Bacteria populate our bodies, our food and our habitats and the balance between a symbiotic and a parasitic relationship is an important one. In an attempt to have some kind of control over the spread of our microbial friends and foes, we have devised a number of methods of identifying it. Due to the man ways bacteria have of spreading, the most dangerous locations where risk of a bacterial infection is highest are places with a relatively close group of individuals in close contact (Barbosa, 2000).
Therefore hospitals, prisons, nursing homes, child care institutions, homeless shelters are at the highest risk for disease outbreaks caused by bacteria. Additionally, some of these places such as hospitals have individuals with a weakened immune system, whether from disease, from old age or because it has yet to develop (Canard, 1992, 1421-1429). Hospitals are even a greater threat since surgical wounds, IVs and catheters offer bacteria free entrance past the number one protector, the skin. A bacterial infection can spread rapidly in such a setting. In the case of a suspected outbreak, it can be important to determine whether the outbreak is coming from a single source that can be a hospital worker or even food being served in the hospital. Pulsed field gel electrophoresis (PFGE) is proving to be a useful way to detect an outbreak caused by a single strain and trace it to its source.
How Pulsed-Field Gel Electrophoresis Is Done
The process begins by growing a pure culture of the desired bacterial cells overnight and harvesting a loopful of culture to make a buffered suspension. It is first necessary to release DNA from the cells without shearing the DNA into small pieces. To do this, the cell suspension is mixed with a protease (an enzyme that disrupts the cell membrane by attacking the membrane proteins) and with agarose mixed with SDS (a detergent that unfolds proteins). The enzyme-detergent mixture will denature the cell membrane proteins thus forming holes in the cell through which the chromosomal DNA will be released. The agarose will keep the DNA embedded in its gel matrix (Cano, 2000, 297-309). Next, the plug is washed several times to remove cell debris and proteases4. These will diffuse out of the agarose gel matrix more easily than the large DNA molecules. It is important to remove the proteases so as not to harm the restriction enzymes that will cleave the DNA in the next step of the process (Richardson, 2001).
The critical stage during preparation for PFGE is incubation of the agarose slices (the plug is sliced after the washes) with restriction enzymes. Restriction enzymes recognize a specific nucleotide sequence and cut the double stranded DNA wherever that sequence occurs. Restriction digestion is important during PFGE because the length of the pieces of DNA resulting from this digestion will provide a pattern that differentiates between two bacterial ...