Isolation and Cultivation of Caenorhabditis elegans-Associated Bacteria

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 us