Evaluating Bacterial Virulence Using the C. elegans Liquid Toxicity Assay

Abstract

Source: Chen, Y., et al. Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model. J. Vis. Exp. (2018).

This video demonstrates the C. elegans Liquid Toxicity Assay with Aeromonas, a method used to evaluate bacterial virulence by measuring worm survival in a nutrient-limited liquid environment. The assay reveals strain-specific pathogenicity based on the extent of toxin-induced damage and mortality in C. elegans.

Protocol

1. C. elegans Liquid Toxicity Assay with Aeromonas

NOTE: To avoid contamination, steps should be performed in a laminar flow hood.

1. The day after seeding the L1 worms on the ENGM (Enriched nematode growth medium) plate spread with E. coli OP50, pick a single colony of each of the four Aeromonas strains and E. coli OP50, and culture with 5 mL LB broth each at 37 °C for 16 h.
   NOTE: In the protocol, the following bacteria strains were used: E. coli OP50, the normal food source of C. elegans. A. dhakensis AAK1, the first fully-sequenced pathogenic clinical A. dhakensis isolate. A. hydrophila A2-066, A. veronii A2-007, and A. caviae A2-9307121 are representatively clinical isolates from National Cheng Kung University Hospital. 
   NOTE: To prepare enriched NGM (ENGM), dissolve 3 g NaCl, 5 g bacterial peptone, 1 g yeast extract, and 30 g agar in 1 L of deionized water. Autoclave at 121 °C for 20 min. Wait until cooled to 55 °C in a water bath, and add 1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄, 1 mL of 5% cholesterol in ethanol, and 25 mL of phosphate buffer. Swirl to mix well.
2. Measure the OD₆₀₀ absorbance of the bacteria broth.
3. Centrifuge the bacterial broth at 3,500 x g for 15 min to remove the LB broth. Resuspend the bacteria and adjust the bacterial broth to OD₆₀₀ = 3.0 with S medium.Add 195 µL of bacterial broth in S medium to at least 8 wells of a 96-well plate.
   NOTE: The composition of S medium: To prepare S medium, mix 40 mL S Basal, 0.4 mL 1 M potassium citrate, 0.4 mL trace metals solution, 0.12 mL of 1 M CaCl₂, 0.12 mL of 1 M MgSO₄, 40 µL of 5% cholesterol in ethanol and 1 mL of 8 mM FudR before use.
4. Wash off the L4 worms on the ENGM plate with E. coli OP5 using M9 medium. Adjust the worm solution to a concentration of 5 worms per µL and add 5 µL of the worm solution to each well of the 96-well plate. Ensure that there are approximately 25 worms per well.
5. Incubate the 96-well plate on a shaker at 200 rpm at 25 °C.
6. Count the number of live worms and dead worms after 24, 48, and 72 h. Calculate the survival rates of each well with the following formula: (Live worms/Total worms) × 100%.
7. Draw a scatter plot graph with the daily data calculation.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Shaker incubator	YIH DER	LM-570R	Bacteria incubation
NaCl	SIGMA	31434	Culture medium preparation
MgSO₄	SIGMA	M7506	Culture medium preparation
Agar	Difco	214530	Culture medium preparation
CaCl₂	SIGMA	C1016	Culture medium preparation
Cholesterol	SIGMA	C8503	Culture medium preparation
Ethanol	SIGMA	32205	Culture medium preparation
KOH	SIGMA	P5958	Culture medium preparation
6 cm Petri plate	ALPHA PLUS	46	agar plate preparation
96-well plate	FALCON	353072	liquid assay
Bacterial peptone	Affymetrix/USB	AAJ20048P2	Culture medium preparation
Yeast extract	SIGMA	92144	Culture medium preparation
Citric acid•H₂O	SIGMA	C1909	Culture medium preparation
Tri-potassium citrate•H₂O	SIGMA	104956	Culture medium preparation
FudR	SIGMA	1271008	Culture medium preparation
Disodium EDTA	SIGMA	E1644	Culture medium preparation
Tryptone	SIGMA	16922	Culture medium preparation
Microscope system	Nikon	Eclipase Ti inverted	microscope imaging
Scientific CCD Camera	QImaging	Retiga-2000R Fast 1394	microscope imaging