Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Summary

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Abstract

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Protocol

1. Establishing a calibration curve for the estimation of bacteria concentrations

This section describes a method for the estimation of bacteria concentration for any given bacteria strain (e.g. OP50, HT115) that will be used as a food source for Dietary restriction experiments in C. elegans. Separate calibration curves should be established for each bacteria strain used.

1. Inoculate a single colony of E. coli OP50 bacteria picked from a fresh streak of bacteria on LB agar in 3 mL liquid LB broth.
2. Place OP50 culture in 37 °C shaker and allow bacteria to grow overnight.
3. Prepare serial 2-....

Discussion

Dietary restriction (DR) is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals ^5-7. Both environmental and genetic manipulations have been used to model DR in C. elegans, including culturing worms in a semi-defined liquid medium ^8,9, dilution of the bacterial food source in liquid medium ^10-12, and use of behavior mutants that feed at a slower rate ¹³. However, genetic mimics of DR may produce .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Carbenicillin	Duchefa Biochemie	C0109.25	Dissolved in water.
Kanamycin	Roche Group	10106801001	Dissolved in water.
Bacto-peptone	BD Biosciences	DF0118072
Bacto-tryptone	BD Biosciences	DF0123075
Bacto-yeast extract	BD Biosciences	DF0127071
Agar	BD Biosciences	DF00145170
Sterile culture tube	USA Scientific, Inc.	1485-0810
Spectrophotometer	Amersham	Gene Quant pro	Amersham is now part of GE Healthcare.
Dissecting Microscope	Carl Zeiss, Inc.	Stemi 2000
Centrifuge	Beckman Coulter Inc.	Accuspin FR