Community DNA Extraction from Bacterial Colonies

Overview

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner

Traditional methods of analysis for microbial communities within soils have usually involved either cultural assays utilizing dilution and plating methodology on selective and differential media or direct count assays. Direct counts offer information about the total number of bacteria present, but give no information about the number or diversity of populations present within the community. Plate counts allow enumeration of total cultural or selected cultural populations, and hence provide information on the different populations present. However, since less than 1% of soil bacteria are readily culturable, cultural information offers only a piece of the picture. The actual fraction of the community that can be cultured depends on the medium chosen for cultural counts. Any single medium will select for the populations that are best suited to that particular medium.

In recent years, the advantages of studying community DNA extracted from soil samples have become apparent. This nonculture-based approach is thought to be more representative of the actual community present than culture-based approaches. In addition to providing information about the types of populations present, this approach can also provide information about their genetic potential. As with any technique, there are limitations to the data that can be obtained with DNA extraction. Therefore, many researchers now use DNA extraction in conjunction with direct and cultural counts to maximize the data obtained from an environmental sample.

Procedure

1. Bacterial Community DNA Extraction

To begin the procedure, weigh out 100 g of sieved soil. Add this to a polypropylene vessel, and add 100 mL of extraction buffer comprised of Tris buffer amended with EDTA to promote the release of bacteria from the soil matrix, then shake by hand.
Next, weigh 100 g of glass beads, and add these to the mixing vessel. Agitate the sample for 5 min using a bead beating device or mechanical wrist-action shaker for 15 min. Add 10 mL 20% sodium dodecyl sulfate, or SDS,

Application and Summary

Community DNA from cultured colonies or extracted from soil can be subjected to bioinformatics and “omic” approaches that allow for characterization of the original bacteria within the sample. The omic approaches include metagenomics – determination of “who” is within the community via 16S rRNA sequencing. This gives an estimate of the diversity within the community.

The number of bacterial cells in the original soil sample can also be calculated. Community D