Muscle Necrosis Assay in C. elegans Induced by a Pathogenic Aeromonas Strain

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 a muscle necrosis assay in C. elegans (Caenorhabditis elegans) using pathogenic Aeromonas strains. By feeding green fluorescent protein (GFP)-expressing worms on Aeromonas-seeded plates, the assay models bacterial infection and reveals tissue damage through fluorescence imaging. The progressive loss of muscle structure and GFP signal indicates an Aeromonas-induced necrotic response.

Protocol

1. Caenorhabditis elegans Muscle Necrosis Assay with Aeromonas

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

1. On the day the L1 worms are seeded on the enriched nematode growth medium (ENGM) spread with Escherichia coli OP50, pick a single colony of each of the four Aeromonas strains and E. coli OP50 and culture with 2 mL Luria-Bertani (LB) broth each at 37 °C for 16 h.​NOTE: Here, 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 representative clinical isolates from National Cheng Kung University Hospital.
2. Measure the OD₆₀₀ absorbance of the bacteria broth and adjust the bacteria broth to OD₆₀₀ = 2.0 with LB broth. Spot and spread 30 µL of bacterial broth on 6 cm nematode growth medium (NGM) plates. Culture the plates at room temperature overnight.
3. On the day the L1 worms grow to the L4 stage, transfer 50 worms to each NGM plate with each of the four Aeromonas strains or E. coli OP50. Incubate the NGM plates at 20 °C.Transfer worms every 24 h to newly prepared NGM plates with bacteria.
4. Take muscle images using a fluorescence microscope.
   1. Randomly pick 10 worms onto a 2% agarose gel in M9 medium on a slide. Paralyze the worms using 2 µL of 1% sodium azide in M9 medium on agarose for less than 5 min before taking images.
   2. Place a cover slip and capture the muscle images using a green fluorescent protein (GFP) filter on a fluorescent microscope with charge-coupled device camera. Capture the muscle images at 24, 48, and 72 h post the L4 stage.

Conduct image analysis and figure preparation with an image processing software.
NOTE: The definitions of the scores and the muscle damage levels are shown in Figure 1, for which the criteria are listed as follows: 3 points for missing muscle fibers; 2 points for ruptured or broken muscle fibers; 1 point for bent muscle fibers; 0 points for healthy muscle fibers.

Results

Figure 1: The corresponding scores for muscle necrosis in C. elegans induced by Aeromonas infection. The criteria for muscle necrosis and corresponding scores are as follo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Shaker incubator	YIH DER	LM-570R	Bacteria incubation
K₂HPO₄	J.T.Baker	MP021519455	Culture medium preparation
KH₂PO₄	J.T.Baker	3246-05	Culture medium preparation
Na₂HPO₄	J.T.Baker	MP021914405	Culture medium preparation
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
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
FeSO₄·7H₂O	SIGMA	215422	Culture medium preparation
MnCl₂·4H₂O	SIGMA	221279	Culture medium preparation
ZnSO₄·7H₂O	SIGMA	204986	Culture medium preparation
CuSO₄·5H₂O	SIGMA	C8027	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