TA: Vasant Chary
March 21, 2012
Phage Titering of a Bacterial Culture and Recombination of Bacteriophage
The main objectives of this experiment included making dilutions of solutions, plating phage or bacteria, and determining the number of bacterial viruses or phage in a suspension. It was also conducted to demonstrate that two different mutants of phage T4 can exchange genetic material to give rise to wild-type phage. The experiment was used to distinguish mutants from wild-type by their host specificity. The recombination in bacteriophage was performed to determine the concentration of unadsorbed phage from the U series plates, total concentration from B series, and concentration of wild-type phage from K series. Finally, the frequency of recombination was calculated. MATERIALS & METHODS:
Since bacterial cultures are so difficult to see even under a microscope, scientists found that mixing it with E. Coli and allowing clear plaques to grow on it on petri dishes makes it simpler to view groups of bacteriophage. First, bacteriophage was titered to determine the number of bacterial phages in a suspension. The bacteriophages used in this experiment were phage 29 and phage 31 T4 rII mutants. A series of E. Coli and KCN dilutions were made and mixed with bacterial cultures and soft agar to observe the amount of resulting colonies. Since phage 29 and phage 31 grew on E. Coli B, it was the permissive host whereas they did not grow on E. Coli K making it the non-permissive host. The ratio of phage to bacteria in the initial infection was 20: 1.
The procedure was rather straightforward however proper and adequate dilutions were to be conducted. 0.5 mL of log phase E. Coli and 0.5 mL mutant phage mixture were added to a test tube with KCN. KCN was used in order to dissipate the potential across the bacterial plasma membrane and permitting adsorption of the phage particles to the bacterial wall and preventing the injection of the phage DNA into the cell. It was removed by performing dilutions which also permitted infection and allowed the lytic cycle to proceed. A 100-fold cyanide dilution was performed in an Erlenmeyer flask which was then incubated in a 37o water bath. The unabsorbed series of dilutions were done with 0.1 mL from the Erlenmeyer flask, 9.9 mL diluent, and 5 drops of chloroform which was the incubated for 15 minutes at room temperature. Chloroform was used to cause the infected cells to lyse therefore releasing newly formed intracellular progeny phage. Titering the unadsorbed phage was necessary to prevent any phage DNA and immature phage particles from developing into complete phage particles. Chloroform was added before titering the progeny phage after 60 minutes of infection to liberate any mature phage particles still inside the bacteria. The mixture from 10-2 was diluted into 10-3 which was then diluted into 10-4. Four tubes were created and two drops of E. Coli were added to each tube to titer the unabsorbed phage. Soft agar was added to each tube and spread over the petri dishes. Finally, the plates were stacked and inverted and placed to incubate at 37oC for one day and 4oC for 6 days.
While wild-type phage could grow on both E. Coli K and E. Coli B, mutant strains were only able to grow on E. Coli B. Furthermore, the plaques on E. Coli B were in deed much larger than those on E. Coli K. The three types of colonies that formed included unabsorbed rII mutants, infected total progeny, and infected wild-type progeny. The total progeny phage was measured by B series and the wild-type progeny phage was measured by K series. Recombination frequency was found along with reversion rates which were measured by dividing the number of wild-type titer over the number of mutant stock titer. RESULTS/DATA:
Table 1: Phage 31 Plaques
Phage 31| Group 2| Group 1|
10-1| 0| 0|
10-2| 0| 0|
10-3| 0| 0|
10-5| 0| 0|
10-6| 1 x 108| 0|...