Evaluating Intestinal Bacterial Colonization in Germ-Free C. elegans

Abstract

Source: Taylor, M. N., et.al. Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions. J. Vis. Exp. (2022)

This video demonstrates the exposure of germ-free, age-matched Caenorhabditis elegans worms to a bacterial strain for intestinal colonization. It explains the selective removal of non-adhered and external bacteria, leaving only viable bacteria adhered to the gut epithelium for quantification.

Protocol

1. Feeding worms on live bacteria in liquid culture

NOTE: This protocol is used to colonize worms with laboratory-grown bacteria in well-mixed conditions in liquid culture. Worms can be colonized with individual isolates from pure culture (e.g., pathogens such as Enterococcus faecium) or mixtures of isolates (e.g., minimal microbiome communities).

1. Start with sucrose-washed synchronized adult worms in a 15 mL conical tube. Wash the worms once in 12 mL of S buffer and discard the supernatant.
2. Resuspend the washed worms in the volume of S medium needed for the experiment. Consider the volume of experimental conditions, the num....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bleach, commercial, 8.25% sodium hypochlorite	Clorox
Calcium chloride dihydrate	Fisher Scientific	AC423525000
Cholesterol	VWR	AAA11470-30
Citric acid monohydrate	Fisher Scientific	AC124910010
Copper (II) sulfate pentahydrate	Fisher Scientific	AC197722500
Corning 6765 LSE Mini Microcentrifuge	Corning	COR-6765
Eppendorf 1.5 mL microcentrifuge tubes, natural	Eppendorf
Eppendorf 5424R microcentrifuge	Eppendorf	5406000640	24-place refrigerated benchtop microcentrifuge
Eppendorf 5810R centrifuge with rotor S-4-104	Eppendorf	22627040	3L benchtop centrifuge with adaptors for 15-50 mL tubes and plates
Eppendorf plate bucket (x2), for Rotor S-4-104	Eppendorf	22638930
Ethanol 100%	Fisher Scientific	BP2818500
Iron (II) sulfate heptahydrate	Fisher Scientific	423731000
Magnesium sulfate heptahydrate	Fisher Scientific	AC124900010
Manganese(II) chloride tetrahydrate	VWR	470301-706
Peptone	Fisher Scientific	BP1420-500
Phospho-buffered saline (1X PBS)	Gold Bio	P-271-200
Polypropylene autoclave tray, shallow	Fisher Scientific	13-361-10
Potassium hydroxide	Fisher Scientific	AC134062500
Potassium phosphate dibasic	Fisher Scientific	BP363-1
Potassium phosphate monobasic	Fisher Scientific	BP362-1
Scoop-type laboratory spatula, metal	VWR	470149-438
Sodium hydroxide	VWR	BDH7247-1
Sodium phosphate dibasic anhydrous	Fisher Scientific	BP332-500
Sodum chloride	Fisher Scientific	BP358-1
Tri-potassium citrate monohydrate	Fisher Scientific	AC611755000
Triton X-100	Fisher Scientific	BP151-100
Zinc sulfate heptahydrate	Fisher Scientific	AC205982500