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.

Preparation and Maintenance of Age-synchronous Mass Cultures

M. Cruzen and T. Johnson

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.