9/17/18

Rational:

To do a second plaque assay with soil B. The plate for the first plaque assay did not have arthro added by mistake so it was not possible to determine whether the sample contained arthro or not.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Set up an aseptic technique
• Added 10 ML FS lysate to 0.5 arthro
• Added 2 mL LB broth to control TA mixture
• Added 2 mL LB broth to TA 2 soil B mixture
• Added ML 1 M CaCl2 to TA mixture for the control and soil B
• Added arthro and FS lysate to TA soil B mixture
• Added TA (2.5 ML) to control TA and soil B mixture
• Poured control TA onto the control plate and poured soil B TA onto another plate
• Waited 10 minutes
• Placed plates into incubator at 26 C at 3:30 9/17 – 2:30 9/19

Conclusion:

The plate from 9/12 had no arthro added by mistake so as a result there was not way to confirm the presence or absence of arthrobacter phage. As a result a new plaque assay was done for soil B. Next lab the plate will be checked for the presence of plaques. If there are any plaques then purification will be done on the plaques, if there is contamination then the plaque assay will be redone, and if there are no plaques then new soil will be collected so that a new plaque assay can be performed.

Questions:

1. Group 4 all had plaques on their plaque assays. Justin had the most and well defined plaque (but all three got plaque). They each did a spot test in addition to their plaque assays, but only Justin had plaque on his spot. What do you think is going on?                                                                                                                                                  It is possible that the sample that Justin had contained more phage which could lead to him having more plaque than the rest of his group. The rest of group 4 on the other hand could have had less phage so that the reduction of time for the phage to infect arthro as well as the smaller sample size could have cause no visible plaques to form.
2. Lathan checked a purified lysate by doing a plaque assay (10 mL of lysate) of a 10^-3 lysate He counted 14 plaques. How many ML of Lathan’s 10^0 lysate should he add to web the plate (75 mm diameter) if his plaque diameter is 1 mm?                                                                                                                                                                         A(plate) = π(75 mm)^2 = 5625π mm^2     A(plaque) = π(1 mm)^2 = π mm^2                                                              5625π mm^2/π mm^2 = 5625 plaques to web plate                                                                                                        (14 pfu/1 ML * 1000 ML/1 mL) * 10^3 = 1.4 * 10^4                                                                                                     5625 pfu/1.4*10^4 pfu/mL = .40 mL to web plate

Fig.5.B – Plaque assay 2 of soil B the yellow circle                      Fig.6.B – Control plate for plaque assay 2 soil B the      indicates the location of a bubble on the plate.                           yellow circle indicates the location of bubles on the                                                                                                                         plate.