High-Throughput Screening using C. elegans

Chemical List

Salts & Buffers

• NaCl - Fisher Chemical, S271-1
• KH₂PO₄ - Fisher Scientific, P288-500
• Na₂HPO₄ - Sigma Aldrich, 8210-500GM
• KOH pellets (for 5M KOH prep) - Thermo Scientific, 437135000
• Potassium phosphate monobasic - Fisher Scientific, P285-500
• Tripotassium citrate - Thermo Scientific, 033222.36
• Citric acid monohydrate - Fisher Scientific, A104-500
• CaCl₂ (1M stock) - Fisher Scientific, C77-500
• MgSO₄ (1M stock) - Sigma Aldrich, M-1880
• Sodium EDTA (Na₂EDTA) - VWR Life Science, 0105-500G
• FeSO₄·7H₂O - Sigma Aldrich, F8633-250G
• MnCl₂·4H₂O - Sigma Aldrich, M5005-100G
• ZnSO₄·7H₂O - Sigma Aldrich, Z0251-100G
• CuSO₄ - Fisher Scientific, C493-500

Other Chemicals

• Cholesterol (5 mg/ mL in ethanol, filter sterilized) - Thermo Scientific, A11470.18
• Nystatin (10 mg/ mL stock) - Thermo Scientific, J62486.09
• Streptomycin (200 mg/ mL stock) - Gibco, 11860-038
• FuDr (7.456 mg for 12.5x solution) - Sigma Aldrich, F0503-100MG

Media & Growth Supplies

• LB Broth - Fisher Scientific, BP1426-2
• Peptone - Gibco, 211677
• Low melt agar - RPI, A20070-100.0

Media Recipes

• Trace metal solution
• 1.86 g Na2EDTA
• 0.69 g FeSO4 ∙7H2O
• 0.20 g MnCl2 ∙ 4H2O
• 0.29 g ZnSO4 ∙7H2O
• 0.016 g CuSO4
• 1000 mL deionized water
• Autoclave and store in the dark
• Name, Date, Label
• 5M KOH solution (do not autoclave)
• To prepare a 5M KOH solution, dissolve 280 grams of potassium hydroxide (KOH) in enough deionized water to reach a final volume of 1 liter in a volumetric flask; ensure proper safety precautions are taken as KOH is a strong base. Key points:
• Calculation: 5M KOH means 5 moles of KOH per liter of solution, which translates to 280 grams of KOH (based on the molar mass of KOH being 56 g/mol). Safety: Wear appropriate protective gear like gloves, goggles, and lab coat when handling KOH.
• Always add solid KOH to water slowly and with stirring to avoid excessive heat generation.
• Procedure:
• Calculate required mass: Determine the amount of KOH needed based on the desired volume of solution (e.g., for 1 liter of 5M KOH, you need 280 grams).
• Weigh KOH: Accurately weigh the calculated amount of KOH on a balance.
• Add to water: Carefully add the weighed KOH to a 1L bottle containing a small amount of deionized water, stirring continuously to dissolve.
• Once the KOH is completely dissolved, Dilute to volume: Add deionized water to the bottle until the desired final volume is reached
• Name, Date, Label
• Potassium citrate, 1M solution
• 268.8 g tripotassium citrate
• 26.3 g citric acid monohydrate
• Add 900 mL of deionized water
• Adjust pH to 6.0 with 5M KOH
• Add deionized water to 1000 mL
• Autoclave
• Name, Date, Label
• 1M Phosphate buffer 6 pH
• 136 g potassium phosphate monobasic
• add to 900 mL DI water
• Adjust pH to 6 with 5M KOH
• Autoclave
• Name, Date, Label
• M9 Buffer (can be made and autoclaved at the same time as S-Basal for efficiency)
• Dissolve 3 g KH₂PO₄, 6 g Na₂HPO₄, and 5 g NaCl in approximately 800 mL of distilled water.
• Adjust the volume to 1 liter with distilled water.
• Autoclave the solution to sterilize it, along with 4 125 mL bottles.
• Cool overnight on the bench and seal 125 mL bottles for sterility
• Once cooled, add 1 mL of sterile 1M MgSO₄. (next day)
• Aliquot M9 into 125 mL bottles using serological
• Name, Date, Label
• S-Complete Medium, 1000 mL
• Make 1 L of S-basal
• To 1 L of water in a glass bottle, add 5.9 g of NaCl and 50 mL of 1M Phosphate Buffer 6 pH
• Get 4 125 mL glass bottles and tape for autoclaving
• Autoclave this base solution and the 4 125 mL glass bottles in a liquid cycle for 35 minutes.
• Remove from the autoclave, set media and bottles on the lab bench, and close 125 mL glass bottles so they remain sterile
• Let S-Basal cool overnight with the lid loosened!!
• The next day, add the following
• 10 mL 1M potassium citrate pH 6 (sterile)
• 10 mL Trace metals solution (sterile)
• 3 mL 1M CaCl2 (sterile) (add slowly)
• 3 mL 1M MgSO4 (sterile)
• 1 mL of 5 mg/ mL filter-sterilized cholesterol in ethanol. (add slowly)
• 0.5 mL of 200 mg/ mL filter-sterilized Streptomycin
• 5 mL of 10mg/ mL Nystatin so the amount in the media is 50 ug/ mL
• Used to increase the shelf life of media and of C. elegans in liquid
• Aliquot 125 mL into each of the 4 bottles for use on the bench so that the main bottle is not repeatedly opened and closed.
• Name, Date, Label

