Materials and Reagents

Note: Unless otherwise specified, all reagents are stored at room temperature.

Erlenmeyer flasks (2,000 mL and 1,000 mL)
6 cm Petri dishes (Genesee Scientific, catalog number: 32-105G)
Magnetic stir bar
Autoclave tape
Caenorhabditis elegans
500 mL Olympus vacuum filter flasks (Genesee Scientific, catalog number: 25-227)
Agar (Fisher Scientific, catalog number: BP1423)
Double distilled water (ddH₂O)
NaCl (Fisher Scientific, catalog number: S671-500)
Trypticase-peptone (Gibco, catalog number: 211921)
Cholesterol (MP Biomedicals, catalog number: ICN10138201)
Ethanol, 200-proof (Decon Laboratories, catalog number: 04-355-223)
CaCl₂·2H₂O (Fisher Scientific, catalog number: C79-500)
MgSO₄·7H₂O (Fisher Scientific, catalog number: A14491)
KH₂PO₄ (Fisher Chemical, catalog number: P285-3)
NA₂HPO₄·7H₂O (Fisher Scientific, catalog number: S373-500)
M9 Minimal Medium (see Recipes)
Cholesterol Stock Solution (see Recipes)
1 M CaCl₂ Stock Solution (see Recipes)
1 M MgSO₄Stock Solution (see Recipes)
1 M KH₂PO₄, pH 6.0 Stock Solution (see Recipes)
Eyebrow hair pick (see Recipes)
NGM Plates (see Recipes)

Equipment

Zeiss Stemi 305 stereo microscope (Zeiss, catalog number: 435063-9010-000)
Incubators for C. elegans (15–25°C)
Ethanol candle (DWK Life Sciences, catalog number: 04-245-1)
Autoclave
pH meter (Thermo Electric Corporation)
Fisher Scientific Isotemp Stirrer (Fisher Scientific, catalog number: 11-100-49S)
Pasteur pipette (Fisher Scientific, catalog numer: 22-378893)
Soft and thin eyebrow hair
Tape
Second counter (such as a YouTube video or app that beeps every second)
Platinum wire worm pick (made in-house as described in Wollenberg et al., 2013)

Software

GraphPad Prism v8.4.3. (GraphPad Software, Inc.)
BioRender (www.biorender.com )

Procedure

Notes:

Unless otherwise specified, all steps are conducted outside of a biological safety cabinet (BSC) using aseptic techniques.
This protocol assumes nematodes have already been cultured and grown to the age of interest on solid nematode growth media (NGM) plates.
Nematodes used for the data presented herein were cultured and maintained on E. coli OP50 and in accordance with methods we have used previously (Walker et al., 2021) .
C. elegans size will affect TOP measurements; therefore, we recommend using this assay on adult worms only. Also, the experimenter has to use proper size-matched controls when testing conditions that affect worm size.

Worm preparation
1. To reduce error, transfer worms using platinum wire pick onto a new NGM plate seeded with bacteria of interest. Make sure there is an equal number of worms per plate as well as an equal amount of bacteria on each plate that is consistent between test and control worms.
  Note: Make sure to flame the worm pick between samples to avoid cross-contaminating with bacteria or worm strains.
Motility assessment
Note: This step is performed using a dissection microscope and a ticking second timer.
1. Using a pick with an eyebrow hair taped to it (Figure 2), put an NGM plate that contains worms in view. Slide the eyebrow hair under the mid-section of a worm and count the number of seconds it takes for the worm to crawl off entirely.
  Note: It is important to make sure that the worm is not poked in the side with the eyebrow hair; instead, the hair should delicately be placed gently underneath the worm. Additionally, ensure that the eyebrow hair is not going into the agar, is on top of the agar at all times, and remains stationary between the surface of the agar and the worm. Do not lift the worm with the eyebrow hair.
2. Record the number of seconds it takes the worm to crawl off. Continue with worms and samples as desired.

Figure 2. Picture of the eyebrow hair-pick used in the TOP assay.

Data analysis

The TOP assay allows the experimenter to assess changes in motility by measuring the number of seconds it takes for a worm to completely move off an eyebrow pick. The TOP measurement is proportional to motility—the more time it takes, the larger the defect. The results are analyzed by comparing the measurements of the experimental group with those of the control. We used various C. elegans strains to demonstrate the feasibility of the TOP assay. Data are representative of two independent experiments with a minimum of 30 worms total. The data are represented as standard error of the mean (SEM), and the statistical significance was calculated using student’s t-test (see legend in Figure 1).

