Volume XXXV, No. 23 | October 18, 2013

The Use of DNA Sequencing in the Microbiology Lab

Most of the students enrolled in microbiology at Gateway Technical College wish to enter the nursing program. One of the objectives of the course is learning how to identify bacteria properly. There are different approaches to accomplishing this, but the gold standard is using DNA sequencing. In the laboratory, the students identify bacteria present in/on an environmental sample using two different approaches.

The Culture Independent Approach
Most bacteria in an environmental sample cannot be cultivated (grown in the laboratory on artificial media). As a result, genomic DNA from the sample is isolated by the students using kits purchased commercially. The students often feel like they are in an episode of CSI. The DNA is then subject to polymerase chain reaction (PCR) amplification using universal primers to the 16S rRNA gene. The PCR product is cloned. Entire E. coli colonies containing the cloned DNA are sent for traditional Sanger sequencing. The sequences generated (approximately 650 bp) are trimmed to remove vector and primer sequences and are then compared to known sequences found in the GenBank nucleotide database using the BLAST algorithm.

The Culture Dependent Approach
Bacteria from the environmental sample are plated onto artificial media, such as TSA agar, blood agar, and cooked meat broth. Antibiotics can also be added as a selective agent. Media is incubated both aerobically and anaerobically at body temperature for several days, after which time the media is examined for bacterial growth. A pure culture of the bacteria cultivated is then made and colonies of the environmental isolates are sent for DNA sequencing. This approach is far easier and less expensive than the culture independent approach. However, if the bacterium cannot be grown on artificial media, the bacterium cannot be identified.

The Goal
The goal is that students will have an even better understanding of how DNA is sequenced than just lecturing on the topic. Students will be able to use computers to identify bacteria based on their DNA sequences. This has a direct application to the medical field. The same methods used here are used in hospitals across the country to determine the bacteria that are causing an infection in a patient.
Since nurses often have to explain medical tests to patients, a more complete understanding of this technology will enable them to describe how the procedure is done and why this procedure will benefit the patient.

Richard McLaughlin, Instructor, Natural Sciences

For further information, contact the author at Gateway Technical College, 3520 – 30th Avenue Kenosha, WI 53144. Email: mclaughlinr@gtc.edu