Microbial Analyses

Microbial analyses were performed in accordance with accepted methods as summarized in Table 3-1. Microbial parameters included total coliforms, E. coli, enterococci, and coliphages. All microbial analyses were performed using aseptic techniques with autoclaved or pre-sterilized materials and supplies.

3.2.3.1 Total Coliforms and E. coli

Total coliforms and E. coli were enumerated for each sample in accordance with Standard Method 9223, the enzyme substrate test (APHA et al., 2012). This method was performed using Colilert®, a commercially available enzyme-substrate liquid-broth medium that allows the simultaneous detection of total coliforms and E. coli, and the 97-well Quanti-Tray® method (IDEXX Laboratories Inc., Westbrook, ME). Each sample processed was 100 mL. Undiluted samples were composed of 100 mL of sample water, and diluted samples included sample water and buffered water as summarized in Table 3-3. Diluted samples were analyzed when the most probable numbers (MPN) for the undiluted samples were greater than the method upper limit of 2,419 MPN/100 mL. The appropriate volumes of sample water and buffered water were transferred to 250 mL, autoclaved dilution bottles.

Buffered water was used for sample dilutions and for positive and negative controls described below. Buffered water was made according to Standard Method 9050c.1a (APHA et al., 2012), by diluting 5 mL of stock magnesium chloride and 1.25 mL of stock phosphate buffer up to 1 L with reagent grade water. The stock magnesium chloride was made by dissolving 20.275 g of MgCl2·6H2O to a total volume of 250 mL with reagent grade water, and the stock phosphate buffer was made by dissolving 8.5 g of KH2PO4 to a total volume of 125 mL with reagent grade water.

If necessary, pH was adjusted to 7.2 + 0.5 with sodium hydroxide.

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Table 3-3: Microbiological Dilutions

Dilution Volume Sample Water

(mL) Volume Buffered Water (mL)

4X 25 75

10X 10 90

100X 1 99

Once the samples and dilutions were prepared, one Colilert® packet was added to each 100 mL sample, which was then shaken to dissolve the media. The mixture was then poured into a 97 well Tray® consisting of 49 large wells and 48 small wells and sealed in an IDEXX Quanti-Tray Sealer (IDEXX Laboratories Inc., Westbrook, ME). The Quanti-Quanti-Tray was then incubated for 24 hours at 36^oC + 0.5^oC. A yellow color indicates positive for total coliforms and fluorescence under UV light (Entela, UVL-23RW, Upland, CA) in a dark room indicates positive E. coli presence. Positive cells were counted and then compared to an MPN table to determine the MPN/100 mL of total coliforms and E. coli in each sample. The lower limit MPN/100 mL of the test is less than 1 (for 0 positive wells), and the upper limit is greater than 2,419 (for 97 positive wells). Duplicate samples were analyzed for each test including each dilution. The results from the duplicate samples were averaged, and the values were adjusted to account for the dilution of the sample to determine the MPN/100 mL in the original sample as collected.

One positive and one negative control were prepared for total coliforms and E. coli for each sampling event. For the positive control, E. coli (ATCC 11775, American Type Culture Collection, Manassas, VA) was cultured in the laboratory the day before sampling. Tryptic soy broth (TSB; Bacto 211825, Sparks, MD) was prepared by weighing 3 grams of tryptic soy broth powder and dissolving it into 100 mL of reagent grade water. Two 250-mL labeled shaker flasks were filled with 50 mL of TSB and autoclaved. The shaker flasks were allowed to cool and were each inoculated with one loop of frozen (-80^oC) ATCC 11775 E. coli stock. The flasks were then incubated at 36^oC on a shaker table set at 100 revolutions per minute for 18-20 hours. After incubation, 1 mL from one of the two flasks was added to 99 mL of autoclaved buffered water in a dilution bottle. This positive control was enumerated using the 97-well procedure described above. For the negative control, a dilution bottle with 100 mL of buffered water was processed using the 97 well procedure.

3.2.3.2 Enterococci

Enterococci were enumerated for each sample in accordance with Standard Method 9223, the enzyme substrate test (APHA et al., 2012). This method was performed using Enterolert®, a commercially available enzyme-substrate liquid-broth medium that allows the detection of enterococci, and the 97 well Quanti-Tray® method (IDEXX Laboratories Inc., Westbrook, ME).

Samples were processed and analyzed as described in Sub-section 3.2.3.1, except using Enterolert® instead of Colilert®. No positive control was prepared for enterococci; however, a negative control was prepared as described above. For enterococci, fluorescence under UV light (Entela, UVL-23RW, Upland, CA) in a dark room indicates positive enterococci presence. MPN values were calculated as discussed in Sub-section 3.2.3.1.

