Worm Breeder's Gazette 3(1): 9
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.
This procedure utilizes nylon filter screens of specific mesh sizes available from: Tetko, Inc., 420 Saw Mill River Road, Elmsford, New York 10523. (Polyester screens are also available and work as well.) The procedure works best with a synchronous population and therefore starts with an egg isolation by the hypochlorite method. Approximately 40,000 him-1 eggs are placed on NGM plates that have been seeded with 2-3 ml of concentrated E. coli (~5 x 1O+E11 /ml) and dried before use. This concentrated E. coli solution is used instead of chicken egg plates because the particulate matter from chicken eggs clogs the filters. These cultures are grown at 20 C and are checked periodically to see if they need refeeding. If the plates need to be refed, an additional 2 ml of concentrated E. coli is added and the plates are again dried. After three days the adult worms, approximately 20% males using him- 1 are floated off the plates by gently pouring on M-9 salts. Worms are collected and washed 3 times in M-9 salts to remove excess E. coli. I use the resettling technique with large 125 ml oil centrifuge tubes and simply let the worms settle to the bottom. The average diameter of the males is then measured and a Nitex filter with a suitable mesh size is selected. The males, for some erotic reason, like to crawl through holes approximately 20% smaller than their diameter. The following table shows the average male diameter and mesh size that I have found most suitable. [See Figure 1] (The filters can be washed in warm water with 7X detergent and reused.) The 40 m filter allows 90-95% of the males through, but also allows many young adults and L4 hermaphrodites. This filter can be used as a prescreening filter followed by a 30 or 35 m filter. I routinely use a 30 m filter because I harvest my cultures before the males get much larger than 35 m in diameter. I use large (10' diameter) plastic embroidery hoops (hereafter known as filter hoops!) to hold the Nitex filters. This allows the routine separation of 1 - 5 x 10+E5 males from cultures with 10+E6 worms. The bottoms of the filter hoops are supported by thin glass rods 1/8' in diameter in shallow trays. The worms are layered on top of the filter with a minimum of liquid. You should be able to see the worms crawling on the filter and not swimming. A thin layer of M-9 salts is placed between the filter and the collecting tray. Allow the males to crawl through for at least 2 hours. They seem to peak in terms of percentage of males coming through at 2-3 hours. If you start with a synchronous culture you can routinely get 70-80% males. The percent yield can be improved by repeating this procedure and with proper care I have obtained cultures of 90% males. If the culture is contaminated with larvae (L1's, L2's and L3's) they can be washed away from the males by suspending a 20 m Nitex filter in a Buchner funnel and washing with 4 liters of M-9 (more or less M-9 depending on the size of the culture). This also removes the E. coli. The males will remain on top of the 20 m filter and can be collected there. I have used this technique on a smaller scale to purify males from mating cultures with satisfactory results. Some additional information for the collection or purification of eggs and L1's is given in the following table: [See Figure 2] We are experimenting with 10 m filters for the continuous collection of newly hatched larvae by layering eggs on the filter fitted to a Buchner funnel. The filter is then washed with a constant flow of M-9 salts at a very low flow rate.
