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Recovery of Viable Legionella pneumophilia from Desiccated Food Vacuoles of the Ciliate Tetrahymena Jonathan Thomas Department of Biology Tennessee Technological University Cookeville, TN 38505. Key Words:. Legionella pneumophilia , Tetrahymena , vesicles, vacuoles, desiccation.

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  1. Recovery of Viable Legionella pneumophilia from Desiccated Food Vacuoles of the Ciliate TetrahymenaJonathan ThomasDepartment of BiologyTennessee Technological UniversityCookeville, TN 38505

  2. Key Words: Legionella pneumophilia, Tetrahymena, vesicles, vacuoles, desiccation

  3. Project Summary • Objective:Test the hypothesis that there is no difference in the number of recoverable viable Legionella pneumophilia between those encased in food vesicles after ingestion and release by Tetrahymena and those living free in the water of cooling towers. • Laboratory proceedings performed at TTU Water Center in conjunction with Dr. Sharon Berk and Dr. John Gunderson • Feed Legionella pneumophilia to Tetrahymena

  4. Project Summary • Allow Tetrahymena to secrete vesicles for 24 hours • Use Gunderson’s method to separate the vesicles from the Tetrahymena • Divide into two sets: Legionella within vesicles, free-living Legionella • Dry both sets • Attempt to resuscitate both sets by adding water over planned time intervals

  5. Project Summary • Expect recovery of Legionella within vesicles, no recovery of free-living Legionella • Calculate percent recovery of Legionella within vesicles

  6. Introduction • Legionnaire’s Disease • Robotham studied Legionella-amoebae interactions in cooling towers (Neumeister et al 2000) • Berk et al (1998) currently researching on vesicles secreted by amoebae

  7. Introduction • Kwaik (1996) and McNeely et al (2000) sparked new interest in the ciliate Tetrahymena secreting vesicles containing Legionella • Barker (1999) Demonstrated that Legionella within vesicles exhibit resistance to biocides and high temperatures • What about resistance to desiccation?

  8. Introduction • The objective of this study is to test the hypothesis that there is no difference in the number of recoverable viable Legionella pneumophilia between those enclosed within food vacuoles released from the ciliate Tetrahymena and those living free in the environment

  9. Methods and Materials • Create graph of number of Legionella vs. optical density • Dilutions in microwells • Optical density reader • Acridine orange and fluorescence microscopy

  10. Methods and Materials Figure 1. Number of Legionella bacteria present for a given optical density

  11. Methods and Materials • Feed Legionella to Tetrahymena • Allow Tetrahymena to secrete vesicles • Use Gunderson’s method to separate the vesicles from the Tetrahymena • Divide into two sets: Legionella within vesicles, free living Legionella • Dry out both sets using desiccator • Attempt to resuscitate by adding water over planned time

  12. Table 1. Record of Legionella recovered at planned time intervals

  13. Expected Results and Benefits • Expect recovery of Legionella within vesicles, no recovery of Legionella not within vesicles • Measure how long Legionella remain viable within the vesicle • Calculate percent recovery of both groups

  14. Expected Results and Benefits • Show importance of Legionella-Tetrahymena interactions • Explain many case histories of Legionnaire’s Disease • Show the dire need for new ways to treat Legionella-Tetrahymena interactions in cooling towers

  15. Project Timeline • One day = set up • One day = feed the ciliates • One day = allow ciliates to produce vesicles • One day later = separate into groups, place them in desiccator

  16. Project Timeline • Plate out and attempt to recover both groups • 24 hours after placing in desiccator • 72 hours later • 22 days later • And so on or until bacteria cannot be recovered

  17. Project Timeline • If vesicle-bound Legionella is recovered, record how long it remains viable • Calculate percent recovery • Times not exact due to the nature of the experiment

  18. Budget • $48 = BCYE plates • $40 = PCB media, pipette tips, buffer solution, and Acridine orange stains • $1000 = Present at ASM meeting, Washington D.C., November 2002 • $1088 = Total estimated expenses

  19. Personnel • Jon Thomas = 30 hours • Dr. Sharon Berk = 12 hours • Dr. John Gunderson = 8 hours

  20. Facilities • Center for Management, Utilization and Protection of Water Resources Cookeville Tennessee, 38505

  21. Questions?

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