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Growth of Nematodes
Wild-type C. elegans strain N2 was grown routinely at 15°C on NGM agar plates seeded with E. coli strain OP50 (Brenner, 1974). Synchronous second-stage larvae (L2s) were obtained by one of two methods. Eggs purified by alkaline hypochlorite treatment (Emmons et al, 1979) were rinsed, suspended in 1.0 ml M9 buffer (Brenner, 1974), and placed on a shaker at 25°C for 12-15 hr. The hatched first-stage larvae (L1 s) then were collected by centrifugation and put on petri plates with E. coli at 25°C. Alternatively, synchronous L2s were obtained from eggs laid by gravid adults during a 2-hr period. Larvae were fixed 2.5-3.0 hr after the L1-L2 molt, which was monitored by observation of pharyngeal pumping (Cassada and Russell, 1975). Starved L2s were prepared by suspending M9 buffer-rinsed larvae (collected 1 hr after the L1-L2 molt) in 1 ml buffer at 25°C for 3 days.
Starvation-induced dauer larvae were removed from NGM
agar plates for fixation after 5 days of starvation. Nonstarved,
pheromone-induced dauer larvae developed from eggs hatched at 25°C on agar
medium made from autoclaved, clarified S-medium obtained from a previous
nematode culture (Sulston and Brenner, 1974; Golden and Riddle, 1982). Any
nondauers were removed after 2 days. Pheromone-induced dauer larvae were fixed
2-3 days after the L2-dauer molt. To obtain fourth-stage larvae (L4s),
starvation-induced dauer larvae were put on a plate spread with E. coli
at 25°C and allowed to recover. The L4s were fixed 3.0-3.5 hr after the
Larval stages or adults picked from an NGM plate were fixed with
1.0% OsO4 in 0.1 M sodium cacodylate-HCl, pH 7.3, for 1.5
hr at 28-30°C. Buffer-rinsed worms were cut in half and embedded in agar (Ward
et al, 1975). Small agar blocks, each containing two specimens, were
dehydrated in ethanol and embedded in Spurr's resin (Spurr, 1969). Transverse
serial sections approximately 60 nm thick were picked up on unsupported slot
grids, stained with uranyl acetate and lead citrate (Reynolds, 1963), and placed
on lightly carbon-coated formvar films (Albert et al., 1981). Sections
were photographed on a Philips 300 electron microscope.
reconstruction of the anatomy of C. elegans is simplified when the
fixation method accentuates cell boundaries, fixation in osmium was preferred to
the standard glutaraldehyde fix, osmium postfix method.
Glutaraldehyde-fixed specimens, however, were also examined to
eliminate possible ambiguities in subcellular morphology. Larvae were fixed
in 3% glutaraldehyde in .07 M sodium cacodylate-HCl, pH 7.3, at 4°C for
6 hr except for the first hour, which was at 20°C. Buffer-rinsed specimens were
postfixed in cold 1% buffered OsO4 for 2.5 hr, during which time
the temperature of the fixative was allowed to warm to room temperature (20°C).
The reconstruction was prepared as a projection to a plane through the center of the organism. The ventral view was prepared by measuring the left-to-right distances of cell boundaries in the ventral half of the nematode from the sagittal plane, and projecting them onto the horizontal plane. The lateral view was prepared in the same way except dorsal-ventral distances were projected onto the sagittal plane. The measurements were taken on every twelfth section (every 720 nm) from the serial cross-section electron micrographs of a single L2 larva. The measurements were reproduced as points on paper, and continuous lines were drawn through the points representing the boundaries of cells or nuclei.
Well-fed worms were washed off an NGM plate and transferred in 5 milimeter M9 buffer to a 5% agar pad supported on a glass microscope slide (Sulston and Horvitz, 1977). Before the worms were transferred to the slide, 5 milimeter of 100 mM NaN3, an anesthetic, were allowed to diffuse into the agar. The nematodes were observed through the differential interference-contrast optics of a Zeiss Universal microscope and photographed with an Olympus PM-10A camera. Kodak Technical Pan or Tri-X film was used, and all photographs were taken with a heat filter and a green filter in the light path.
A mixed population of worms was washed from an NGM plate with M9 buffer and freed of bacteria by low speed centrifugation in distilled water. The animals were fixed for 3-7 days in modified Bouin fixative which was prepared with trichloroacetic acid. Staining was as described by Cameron and Steele (1959) except the oxidation was for 3 min in hot potassium permanganate.
The correlation of PAF staining with nutritional state of the animals was tested using L2 larvae synchronized as above by the alkaline hypochlorite method. One hour after the L1-L2 molt the worms were suspended in M9 buffer and incubated at 20°C for 20 hr without food. Half of the animals then were placed in fixative, and the other half were returned to food for 3.5 hr, washed free of bacteria, and fixed. Both the starved and the fed animals were PAF-stained. As a positive control in the staining procedure, a morphologically distinguishable C. elegans strain, CB61 (dpy-5 I), was mixed with each test sample at the time of fixation. In the mixed population with starved worms, about 90% of the positive control animals exhibited glandular staining, whereas none of the starved worms stained.
Adapted by Yusuf KARABEY for WORMATLAS, 2003