August
31
AUGUST 27TH 2018- Lab
- AUGUST 27, 2018
- PLATING SPOT TEST + FILTRATION OF DIRECT SAMPLE
- OBJECTIVE: Be able to a plate spot test without contamination to test for phage presence
- PROCEDURE:
- Tables were sanitized and lamps were lit to create an aseptic environment
- 4 plates were obtained, one for each group members as well as the broth TA control
- The plates were then labeled
- Next, a 50mL test tube for the control plate was filled with:
- .5mL Arthro
- 4.5mL LB broth
- 45𝝁L CaCl2 1M
- Then another 50mL test tube was filled (for the spot test, not control) with:
- 1.5mL arthro
- 13.5mL LB broth
- 15mL 2X TA
- 135𝝁L CaCl2 1M
- Then the direct sample was filtered using a syringe:
- 2mL was taken up into the syringe
- A .22𝝁m filter was attached to the end of the syringe
- Pressure was applied to the top of the syringe, slowly pushing the solution through the filter and into a micro-centrifuge tube
- 10mL from the control test tube was pipetted onto the control plate
- Then 10mL from the mixture for the spot test was then pipetted onto each group members’ plate one at a time
- 10 minutes was allowed for the plates to solidify
- Each group then took their enriched sample and filtered it:
- 2mL was taken up into the syringe
- A .22𝝁m filter was attached to the end of the syringe
- Pressure was applied to the top of the syringe, slowly pushing the solution through the filter and into a micro-centrifuge tube
- Each group member, one at a time, pipetted 10𝝁L of their direct sample onto the designated spot on their plate
- The previous step was conducted for the phage buffer and direct sample as well
- The plates were then left to absorb the samples for 15 minutes before being placed in the incubation cabinet (kept at room temperature)
- POTENTIAL RESEARCH QUESTIONS:
- Do phages act as an indicator for wilted oak disease?
- Do trees suffering from wilted oak lack phages in their soil? If so why?
- Do trees with wilted oak have less or more phages in their soil?
- Do trees suffering from wilted oak have greater or lower amounts of phages in the soil compared to healthy oaks?
- Does having a water source near an oak affect the amount of phages in the soil?
- Does the amount of phages in different oak species vary?
- RESULTS:
- Plates collected after incubation period were inconclusive due to contamination as seen in figure 3
- The control came out clear as seen in figure 4
- This is an indication that there is more than likely no phage presence in the soil sample collected
- Further tests will be run in a plaque assay to be fully sure that there are no phages present
- NEXT STEPS:
- Conduct a plaque assay to test for phage presence again
- If no phages are found again, then we will go back to soil collection phase
