Table 1.

C. elegans culture standardization factors for toxicology and GCeCP

Factor	Details
Temperature	Small temperature differences have a large effect on C. elegans growth rate, motility, lifecycle, lifespan, and gene expression. The method and duration of vessel contact with human hands and metal surfaces can significantly affect many endpoints. Handling culture vessels by edges that are not in contact with medium, and a layer of Styrofoam on work surfaces will reduce heat exchange.
Humidity	Low humidity will alter test article and nutrient concentration, the smaller the volume the larger the effect. Unless equipment is enclosed and carefully climate controlled, it is unlikely that the small volumes used in HTS will work well with C. elegans assays. For larger test volumes, incubators can be humidified with an open vessel of water that is cleaned and refilled regularly.
pH	Extreme pH is required to alter adult C. elegans viability, but other endpoints are more sensitive to pH. The appropriate pH range should be determined for each assay, and the pH of test articles in assay medium must be assessed and reported.
Worm Density	C. elegans gene expression and life cycle respond to nutrient availability and secreted hormones. Given a 3‐day generation time and ~300 progeny per worm, cultures can easily outstrip nutrients if they are not consistently monitored. Conversely, C. elegans do not grow well if maintained too sparsely. Note that the worms will not necessarily die in these conditions, instead they will adapt epigenetically (Hall et al., 2010), potentially resulting in altered toxicity test results for several generations.
Cohort Synchronization	A cohort of 1st larval stage (L1) C. elegans can be isolated by hypochlorite treatment of gravid hermaphrodites (an egg prep) followed by hatching of the released eggs in non‐nutrient buffer. In the absence of nutrients, these L1 s halt development just after hatching. At 20 °C, about 12 hours are required for all the eggs to hatch. After more than 18 hours in buffer, gene expression is altered resulting in delayed and unsynchronized development, and increased stress resistance (Jobson et al., 2015, Nass and Hamza, 2007). Some genetics protocols state that hatched L1 s can be maintained in non‐nutrient buffer and used for a week or more, but this will result in variable toxicity outcomes.
Dauers	The C. elegans dauer larva is a stress resistant, long‐lived alternate to the 3rd larval stage (L3). Dauers must revert to the normal lifecycle in order to grow and reproduce. C. elegans dauers secrete dauer pheromone, which promotes conversion to the dauer state in other larvae and induces increased stress resistance in exposed adults. This will both reduce apparent growth rates as measured by worm length (dauers can remain at the L3 length for months or even years), and increase viability in the presence of many toxins. Dauers are thinner and darker than L3s of similar length, and lack the clearly defined gonadal region and visible intestinal lumen identifiable in developing C. elegans. Liquid cultures are unlikely to produce dauers if they are consistently maintained with adequate nutrient supply and are started from agarose cultures that were well fed for at least 3 generations. Daily media exchange along with a few sequential egg preps as soon as each generation becomes gravid can sometimes free a culture of dauers.
Genetic Drift	Genetics labs often maintain commonly used C. elegans strains at room temperature as dauers. If C. elegans cultures are consistently well fed for optimal toxicity studies, use of frozen stocks must be scheduled in order to prevent genetic drift.
Males	Non‐disjunction of the X chromosome results in XO males. This happens rarely in nature, and is induced by toxins and stress. C. elegans males can be identified by their flared tail and single gonad arm. Males are smaller than the XX hermaphrodites, which will result in apparent reduced growth if automated methods are used and technicians are not trained to recognize males. Mating with males more than doubles the progeny per hermaphrodite relative to selfing, so males in a culture will increase reproductive output. Removing males from a culture requires isolating developing hermaphrodites away from the males.
Solid vs. Liquid Medium	When the test article is mixed into molten agar, or spread in solution onto solidified agar, and then the E. coli feeder organism is grown in a lawn on top of the dosed agar, the true exposure will depend on many factors such as humidity, compound solubility, compound‐agar interaction, and feeder organism uptake and metabolism. Dosing in liquid medium provides a measureable exposure, but limits the test to water‐soluble compounds.
E. coli vs. Axenic Medium	Axenic medium avoids the complicating factor of the metabolic response of the feeder organism, which is especially important for test articles with antibiotic activity. Killed E. coli are sometimes used (usually heat or UV), but the method and exposure time must be carefully controlled to avoid the bacteria producing toxins which will alter assay results.