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3.2.3.3 Coliphages

The single agar layer (SAL) method (EPA Method 1602) was used to enumerate F+, or male-specific, coliphages. Ampicillin and streptomycin resistant E. coli Famp (ATCC 700891, American Type Culture Collection, Manassas, VA) was cultured as the host for the F-specific phages to infect. The host broth used for growing E. coli Famp consisted of 3 g of tryptic soy broth (TSB, Difco Laboratories, Detroit, MI) per 100 mL of reagent grade water, which was autoclaved in 50 or 100 mL volumes. In order to select for the male-specific host bacteria, 150 mg ampicillin sodium salt and 150 mg of streptomycin sulfate (Sigma Chemical Company, St. Louis, MO) were dissolved in 100 mL of reagent grade water. The solution was filter sterilized using a sterile 0.22 μm pore size membrane filter assembly. The filter sterilized antibiotics were added to the TSB at 0.5 mL antibiotics per 50 mL of TSB. Next, one loop of frozen E. coli Famp, stored at -80^oCwas transferred to 50 mL of TSB containing the antibiotics. The inoculated broth was then incubated at 36^oC for 16-18 hours. One mL of this culture was then transferred to a flask containing 100 mL of TSB and 1 mL of antibiotic (streptomycin/ampicillin solution). This flask was then incubated at 36^oC for 4-5 hours to create a log-phase host bacterial culture. After incubation, the log-phase host was placed on ice and used within two hours. Typically, this transfer procedure was used to create up to five log-phase host cultures so that an adequate volume of host was prepared and that the window of time available for phage enumeration was adequate to process all samples.

Agar was prepared prior to sample processing. The agar was a 2X tryptic soy broth (TSB, Difco Laboratories, Detroit, MI) with 1.5 percent agar (Bacto™ agar, Difco Laboratories, Detroit, MI).

The 2X tryptic soy agar (2X TSA) contained 6 g TSB and 1.5 g agar for every 100 mL of reagent grade water. The TSB and agar were added to the reagent grade water, and the solution was heated and stirred until the TSB and agar were dissolved. The agar was then autoclaved at 121^oC for 30 minutes. Next the agar was either transferred to a 48^oC water bath for use or cooled and refrigerated at 4^oC for later use. Refrigerated agar was re-autoclaved prior to use.

Duplicate 100 mL volumes of each sample were aseptically transferred into separate sterile 250 mL screw-cap polypropylene bottles (Nalge Nunc International, Rochester, NY). At least one temperature blank was also made by pouring 100 mL of sample water into another 250 mL screw-cap bottle. Then, 0.5 mL of 4 M MgCl2 (81.4 g of MgCl2 6H2O per 100 mL reagent grade water, autoclaved prior to use) were added aseptically to each bottle. The bottles were then placed in a 48^oC water bath and heated until the temperature blank reached 36^oC. Then, 10 mL of log-phase E. coli Famp were added to each 250 mL sample bottle and the temperature blank bottle. The bottles were returned to the 48^oC water bath and heated until the temperature blank reached 43^oC. The samples were then removed from the 48^oC water bath and placed in a 43^oC water bath.

Ampicillin/streptomycin was then added to the 2X TSA. Two mL of antibiotics were added for each 100 mL of agar. Typically, 550 mL bottles of agar were used, thus 11 mL of ampicillin/streptomycin solution were added. The antibiotics were added along the inside of the bottle to avoid forming bubbles in the agar. The bottle of agar and antibiotics was then rocked gently to mix. Approximately 110 mL of 2X TSA was then added to each sample bottle. The sample bottle was then rocked gently to mix the sample with the agar and poured approximately equally into each of five 150 mm sterile plastic petri dishes (Fisher Scientific, Fair Lawn, NJ). The tops of the petri dishes were left askew for approximately five minutes, allowing the plates to cool

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and solidify. The plates were then covered, stacked upside down, and moved aside to proceed with the remaining samples. The plating method described above was repeated for each sample.

An MS2 positive control and a matrix spike were also plated. The MS2 positive control was created by spiking 100 mL of phage phosphate buffered saline (PBS) solution with a known concentration of MS2 stock coliphage (ATCC 15597-B1, American Type Culture Collection, Manassas, VA). The phage PBS solution was created by dissolving 80 g of NaCl, 2.0 g KH2PO4, 29 g Na2HPO4 · 12H20, and 1.0 g KCl to a total volume of 1 L with reagent grade water. The matrix spike control was created by spiking a 100 mL surface water sample with the same volume of MS2. The positive control and matrix spike were mixed with agar and poured into five plates consistent with the surface water samples.

Also plated were an agar-only (negative) control and a host and agar only (host positive) control.

These controls were prepared in sterile plastic 50 mL centrifuge tubes (Fisher Scientific, Fair Lawn, NJ). The negative control contained 15 mL phage PBS, and the host positive control contained 15 mL phage PBS and 1.5 mL of log-phase E. coli Famp. Both of the control tubes were filled to the 30 mL line with agar, rocked gently, and then poured into individual 150 mm petri dishes.

All plates were inverted and incubated for 16-24 hours at 36^oC. The agar only plate appeared as clear agar with no E. coli or coliphages present. The host positive plate appeared as a clean lawn with visible E. coli present uninterrupted covering the agar in the plate. For samples and positive controls, plaque forming units were counted, and bacterial colony contamination was noted.

Plaque forming units appeared as relatively small, circular clearings in the E. coli lawn.

Due to low detection rates observed for the male specific coliphages, somatic coliphages were measured starting on July 30, 2014 and analysis of male specific coliphages was terminated.

Somatic coliphages were enumerated using the SAL method (EPA Method 1602) consistent with male-specific coliphages. The primary differences to the method were that E. coli CN-13 (ATCC 700609, American Type Culture Collection, Manassas, VA), resistant to nalidixic acid, was cultured as the host, and nalidixic acid sodium salt (1 g/100 mL) was used in the host broth and agar. Also, positive control and matrix spike samples were inoculated with ϕ-X 174 stock coliphage (ATCC 13706-B1, American Type Culture Collection, Manassas, VA).