection previously accepted for thermotolerant coliforms. EPA-recommended water quality criteria are based on these findings. 10 While the primary indicators of water quality

are E. coli and enterococci, the enumeration of P. aeruginosa
, Aeromonas hydrophila

, Klebsiella spp., and Staphylococcus aureus 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

a. Containers: Collect samples as directed in Section 9060A. Select container size according to the number and variety of tests to be performed. Adding Na2 S 2 O 3 to the bottle is unnecessary.

b. Sampling procedure: Collect samples 0.3 m below the water surface in the areas of greatest bather load. Samples may be taken ankle deep (at
 0.075 m below water surface). In deeper waters, if desired, take another sample approximately 0.075 m below the water surface. This area may be somewhere between the knees and the chest, depending on how deep the water is where the sample is taken. Take samples over the range of environmental and climatic conditions, especially during times when maximal pollution can be expected (i.e., periods of tidal, current, and wind influences; stormwater runoff; and wastewater treatment bypasses). See 9213B.2 b for methods of RECREATIONAL WATERS (9213)/Natural Bathing Beaches

5sample collection and Section 9222 for suggested sample volumes.

c. Sample storage: See Section 9060B.

3. Tests for Escherichia coli

a. Media: Media described in this section are available commercially. Manufacturer’s instructions should be followed for storage and disposal after preparation. The formulation may be available commercially for use in the multiple-tube procedure, the multi-well procedure, or the presence–absence procedure. The need for good quality assurance and uniformity requires the use of a commercial substrate media.

1) mTEC agar: * 2

Proteose peptone No. 3 ......................................................... 5.0 g Yeast extract .......................................................................... 3.0 g Lactose................................................................................. 10.0 g Sodium chloride, NaCl.......................................................... 7.5 g Dipotassium phosphate, K2HPO 4 ......................................... 3.3 g Monopotassium phosphate KH 2PO 4 .................................... 1.0 g Sodium lauryl sulfate ............................................................ 0.2 g Sodium desoxycholate............................................................0.1 g Bromcresol purple ................................................................. 0.08 g Bromphenol red..................................................................... 0.08 g Agar..................................................................................... 15.0 g Reagent-grade water .............................................................. 1 L Sterilize via autoclaving; pH should be 7.3 0.2. Pour 4 to 5 mL liquefied agar into culture dishes (50  100 mm). Store in refrigerator.

2) Urea substrate: * 2

Urea ...................................................................................... 2.0 g Phenol red .......................................................................... 10 mg Reagent-grade water ........................................................ 100 mL 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 (see Section 9222), place membrane on mTEC agar, incubate at 35 0.5°C for 2 h to rejuvenate injured or stressed bacteria, and then incubate at 44.5 0.2°C for 22 h. Transfer filter to a filter pad saturated with urea substrate. After 15 min, count yellow oryellow-brown colonies using a fluorescent lamp and a magnifying lens. Verify a portion of these differentiated colonies via a commercial multi-test system [see Section 9222B.5 f

2)b)]. b. Modified mTEC method :

1) Medium—Modified mTEC agar: Prepare in same mannerand with same ingredients as for mTEC agar, but omit bromcresol purple and bromphenol red and add 0.05 g 5-bromo-6- chloro-3-indoxyl- - D -glucuronide chromogen. Sterilize via autoclaving; final pH should be 7.3 0.2. Pour 4 to 5 mL liquefied agar into culture dishes (50  40 mm). Store in refrigerator. 2) Procedure—Filter sample through a membrane filter (seeSection 9222), place membrane on modified mTEC agar, incubate at 35 0.5°C for 2 h to rejuvenate injured or stressed bacteria, and then incubate at 44.5 0.2°C for 22 h. After incubation, count red or magenta colonies (E. coli) using a fluorescent lamp and a magnifying lens. Verify a portion of these differentiated colonies via a commercial multi-test system [see Section 9222B.5 f

2)b)]. c. Enzyme Substrate test: See Section 9223B.

4. Tests for Enterococci

Perform tests for enterococci using the multiple-tube technique (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 procedures 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.6). 11 This method adds 0.005% sodium azide to Tellurite Glycine Agar (TGA) or Vogel-Johnson Agar (VJA) as an additional inhibitor to increase the selectivity of these media to enumerate concentrations of total staphylococci and S. aureus from natural coastal marine waters. TGA and VJA use the same basic ingredients (lithiumchloride, glycine, and tellurite) as selective agents against nonstaphylococci bacteria, which form black target colonies on these media. However, supplementing TGA or VJA 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 bacteria found in freshwater streams. All black colonies are presumptive counts oftotal staphylococci colonies and must be verified as total staphylococci or S. aureus by further testing Gram staining (grampositive cocci), catalase (positive), or lysostaphin (positive) to confirm as total staphylococci and via coagulase (positive) to confirm as S. aureus

.

