Application of flow cytometry to detect Staphylococcus aureus in milk samples
Open Access
- Author:
- Piergiovanni, Jessica A
- Area of Honors:
- Veterinary and Biomedical Sciences
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Bhushan M Jayarao, Thesis Supervisor
Dr. Lester C Griel Jr., Thesis Honors Advisor - Keywords:
- Staphylococcus aureus
flow cytometry
Protein A antibody
milk
mastitis - Abstract:
- Mastitis is inflammation of the udder typically caused by bacteria, fungi and yeast. Of the various pathogenic organisms that cause mastitis, Staphylococcus aureus is one of the most commonly isolated pathogens from mastitic milk. In this study, flow cytometry was explored as a new diagnostic tool for detection of S. aureus in milk samples. The study was conducted in two phases. In the first phase, S. aureus, Escherichia coli, Staphylococcus warneri and Staphylococcus epidermidis were inoculated in Brain-Heart Infusion (BHI) broth then serially diluted. These serial dilutions were then combined with Protein A antibody marked with a FITC fluorescent tag, specific for S. aureus. The specificity of the antibody was tested with S. aureus and against the other bacteria species. The findings of this part of the study showed that a 1:500 dilution of Protein A antibody was the optimal dilution for the flow cytometric assay. The study also showed that the antibody was highly specific for S. aureus. In the second phase of the study, milk samples were inoculated with S. aureus and washed with Triton X-100 and Proteinase K to remove the milk lipids and proteins prior to the addition of the Protein A antibody. Based on the fluorescence frequency it was difficult to discern the inoculated milk from the un-inoculated milk samples. In summary, it can be inferred that flow cyotmetry can be used to detect S. aureus in BHI broth, however detection of S. aureus directly from milk is hindered perhaps due to the inability of Protein A antibody to bind to S. aureus due to background interference of milk proteins and fat. It is recommended that a more effective washing step be developed to remove milk proteins and fat, in particular proteins that could bind to protein A, to improve the diagnostic sensitivity of flow cytometry for detection of S. aureus directly from milk samples.