C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Protocolo

The following protocol is an excerpt from Campbell et al, A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay, J. Vis. Exp. (2017).

1. Preparation of Experimental Organisms
   1. Pick 10 L4 staged nematodes per strain 24 h prior to commencing the assay to ensure that organisms are young adults when tested. For each mutant or control nematode tested, pick 10 L4s (10 for the control and 10 for the assay).
      1. Maintain L4 organisms using standard methods for 24 h on standard agar plates seeded with OP50 Escherichia coli. If organisms are lost during the subsequent wash steps, compensate by increasing the starting sample size (i.e. pick 20 worms rather than 10).
         NOTE: Behavior is an innately variable phenotype. Perform the method in triplicate for each strain on three separate days. Include additional control strains and conditions for novel strains, as highlighted in the discussion section.
   2. Immediately prior to assay, transfer experimental organisms to an agar plate with no bacteria and allow nematodes to move freely for 1 min to remove excess bacteria.
      1. If experimental organisms experienced contamination conditions during the 24 h prior to the assay, discard them.
   3. Pipette 1 mL of M9 onto the bacteria-free plate to wash worms into a microcentrifuge tube.
   4. Centrifuge at 3,000 x g for 1 min. Worms should form a pellet at the bottom of the tube. Aspirate M9 solution without disrupting the worm pellet. Add 1 mL of M9 to the worm pellet, invert tube to mix worms with the solution.
   5. Repeat steps 1.4 three more times.
      1. If excess bacteria was initially transferred with the worms, repeat for a total of 5 times. No bacteria should be transferred to the copper food race plates. If food is transferred to the assay plate, it will interfere with accurate data collection.
   6. After the final wash, aspirate supernatant until 100 µL of M9 and the worm pellet remains.
      Caution: Starvation related behaviors become evident after 30 min. Consequently worms should be immediately transferred from solution once the wash steps have been completed.
2. Preparation of Assay Plates
   1. Prepare standard NGM agar plates two days prior to the assay.
      1. If plates are kept in a humid environment, make agar plates 3 days prior to assay. Alternatively, remove the lid of the plate for 3 - 6 h to ensure proper dryness (if in a sterile environment).
   2. With a thick permanent marker, make a line on the underside of the plate along the outer edge and another to form a mid-line barrier (Figure 1). The mid-line barrier should be equidistant from each edge of the plate. Use a ruler to ensure precise measurements. These lines will serve as a guide when transferring bacteria and the copper solution. Provided that E. coli is transferred before the copper solution, these lines will serve as indicators.
   3. Seed plate with 50 µL of OP50 E. coli on only one side of the copper barrier to create a uniform lawn (Figure 2). Bacterial concentration should remain consistent across assays; however, little variability has been observed in response to mild differences in concentration.
      1. Use the marked lines on the underside of the plate to ensure the bacteria do not come into contact with the copper solution. Provided that the copper solution lines the edge of the plate and forms a mid-line barrier, transfer the bacteria so that the copper solution will not come into contact with the food source.
   4. Mark a second set of plates and transfer no OP50 E. coli to them (Figure 2). These plates will serve to evaluate the negative control. These plates should also have a marking on one half of the plate to denote the initial transfer origin.
   5. Allow bacteria to dry then incubate plates at 37 °C overnight. Ensure that bacterial patches are not disturbed when transferring to a 37 °C incubator or room. Excessive disturbance could alter the location or shape of the food patch.
3. Chemotaxis Assay
   1. Freshly prepare a 0.5 M copper (II) sulfate solution prior to assay start time. Provided that 125 µL of the solution is used per plate, scale up this volume dependent on the number of assay plates used (e.g. 5 assay plates, 625 µL).
   2. Pipette 100 µL of the copper (II) sulfate solution on the edge of the agar to create an outer copper barrier. The marked underside of the plate should serve as a guide.
   3. Pipette 25 µL of the copper (II) sulfate solution to create a midline barrier.
      1. Ensure that the copper (II) sulfate solution does not come into contact with the bacterial patch. Use a spotted technique as streaking may affect locomotion due to indentations/scratches on the agar.
   4. Allow the copper solution to dry onto plate. Time period can vary depending on plate and lab conditions. Visually check for dryness every 5 min after transfer.
      NOTE: The copper solution displays a blueish tint and is easily identifiable. Use a laboratory tissue to lightly dab the solution near the edge of the plate to discern dryness.
   5. Pipette 20 µL of the worm pellet from the bottom of the tube onto the bacteria-free half of the assay plate.
      1. Ensure that 10 worms are transferred to the assay plate. If extra worms were accidentally included, remove them by picking with halocarbon oil to ensure that no bacteria are added to the plate. Each assay should have a consistent number of nematodes during the assay.
         NOTE: If too few worms are transferred during assays, increasing the initial sample size will mitigate potential losses during washes and transfers.
   6. Remove excess M9 from the plate with a laboratory tissue. M9 should not come into contact with the copper (II) sulfate solution.
      Caution: Ensure that worms and the agar surface remain unaffected while performing this step. If used too harshly, the laboratory tissue can create indentations to the agar surface of the plate and can remove worms. Worms accidentally removed via KimWipe should be discarded.
   7. Once the M9 solution has been removed and all worms have commenced non-liquid locomotory patterns, start the assay stopwatch.
      1. Optimally, remove the M9 solution within a min. The essential parameter is the identification of sinusoidal locomotion. Worms locomote differently, e.g. thrashing rather than sinusoidal, when in liquid. Start the assay stop watch once each of the experimental organisms stops thrashing.
   8. Check assay plates every 30 min.
      1. For the assay plates with bacterial patches, positively score organisms if they reach the food patch over a 4 h period. For the negative control plates, positively score organisms if they have crossed the barrier.

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Resultados

Figure 1: A Visual Representation of Markings to Indicate Copper Solution Placement on the Underside of a 5 cm Petri Dish. These indications are used to ensure that the sections of the plate have been appropriately measured and serve as a guideline when transferring the bacterial patch and then copper solution onto the agar surface.

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Materiales

Name	Company	Catalog Number	Comments
M9 Solution [3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Autoclave to sterilize before use.]			Produced in lab
Cupric Sulfate	Sigma	C-1297	Use water to appropriately suspend to a concentration of 0.5M