9. References

1. C ABELLI , V.J. 1980. Health Effects Criteria for Marine Recreational

Waters. EPA-600/1-80-031, U.S. Environmental Protection Agency, Research Triangle Park, N.C. 2. D UFOUR , A.P. 1984. Health Effects Criteria for Fresh Recreational Waters. EPA-600/1-84-004, U.S. Environmental Protection Agency, Research Triangle Park, N.C. * Difco or equivalent. RECREATIONAL WATERS (9213)/Natural Bathing Beaches

63. KESWICK , B.H., C.P. GERBA & S.M. G OYAL . 1981. Occurrence of enteroviruses in community swimming pools. Amer. J. Pub. Health 71:1026. 4. D UTKA , B.J. & K.K. KWAN . 1978. Health indicator bacteria in water surface microlayers. Can. J. Microbiol . 24:187. 5. C ABELLI , V.J., H. KENNEDY & M.A. LEVIN . 1976. Pseudomonas aeruginosa and fresh recreational waters. J. Water Pollut. Control Fed. 48:367. 6. S HERRY , J.P., S.R. KUCHMA & B.J. D UTKA . 1979. The occurrence of Candida albicans in Lake Ontario bathing beaches. Can. J. Microbiol . 25:1036. 7. S TEVENS , A.R., R.L. T YNDALL , C.C. C OUTANT & E. WILLAERT . 1977.Isolation of the etiological agent of primary amoebic meningoencephalitis from artificially heated waters. Appl. Environ. Microbiol

. 34:701. 8. WELLINGS , F.M., P.T. AMUSO , S.L. C HANG & A.L. LEWIS . 1977. Isolation and identification of pathogenic Naegleria from Florida lakes . Appl. Environ. Microbiol . 34:661. 9. N’D IAYE , A., P. GEORGES , A. N’G O & B. FESTY . 1985. Soil amoebas as biological markers to estimate the quality of swimming pool
waters. Appl. Environ. Microbiol . 49:1072. 10. U.S. ENVIRONMENTAL PROTECTION A GENCY . 1986. Ambient WaterQuality Criteria for Bacteria—1986. EPA-440/5-84-002, U.S. Environmental Protection Agency, Washington, D.C. 11. C HAROENCA , N. & R. FUJIOKA . 1993. Assessment of Staphylococcus bacteria in Hawaii’s marine recreational waters. Water Sci. Technol

. 27:283.

10. Bibliography

O LIVIERI , V.P., C.W. D RUSE & K. KAWATA . 1977. Microorganisms inUrban Stormwater. EPA-600/2-77-087, U.S. Environmental Protection Agency, Cincinnati, Ohio. RICE , E.W., T.C. C OVERT , D.K. WILD , D. B ERMAN , S.A. J OHNSON & C.H. J OHNSON . 1993. Comparative resistance of Escherichia coli and Enterococci to chlorination. J. Environ. Health A28:89.



## 9213 E. Membrane Filter Technique for Pseudomonas aeruginosa

1. 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.

L -Lysine HCl ......................................................................... 5.0 g Sodium chloride, NaCl.......................................................... 5.0 g Yeast extract .......................................................................... 2.0 g Xylose .....................................................................................2.5 g Sucrose................................................................................... 1.25 g Lactose................................................................................... 1.25 g Phenol red.............................................................................. 0.08 g Ferric ammonium citrate....................................................... 0.8 g Sodium thiosulfate, Na2 S 2 O 3 ................................................ 6.8 g Agar ..................................................................................... 15.0 g Reagent-grade water.............................................................. 1 L
Adjust pH to 6.5 0.1 and sterilize via autoclaving. Cool to 55 to 60°C; readjust pH to 7.1 0.2 and add the following dryantibiotics per liter of agar base: sulfapyridine,* 176 mg; kanamycin,* 8.5 mg; nalidixic acid,* 37.0 mg; and cycloheximide,* 150 mg. After mixing, dispense in 3-mL quantities in 50  12 mm petri plates. Store poured plates at 2 to 8°C. Discard unused medium after 1 month.

b. Modified M-PA agar† c. Milk agar (Brown and Scott Foster Modification): Mixture A:

Instant nonfat milk ‡ ............................................................ 100 g Reagent-grade water ........................................................... 500 mL

Mixture B:

Nutrient broth .................................................................... 12.5 g Sodium chloride, NaCl........................................................ 2.5 g Agar .................................................................................... 15.0 g Reagent-grade water......................................................... 500 mLSeparately prepare and sterilize Mixtures A and B; cool rapidly to 55°C; aseptically combine mixtures and dispense 20 to 25 mL per petri dish.

3. Procedure

a. Presumptive tests : Filter 200 mL or less of natural waters orup to 500 mL of swimming pool waters through sterile membrane filters. Place each membrane on a poured plate of modified M-PA agar so there is no air space between the membrane and the agar surface.

