Assessing Pathogenic Bacteria–Induced Mortality in C. elegans Larvae

Abstract

Source: Anderson, Q. L., et al. A High-throughput, High-content, Liquid-based C. elegans Pathosystem. J. Vis. Exp. (2018)

The video demonstrates a liquid-based assay where genetically modified C. elegans larvae are exposed to pathogenic bacteria in a microplate. Bacterial toxins secreted into the medium induce host stress and death. A fluorescent dye stains dead worms, allowing imaging-based quantification of mortality to assess genetic susceptibility or resistance.

Protocol

1. Liquid Killing Assay Setup (Basic Protocol)

Note: This is a protocol for one bacterial strain and one source of worms.

1. Using a cell scraper, remove the Pseudomonas aeruginosa from a Slow Killing or SK plate and resuspend in ~ 5 mL of S Basal. Scale up if needed. Measure the optical density of the bacterial suspension using a spectrophotometer (OD₆₀₀)
2. Prepare 24 mL of diluted stock of P. aeruginosa in S Basal at OD₆₀₀ ≈ 0.09 (3X final concentration). See 4.6.2 for final content per well, and scale volume of bacterial dilution accordingly.
3. Add 21 mL of liquid Slow Kill media (3 g of NaCl, 3.5 g of peptone/L supplemented with CaCl₂ and MgSO₄ to a final concentration of 1 mM). Using a multichannel pipette, transfer 45 µL of bacteria and media to each well of a 384-well plate.
4. Wash worms from their source into a 50 mL conical tube and allow worms to settle under gravitational force. Aspirate supernatant to 5 mL. Resuspend in a total of 50 mL S basal.
5. Repeat step 1.4 twice.
6. Using a worm sorter, sort approximately 22 worms into each well of the 384-well plate.
   Note: The setup drops each worm in ~1.1 µL of liquid. 22 worms will amount to 25 µL, bringing total assay volume to 70 µL/well.
   1. The final composition of each well consists of 70 µL. 45 µL of this volume is added as bacterial medium (0.03 OD₆₀₀ bacteria in 24 µL S Basal, and 21 µL SK supplemented with CaCl₂ and MgSO₄). The other 25 µL is added with the 22 worms.
   2. If the sorter used here (see Table of Materials) is unavailable, worms can be diluted to a final concentration of 1 worm/µL in S Basal and pipetted 25 µL/well using a multichannel pipette. However, results may exhibit increased variability. Alternatively, it is possible to use a peristaltic liquid dispenser, as described by Leung and colleagues.
7. After worms have been sorted into the 384-well plate, seal the plate with a gas-permeable film and incubate plates at 25 °C for 24 - 48 h.
8. At the desired time, use a microplate washer to wash the 384-well plate with S Basal a total of 5 times.
   1. After the second wash, aspirate most of the media, leaving ~20 µL. Vigorously shake plates using a microplate vortexer for at least 30 seconds to loosen any debris from the bottom of the wells).
9. After the final wash, aspirate supernatant down to 20 µL. Add 50 µL of 0.98 µM nucleic acid stain (see Table of Materials)/well of the 384-well plate, for a final concentration of 0.7 µM.
10. Incubate at room temperature for 12 - 16 h. This dye will only stain dead worms. After the desired incubation period, wash plates using the microplate washer to remove any excess stain (a minimum of 3 washes).
11. For data acquisition, use a spectrophotometer or an automated microscope to image both transmitted light and fluorescence (531 nm excitation and 593 nm emission).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
COPAS FP BioSorter	Union Biometrica		Large object flow cytometer/worm sorter
Cytation 5	BioTek
EL406 Washer Dispenser	BioTek
Multitron Pro	Infors HT
24 Deep-Well RB Block	Thermo Fisher Scientific	CS15124
384-Well plate	Greiner Bio-One	MPG-781091
Nematode Growth Media (NGM)			Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl₂ (1 M), 1 mL MgSO₄ (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water
Slow Killing (SK) plates			Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl₂ (1 M), 1 mL MgSO₄ (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water
Slow Killing (SK) media			Amount per liter: 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl₂ (1 M), 1 mL MgSO₄ (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water
Lysogeny Broth (LB)	USBiological Life Sciences	L1520
Brian Heart Infusion broth (BHI)	Research Products International Corp	50-488-526
Worm Bleach Solution			Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water
S Basal			Amount per liter: 5.85 grams NaCl, 6 grams KH₂PO₄, 1 gram K₂HPO₄, and 1 Liter of milli-Q water
Agar	USBiological Life Sciences	A0930
NaCl	USBiological Life Sciences	S5000
Peptone	USBiological Life Sciences	P3300
CaCl₂	USBiological Life Sciences
MgSO₄	Fisher Scientific	M63-500
Phospate buffer			amount per liter: 132 mL of K₂HPO₄ (1M) and 868 mL of KH₂PO₄ (1M)
KH₂PO₄	Acros Organics	7778-77-0
K₂HPO₄	USBiological Life Sciences	P5100
5% Sodium Hypochlorite Solution	BICCA	7495.5-32
NaOH solution	Fisher Scientific	SS255-1
Breathe-easy	Diversified Biotech	BEM-1
SYTOX Orange Nucleic Acid Stain	Fisher Scientific	S11368
Bacterial Strains
P. aeruginosa (PA14)
E. faecalis (OG1RF)
E. coli superfood (OP50)
E. coli RNAi expressing bacteria (HT115)
Worm Strains
glp-4(bn2) (Beanan and Strome, 1992, PMID: 1289064)
PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717)