1. Preparation of buffers and media
2. Preparation of environmental samples (Figure 1)
Figure 1: Isolation of nematodes from substrates. Substrate samples are placed in empty Petri dishes and covered with viscous medium to flush out nematodes. Nematodes are transferred to M9-T and repeatedly washed to remove bacteria from the outside. Individual nematodes can be used for DNA isolation, isolation of associated bacteria, or placed on agar plates to culture worm populations. Figure created with BioRender.com. Please click here to view a larger version of this figure.
3. Isolation of Caenorhabditis nematodes (Figure 1)
4. Preparation of the worms for molecular identification of C. elegans and microbes
5. Isolation and cultivation of nematode-associated bacteria (Figure 2)
Figure 2: Species identification and isolation of individual bacteria. Individual nematodes are broken up using a bead homogenizer, and DNA is isolated for species determination via PCR or sequencing. Alternatively, the broken-up nematode material is serially diluted and plated onto growth medium plates. Plates are incubated until bacterial colonies appear, and single colonies are streaked to new plates to obtain pure cultures. Single colonies of the pure cultures are used to grow liquid bacterial cultures for the preparation of bacterial stocks for long-term storage at -80 °C. Figure created with BioRender.com. Please click here to view a larger version of this figure.
The nematode C. elegans is frequently found in decomposing fruits, such as apples, and also compost samples. In Northern Germany, C. elegans as well as congeneric species (particularly C. remanei but also C. briggsae) are mainly found from September until November2. The nematodes are most commonly found in decomposing plant matter, especially rotting fruits such as apples or pears, and also compost, particularly material that shows a high grade of decomposition. The fruit and compost samples can be taken into the lab (Figure 3A,B), distributed in Petri dishes (Figure 3C,D), and subsequently submerged in a liquid or viscous medium to force the worms to leave the substrate material and swim to the medium's surface (Figure 3E,F). Thereafter, the worms can be collected, transferred onto agar plates or microcentrifuge tubes, and the species identity determined using diagnostic PCRs2,6,7,22.
Figure 3: Preparation of apples for isolation of nematodes. (A,B) Nematodes can be isolated from environmental samples like compost or rotten fruits and additionally be attracted by placing apples on compost. (C,D) The samples are divided, and small pieces are placed in a Petri dish. (E,F) Viscous medium is added to the samples, which usually leads to nematodes leaving the substrate material and swimming to the surface of the medium. Please click here to view a larger version of this figure.
PCR using the C. elegans specific primers nlp30-F and nlp30-R results in a PCR product of 154 bp length for C. elegans and no product for other nematodes (Figure 4). To confirm the successful performance of the PCR, DNA of a laboratory strain such as N2 should be run as positive control concurrently with the DNA of the isolated nematodes. Small amounts of biological material, such as small larval stages, can result in weak PCR bands (Figure 4A). The visibility of the PCR bands can be improved by using more DNA in the PCR (note that this will minimize the amount of DNA left for bacterial 16S rDNA amplicon sequencing, e.g., 2 µL of DNA instead of 1 µL), by applying a larger amount of the PCR product to the gel (e.g., 10-15 µL), or by increasing the number of PCR cycles. In comparison to single worms, worm populations consisting of at least 50 worms usually yield a larger amount of DNA, and the bands in the electrophoresis gel are clearly visible (Figure 4B).
Figure 4: PCR product of C. elegans-specific PCR. (A,B) The C. elegans-specific primers nlp30-F and nlp30-R produce a PCR product of 154 bp length. As an example, PCRs were performed on (A) single worms and (B) worm populations (e.g., 300-500 individuals), all isolated with the described protocol from rotting plant matter from the compost of the Botanical Garden in Kiel, Germany. (A) DNA of single worms and, in particular, of small larval stages often results in weak PCR bands. (B) DNA isolated from worm populations (e.g., 300-500 individuals) results in clearer bands. DNA of C. elegans N2 was used as positive control (+), no template was used as negative control (-). The smears below PCR bands are gel artifacts. Please click here to view a larger version of this figure.
The microbes, which are associated with C. elegans, can be isolated from worms and also the worm-containing substrate samples. Microbe isolation follows general microbiology standards, including isolation of pure cultures from agar plates, their growth in liquid media, and their subsequent characterization (e.g., using the sequences of the 16S ribosomal RNA gene for bacteria). It is critical that the microbes are isolated as pure single colonies from plates, using standard microbiology procedures (Figure 5). This is important because some of the C. elegans-associated bacteria can form biofilms and/or easily aggregate, resulting in multi-species cultures (Figure 5C,D). The purity of the isolated bacterial cultures can be confirmed through the sequences of the 16S rRNA gene, as done for previously isolated C. elegans-associated microbes6,7. The obtained microbes can subsequently be used in experiments with C. elegans.
Figure 5: Bacterial isolates on agar plates. Individual bacterial isolates are streaked out onto agar plates, in this case, tryptic soy agar (TSA) plates, showing as examples an isolate of (A) Pseudomonas lurida, (B) Pseudomonas fluorescens, and an example with several bacterial species, shown as an (C) overview of the entire plate and (D) magnification of a section of this plate, in which distinct colony types are visible. Please click here to view a larger version of this figure.
Table 1: List of primers used in this study Please click here to download this Table.
COMSOL | COMSOL | multiphysics simulation software |
The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode’s microbiota as a basis for future functional research.
The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode’s microbiota as a basis for future functional research.
The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode’s microbiota as a basis for future functional research.