Culturing Bacteria from Caenorhabditis elegans Gut to Assess Colonization Proficiency   

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Original research article

A brief version of this protocol appeared in:
Nature Communications
Jan 2017


Determining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut worm, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original intestinal sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the intestinal sample.

Keywords: Caenorhabditis elegans, Bacillus subtilis, Escherichia coli, Culture


Animals rarely live in isolation but rather exist in association with microorganisms. The more characteristic host-microbe interaction in nature is the symbiotic relationship between host and intestinal microbiota (Rosenberg and Zilber-Rosenberg, 2011). In mammals, host-microbe symbiotic interactions mainly occur along mucosal surfaces, with the most important one being the intestinal mucosa. When freshly isolated from the wild, C. elegans often harbours a diverse bacterial flora in its gut lumen, reminiscent of the microbial communities of higher organisms (Duveau and Felix, 2012; Bumbarger et al., 2013). By contrast, in the laboratory C. elegans is typically maintained in the presence of single bacterial strain (Brenner, 1974). Most often, this is the Gram-negative bacterium Escherichia coli. However, other species are sometimes used, such as the Gram-positive Bacillus subtilis (Garsin et al., 2003). An adult worm contains approximately 10,000 bacterial cells, a number 10-times greater than that of host worm somatic cells (Portal-Celhay and Blaser, 2012): perhaps coincidentally, this microbiota-to-host cell ratio is similar to that found in humans.

Different strategies are being used to measure the colonization proficiency of C. elegans gut by a bacterium such as fluorescein isothiocyanate (FITC)-labelled bacteria, bacteria expressing a fusion reporter (green fluorescent protein [GFP] or β-galactosidase). However, the more accurate method is to measure the number of CFU isolated from the worm intestine. In this protocol, we show how to isolate and count E. coli and B. subtilis strains from C. elegans gut. In the case of B. subtilis, we also show how to distinguish vegetative forms from highly resistant spores formed by this bacterium inside the C. elegans gut.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Rodriguez Ayala, F., Cogliati, S., Bauman, C., Leñini, C., Bartolini, M., Villalba, J. M., Argañaraz, F. and Grau, R. (2017). Culturing Bacteria from Caenorhabditis elegans Gut to Assess Colonization Proficiency. Bio-protocol 7(12): e2345. DOI: 10.21769/BioProtoc.2345.

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