This protocol allows new insights into the natural microbiota of C.elegans. Simultaneously, the isolated microbes can be used in controlled laboratory experiments to characterize microbiota-host interactions and C.elegans biology. This protocol focuses on isolating microbes associated with C.elegans in nature, which are therefore key for understanding the biology of this important model host in a more natural context.
It will be helpful to be familiar with the appearance of Caenorhabditis compared to other nematodes. Pipetting under the dissecting scope can initially be challenging, but practice will be helpful. Begin by collecting the environmental samples like compost or rotten fruits in separate plastic bags, tubes, or clean containers.
Evenly spread the pieces of the collected environmental samples in a sterile nine centimeter empty Petri dish and add approximately 20 milliliters of sterile viscous medium to cover the sample with the medium. Under the dissecting microscope, search for Caenorhabditis nematodes following the guidelines on isolating Caenorhabditis elegans and related nematodes. Using a 20 to 100 microliter pipette, collect the smallest possible amount of liquid containing the Caenorhabditis nematodes from the plate and transfer it to the three centimeter Petri dish containing one to three milliliters of sterile M9T.
To establish a worm population, the individual worms can be transferred onto a plate with a nematode growth medium or NGM. To wash the nematodes and remove the microbes present outside the nematodes, incubate them in M9T for 10 to 15 minutes. Pipette out the smallest possible amount of liquid containing the nematodes and transfer it to a new sterile three centimeter Petri dish containing one to three milliliters of fresh M9T.
For unbiased identification of nematode-associated microbes, prepare a 96 well-plate with approximately three sterile beads of one millimeter diameter and add a mixture of 19.5 microliters of PCR buffer and 0.5 microliters of proteinase K per well. Transfer a single washed nematode to each well with as little liquid as possible. Break up the transferred nematodes using a bead homogenizer for three minutes at 30 hertz and centrifuge the plates or tubes at 8, 000 G for 10 seconds at room temperature to get the liquid to the bottom.
To identify C.elegans, heat the samples in a PCR cycler for one hour at 50 degrees Celsius and 15 minutes at 95 degrees Celsius to isolate DNA of individual nematodes, or use commercial kits to isolate DNA of worm populations. Freeze the isolated DNA at minus 20 degrees Celsius for long-term storage. Use the DNA and the primer pair NLP 30 forward and NLP 30 reverse and produce a 154 base pair PCR product.
To isolate the bacteria, prepare a 96-well plate with approximately three sterile one 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 10 proportion.
Plate up to 100 microliters of the diluted suspension onto nine 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 1/3 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 1/3 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. Next, characterize the bacteria by extracting the bacterial DNA from pure liquid cultures amplifying the 16S ribosomal RNA using 27 forward and 1495 reverse primers and performing the PCR as described in the text.
When the collected fruit and compost samples were distributed in Petri dishes and submerged in a liquid or viscous medium, it resulted in the worms leaving the substrate material and swimming to the medium's surface. The microbes were isolated as pure single colonies and the purification played an essential role as some C.elegans associated bacteria can form biofilms or easily aggregate, resulting in multi-species cultures. The PCR using the C.elegans specific primers, namely NLP 30 forward and NLP 30 reverse, resulted in the amplification of a 154 base pair product for C.elegans.
The DNA isolated from single worms resulted in weak PCR bands, whereas DNA extraction from the worm populations consisting of at least 50 worms yielded a larger amount and clearer bands. The success of C.elegans specific PCR was confirmed by running the DNA of a laboratory strain namely N2 as a positive control simultaneously with the DNA of the isolated nematodes Microbes are everywhere. Hence, it is essential to work under sterile conditions to ensure that those microbes that are isolated are truly associated with the worm in nature.
The isolated microbes can be used to study host-microbiota interactions or the role of microbes on host biology, for example, aging, development, or immunity in controlled laboratory experiments. Microbes isolated with this technique are currently used by the C.elegans community to improve the understanding of microbiota diversity on host fitness or the role of microbes on immunity.