Agar Plates for Growing C. elegans

• Get 2 L Erlenmeyer flask
• Add in this order:
• 4.5 g NaCl
• 25.5 g Low melt agar
• Allows for the most efficient C. elegans growth to adulthood and the fastest rate of starvation
• 3.75 g Peptone
• 1462.5 mL DI water
• 750 µL Cholesterol in EtOH
• Add 1.5 mL 1M CaCl2
• Add a large stir bar and mix on a stir plate. The solution will be autoclaved with the stir bar in the bottle for later sterile stirring
• Cover and tape with foil, Autoclave for 30 minute liquid cycle
• Cool to 52°C in a water bath. Change the water first after 5 minutes. After changing the water, wait another 5 minutes. The agar should now be at 52°C
• Add the following once at 52°C (keep hotplate on at setting 3 so that agar is warm but not boiling):
• 18.75 mL phosphate buffer
• 750 µL 1M MgSO4
• 750 µL 200mg/ mL filter-sterilized Streptomycin
• 7.5 mL 10mg/ mL Nystatin
• Aliquot 25 mL per 100mm plate, ensure even plate stacking. Slightly tilted plates can cause uneven solidification
• Let the plates sit near the flame for 1 hour to cool
• Turn the flame off, leave to cool overnight at room temperature
• Store these plates at 4°C lid side down

Making OP50 Bacteria Back Stocks

• Pick a colony of OP50 bacteria and streak on an agar plate.
• Incubate agar plate overnight at 37°C
• Make LB Broth and 200mg/ mL 0.22µm filter-sterilized Streptomycin in water
• Add 25 mL of LB broth and 6.25 µL of Streptomycin to a 50 mL conical
• Repeat 3 times for 3 stocks
• Pick a bacterial colony with a 200 µL pipette tip from the incubated plate and add the tip to 25 mL strep + LB broth in a 50 mL conical and incubate on a shaker overnight at 37°C
• Repeat this for the 3 50 mL conicals, totaling to 75 mL of bacteria cultured
• store at 4°C the next morning
• Repeat this each month to refresh the back stocks

Making Bacterial Plates for C. elegans Growth

• Turn on the Bunsen burner
• Warm plates in the incubator before adding bacteria
• Add 1 mL of OP50 bacteria from backstocks to the agar plate
• Spread around the plate by tilting, small uncoated areas are okay
• Let the plate soak with the lid closed for 5 minutes near the flame
• Label “OP50 + Bacteria plate”, name, and date
• Place lid side down in the incubator overnight to grow bacteria on the plate
• The next day, there should be a thin film of bacteria visible when put to light
• Parafilm these plates and store at 4°C until it is time for chunking to begin growing C. elegans

