Jul 12, 2023
Water Sampling and Plating Generations (Antimicrobial Group)
- Melissa B Duhaime1,
- Aubrey Reed1,
- Nicole Sunshine1,
- Renae Lyons1
- 1University of Michigan
Protocol Citation: Melissa B Duhaime, Aubrey Reed, Nicole Sunshine, Renae Lyons 2023. Water Sampling and Plating Generations (Antimicrobial Group). protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1yd87vr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 09, 2018
Last Modified: July 12, 2023
Protocol Integer ID: 18493
Abstract
Protocol developed by the Antimicrobial Group class project. The goal was to isolate microbes from freshwater lakes that produce antimicrobial compounds in order to study antibiotic production in nature and environmental reservoirs of antibiotic resistance.
Protocol for class project in Microbes in the Wild: Advanced Environmental Microbiology Lab (EEB 447) course at University of Michigan Biological Station and on campus.
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
Sites samples and description
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.
4. Label these plates "generation 1"
5. Continue until generation 3 is obtained.
6. Use generation 3 for testing.