rolling the tube between his/her palms and then layering the soft agar over the surface of the tryptic soy agar plate labeled "Control."The plate should then be rotated and tilted so that the soft agar is spread evenly across the surface of the firmer agar plate. Set the plate aside until the soft agar hardens.
10. Using the same 1.0-ml pipette as in step 9, remove 0.1 ml of the coliphage suspension from the tryptic soy broth tube labeled 10 9 and transfer it to the tube of soft agar labeled 10 10. Note that the labels on the broth and soft agar tubes are not the same (but both are correct). Hand the tube to your partner who will layer the contents carefully over the agar plate labeled 10~10.
11. Continue to repeat step 10, transferring 0.1 ml from each coliphage dilution to the soft agar tube labeled with the next higher dilution, until you end with the 10 3 dilution in the soft agar tube labeled 10 4. You can use the same pipette throughout unless you think you have contaminated it by touching it to a surface. In each instance, your partner should layer the contents of the tube of soft agar over the corresponding plate of tryptic soy agar.
12. After all plates have hardened, incubate them at 37°C until the next session.
13. Examine the plates for evidence of the lytic activity of the coliphage on the strain of E. coli. A clear area or "plaque" will appear at each spot where one viral particle attached to and entered one bacterial cell, lysed the cell, and invaded adjacent bacteria.
14. Compare the plates that show plaques with the control plate, which should show an even "lawn" of bacterial growth.
15. Choose a plate on which the number of plaques is between 30 and 300 and count them. Calculate the original concentration (plaque-forming units, or PFU) of the coliphage by using the following formula.
PFU/ml of original suspention = number of plaques X 1/plate dilution
16. Record your results and compare them with those of other groups.
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