Date of Award

12-2013

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Animal Sciences

Advisor

Anne Lichtenwalner

Second Committee Member

David Marcinkowski

Third Committee Member

James Weber

Abstract

Prototheca, an alga, can cause mastitis in cattle, does not respond to routine therapy and necessitates culling of productive animals. The resazurin microbroth dilution assay was adapted for use in a prototheca susceptibility trial. Using this assay, the susceptibility of prototheca against an antibiotic (Amphotericin B {AB}) and 2 plant hormones, Indolyl propionic acid (IPA) and Indolyl butyric acid (IBA), all prepared in deionized water, was tested. P. zopfii GT2 solutions were calibrated and found to grow better in RPMI-1640 broth versus prototheca isolation medium (PIM) at 48 hours. RPMI-1640 broth was found to be compatible with the full spectrum of resazurin color changes, as compared to PIM broth. Dye reduction (pink color change) was seen by 22 hours of incubation at the highest concentration of prototheca used. The amount of dye (2 or 4 μg using visual scoring) and concentration of prototheca (250 CFU per well) were optimized to allow results to be obtained at 48 to 72 hours. A visual scoring system was validated using prototheca culture, based on the different reduction stages of the dye. Spectrophotometric wavelengths of 575 and 610 were contrasted for use with this assay. Using the optimized assay, several concentrations of AB, IPA and IBA were tested. Resazurin was found to increase protothecal growth or counts on hemocytometer; this finding was unexplained. The dye may promote growth or increase prototheca solubility in the wells. AB was inhibitory to prototheca at 0.25-1 μg per ml; the color change (blue) was stable for 72 hours. IPA and IBA were non-inhibitory at the concentration utilized (242 pg per ml; a range of 6, 12 and 24 pg per well was tested). Alternative solvents may be needed to achieve effective (higher) concentrations.

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