Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

Summary

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

Abstract

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Introduction

Heterogeneity in host-microbe associations is observed ubiquitously, and variation between individuals is increasingly recognized as a contributing factor in population-level processes from competition and coexistence¹ to disease transmission^2,3,4. In C. elegans, "hidden heterogeneity" within isogenic populations has been observed repeatedly, with sub-populations of individuals showing distinct phenotypes in heat shock response^5,6, ageing, and lifespan

Protocol

Worms used in these experiments were obtained from the Caenorhabditis Genetic Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Bristol N2 is the wild-type. DAF-2/IGF mutants daf-16(mu86) I (CGC CF1038) and daf-2(e1370) III (CGC CB1370) are used to illustrate differences in intestinal bacterial load.

HT115(DE3) E. coli carrying the pos-1 RNAi vector is from the Ahringer library²¹. The MYb collection of C. elegans native gut bacteria²² was obtained from the Schulenburg lab. Salmonella enterica....

Results

Bleach sterilization of live worms
Surface-bleached worms are effectively free of external bacteria until motility returns and excretion resumes. Under the conditions used here, rapid extinction of bacteria in buffer is observed (Figure 1A-C, Supplementary Figure 2, Video 1) without disturbing the gut-associated bacteria in cold-paralyzed worms (Figure 1D-F, Video 2). Thes.......

Discussion

Here data are presented on the advantages of single-worm quantification of bacterial load in C. elegans, along with a 96-well disruption protocol to allow the rapid and consistent acquisition of large data sets of this type. As compared with existing methods³³, these protocols allow higher-throughput measurement of intestinal microbial communities in the worm.

This approach has plating as a rate-limiting step and is not truly "high-throughput". Large-ob.......

Materials

Name	Company	Catalog Number	Comments
96-well flat-bottom polypropylene plates, 300 uL	Evergreen Labware	290-8350-03F
96-well plate sealing mat, silicon, square wells (AxyMat)	Axygen	AM-2ML-SQ
96-well plates, 2 mL, square wells	Axygen	P-2ML-SQ-C-S
96-well polypropylene plate lids	Evergreen Labware	290-8020-03L
Agar	Fisher Scientific	443570050
Bead mill adapter set for 96-well plates	QIAGEN	119900	Adapter plates for use with two 96-well plates on the TissueLyser II
Bead mill tissue homogenizer (TissueLyser II)	QIAGEN	85300	Mechanical homogenizer for medium to high-throughput sample disruption
BioSorter	Union Biometrica	By quotation	Large object sorter equipped with a 250 micron focus for C. elegans
Bleach, commercial, 8.25% sodium hypochlorite	Clorox
Breathe-Easy 96-well gas permeable sealing membrane	Diversified Biotech	BEM-1	Multiwell plate gas permeable polyurethane membranes. Thin sealing film is permeable to O2, CO2, and water vapors and is UV transparent down to 300 nm. Sterile, 100/box.
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
Disodium EDTA	Fisher Scientific	409971000
DL 1,4 Dithiothreitol, 99+%, for mol biology, DNAse, RNAse and Protease free, ACROS Organics	Fisher Scientific	327190010
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
Glass beads, 2.7 mm	Life Science Products	LS-79127
Glass beads, acid-washed, 425-600 µm	Sigma	G877-500G
Glass plating beads	VWR	76005-124
Hydrochloric acid	VWR	BDH7204-1
Iron (II) sulfate heptahydrate	Fisher Scientific	423731000
Kimble Kontes pellet pestle motor	DWK Life Sciences	749540-0000
Kimble Kontes polypropylene pellet pestles and microtubes, 0.5 mL	DWK Life Sciences	749520-0590
Leica DMi8 motorized inverted microscope with motorized stage	Leica	11889113
Leica LAS X Premium software	Leica	11640687
Magnesium sulfate heptahydrate	Fisher Scientific	AC124900010
Manganese(II) chloride tetrahydrate	VWR	470301-706
PARAFILM M flexible laboratory sealing film	Amcor	PM996
Peptone	Fisher Scientific	BP1420-500
Petri dishes, round, 10 cm	VWR	25384-094
Petri dishes, round, 6 cm	VWR	25384-092
Petri dishes, square, 10 x 10 cm	VWR	10799-140
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
R 4.1.3/RStudio 2022.02.0 build 443	R Foundation	n/a
Scoop-type laboratory spatula, metal	VWR	470149-438
Silicon carbide 36 grit	MJR Tumblers	n/a	Black extra coarse silicon carbide grit. Available in 0.5-5 lb sizes from this vendor.
Sodium dodecyl sulfate	Fisher Scientific	BP166-100
Sodium hydroxide	VWR	BDH7247-1
Sodium phosphate dibasic anhydrous	Fisher Scientific	BP332-500
Sodum chloride	Fisher Scientific	BP358-1
Sucrose	Fisher Scientific	AC419760010
Tri-potassium citrate monohydrate	Fisher Scientific	AC611755000
Triton X-100	Fisher Scientific	BP151-100
Zinc sulfate heptahydrate	Fisher Scientific	AC205982500