Optimization of Nucleic Acid Staining For Accurate Determination of Mycobacterium Avium Paratuberculosis Viability by Flow Cytometry

Presenter Information

Dylan Eaton, Minnesota State University - Mankato

Location

CSU Ballroom

Start Date

21-4-2014 10:00 AM

End Date

21-4-2014 11:30 AM

Student's Major

Biological Sciences

Student's College

Science, Engineering and Technology

Mentor's Name

Timothy Secott

Mentor's Email Address

timothy.secott@mnsu.edu

Mentor's Department

Biological Sciences

Mentor's College

Science, Engineering and Technology

Description

Johne’s disease is a chronic infection of the small intestine of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Mpt). This disease greatly affects the profitability of dairy farming in the US, and Mpt has been linked by some to Crohn’s disease in humans. Currently, there are many problems with detecting and diagnosing Johne’s disease. We have been working to improve culture sensitivity by attempting to quantitate the number of viable organisms present in a sample via flow cytometry. LIVE/DEAD Bacterial Viability Kits provide a fluorescence assay of bacterial viability. While the suggested dye ratios in the LIVE/DEAD kits work for many different species of bacteria, previous work in this laboratory using the suggested dye ratios with Mpt greatly underestimated the number of viable cells in cultures with high viability and overestimated that number in low viability cultures. We will present data demonstrating the effect of altering the dye ratios on the accuracy of estimates of Mpt viability, as measured by flow cytometry. Improving the accuracy of real-time enumeration of this organism will ultimately lead to improved diagnostic testing protocols for Johne’s disease.