• SEPTEMBER 17TH 2018
  • RESULTS: (following results and conclusion are from 9/12/18 and were not viewed until 9/17/18)
    • Spot test, seen in figure 7, was cloudy, had a yellow liquid in the bottom of dish, and was contaminated due to it not being inverted 
    • The control, seen in figure 8, was cloudy from contamination, and also had yellow liquid  gathered in the bottom of the dish, was most likely contaminated from not being inverted 
  • CONCLUSION: 
    • As a result of the contamination the plates were cloudy and had no signs of plaques being present. A plaque assay will be conducted next to determine if there are plaques or not. One group member did have plaques present, and her sample was obtained from Baylor’s campus meaning her soil had been exposed to pesticides. 
  • SPOT TEST ANALYSIS + PLAQUE ASSAY 
  • OBJECTIVE: Conduct a plaque assay with no contamination 
  • PROCEDURE:
    • Tables were cleaned and lamp was lit
    • .10𝝁L of lysate was pipetted into a test tube containing .5mL of arthrobactor and was left to sit for 15 minutes
    • A large test tube (50mL)  was then filled with: 
      • 8mL LB broth
      • 90𝝁L CaCl
      • 10mL 2X TA
    • The control plate accidentally had 9.5 mL of the top agar solution added to the plate, therefore there was only enough for 2 group members to have the solution added to their lysate
    • Next the 4.5mL of the original solution was added to the test tube containing the lysate and arthrobactor 
    • The test-tube was then poured onto the plate and left to solidify for 15 minutes 
    • During the 15 minutes a different group member (SA) made a double batch of the TA solution and conducted a separate control
  • RESULTS: 
    • The control plate, seen in figure 9 had many spots on it from contamination, also had yellow 
    • The spot test is cloudy from contamination as seen in figure 10
    • As seen in figures 9 and 10 there is contamination on both plates. Contamination could possibly be from Micropipettes or LB.   
  • CONCLUSION: 
    • Due to the plates showing no plaques after a spot test and plaque assay, the group had decided to collect new soil samples, and keep continuing investingng the question: Do trees sprayed with pesticides have a higher concentration of phage than trees that have not been treated? (species of tree being test has yet to be determined). Also due to contamination from now on, LB bottles used will be labeled by groups, and all micro pipettes have been cleaned with ethanol to help diminish the chanced of future contamination. 
  • NEXT STEPS:
    • Collect new soil samples from Cameron Park and the Baylor campus 
  • EXTRA- Critical Thinking Problems:
    • 1)Group 4 all had plaques on their plaque assays. Justin had the most and most well defined  plaques (but all 3 had plaques). They each did a spot test in. Addition to their play assays but only Justin had plaque on his spot…what do you think is going on? I think that since Justin had the most, and most well defined plaques that his phages were probably very stable when put into the spot test so his pages were probably the most likely to survive since he had a higher concentration of pace and had “stronger” or more well preserved plaques. The other group members phages probably weren’t in high enough concentrations, or were in a “weaker” state and were unable to be put into a spot test with out falling apart.
    • 2) Lathan Checked a purified lysate by doing a play assay of a 10^-3 lysate. He counted 14 plaques. How many 𝝁L of Lathan’s 10^0 lysate should he add to web a plate (75mm in diameter) if his average plaque diameter is 1mm? .004017 mL
• SEPTEMBER 19TH 2018
  • GRAM STAINING OF CONTAMINANTS 
  • OBJECTIVE:
    • Determine the source of contamination 
  • PROCEDURE: 
    • .10 𝝁L of water was put on 2 spots onto a slide
    • Inoculating loop was dragged across plate and mixed into one water spot
      • Was done for control and play assay 
    • Water was then left to air dry
    • Then the slides were dragged over a flame multiple times to heat fix the bacteria 
    • Then crystal violet was applied to both samples for 1 minute and were then rinsed 
    • Next, potassium iodine was applied to both samples for one minute and was the washed away with water
    • Then ethanol was applied to the samples for 30 seconds, and was then washed away with water 
    • Lastly, Zafrin was applied to both sampled for 1 minute and was then washed away by water 
    • Samples were dried, then viewed under a compound microscope
  • RESULTS: 
    • As seen in figures 11 and 12 the control and plaque assay were both contaminated by a cocci gram-negative bacteria 
  • CONCLUSION:
    • It appears that arthrobactor was the possible bacteria present on both plates. It was a gram negative cocci, which correlates with arthrobactor. 
  • NEXT STEP: 
    • Collect new soil sample from Cameron Park.

FIGURES 7-12: