Previous Results:

• The plaque assays with the serial dilutions of Lysate #6 were examined. The control was not contaminated and plates 10^-1 and 10^-2 were completely lysed. Plates 10^-3 and 10^-4 had a good number of plaques that can be used in a titer calculation.

Objective:

• To calculate the titer of Lysate #6
• Run TEM on the lysate and obtain a picture of the phage in the sample

Procedure: Titer Calculation

1. Plate “10^-3” had 680 plaques on the plate
   1. (680 pfu/10 microliters) x (10^3 microliters/mL) x (10^3) = 6.8 x 10^7 mL = Medium Titer

Procedure: TEM

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. 0.5 mL of Lysate #6 was obtained and put into a microcentrifuge tube
3. Parafilm was placed on the cover of a plate and 1 drop (20 microliters) of lysate, 2 drops H2O, and 1 drop uranyLess were put on the parafilm in a line
4. EM grid taken from the well labeled A7 was placed shiny side down in lysate for 5 min
5. EM grid placed on each drop of water for 2.5 min
6. EM grid placed on uranyLess drop for 1 min
7. Liquid was wicked away from EM grid using paper, then the grid was placed back into well A7
8. Grid was looked at under TEM, picture taken

Results:

• It was difficult finding any phage using the microscope. After searching the grid it was noticed that there was phage, but only heads, no tails. Clusters of material were found all over the grid.

Picture of phage using TEM

Next Steps:

• During the next lab, a TEM will be run again but using a dye other than uranyLess to try and stain the phage differently. The same procedure will be used. Since the titer of the lysate is still not high enough, plaque assays and plate flooding will continue.