Title

Proteinase K's effect on C. elegans and C. burnetti

Presentation Type

Poster

Abstract

The objectives of my study examined the effects of the enzyme proteinase K, and how it affects the viability of a wild type strain of Caenorhabditis elegans and a red-fluorescently tagged strain of Coxiella burnetii. The research that was conducted including developing a large sample of C. elegans grown on Nematode Growth Media and fed a strain of E. coli (OP50). In duplicate form, four samples of 10 nematodes were “picked” from the media plates using platinum wire, and transported into Worm Lysis buffer (WLB) solution. Another sample contained only Coxiella, which was then serially diluted to 104, 103, and 102 cell concentrations. At this point a low concentration of Proteinase K was added to all viles except for one of each to account for a negative control, and the mixture was then incubated at 60’C for 1 hour on a dry bath. The Coxiella samples were then spun down using a centrifuge at 10,000 rcf for 10 minutes, resuspended with PBS-S, and plated on Coxiella growth media to account for viable colony forming units. C. elegans were centrifuged at 700rcf for 10 minutes, and resuspended in M9 salt solution. Then placed on OP50 enriched media, to examine viability, and while another was transferred to glass slides for viability under a microscope. The Pilot experiment concluded that Proteinase K in low concentrations is efficient at disrupting the cell membrane of C. elegans, while not disrupting the membranes of Coxiella. The significance of these findings will allow for pure isolation of Coxiella from the gastrointestinal tract of C. elegans for future research looking at vector transmission of Coxiella between nematodes.

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Apr 12th, 11:00 AM Apr 12th, 12:00 PM

Proteinase K's effect on C. elegans and C. burnetti

UC Ballroom

The objectives of my study examined the effects of the enzyme proteinase K, and how it affects the viability of a wild type strain of Caenorhabditis elegans and a red-fluorescently tagged strain of Coxiella burnetii. The research that was conducted including developing a large sample of C. elegans grown on Nematode Growth Media and fed a strain of E. coli (OP50). In duplicate form, four samples of 10 nematodes were “picked” from the media plates using platinum wire, and transported into Worm Lysis buffer (WLB) solution. Another sample contained only Coxiella, which was then serially diluted to 104, 103, and 102 cell concentrations. At this point a low concentration of Proteinase K was added to all viles except for one of each to account for a negative control, and the mixture was then incubated at 60’C for 1 hour on a dry bath. The Coxiella samples were then spun down using a centrifuge at 10,000 rcf for 10 minutes, resuspended with PBS-S, and plated on Coxiella growth media to account for viable colony forming units. C. elegans were centrifuged at 700rcf for 10 minutes, and resuspended in M9 salt solution. Then placed on OP50 enriched media, to examine viability, and while another was transferred to glass slides for viability under a microscope. The Pilot experiment concluded that Proteinase K in low concentrations is efficient at disrupting the cell membrane of C. elegans, while not disrupting the membranes of Coxiella. The significance of these findings will allow for pure isolation of Coxiella from the gastrointestinal tract of C. elegans for future research looking at vector transmission of Coxiella between nematodes.