Worm Breeder's Gazette 5(2): 10
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.
After much frustration trying to grow enough males for sperm biochemistry, we spent several weeks experimenting with liquid growth conditions and have improved our yields by a factor of ten. More than 10 ml of healthy adult worms can be obtained from a 130 ml culture. The key parameters appear to be adequate aeration and the absence of contamination. The first is achieved by using deep baffled shake flasks and the second by careful sterile technique. Our present protocol adapts previous procedures and methods published in the Newsletter (Hecht, Vol . 1, 2; Klass, Vol . 3, 1; Sulston and Brenner, Genetics 77: 95-104). 1. Growth of E. coli for worm food As noted previously (Newsletter 3, 2, p. 14) commercial bacteria are unsatisfactory in our hands. We grow our own in a Lab Line Model 29500, High-density Fermentor from which we obtain 180g wet weight of E. coli from 3L of culture. The baffled flasks described below would presumably give less yield per volume, but might work better than bubbling. E. coli strain NA22 is grown in the following medium: [See Figure 1] Add 3 ml trace metal solution (Sulston and Brenner, Genetics 77: 95- 104, 1974), 25 ml 1M MgS04. Grow overnight at 37 C. Harvest cells aseptically by centrifugation and resuspend in S medium at 2-3 X 10+E11 cells/ml (OD550 ~200). 2. Growth of Synchronized Worm Population and Isolation of Males General Comments Contamination of cultures by foreign bacteria or yeasts must be avoided, Tissue culture handling procedures are essential. These should include the following: 1. All glassware should be autoclaved. Growth flasks should be well sealed and dry. 2. Wash your hands with prep-o-dyne before handling cultures, especially for filtration. 3. Open flasks and cultures only in the laminar flow hood- Wipe hood surface with 70% ETOH before use. Flame all openings before transfers. 4. Use only newly opened sterile pipettes for adding to cultures or sampling. 5. Plug all flasks with sterile gauze-wrapped cotton plugs. Handle from outside only. Use sterile silicone rubber coverings for 1L and 2L flasks. Equipment and Solutions Needed Glassware - all sterile 1L and 2L baffled flasks (Bellco 2542 series) This is important 200ml screw cap centrifuge bottles (Bellco 3045 series) sterile cotton or silicone plugs for flasks sterile 25 and 10 ml pipettes. [See Figure 2] Distribute WG medium, 250 ml/bottle Bacteria - NA22 in WG or S medium at 2-3x10+E11 cells/ml. Uncontaminated. Solutions for washing and Isolating Worms 1.25 M-9 salts, autoclaved 30% Ficoll in 1.25 M-9 salts, autoclaved. Worms survive Ficoll floatation better than sucrose flotation. Filters Nitex nylon filters should be stretched tightly over 13 cm embroidery hoops with stainless steel screws. Label size in pencil on the surface. One 35 M and one 20 M will be needed for each 300 ml culture. Wash them out after use. Soak in 70X ETOH then set in sterile glass petri dish lids with support rods in UV hood. Keep them sterile. Starting Cultures For growth of males E1490, him-5(e1490) is the mutant of choice. Doubles with e1490 are used for most experiments with fer mutants. 1. Start from a clean, uncontaminated master plate. Transfer a chunk to a large growth plate. As this plate reaches starvation, rinse the larvae off with several changes of M-9 salts. Work in the laminar flow hood to minimize contamination. Transfer a plateful of worms to 100 ml of WG medium in a baffled flask. Add 10 ml bacteria. Grow two generations. isolate hermaphrodites and eggs as described in the following. If culture is not synchronous, isolate eggs and grow another generation. Preparing Eggs to Start Synchronous Culture 1. Concentrate adults by letting them settle 10-15 minutes or spin at 800 RPM at room temperature for 5 minutes. (About 400 x G). 2. Aspirate off bacterial supernatant. Resuspend in 1.25 x M-9 buffer. Transfer to 40 ml conical bottom tubes. 3. Repeat spin at 1/2 speed in clinical centrifuge 5 minutes. 4. Aspirate supernatant. Add ~25 ml of 30% Ficoll in 1.25 x M-9, mix. 5. Spin at 3/4 speed (~1000 RPM) in clinical for 10 minutes. 6. Withdraw worms from top of tube. Dilute with 1.25 M-9. Spin again at 1/2 speed 5 minutes. 7. Chlorox hermaphrodites. Make up fresh: 10 ml Bleach 25 ml 1N NaOH 15 ml H20 Add 20-25 ml to 3-4 ml hermaphrodites. Vortex occasionally for 6-9 minutes, until you see that all worms have dissolved. Spin down at 1/2 speed for 4 min. Aspirate chlorox sterilely. Dilute with WGM. Spin again. Resuspend WGM. Check a sample under the microscope to be sure all carcasses have dissolved. 8 ml of hermaphrodites should give 1.5 ml of eggs. 1 ml of eggs = ~4 x 10+E6 worms. 8. Place ~1 ml of eggs/250 ml of WG medium in 2L baffled flask. Add 40 ml bacteria. 9. Leave on shaking table at 20 C. Eberbach 25 mm diameter rotation. 150 RPM. The diameter of rotation is important, shaking water baths don't work well. Growth and Isolation of Males 1. Count the number of worms hatched 12-24 hrs after starting the culture and check their motility and condition. Smell the flask to detect contamination. If contamination is detected at this early stage it is probably best to clean the worms by centrifugation then flotation, return them to culture and rechlorox the hermaphrodites. 2. Adjust culture concentration from ~7500/ml to 25,000/ml. Add 12 ml bacteria for each 10+E6 worms total. We use 250 ml total volume in 2L flasks and 120ml in 1L. 3. After 48-60 hrs culture, feed again 12 ml bacteria/10+E6 worms. 4. After 80-96 hrs culture, spin down or let settle. Aspirate bacteria. Rinse with M-9 spinor settle again. Float with 30% Ficoll as in steps 2-6 of egg isolation. Keep culture sterile. 5. Have sterile 35 and 20 filters ready. 6. Distribute worms on 35 up to 10 ml worms per 13 cm filter. Add 1.25 x M-9 until liquid is even with bottom of filter. After 1/2 hr transfer filtrate to 20 . Add fresh 1.25 x M-9 and mix up worms on 35 filter. Check after ~1/2 hr to see what comes through. 7. If more males,add to 20 . After ~1 hr hermaphrodites will crawl through the 35 filter so don't add filtrate without checking. 8. Collect males with conical centrifuge tube from top of filter by rinsing with sterile 1.25 x M-9. This is the most difficult to keep sterile. Wash bands with prep-o-dyne before collection and handle worms as carefully as possible. 9. Spin down. Note volume. Resuspend in WG medium at <10,000/ml. At this age 2 ml males (= 6 x 10+E5) can go into 100 ml of culture. Add 10 g/ml Gentamycin to culture to minimize contaminants. Add 20 ml bacteria/10+E6 Worms. 10. Feed every 24 hours, ~20ml/10+E6 worms. 11. Prepare worms for squash 48 or 72 hrs after fractionation = 6 days or 7 days total growth at 20 C. 12. Spin worms and float as in egg isolation steps 2-6. Rinse final pellet in squash medium. Let males settle again before squashing. If there are many larvae or hermaphrodites, fractionate again in 35 and 20 filters.
