Isolation and Cultivation of Caenorhabditis elegans-Associated Bacteria

Abstract

Source: Petersen, C. et. al., Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans. J. Vis. Exp. (2022)

This video demonstrates the isolation and cultivation of bacterial species associated with Caenorhabditis elegans. A bead-based lysis of C. elegans, followed by selective culturing, enables the isolation of diverse microbial strains for further analysis.

Protocol

1. Isolation and cultivation of nematode-associated bacteria (Figure 1)

1. To isolate bacteria, prepare a 96-well plate with three sterile 1 mm beads, 20 µL of M9 minimal medium salts (M9) buffer per well, and pipette an individual, washed nematode to each well, transferring as little liquid as possible.
   NOTE: Alternatively, worm populations can be washed from the plates with M9-buffer, and ~300 µL of worm-containing M9-buffer can be transferred to 2 mL tubes with 10-15 beads.
2. Break up the nematodes using a bead homogenizer (e.g., bead-beating for 3 min at 30 Hz). Centrifuge the plate or tubes briefly to get the liquid to the bottom (e.g., for 10 s at 8000 x g at room temperature, RT).
   NOTE: The usage of this method led to the isolation of bacterial taxa similar to those revealed by 16S ribosomal DNA (rDNA) amplicon sequencing, suggesting that the described bead-beating leaves most bacterial cells intact.
3. Collect the supernatant, serially dilute it at 1:10, and plate up to 100 µL onto 9 cm agar plates.
   1. To ensure that most bacteria can be cultivated, use a variety of alternative media with different nutrient compositions, including diluted trypticase soy agar (TSA, 1:10 dilution), MacConkey agar, Sabouraud glucose agar, potato dextrose agar, or yeast peptone dextrose agar.
4. Incubate the plates at the average temperature conditions of the sampling location (e.g., temperatures between 15-20 °C for temperate locations) for 24-48 h.
5. Use the standard three-streak technique and a sterile loop to obtain pure bacterial cultures (Figure 1).
   1. Pick a single colony from a plate using a sterile loop or toothpick and streak it out onto a new agar plate containing the same agar medium as used for purification. Make sure to use only roughly 1/3 of the plate.
   2. Either use a new sterile loop or sterilize a reusable loop and drag it through the first streak to create a second streak on another 1/3 of the same plate.
   3. Repeat this step by dragging a sterile loop through the second streak.
   4. Incubate the plate under the same growth conditions used for isolation. This technique must result in single colonies growing in the area of the third streak.
      NOTE: It might be necessary to repeat the purification step multiple times as natural isolates tend to form biofilms and/or aggregates.
6. Grow the pure colonies in a liquid medium (of the same type as the agar medium) using the same temperature and growth time as above (step 1.4)

Results

Figure 1: Species identification and isolation of individual bacteria. Individual nematodes are broken up ...