Polyunsaturated fatty acids are sensitive to heat, light and oxygen. Therefore, care must be taken when preparing fatty acid supplementation plates such that fatty acids are not exposed to excess heat and light. NGM media containing 0.1% Tergitol (NP-40) is autoclaved and partially cooled, after which fatty acid sodium salts are added with constant stirring. The plates are allowed to dry in the dark. Uptake of fatty acids by C. elegans cultured on these plates can then be monitored by gas chromatography.

1. Preparation of Fatty Acid Supplemented Media

1. Measure out media components into an appropriately sized flask. Per 1 L, add 17 g Bacto-agar, 2.5 g tryptone, 3 g NaCl, 1 ml 5 mg/ml cholesterol dissolved in ethanol, and 10 ml 10% Tergitol dissolved in water.
2. Add 80% of final desired volume of Millipore water and autoclave the media along with empty glass bottles (equal to the number of different fatty acid concentrations to be tested), as well as appropriate graduated cylinders.
3. Cool media in a water bath set to 55 °C. While the media is cooling prepare the working stock solution of the fatty acid sodium salt by breaking open the glass vial, using safety precautions and ensuring glass particles do not mix in with the fatty acids. Weigh out enough fatty acid to make a 100 mM working stock.
4. Bring the fatty acid solution to a final concentration of 100 mM with purified water. Fully dissolving the fatty acid sodium salt typically takes about 20-30 min. Purge the working stock of fatty acid with argon or nitrogen, because contact with an inert gas prevents oxidation. Cap the vial and store the working stock in the dark until media has cooled.
5. (Optional) If RNAi media is desired, measure ampicillin and Isopropyl β-D-1-thiogalactopyranoside (IPTG) solutions here.
6. When the agar has cooled to 55 °C add per 1 L: 1 ml of 1 M MgSO₄, 1 ml of 1 M CaCl₂, and 25 ml of phosphate buffer (108.3 g KH₂PO₄ and 35.6 g K₂HPO₄ per 1 L autoclaved). Add the filter sterilized ampicillin and IPTG solutions if making RNAi plates. For all types of plates, add sterile water to bring the final media volume to 1 L.
7. Near a flame, transfer media to an autoclaved and appropriately sized graduated cylinder and then add warm sterile water to desired volume. Transfer media back to initial flask and mix by stirring.
8. Near a flame, transfer media into a number of bottles equal to the number of concentrations to be tested by measuring with an autoclaved and appropriately sized graduated cylinder. Maintain the aliquoted media as liquid using a water bath until the fatty acid working stock has fully dissolved.
9. Place one bottle on a stir plate and stir until the media is warm to the touch, but not hot. Make sure to leave yourself enough time to stir in the fatty acid stock solution and pour plates before the media begins to solidify. If stir plate has temperature control, set it to 55 °C.
10. Dilute the 100 mM fatty acid working stock into the media for the final concentration desired. Stir media until the white precipitate is in solution (approximately 1 min). Media may still be slightly cloudy afterwards.
11. (Optional) If RNAi media is desired add ampicillin to 0.1 mg/ml and IPTG to 2 mM final concentration.
12. Pour media using an automated pipette aid and a sterile 25 ml pipette, adding 8 ml of media per 60 mm plate or 25 ml of media per 100 mm plate. Repeat fatty acid addition and plate pouring steps for the remaining bottles.
13. After the agar has solidified, cover the fatty acid-supplemented plates with a box or store at room temperature in a well-vented drawer to protect from light oxidation.
14. Seed E. coli OP50 onto plates two days after plates have dried. For 60 mm plates, pipette 300 µl of an overnight OP50 culture (grown at 37 °C). If RNAi plates were poured, seed with 300 µl of RNAi bacteria after the plates have dried for four days. Plate drying time may need to be adjusted due to differences in humidity in various lab environments.
15. Incubate the plates in a dark environment at room temperature while the bacterial lawn is drying.

2. Inducing Germ Cell Destruction by Supplementation of DGLA

1. C. elegans grown on plates containing 0.3 mM DGLA (20:3n-6) become sterile due to the destruction of germ cells in both larval stage and adult nematodes.
   1. Prepare a synchronized population of L1 larvae by treating gravid hermaphrodites with alkaline hypochlorite solution(for a 10 ml solution: 0.5 ml of 5M NaOH, 2.5 ml of household bleach, and 7 ml H₂O). Gently rock the gravid hermaphrodites in this solution until the adult worms dissolve. Eggs will be preserved, and can be pelleted by low speed centrifugation.
   2. Wash egg preparation 3 times in M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 ml 1 M MgSO₄, H₂O to 1 L. Add MgSO₄ after autoclaving). To provide enough oxygen, resuspend the eggs in a 15 ml plastic tube to a final volume of 5 ml of M9 buffer and rock on a shaker overnight.
   3. L1 larvae should be added to the supplemented plates two days after seeding with OP50, or one day after plating the RNAi bacteria. Incubate worms at 20 °C for three days or until they reach the adult stage.
2. Adult worms can be scored for sterility using a dissecting microscope with high enough magnification to visualize embryos developing in the uterus. Successful germ cell loss will appear as a clear uterus void of eggs.
3. Worms can also be scored by fixing and staining with the nucleic acid dye diamindinophenylindole (DAPI), which facilitates the visualization of nuclei in germ cells and developing embryos. A rapid DAPI stain is achieved by picking worms into a drop of M9 buffer on a watchglass⁹.
   1. Flood watch-glass with 1 ml of a 0.2 ng/m. DAPI in 95% ethanol solution, let sit for approximately 5 min.
   2. Pick worms into a drop of VectaShield on a slide, and then cover with a coverslip.
   3. Seal coverslip and store at 4 °C in the dark overnight for optimum staining, however slides can also be viewed immediately. Score for presence or absence of germ cell nuclei using a fluorescence microscope equipped with a UV lamp and filter.

3. Confirming Fatty Acid Uptake by Gas Chromatography

Overall fatty acid composition of C. elegans can be determined by producing fatty acid methyl esters (FAMEs) which are then separated and quantified using gas chromatography⁴.

1. Collect 500-1,000 adult worms by washing them off of the plates with water and transferring to a silanized 13 mm x 100 mm glass screw top tube.
2. Let worms settle by gravity, and then remove as much water as possible with a glass Pasteur pipette.
3. Wash worms once with water, and then again remove as much water as possible.
4. FAMEs are formed by adding 1 ml of 2.5 % H₂SO₄ in methanol, and then heating at 70 °C for 1 hr in a water bath.
5. Remove the tubes from the water bath and cool for 1 min.
6. Extract FAMEs by adding 1.5 ml water and 0.25 ml of hexane.
7. Recap tubes and shake vigorously.
8. Centrifuge tubes in a tabletop clinical centrifuge for 1 min at maximum speed to separate hexane from aqueous solvent.
9. Transfer hexane (top layer) to a GC vial insert within a GC vial, being careful not to transfer any of the aqueous phase. For GC analysis, 1-2 μl of FAMEs in hexane is injected onto a polar capillary gas chromatography column suitable for FAMEs analysis. The Agilent 7890 GC injector is set at 250 °C, with a flow rate of 1.4 ml/min, and the GC oven is programmed for an initial temperature of 130 °C, which is held for 1 min. Subsequently, the temperature is ramped 10 °C/min until 190 °C, and then ramped again at 5 °C/min until 210 °C and held for an additional 1 min.
10. To ensure that uptake of DGLA has occurred, analyze FAMEs by flame ionization detection (FID) or mass spectrometry (MS) detection, using authentic standards for the identification of the C. elegans fatty acids.