C. elegans Growth Stages

Chunking C. elegans

• Retrieve bacteria + agar plates made previously from the 4°C and place them in the incubator at 37°C to warm them up
• Remember to remove the parafilm before warming them
• Once warm, get a spatula and sterilize it in ethanol
• A spatula can be stored in a small flask of ethanol; change ethanol every 2 weeks
• Flame sterilize the spatula with Bunsen burner
• Pick a plate with a good concentration of starved C. elegans*
• Cut out 2 chunks from this plate, each 1 cm x 1 cm, for increased C. elegans populations for age sync.
• If chunking for a starve longer than a week, use 1 chunk
• Add a chunk to the new plate face down, and move the chunk down the plate slightly to spread the C. elegans around on the plate
• Verify on the scope that C. elegans leave the chunk and are moving around the new plate
• Parafilm the plate and twist the lids slightly to loosen the film to allow gas exchange to prevent hypoxia
• Let the plate sit face up when storing
• Flame sterilize the spatula again before chunking another plate
• Repeat this for 5 100 mm bacteria plates, 4 for age sync, and 1 to starve for the next age sync.
• IMPORTANT: Allow the 4 plates for age sync to grow for 2 days, until C. elegans reach adulthood, and proceed with age synchronization at 10 am on the second day
• Day 1: @ 10 am-12 pm Chunk C. elegans
• Day 2: @ 10 am-12 pm check their growth (~L3 stage)
• If C. elegans are not moving, they are hypoxic
• To fix this, remove the parafilm and let the plate sit for 5 minutes
• The C. elegans should begin moving again
• Consider this plate a starving plate now, as the C. elegans will not reach adulthood in time for age synching the next day
• Day 3: @ 10 am sharp, Age sync. C. elegans should be adults and be laying eggs

Age Syncing

• At 10 am on day 3, get C. elegans off of the 4 100mm plates by washing them off the plate with 10 mL M9 each. Collect the 10 mL C. elegans/M9 solution into a 15 mL conical
• Wash the plate with the 10mL of M9 from the top before collecting
• You should have 1 15 mL conical per plate of C. elegans washed off.
• eggs are collected from inside of gravid C. elegans once they disintegrate
• Wash all 4 tubes of C. elegans by centrifuging for 2 min in a tabletop centrifuge at 2000 rpm. Discard supernatant and add 10 mL M9. Repeat this wash again for all 4 tubes.
• Be sure not to disturb the pellet when removing supernatant, if disturbed, just add fresh media and centrifuge
• Once new media is added shake tubes to suspend pelleted C. elegans in solution.
• Don’t risk losing C. elegans
• While washing with M9, Prep bleach solution for 4 tubes in a 50 mL conical (4 mL household bleach, 2 mL 5M NaOH, 14 mL H2O).
• Remove supernatant and add 5 mL of freshly prepared bleach/NaOH solution per tube. Incubate at RT and invert/lightly shake throughout until half the C. elegans break down
• You should be able to see the little C. elegans bodies floating in the liquid decrease as they break down (visible with the eye and with a scope)
• As soon as half the adults dissolve, add 5 mL of M9 buffer to each tube to neutralize the reaction. Centrifuge and remove supernatant, and then swap to a new 10 mL serological.
• With a fresh serological for removing supernatant, wash the 4 tubes, now containing eggs, 3-5 times with 10 mL M9 buffer by centrifuging 2 min at 2500 rpm (1100 x g).
• When removing supernatant, be careful not to suck up eggs in the pellet, as they are very tiny
• It is okay for ~1 mL of old liquid to be left behind between washes, so long as the pellet isn’t disturbed while removing supernatant
• Smell to ensure all bleach is removed
• After the bleach smell is gone, get fresh serologicals, and wash the eggs once with 10 mL S-complete by centrifuging for 2 min at 2500 rpm (1100 x g).
• Remove the supernatant, add 10 mL S-complete, and transfer the solution to a fresh 50 mL conical tube. Do this for all 4 tubes
• Confirm eggs are transferred on the microscope (eggs look like small black beans)
• Add 15 mL of S-complete to each of the 4 tubes for a final volume of 25 mL per tube
• Have half of the tube empty
• leave one turn open, then parafilm with one layer to allow gas exchange
• Cap should be loose enough to wiggle a little.
• Wrap in a single layer of parafilm and invert to make sure nothing leaks
• Rotate each of the tubes for 24 hours at RT so C. elegans can hatch

Making Bacteria Feeding Solution for Plating

• Weigh and tare a 50 mL conical
• Get the bacteria back stock tube and vortex to ensure all bacteria are homogeneous
• Remove 10 mL of bacteria stock and add it to a 50 mL conical
• Centrifuge at 3000 rpm for 5 min until pelleted, and the liquid is not hazy
• Remove as much supernatant as possible without sucking up bacteria
• Weigh the tube to find the mass of bacteria and write this down
• Resuspend in 10 mL of water and vortex to homogenize.
• Centrifuge at 3000 rpm for 5 min until pelleted, and the liquid is not hazy
• Remove supernatant
• Resuspend in 10 mL of water and vortex to homogenize.
• Centrifuge at 3000 rpm for 5 min until pelleted, and the liquid is not hazy
• Remove supernatant
• Resuspend in S-complete media to bring the concentration of bacteria to 3mg/ mL
• Label, Name, and Date
• Store at 4°C

Test the bacteria volume in the “counting plate” and ensure the bacteria is homogeneous in solution for the best results.

