This article outlines a method for isolating bacterial communities associated with C. elegans nematodes. The process involves mechanical disruption of the nematodes, followed by centrifugation and plating on selective agar media to obtain pure bacterial cultures.
Begin with a multi-well plate containing sterile beads and buffer in each well.
Add a C. elegans nematode to each well to isolate its bacterial community.
Using a homogenizer, mechanically disrupt the nematodes to release the bacteria into the buffer.
Centrifuge to separate the cell debris and collect the supernatant containing the bacteria.
Plate the diluted bacterial solution onto selective nutrient agar plates with varying compositions to support the growth of diverse bacterial types.
Incubate the plates to allow bacteria to multiply and form visible colonies.
Using a sterile loop, pick a single, isolated colony and streak it onto a fresh plate of the same medium.
Incubate the plates to allow the growth of pure bacterial colonies.
Inoculate each pure colony into the corresponding liquid media and incubate.
These bacteria grow into pure cultures, each representing a bacterial species naturally associated with C. elegans.
To isolate the bacteria, prepare a 96-well plate with approximately three sterile 1 millimeter beads, 20 microliters of M9 buffer per well, and pipette a single washed nematode to each well with as little liquid as possible. Break up the nematodes as demonstrated before using a bead homogenizer, followed by brief centrifugation of the plate to get the liquid to the bottom. Once done, collect the suspension and serially dilute it at one to ten proportion.
Plate up to 100 microliters of the diluted suspension onto 9-centimeter agar plates. Then incubate the plates at 15 to 20 degrees Celsius for 24 to 48 hours. To obtain the pure bacterial culture, pick a single colony from the incubated plate using a sterile loop or toothpick and streak it onto a new agar plate containing the same agar medium used during the purification. Ensure to only use one-third of the plate.
Then, sterilize a reusable loop or use a new sterile loop and drag it through the first streak to create a second streak on another one-third part of the sample plate. Repeat it by dragging a loop through the second streak and creating the third streak to achieve the single colony growth.
Incubate the plate under the same growth conditions used for isolation, and if required, repeat the purification step. Grow the pure colonies in a liquid medium using the same temperature and growth conditions.
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