Worm Breeder's Gazette 8(2): 56
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.
In order to provide sufficient material for partial purification of minor nematode proteins preliminary to immunization and selection of monoclonal antibodies, we have developed a method for growing C. elegans (sometimes referred to as 'the nematode') in the 300 liter fermenter located in the Department of Biochemistry. Briefly, a 250 liter bacterial culture was grown in the fermenter and collected with a continuous flow centrifuge. Two kilograms of the bacteria were resuspended in 250 liter of S medium in the fermenter and seeded with 10 nearly exhausted 1 liter shaker cultures of C. elegans. With stirring and vigorous aeration, the worms grew rapidly and appeared very healthy. After 4 days, as the bacterial count began to decline, we added the remainder (3 kilograms) of the bacterial pellet. We harvested the worms, by continuous flow centrifugation on day 6. Worms were resuspended in ice cold water and recentrifuged. The pellet was resuspended in cold 35% sucrose and the worms collected by flotation. After washing twice with water, the final pellet (1.35 kilograms) was resuspended in a small amount of water, dripped into liquid nitrogen and the nuggets (which resemble garbanzo beans, but taste like chicken) stored at -80 C. Specific Notes: 1) Bacterial Growth Medium: A less expensive modification of 3XD was used. This medium, developed by Paul Gardner and Dick Russell, which we call GaRuM, contains per liter 10.5gm Na2HP04, 4.5gm KH2P04, 3.0gm NH4Cl, 25gm Sigma #C0626 Casein hydrolysate, 25ml glycerol, 0. 1gm uracil and 0.2gm streptomycin sulfate. After autoclaving, 4ml 1M MgS04 and 1.0ml trace metals (1.0mM FeS04, 1mM MnS04, 0.5mM ZnS04, 0. 5mM CuS04, 4 M Na2MoO4, 0.1M KCl, 0.1M NaCl) were added. Sterile antifoam was added automatically throughout the growth to control excess foaming. 2) E. coli strain OP50-1: A variant of OP50, the standard uracil requiring strain, was selected to be resistant to phage phi80 (and therefore cross resistant to phage T1). Phage-resistant OP5O was further selected to be resistant to streptomycin using mutagenesis with nitrosoguanidine. We call the phi80 and streptomycin resistant strain OP50-1. We now use OP50-1 routinely in combination with streptomycin and mycostatin in both liquid cultures and in agar. Contamination problems are greatly reduced. 3) Nematode Growth Medium: S medium with 0.2gm/liter streptomycin and 50,000 units/liter mycostatin. The mycostatin was suspended in 70% ethanol and added after autoclaving. 4) Harvesting: Bacteria were collected with a Sharples centrifuge at 3 liters/minute. Prior to use, the centrifuge bowl was autoclaved and the tubing from the fermenter to centrifuge steam sterilized. Bacteria were scraped into sterile beakers and stored at 4 C. Nematodes were harvested at 15 liters/minute. One ml samples were monitored at ~2minute intervals during the harvest; no worms were observed in any sample. 5) Ciliate Contamination: On day 4 after initiation of the nematode culture, we noticed the presence of small ciliates (possibly the species Colpoda claimed to have air-borne, autoclave-resistant spores). The ciliates grew rapidly and threatened to overgrow the culture. After investigating several possible treatments, we found that a 1/1500 dilution of commercial bleach selectively eliminated the ciliates with no apparent effect on the worms. The final product contained no trace of ciliates. 6) Yield: We harvested the nematodes well before exhaustion of the bacteria in order to obtain healthy well-fed worms since starved worms have lower levels of choline acetyltransferase (one of the enzymes to which we are attempting to generate monoclonal antibodies). Yield would have been substantially larger if grown to exhaustion. 7) Cost: Approximately $200 for media and $265 for use of the fermenter.