Overview
In this video, we show a method of producing a developmentally synchronized population of sterile adult C. elegans. The example protocol synchronizes worms to study oxygen consumption rates in live animals.
Protocol
This protocol is an excerpt from Sarasija and Norman, Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans, J. Vis. Exp. (2019).
1. Growth and synchronization of nematode population
These animals will reach the L4 larval stage after ~42 h. At this time, move the L4 larvae using a platinum pick to OP50 seeded NGM plates containing 0.5 mg/mL 5-fluoro-2’-deoxyuridine (FUdR) to prevent them from producing progeny.
NOTE Make sure that within an experiment all strains are synchronized for a similar amount of time. FUdR treatment sterilizes the animals and prevents egg laying and progeny production, which could influence OCR. These sterilized animals will be analyzed the next day (day 1 adult animals at ~66 h) for the assay. FUdR has been reported to impact physiology and lifespan of certain mutant worms. Therefore, this should be taken into consideration when the drug is used for sterilization. Worms can also be sterilized by the means of feminizing mutations such as fem-1(hc17) or fem-3(e2006). However, these mutations might impact mitochondrial function.
- Transfer L4 larvae of desired genetic backgrounds (e.g., N2 [wild type] and sel-12 animals) onto nematode growth media (NGM) plates (see Table 1 for recipe) freshly seeded with a lawn of Escherichia coli (OP50). Use at least two 100 mm or three 60 mm plates for each strain. Incubate the worms at the appropriate growth temperature (between 15–25 °C) for 3–4 days or until plates are concentrated with large number of eggs and gravid worms.
- Wash the eggs and worms off the plates using approximately 6 mL of M9 buffer (see Table 1 for recipe) per 100 mm plate of nematodes and transfer them with glass Pasteur pipettes into individual 15 mL centrifuge tubes for each strain. Spin these tubes down for 3 min at 6,180 x g and aspirate out the M9 buffer, retaining just the animals and eggs pellet.
- Add 3–4 mL of bleach solution (see Table 1 for recipe) to each tube and intermittently vortex for 6 min. Add M9 buffer to fill each tube and spin at 6,180 x g for 1 min and aspirate supernatant. Repeat this wash with M9 three times and move the egg pellet to a fresh 15 mL tube containing approximately 9 mL of M9 buffer.
- Synchronize the freshly hatched worms by nutating at 20 °C for 16–48 h. Spin these tubes down at 6,180 x g for 1.5 min and put the synchronized L1 animals down on individual NGM plates freshly seeded with a lawn of OP50 (approximately 6,000–10,000 animals per 100 mm plate) and keep at 20 °C.
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Representative Results
Table 1: Recipes for NGM plates, M9 buffer, and bleach solution.
| M9 buffer (1 L) | |||
| dH2O | 1,000 mL | ||
| NaCl | 5 g | ||
| KH2PO4 | 3 g | ||
| Na2HPO4 | 6 g | ||
| 1 M MgSO4 | 1 mL *add after autoclaving | ||
| Split between 2 bottles: 500 mL each. Autoclave on liquid cycle (15 min exposure) | |||
| Bleach solution (50 mL) | |||
| dH2O | 36 mL | ||
| Bleach | 14 mL | ||
| 10 N NaOH | 800 µL | ||
| Standard Nematode Growth Media (NGM) plates | 1 L | 0.5 L | 0.25 L |
| NaCl | 3 g | 1.5 g | 0.75 g |
| Bacto-agar | 17 g | 8.5 g | 4.25 g |
| Bacto-peptone | 2.5 g | 1.25 g | 0.625 g |
| a. Autoclave on liquid cycle (45 min exposure), allow to cool to ~60 °C and then use sterile technique and add the following: |
|||
| 1 L | 0.5 L | 0.25 L | |
| 1 M CaCl2 | 1 mL | 0.5 mL | 0.25 mL |
| 1 M MgSO4 | 1 mL | 0.5 mL | 0.25 mL |
| 1 M KPO4 | 25 mL | 12.5 mL | 6.25 mL |
| 5 mg/mL cholesterol | 1 mL | 0.5 mL | 0.25 mL |
| b. Swirl to mix thoroughly after each addition. After all additions are made, pour plates |
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Materials
| Name | Company | Catalog Number | Comments |
| 100 mm; 60 mm Petri dishes | Kord-Valmark Labware Products | 2900; 2901 | |
| 15 mL conical tubes | Corning | 430791 | |
| 50 mL conical tubes | Corning | 430829 | |
| Agar | Fisher Scientific | BP1423-2 | |
| Bacto peptone | BD; Bacto | 211677 | |
| Bacto tryptone | BD; Bacto | 211705 | |
| Bacto yeast extract | BD; Bacto | 212705 | |
| Bleach | Generic | ||
| Calcium chloride dihydrate (CaCl2·2H2O) | Fisher Scientific | C79-500 | |
| Cholesterol | Fisher Scientific | C314-500 | |
| Deionized water (dH2O) | |||
| Glass Pasteur pipettes | Krackeler Scientific | 6-72050-900 | |
| Magnesium sulfate heptahydrate (MgSO4·7H2O) | Fisher Scientific | BP213-1 | |
| Potassium phosphate dibasic (K2HPO4) | Fisher Scientific | BP363-1 | |
| Potassium phosphate monobasic (KH2PO4) | Fisher Scientific | P285-500 | |
| Sodium chloride | Fisher Scientific | BP358-10 | |
| Sodium hydroxide (NaOH) | Fisher Scientific | BP359-500 | |
| Sodium phosphate dibasic anhydrous (Na2HPO4) | Fisher Scientific | BP332-1 |