Plating C. elegans Overview:

General Workflow

Day 1: Plate C. elegans and Add Feeding Solution

Day 2: C. elegans growing (should be late L2, early L3), prepare 12.5x FuDr and drugs for treatment

Day 3 @ 10 am sharp: Add 1µL of 12.5x FuDr to each well to sterilize C. elegans at L4

Day 4: Add drugs and image from here on out.

Determining C. elegans Concentration

• Find the number of C. elegans per 50 µL pipette of C. elegans solution and dilute to get around 5 C. elegans per well
• Shake the tube of C. elegans and pipette 10 wells with 50 µL of hatched C. elegans in a dedicated “counting plate” and wait 1 min to let C. elegans settle at the bottom of the wells
• On the scope, count the C. elegans per well and find the average C. elegans per well per 50 µL
• Dilute the solution of C. elegans so that there would be ~5 C. elegans per well, as opposed to how many are in the hatching tube, determined by the average C. elegans per well per 50 µL pipette
• EX:
• If the average C. elegans in 50 µL is 20 C. elegans, take the 25 mL tube of C. elegans solution and evenly split it into 4 different tubes, and dilute each tube to 25 mL (4-fold dilution)
• If the average C. elegans in 50 µL is ~10 C. elegans, take the 25 mL tube of C. elegans solution and evenly split it into 2 different tubes, 12.5 mL each, and dilute each tube to 25 mL (2-fold dilution)
• Note: The number of C. elegans that actually end up in the well after the dilution will be slightly higher than 5 due to adding C. elegans via the reservoir
• Once diluted to around 5 C. elegans per well, the C. elegans are ready for plating
• Add 2 µL of Bacteria feed to the wells used for counting in the “counting plate”.
• Doing this will simulate what entire plates will look like.
• 2 µL should give sparse speckles of bacteria added to each well, but you may have to add slightly less or more depending on how these wells look. Too much bacteria can outcompete the C. elegans.
• To find the correct amount of bacteria feed to use, adjust the volume of pipetting so that speckles of bacteria are sparse in the wells when pipetting from a reservoir.

Plate Making

• Seed C. elegans into a 384-well plate, 50 µL per well
• Grab a reservoir and bring it back to the bench
• Start flame
• Shake and pour out the diluted C. elegans into the reservoir
• Using the 50-300 µL multiasp pipette
• Set the pipette to 50 µL if not already
• Add 50 µL to all wells of the plate
• Counting columns 1, 1.5, 2, 2.5…. 21.5, 22, 22.5, etc. out loud helps keep track, as you will be pipetting twice per column to fill all wells
• Repeat for the rest of the plates
• Add bacteria feeding solution (2 µL per 50 µL well)
• Grab a fresh reservoir and bring it back to the bench
• Start flame
• Pour out the feeding solution into the reservoir
• Using the pink multiasp pipette
• Set the pipette to 2 µL if not already
• Pipette ALL the way down to collect extra liquid in the tips (to prevent bubbles and ensure exact volume is transferred at low volumes)
• Add 2 µL to all wells of the plate, touching the bottom of the well to ensure transfer of liquid
• Counting columns 1, 1.5, 2, 2.5…. 21.5, 22, 22.5, etc. out loud helps keep track, as you will be pipetting twice per column to fill all wells
• Close the plate, label it with the experiment description, and write down that the bacteria feed was added on the plate lid with the date.
• Repeat for the rest of the plates. Leftover bacteria feed can be reused.
• Wait until C. elegans are L4 @ 10 am on day 3 to add FuDr to prevent offspring
• Make 12.5x FuDr (7.456mg FuDr + 4 mL water)
• Grab a reservoir and bring it back to the bench, and start the flame
• Pour out the FuDr into the reservoir
• Using the pink multiasp pipette
• Set pipette to 1 µL and Pipette ALL the way down to collect extra FuDr in the tips (to prevent bubbles and ensure exact vol is transferred)
• Add 1 µL to all wells of the plate
• Counting columns 1, 1.5, 2, 2.5…. 21.5, 22, 22.5, etc. out loud helps keep track, as you will be pipetting twice per column to fill all wells
• Close the plate and write that FuDr was added on the plate lid with the date
• Repeat for the rest of the plates. Leftover FuDr can be reused.
• 24 hours after adding FuDr, add drugs, date when they were added, and image to collect data.