Culture Conditions For S Ratti

Parasitic S. ratti The parasitic stages of S. ratti are maintained in the laboratory by infecting rats. Immunologically normal animals mount an immune response against these infections, such that the infections are lost after four to six weeks (Wilkes et al., 2004). However, long-term infections can be established in immunologically deficient (nude) rats (Gemill et al., 1997). Feces collected from infected rats contain the eggs or first-stage larvae. These feces can be collected and used to make fecal cultures, from which all the free-living stages of the life cycle can be grown. Infective L3s obtained from such cultures are used to initiate new infections, which is done by the subcutaneous administration of iL3s to naive rats (Gemill et al., 1997; Wilkes et al., 2004).

Free-living S. ratti For the analysis of aging and lifespan, the culture conditions used for the free-living adult S. ratti are based on those used for C. elegans. The latter are typically maintained on NGM agar seeded with Escherichia coli strain OP50 (a uracil auxotroph) as a food source (Sulston and Hodgkin, 1988). For S. ratti, L4 female and male worms are taken from 2-day old rat fecal cultures (19°C), cleaned by repeated washing in sterile distilled water to remove associated bacteria, and then introduced onto NGM plates under optimized culture conditions (see below). To measure lifespan, survival of the free-living stages of S. ratti is measured at least daily, with scoring for death as described for C. elegans (Klass, 1977).

Cultures of C. elegans are monoxenic; that is, only the E. coli food source is present. In contrast, S. ratti larval stages derived from fecal cultures are inevitably contaminated with fecal bacteria, which may reduce their lifespan. To minimize this problem, the agar plates on which these S. ratti free-living stages are cultured contain 400 ^g/ml streptomycin and are seeded with a streptomycin-resistant (SR) E. coli OP50 food source. Under these conditions, visible contamination by other microbes is reduced but not prevented. With these conditions the maximum mean lifespan (± SE) of S. ratti females was 4.2 ± 0.2 days. This lifespan is significantly greater than that of S. ratti maintained without the presence of streptomycin (Gardner et al., 2004; Figure 20.2A).

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