Invert plates and incubate at 41.5 0.5°C for 72 h. Typically, P. aeruginosa colonies are 0.8 to 2.2 mm in diameter and appear flat with light outer rims and brownish to greenish-black centers. Count typical colonies, preferably from filters containing 20 to 80 colonies. Use a 10- to 15-power magnifier as an aid in colony counting.

* Sigma Chemical Co., St. Louis, MO, or equivalent.† Commercially available as M-PA-C agar. Contains magnesium, sulfate, kanamycin, and nalidixic acid. ‡ Carnation or equivalent. RECREATIONAL WATERS (9213)/Membrane Filter Technique for Pseudomonas aeruginosa

7A two-plate method also may be used; this method will help recover chlorine-stressed P. aeruginosa (e.g., from swimming pools and whirlpools). Use membrane filter technique to prepare samples. Place membrane filters on R2A agar (Section 9215A) and incubate at 37 0.5°C for 24 h. Aseptically transfer the membranes to M-PA agar and incubate at 35 0.5°C for another24 h. Random and atypical colonies can be verified using confirmation tests.

b. Confirmation tests : Use milk agar to confirm a number of typical and atypical colonies. Make a single streak (2 to 4 cm long) from an isolated colony on a milk agar plate and incubate at 35 1.0°C for 24 h. P. aeruginosa hydrolyzes casein and produces a yellowish to green diffusible pigment.

4. Interpretation and Calculation of Density

Confirmation is not routinely required. In the absence of confirmation, report results as the number of presumptive P. aeruginosa/100 mL. 5. Bibliography

D RAKE , C.H. 1966. Evaluation of culture media for the isolation and enumeration of Pseudomonas aeruginosa. Health Lab. Sci . 3:10. B ROWN , M.R.W. & J.H. S COTT F OSTER . 1970. A simple diagnostic milk medium for Pseudomonas aeruginosa. J. Clin. Pathol . 23:172. LEVIN , M.A. & V.J. C ABELLI . 1972. Membrane filter technique for enumeration of Pseudomonas aeruginosa. Appl. Microbiol . 24:864. D UTKA , B.J. & K.K. KWAN . 1977. Confirmation of the single-step membrane filter procedure for estimating Pseudomonas aeruginosa densities in water. Appl. Environ. Microbiol . 33:240. B RODSKY , M.H. & B.W. C IEBIN . 1978. Improved medium for recovery and enumeration of Pseudomonas aeruginosa from water using membrane filters . Appl. Environ. Microbiol . 36:26.



## 9213 F. Multiple-Tube Technique for Pseudomonas aeruginosa

1. Laboratory Apparatus

See Section 9221.

2. Culture Media

a. Asparagine broth: This medium may not be available in dehydrated form and may require preparation from the basic ingredients.
Asparagine, DL ......................................................................... 3.0 g Anhydrous dipotassium hydrogen phosphate, K2HPO 4 ......... 1.0 g Magnesium sulfate, MgSO 4  7H 2 O........................................ 0.5 g Reagent-grade water................................................................ 1 L Adjust pH to between 6.9 and 7.2 before sterilization. b. Acetamide broth: This medium may not be available in dehydrated form and may require preparation from the basic ingredients.
Acetamide ............................................................................ 10.0 g Sodium chloride, NaCl.......................................................... 5.0 g Anhydrous dipotassium hydrogen phosphate, K2HPO 4 ....... 1.39 g Anhydrous potassium dihydrogen phosphate, KH 2PO 4 ....... 0.73 g Magnesium sulfate, MgSO 4  7H 2 O ..................................... 0.5 g Dissolve 1.2 g phenol red in 100 mL 0.01 N NaOH and add 1 mL/L of acetamide broth. Use phenol red stock solution within 1 year. Adjust pH to between 7.1 and 7.3 before sterilization. Final pH should be 7.0 0.2. Prepare acetamide broth asdescribed above. If agar slants are preferred, prepare as described above but add 15 g agar/L, heat to dissolve agar, and dispense 8-mL quantities in 16-mm tubes. After autoclaving, incline tubes while cooling to provide a large slant surface.

3. Procedure

a. Presumptive test: Perform a five-tube test. Use 10 mL single-strength asparagine broth for inocula of 1 mL or less and 10 mL double-strength asparagine broth for 10-mL inocula. Higher dilutions may be necessary for swimming pools. Incubate inoculated tubes at 35 to 37°C. After 24 h and again after 48 h of incubation, examine tubes under long-wave ultraviolet light(black light) in a darkened room. Production of a green fluorescent pigment constitutes a positive presumptive test.

b. Confirmed test: Confirm 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.

c. Computing and reporting results: Refer to Table 9221:V and Section 9221D. RECREATIONAL WATERS (9213)/Multiple-Tube Technique for Pseudomonas aeruginosa

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