27 August 2018 ✷ Spot Test

A spot test is being performed to check for the presence of phage.

Scientific Question:

Which types of Oak tree does arthrobacter phage inhabit more frequently?

Procedure:

• Workspace was cleaned with CiDecon and 70% Ethanol and an alcohol burner was lit to promote an aseptic work environment.
• My petri dish was labeled with my initials and the date, in addition to 3 penny-sized circles (one labeled “negative control,” one labeled “enriched,” and the last labeled “direct.”
• An additional petri dish was set up for my lab group; its purpose is to test the agar my group made.
• In the process of making the plate, my group made 2 tubes: one for the control plate, and one with enough for our three plates (both used the same LB broth). SEE TABLE. The Top Agar was added last because it hardens as it cools and it was important to add the other components first as to be time efficient.

Components of Plate

control plate 

3 spot test plates

• The mixtures were poured into their respective plates and allowed to cool and harden.
• The enriched solution was filtered through a 22 micron filter into a microcentrifuge tube in order to remove arthrobacter.
• After plates hardened, a micropipette was used to place 4.4 µL of each sample into its respective circle on the plate (enriched, direct, negative control phage buffer).
• The remaining direct isolation sample was refrigerated and the plates were placed in an incubator for 46 hours.
• The lab space was then cleaned once again with CiDecon and 70% Ethanol Solution.

Observations/results/data:

• After the enriched solution was filtered, it became paler yellow.
• In the process of pouring my plate, my partner burned her finger on the burner flame and a small portion (less than 1 mL) of the agar solution spilled onto the table. Though this didn’t directly cause contamination to my plate, its possible there may be some issues with the data produced from my plate.
• UPDATED: Upon returning to lab on Wednesday, August 29, I removed the petri dish from the incubator and found no plaques, only contamination. However, our agar control plate showed no signs of contamination, thus the error had to have occurred in the process of pouring and storing our plates because each member of my lab group had contamination on her plate (see images)
• A likely source of contamination is that trying to maintain aseptic technique while having 3 people assist led to several moments where the open container we were working with left the aseptic zone and more precaution will need to be taken in future experiments.
• 

Interpretation/Conclusion/Next Steps

• The phage in each sample (if present) would infect the arthrobacter in the plate and kill it, leaving plaques that should be visible.
• If phages are present, the spot test is a way to indicate their presence; however, it does not indicate concentration.
• Instead, the use of a plaque assay next time will give further information in regards to the presence of phages from the soil sample.
• UPDATED: Because no phage was detected in the spot test, only one plaque assay will be run (with the enriched lysate) just to practice the technique. At some point in the future, another soil sample will be collected to attempt to isolate a phage.