The Agricultural Research and Development Center

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The Agricultural Research and Development Center
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OARDC Background

• Mission To enhance the well-being of the people
  of Ohio, the world through research on foods,
  agriculture, family and the environment.
• Location Wooster, Ohio.

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My Duties as a Research Assistant

• Performing projects in a timely and safely manor.
• Preparing different types of media for my use and
  general lab use.
• Preparing primers for PCR, buffers for samples,
  and autoclaving of biohazard waste.

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MAC Media
Gel Electrophoresis
LB Agar Media
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Streaking E. coli. Onto Media
Streaking is a technique that I first learned
working at OARDC. This is also a technique I was
almost able to perfect through PLENTY of practice!
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E. coli. Colonies growing on LB Agar Media
The purpose of streaking is to get isolated
colonies of bacteria growth so that you may
transfer individual colonies for further testing
of the sample
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These are a few members of my laboratory family
members!
Amy
Yehuyn
Megan
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Project Title

• Phage-encoded dissemination of Shiga toxin genes
  among bovine E.coli - Calf Challenge Study

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Background

• Shiga-toxin producing Esherichia coli (STEC) are
  recognized as an important cause of diarreal
  disease worldwide and are usually associated with
  the consumption of bovine origin foods.
• Although bovine foods are the most common source
  of human contamination, very few of the bovine
  0157 bacteria have the potential of causing
  serious human disease.

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Calf Challenge flow chart
Inoculate calf
Fecal samples
Selective media
Characterization and Enumeration of colonies
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Protocol

• Two calves were orally inoculated with strains of
  E. coli 629 (026) and E. coli 687 (0111) which
  have been made Naladixic acid resistant.

Black
White
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Protocol continued

• Inoculation was followed the next day with an
  inoculation of phage from E. coli 632 (0157)
  strain that contains the antibiotic marker
  (chloramphenicol-cat) which has replaced a
  portion of the stx2 gene.
• Fecal samples were tested on days 1, 2, 3, 4, 5,
  7, 9, and11 of trial.
• Sample dilutions were made and plated on MACNal
  and MACChlor . Naladixic acid concentration
  40?g/ml Chloramphenicol concentration 30?g/ml.
• MACNal and MACChlor plates were enumerated and
  lactose fermenting colonies were transferred to
  E. C. MUG Broth.

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Protocol continued

• Broth cultures were transferred to the following
  selective media

- No Fermentation (Clear colony)
Fermentation (Purple colony)
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Results

• Following inoculation we found only E. coli 026
  suspects within the calves. E. coli 0157 and
  0111 exhibited no apparent colonization.
• The calves continued to show colonization of 026
  throughout testing.
• Antimicrobial treatment eliminated the presence
  of chloramphenicol resistant strains.

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Troubleshooting

• Verify 026 suspects
• 1. PCR
• 2. Anti-serum agglutination
• Inoculate with a higher dosage of E. coli 632.
• 1. inoculate with 1x109 (8.2x107 previously
  used)
• Compare colonization of two different 0157
  strains
• 1. Inoculate one calf with 632
• 2. Inoculate other calf with a different 0157

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These are my babies!!
Rest in Peace Children
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How Cool Is That!
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Oink, Oink!! Here we come!!
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Battle of The Beasts!!!
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Presentations
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Highly Recommended!!

• OARDC is a great place to learn from some of the
  best researchers in the country.
• I have obtained skills that I can apply to a
  research position and to many other positions in
  the work force that may come my way.
• This is a great way to network and develop
  relationships with many wonderful people.