eAppendix 1
Additional Description of Beach Sand Collection and Analysis
Wet sand samples were collected by pushing the soil auger into the sand at least 8
inches, capped, labeled with a unique alpha-numeric code, sealed in a zip-loc bag, and
transported to the laboratory for processing on ice in a cooler maintained at 1 to 4°C.
Culture-based tests of Enterococcus were performed by distributing 75 g of sand into 300
ml of phosphate buffered rinse/dilution water (pH 7.2), shaking vigorously by hand 50
times, and membrane filtering aliquots of the supernatant following EPA Method 1600.1
One hundred mL of this supernatant was filtered through 0.4 micrometer polycarbonate
membrane filters for qPCR analysis within 6 hours of collection. See the electronic
supplement to Wade et al., (2010) for further details regarding the qPCR assays and the
quantification approach used.2 To ensure consistency across the 2 beaches, polycarbonate
filters were frozen and sent on dry ice by overnight express for analysis by EMSL
Analytical, Inc. Laboratory (Cinnaminson, NJ). For the F+ coliphage analysis, sand
samples were inverted three times to mix the sample, then three aliquots each of 0.33 g,
3.3 g, and 33 g of sand were weighed and placed in 15-ml conical tubes (for 0.33 and 3.3
g samples) or 125-ml bottles (for 33 g samples). Ten ml of 0.5X tryptic soy broth (TSB)
with enrichment supplements (4M magnesium chloride and 100X
streptomycin/ampicillin) and 1% log-phase E. coli Famp host (ATCC # 700891) was
added to the 0.33 g and 3.3 g sand samples, and 50-ml 0.5X TSB with enrichment
supplements and E. coli Famp was added to the 33 g sand samples. Samples were
incubated on their sides on a rocking platform at 75 rpm for 1 hour at 36C  1C, then
held upright without rocking with loosened lids overnight at 36C  1C. After overnight
incubation, sub-samples were collected and centrifuged in 1.5 ml eppendorf tubes at
10,000 x g for 10 min to clarify samples. A 10-l volume from each sand enrichment was
spotted onto pre-poured 0.75 X tryptic soy agar plates containing E. coli Famp host,
allowed to dry, and incubated inverted at 36C  1C overnight.3,4 Lysis zones were
scored as positive or negative and used to calculate a three dilution, three replicate most
probable number expressed per gram dry weight sand (most probable number/g)5. In
addition, a 25-g portion of each sand sample was dried at 100 °C to determine the dry
weight of the sand samples. All measured concentrations of fecal indicators in sand are
reported per g of dry sand.
References
1.
2.
3.
4.
5.
EPA. Method 1600: Enterococci in water by membrane filtration using
membrane-enterococcus indoxyl-beta-D-glucoside agar (mEI). In: Water Oo, ed
Publication EPA 821-R-02-022, United States Environmental Protection Agency,
Washington, DC, 2002.
Wade TJ, Sams E, Brenner KP, Haugland R, Chern E, Beach M, Wymer L,
Rankin CC, Love D, Li Q, Noble R, Dufour AP. Rapidly measured indicators of
recreational water quality and swimming-associated illness at marine beaches: A
prospective cohort study. Environ Health 2010;9(1):66.
EPA. Method 1601: Male-specific (F+) and somatic coliphage in water by twostep enrichment procedure. In: Water Oo, ed Publication EPA 821-R-01-030
United States Environmental Protection Agency, Washington, DC, 2001.
Love DC, Sobsey MD. Simple and rapid F+ coliphage culture, latex
agglutination, and typing assay to detect and source track fecal contamination.
Appl Environ Microbiol 2007;73(13):4110-4118.
Heaney CD, Sams E, Dufour AP, Brenner KP, Haugland R, Wymer L, Chern E,
Love DC, Wing S, Serre M, Seed JR, Noble R, Wade T. Weather and
environmental factors associated with F+ coliphages and fecal indicator bacteria
in beach sand at two recreational marine beaches. Appl Environ Microbiol
Submitted, under review.