Worm Breeder's Gazette 3(1): 11
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 starts with a predominantly male culture isolated by the Nitex filter method described previously. I usually start with a culture 60-70% males (a total of 1 - 5 x 10+E5 males). These males are washed extensively with 4 liters of M-9 salts over a 20 m Nitex filter to remove the larvae (especially the L1 which can be a problem later!) and to remove the E. coli. The washed male culture is concentrated by settling into 2-3 ml of M-9 salts. The worms are then spread evenly in very small disperse droplets over the surface of an 11'x7'x3/8' Plexiglass plate. Another Plexiglass plate of equal size is placed on top. The worms are then squashed between these plates in a Carver Laboratory Press with a manual hydraulic jack. The worms are subjected to 15,000 psi. This pressure causes the males to eject their sperm as well as their intestinal cells. However the sperm are the only free floating cells. The intestinal cells of both the males and hermaphrodites adhere to the Plexiglass. The plates are then pried apart and rinsed off. The rinse is collected in a glass trough and placed in 125 ml conical centrifuge tubes on ice. Everything is kept on ice from this point on. (If some males don't get squashed in the first run, they can be squashed again, although resquashing usually only gives 10% additional sperm.) The rinse fluid contains the sperm (and worm carcasses) and is filtered through two 10 m Nitex filters fitted to a 25 mm millipore filter holder of the type used on syringes. This removes the worm carcasses and larger debris. The filtrate is centrifuged in a clinical centrifuge on setting 6 for five minutes. The supernatant is discarded and the pelleted sperm are washed in M-9 salts and filtered through an 8 m polycarbonate Nucleopore filter fitted to a 25 mm millipore filter holder. I routinely obtain 10+E7-10+E8 sperm, an average of about 100 sperm per male. One dimensional SDS polyacrylamide gel patterns of these sperm closely match the patterns seen on autoradiographs of hermaphrodites mated with radioactively labeled males. Some additional information: Sperm can be stored overnight in M-9 salts with a minimum of lysis (1%) but they seem more fragile and after 48 hours show 50% lysis. I have found that sonication works best to lyse sperm rather than freeze/thawing. I have used M-9 salts throughout these procedures and have not tried Ascaris Ringer's solution. These purified sperm are currently being used to investigate cell surface proteins, DNA binding proteins, sperm actin and other sperm- specific proteins and their modifications in various spermatogenesis mutants as well as being used as a source of germ line DNA.