Water Sampling and Plating Generations (Antimicrobial Group)

Collect whole water at six sites using a Van Dorn water collector for deep (~122.5-123.7 m) samples and 2 L containers for surface samples (~15 cm below the surface).

Set site names and classifications

3. Prefilter whole water using 20μm mesh.

4. Set up peristaltic pump with 3 µm filter

5. Filter approximately 500mL of sample water.

6. Collect the filter in an eppendorf with 1mL of PBS as the particle-associated large fragment

Collect the <3μm filtered water as the free-living (FL) smaller fraction.

For the particle-associated (PA) fraction, vortex tube, then partially remove filter with an edge under the closed cap

Centrifuge at 1500 RPM for 1 minute

Fully remove filter and resuspend pellet

Pipette 10μL of both PA and FL onto separate plates of 4 agar types (TSA, LB, BG11, and LAKE) and spread.

This is the parent generation.

PROPAGATE NEXT GENERATIONS:

From the lawns initially plated, by sterile loop pick up a single colony.

Select a plate of the same media.

Steak for isolation using a three phase streak pattern as shown below, sterilizing the loop in between each quadrant.

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5. Continue until generation 3 is obtained.

6. Use generation 3 for testing.