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Sampling

Part 1 – Pond & Stream
1. Fresh water samples were taken from a stream and a pond in Lawrenceville, New Jersey on July 17, 2003. The stream was at the influent end of the pond and was free flowing.
2. Two 240 ml water bottles were collected at a depth of approximately six inches below the surface of the pond adjacent to a small island. Stream collection used six 240 ml water bottles taken from a shallow waterfall area and from the middle of a stream at a depth of two feet near the bottom of the stream.
3. At the site, tests were conducted for temperature, pH, dissolved oxygen, nitrate content, and organic phosphorous.
4. Samples were filtered according to filtration protocol below.

Part 2 – Human Saliva
1. Human saliva samples were donated by ten adults (male and female.)
2. Samples were obtained before individuals brushed their teeth by using their personal toothbrushes, swishing tap water and then spitting into a 100ml sterile, capped tube.
3. Total volume of saliva was approximately 200 ml.
4. Samples were filtered according to filtration protocol below.

Filtration

Materials
1 L side arm Erlenmeyer with filter frit
1.2 ?l Millipore filter
0.2 ?l Millipore filter

1. A clean, DNA-free 1 L side arm Erlenmeyer with a filter frit hooked to a vacuum of approximately 5 psi was used to filter each sample.
2. The first filtration setup used a 1.2 um Millipore filter. As the filtration process slowed the filter was replaced with another 1.2 um filter until no more than 6 filters were collected. These filters trapped particle-bound bacteria and larger bacteria as well.
3. The filtrate obtained from the previous runs was re-filtered using .2um Millipore filters until no more than 6 filters were collected. These filters were used to collect the free-floating bacteria.
4. A quarter section of each filter was removed and frozen to become part of the cryogenic archive at the American Museum of Natural History.