Notes

All reagents should be sterile to avoid contamination of samples. All other precautionary notes are contained within corresponding steps of the protocol in “Procedure”.

Recipes

M9 Minimal Medium
1. Combine 5.8 g of Na₂HPO₄·7H₂O, 3.0 g of KH₂PO₄, 5.0 g of NaCl, and 0.25 g of MgSO₄·7H₂O in a 1 L flask.
2. Add a magnetic stir bar and fill to 1 L with ddH₂O.
3. Place on stir plate and let mix until all reagents are dissolved.
4. Filter sterilize into two 500 mL bottles.
5. Aliquot into smaller sterile bottles if desired, and store at RT.
Cholesterol Stock Solution
Dissolve cholesterol in 200-proof ethanol to a final concentration of 5 mg/mL cholesterol. Store at -20°C until use.
1 M CaCl₂ Stock Solution
1. Add 147 g of CaCl₂·2H₂O to a 1 L flask.
2. Fill to 1 L with ddH₂O.
3. Add a magnetic stir bar.
4. Place on stir plate until reagent dissolves.
5. Aliquot and screw caps loosely on bottles.
6. Adhere autoclave tape.
7. Autoclave fluid cycle for 30 min.
8. Let cool, fully screw caps on, and store at RT until use.
1 M MgSO₄Stock Solution
1. Add 246.5 g of MgSO₄·7H₂O to a 1 L flask.
2. Fill to 1 L with ddH₂O.
3. Add a magnetic stir bar.
4. Place on stir plate until reagent dissolves.
5. Aliquot and screw caps loosely on bottles.
6. Adhere autoclave tape.
7. Autoclave using a fluid cycle for 30 min at 121°C.
8. Let cool, fully screw caps on, and store at RT until use.
1 M KH₂PO₄, pH 6.0 Stock Solution
1. Add 136.1 g of KH₂PO₄ to a 1 L flask.
2. Dissolve in 800 mL of ddH₂O.
3. Add a magnetic stir bar.
4. Place on stir plate until reagent dissolves.
5. Adjust pH to 6.0 with 10 g of KOH. Add ddH₂O up to 1 L.
6. After reagents are completely dissolved, and pH is adjusted to 6.0, aliquot and screw caps loosely on bottles.
7. Adhere autoclave tape.
8. Autoclave using a fluid cycle for 30 min at 121°C.
9. Let cool, fully screw caps on, and store at RT until use.
Eyebrow hair pick
Place soft and relatively thin eyebrow hair in the smaller tip of a glass Pasteur pipette, root-first. Secure with tape.
NGM Plates
1. In a 2 L flask, combine 3.0 g of NaCl, 2.5 g of trypticase-peptone, and 17.0 g of agar.
2. Fill ddH₂O up to 1 L.
3. Add a magnetic stir bar.
4. Place on stir plate and let reagents mix for several minutes.
5. Place cap (loosely screwed on) or foil on the opening of the bottle.
6. Adhere autoclave tape.
7. Autoclave using a fluid cycle for 30 min at 121°C.
8. Cool to 50°C.
9. Add 1 mL of 5 mg/ mL cholesterol stock solution, 1 mL of 1 M CaCl₂ stock solution, 1 mL of 1 M MgSO₄stock solution, and 25 mL of 1M KH₂PO₄, pH 6.0 stock solution.
  Note: Let cholesterol stock solution (5 mg/mL) reach room temperature and ensure cholesterol re-dissolves in solution before adding to NGM.
10. Cover and put back on stir plate for one minute.
11. Pour into 60 mm Petri dishes.
12. Let cool and store upside down in a plastic box, with lid at 4°C.

Acknowledgments

This work was supported in part by the Infectious Diseases Society of America, The National Institutes of Health (R03AG070580-01), and Start-up funding provided by the Microbiology and Cell Science Department at the University of Florida Institute of Food and Agricultural Sciences to DMC. Walker et al. (2021) is the original research paper from where this protocol was derived. The Graphical Abstract figure was created using BioRender.com.

Competing interests

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have declared that no competing interests exist.

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