Viral Titration through Plaque Assay

 A summary of Viral Titration

October 16th, 2021

 

Introduction 

    When it comes to viruses, there is a wide variety that could be talked about; few of viruses are pathogenic, others are harmless, while a good amount of them are bacteriophages. In Dr. Deutch's case of study, P1 is used as a bacterial phage to carry out transduction of different strains of E.coli. Most specifically, transduction of the the strains JW5055 (a strain missing the ProY gene) and JW0391 (a strain missing the BrnQ gene) is the project of the upcoming weeks, and the right concentration of P1 is necessary for the success of the procedure. For this experiment, a P1kc virus was obtained by Dr. Deutch as a frozen stock. The P1kc virus is one mutant of the P1 virus which can undergo both the lytic and the lysogenic cycles once it enters the bacterial cell. Although the other mutant of P1, the P1vir, would have been more effective in producing a desirable amount of lysate than P1kc since "the vir mutation prevents [it] from generating P1 lysogens among transductants," P1kc was picked by Dr. Deutch to perform the same job although with less efficiency (Thomason, Costantino, Court, 2007).                                                                                                                                                          

Method Used

  The procedure for P1kc titering is done by plating the virus on the parent E.coli strain BW25113 which starts with making a series of dilutions that later can be mixed with the desired strain to monitor bacterial growth and the appearance of plaques (areas with no bacterial growth due to virus-lysing activity). The dilution series start with a one to one hundred dilution (10^-2) and ends with a one to one billion dilution (10^-9). First, 10 μl of the stock phage solution is added to 990 μl of the MC solution (a growth solution containing magnesium sulfate and calcium chloride) in a microcentrifuge tube and vortexed well to make a one to one hundred dilution. Then, a 100 μl of the newly-made solution is added to 900 μl of MC solution in a separate tube to obtain a new dilution of one to one thousand. This procedure is then repeated continuously until a dilution of one to one billion is reached. Furthermore, the content in the centrifuge tubes is then transferred to bigger tubes to allow for space to fill in top agar made from the regular growth medium LB with calcium added to it. After adding the E.coli cultures from the BW25113 strain to the tubes and mixing the solution well, the top agar is then added to each tube. Subsequently, the whole mixture is transferred into LB plus calcium plates to allow for bacterial growth in correspondence with the virus titer. Results showing plaques of bacterial lysis are first seen after roughly six hours. 

Results and Conclusion

     P1kc virus seems to be functional when entering the lytic cycle since visible plagues prove the lysis of E.coli cells. The plaques in the lawn of bacterial growth on the plates indicate that some bacterial cells were lysed in the process of making new P1 viruses. The results of P1 titering show that the plates which have the viral and bacterial solution with the dilution 10^-5 and 10^-6 yielded a countable amount of plaques (68 plaques for 10^-5 and 5 plaques for 10^-6) while the dilutions 10^-2 to 10^-4 yielded too many plaques to count and the rest from 10^-7 to 10^-9 yielded zero plaques. The titer was then calculated by  dividing the number of plaques for each feasible dilution by the volume of P1 virus in the solution and then multiplying the result by the number of dilutions made for that particular tube of solution. The two titers of the feasible dilutions are then averaged for a single titer of P1. Nonetheless, this process is not perfect, which makes the results desirable. Bacteriophages are known for making phage packaging mistakes, hence P1. The process of packaging viral DNA inside new phages within a bacterial cell could mistakenly package bacterial DNA as well, forming phages carrying desired genes of bacterial DNA which can then be used to infect other strains of bacteria to introduce the new gene in a process called transduction. All in all, viral titering is an important process for determining the right concentration of viruses capable of producing an observable amount of bacterial lysis through the examination of plaques. 

References

Thomason, L.C., Costantino N., Court D.L. (July 2007) E.coli Genome Manipulation by P1 Transduction. Current Protocols in Molecular Biology 1.17.1-1.17.8.

 

Deutch, C.E. (October 2021) Titering the Stock Sample of Phage P1. Put Project at GCC.