10/01/2018

Purification Run 3

Objectives:

The objective for the day was to pick plaques from the plaque assays that were prepared on 09/26/18 and continue the purification process to acquire unique phages and a high titer lysate, which will later be used to web a plate.

Pre- Lab Observations:

The control plate was yet again contaminated. The one plaque that had formed on the plaque assay was noticeably similar to air bubbles but seemed to be plaque. It will be used anyway to test whether it is actually a plaque. If it is not, a new plaque will be picked from the initial plate. The control plates of a lab groups were contaminated in this round. To prevent further contamination, all equipment was thoroughly cleaned and bleached by the lab instructors. Due to some error, the water baths for the top agar was turned off and all the top agar had solidified. So all the 2X TA was autoclaved and the bath was turned on, restoring the stock of 2X top agar.

Procedure:

After following the protocols to set up the aseptic zone, phages were extracted from the plaque assay and transferred to a microcentrifuge tube with phage buffer(100μl). An Arthrobacter culture(0.5ml) was enriched with the extracted phage sample (10μl) for 15 minutes. While the Arthrobacter culture was being enriched, part of the Top Agar mixture was prepared for two group members and a control plate. LB broth (6ml) and 1M CaCl (67.5μl) were added to a conical vial (50ml). After the Arthrobacter culture had been allowed to enrich for 15 minutes, 2X TA (7.5 ml) was added to the conical vial containing LB broth and CaCl. 4.5 ml of the top agar mixture was then added to the culture tube, the contents of which were then poured onto an agar plate. 4.5 ml of the top agar mixture was also used to make a control group for the two group members. After the top agar was allowed to solidify for 15 minutes, the plates were inverted and placed in the incubator, were they will remain for 48 hours.

Analysis and Conclusions

The cause of contamination for the plates continues to be elusive. A non-contaminated  plate did result form the plaque assays prepared on 09/24/18, but the other control plates continue to be contaminated. The recent cleaning and bleaching of equipment may have hopefully removed the cause of contamination. The characteristics of the plaque picked on this day seem to be peculiar and may result in a negative plaque assay, which will then require picking of a plaque from the first round of purification. The procedures were properly performed in the aseptic zone and there were no apparent sources of contamination.