Worm Breeder's Gazette,
A 4 liter glass 'fermenter' has been used instead of Roux bottles in our lab for C. elegans mass liquid cultures. It is made of glass plates stuck together with silicone rubber. It is autoclavable at 120 C, and provided with an aluminium cover containing two noles, into which tubes, are sealed by silicone rubber. One tube (the shorter one) includes a 'weighted' valve, supplied with a movable but hole- covering glass ball. The other an inverted T, the horizontal arms of which are perforated with holes, the prefiltered condensed air entering via the vertical section. This apparatus is practical for both bacteria and C. elegans. New cultures are started by transferring the cover to a new, sterilized culture from an old one. [See Figure 1]
Worm Breeder's Gazette,
For several years we have been studying age-related changes in enzymes isolated from the free-living nematode Turbatrix aceti. This has required the large-scale cultivation, synchronization and aging of nematodes in axenic, chemically defined media. Organisms are grown in one liter Roux bottles containing 50 ml of medium for 14 to 16 days ant then harvested by filtration with a fritted glass Buechner funnel. Small individuals are separated by 'screening' through stainless steel filter cloth and transferred to a medium containing fluorodeoxyuridine (FUDR) plus uridine (100 g/ml and 150 g/ml, respectively). FUDR inhibits reproduction, but not the life-span of the nematodes and thereby permits the development and ageing of cultures without contamination by newborn organisms. Growth of the organisms in length is nearly normal during the aging process. Turbatrix can also be synchronized by increasing the incubation temperature to 36 C or by repeated removal of small larvae from cultures. Biochemical characteristics of nematodes obtained by these methods appear to be similar to those of FUDR-treated worms. The yield of organisms in mixed culture on a wet weight basis is approximately 2 grams per 50 ml of medium in a period of two weeks. Our maximum incubator capacity is 500 Roux bottles, resulting in a potential production of a kilogram of nematodes per fortnight. Quantities available from aged worms are only 10 to 20% of this figure due to losses in screening and to the limitations of the worm concentration which is permissible in 'ageing' cultures. Based on our experience with Turbatrix and Caenorhabditis briggsae and various methods in the published literature, it is reasonable to conclude that C. elegans can be synchronized and aged under similar conditions. An advantage to using this organism is its ability to grow in cultures which are aerated by mechanical agitation. The use of rotary shaker cultures permits a greater yield of organisms and more economy of space than static cultures. Unfortunately, T. aceti will not grow under the former conditions. The culture of nematodes in completely defined medium offers an advantage for certain biological studies. However, C. elegans has a requirement for an unidentified growth factor present in soy-peptone. Elucidation of the structure of this material is therefore of importance if we are to fully utilize this organism for ageing studies. We also believe that the cultivation of C. elegans in an axenic system has definite potential for developmental biology and genetics.