Background

The nematode Caenorhabditis elegans is a bacterivore that is widely used as a model to study host-bacteria interactions, due to its simple morphology, high homology of its genome to the human genome, and the amenability of its microbiome (Zhang et al., 2017). These nematodes use their pharynx to intake bacteria from their surrounding environment (Avery and You, 2018). In a laboratory setting, germ-free larvae are obtained by isolating embryos from gravid adults. Once hatched, larvae (L1s) will begin to feed on bacteria (Brenner, 1973). Hence, it is easy to colonize the C. elegans gut with bacterial strain(s) of interest. Studies involving host-bacteria interactions often call for the enumeration of the bacterial load within the intestine. One of the most common and reliable bacterial quantification techniques is plating for colony forming units (CFUs). Quantification of bacteria within C. elegans is achieved via the physical disruption of worm bodies to release intestinal content, and plating the resulting lysate on solid medium to support microbial growth and subsequent enumeration of CFUs. However, there is variation between existing protocols for the aforementioned assay. For example, prior to the homogenization process, worms are washed several times in a microcentrifuge tube to eliminate external bacteria from the sample. During these washes, worms must be allowed to settle to the bottom of the tube before the supernatant is aspirated, and the researcher must be aware of the location of the worms within the tube, to ensure worms are not aspirated out with the supernatant—because worms tend to stick to pipette tips, this is a critical precautionary measure, which is not emphasized in many existing protocols. Many protocols also call for gentamicin or bleach washes, to eliminate bacteria attached to the cuticle of the worm prior to homogenization. However, if swallowed, bactericidal agents could affect the viability of intestinal bacteria, skewing results. To circumvent these risks, we add levamisole, an alkaline phosphatase inhibitor that reversibly paralyzes C. elegans, to our gentamicin and non-gentamicin washing solutions. This step stops pharyngeal pumping and decreases the risk of the germicidal agent making its way to the intestine. Other techniques used to prevent ingestion of bactericidal agents include lowering the temperature of the samples, and adding reagents that retard motility and minimize pharyngeal pumping (Vega and Gore, 2017; Ortiz et al., 2021). Temperature-mediated inhibition of motility is an attractive approach that can be incorporated into our protocol, for applications that may want to avoid using levamisole. Manual homogenization of worms introduces the most variability. For example, moving the pestle rapidly in the sample tube could cause bubbling and subsequent spillage of the sample. The time and force exerted on the pestle could also affect reproducibility between experimenters. We describe detailed steps in our protocol, such as the position of the pestle and the movement of the hand-held homogenizer, to mitigate the aforementioned risks. Other more hands-off methods of disrupting worm bodies include the automated higher-throughput worm lysis that likely limits the risk of human-induced variation (Vega and Gore, 2017). As such, we describe a benchtop homogenizer-based automated method that diminishes the risk of human error and variation. A flow-chart of the described method is illustrated in Figure 1. Three researchers in our lab performed the protocol described herein on the same day, using worms taken from the same sample. Quantification of the bacterial load within C. elegans varied between researchers using a hand-held homogenizer (Figure 2A), whereas results were nearly identical when a benchtop homogenizer was used (Figure 2B). Hence, option 2 should be the method of choice, as it provides the most consistent results. It is worth noting that this protocol can be modified to permit quantification of other microbes harbored within C. elegans, or any experiment that requires worm lysis to be consistent between samples.

Figure 1. Schematic illustrating the protocol “Quantification of Bacterial Loads in C. elegans”. Detailed methods can be found in “Procedure”. Explanation of CFU calculations can be found in “Data Analysis”.

Figure 2. Quantification of bacteria within C. elegans. Reproducibility between three experimenters conducting the protocol described herein using (A) a hand-held homogenizer, and (B) a benchtop homogenizer to lyse C. elegans. Data are represented as the average bacterial load per C. elegans intestine. Each bar represents one independent experiment with three internal replicates. Error bars represent standard error of the mean (SEM). Significance was calculated using one-way analysis of variance (ANOVA) followed by multiple comparison Tukey’s post-hoc test (*P < 0.05).