Worm Breeder's Gazette 10(1): 141
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
For biochemical analyses of aging it is sometimes necessary to have gram quantities of material. We have devised a protocol for maintaining large numbers of age-synchronous worms in mass culture that provides gram quantities of aged worms. We faced two problems in generating large cultures: 1) worms must be synchronized and maintained in synchrony uncontaminated by progeny; 2) dead worm carcasses and other unwanted contaminants must be disposed of. Cultures are initially grown at 20 C on NGM then transferred to large NGM plates supplemented with egg yolk. Worms on these plates are allowed to grow to confluency and harvested to maximize the number of fecund adults. Age-synchronous cultures are initiated by treating fecund adults with a hypochlorite solution, washing the resultant eggs in S-basal three times, and allowing them to hatch overnight in S- basal. L1's are then counted by making serial dilutions and transferred at a concentration of 3x10+E4 L1's per plate to 2% peptone plates spread with RW2, a prototrophic strain of E. coli. The worms are kept on 2% peptone plates until they are young adults (40 hrs at 25 C after plating as L1's). Worms are suspended in S-basal at 10 worms/ml and transferred to large petri plates in 2x10+E9 E.coli/ml. Synchrony of the mass cultures is maintained by sterilization using fer-15 (b26,ts) grown at 25 C. We are also experimenting with the use of DNA synthesis inhibitors (e.g. FUdR) and brute force filtration to remove contaminant progeny using a 30 nylon mesh filter. Worm carcasses are removed by suspending the worms in S-basal, layering over an equal volume of Sepracell-MN (1.045 g/cm ) and centrifuging for 5 minutes at 500g. Live worms are found in the pellet while the carcasses remain at the interface. When maintaining the worms in liquid medium, ciliate contamination is sometimes a problem. This can be remedied by treating the entire population with . 025% detergent (Dove) and subsequently washing two times in S-basal with no apparent effect on the worms. Mean life spans of populations ( 25 worms) of DH26 with or without detergent treatment were 9.2 and 10. 1 days, respectively at 25 C. We are currently using these techniques to examine lysosomal enzyme specific activity and oxygen consumption in aging worms.