11.2: Plaque Assay (Day 1)
Unlike bacteria, many of which can be grown on an artificial nutrient medium, viruses require a living host cell for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium can be harvested as a source of virus. In this lab, we will be quantifying bacteriophages (viruses that attack bacteria) using a plaque assay. This means that we must grow both bacteria and virus.
As the virus replicates and grows, it will lyse bacteria resulting in an area of clearing that can be observed on a bacterial lawn. These clearings are called plaques. A plaque assay, therefore, is a method by which the number of plaques observed can be used to estimate the amount of virus present in the original culture. The technique required to do so is called the pour-plate technique because you will mix the virus with the bacteria with melted agar and then pour the mixture onto a plate.
The unit of viral titer (concentration) is plaque-forming units per milliliter (pfu/ml).
As we do not know the starting concentration of virus, we will need to do a serial dilution (similar to the standard plate count). After incubation, the number of plaques formed will be used to calculate the original viral (phage) titer.
Plague Assay:
Complete the Following Lab with Your Partner:
1. Obtain 14 microcentrifuge tubes and label either #1-7 or E. coli #1-7.
2. Obtain 7 nutrient agar plates. Label them A-G and place them in the 37°C incubator to warm up until needed.
3. Aseptically add 990 μL of sterile saline to tubes #1.
4. Aseptically add 900 μL of sterile saline to tubes #2-7.
5. Finger vortex the E. coli broth. Then, aseptically add 300 μL to all seven E. coli microcentrifuge tubes.
6. Vortex the T4 bacteriophage suspension and then aseptically transfer 10 μL to tube #1. Close the lid and vortex. This is your 10-2 dilution.
Enterobacteria phage T4 is a bacteriophage that infects Escherichia coli bacteria.
7. Aseptically transfer 100 μL from tube #1 to tube #2, close the lid and vortex. This is your 10-3 dilution.
8. Aseptically transfer 100 μL from tube #2 to tube #3, close the lid and vortex. This is your 10-4 dilution.
9. Aseptically transfer 100 μL from tube #3 to tube #4, close the lid and vortex. This is your 10-5 dilution.
10. Aseptically transfer 100 μL from tube #4 to tube #5, close the lid and vortex. This is your 10-6 dilution.
11. Aseptically transfer 100 μL from tube #5 to tube #6, close the lid and vortex. This is your 10-7 dilution.
12. Aseptically transfer 100 μL from tube #6 to tube #7, close the lid and vortex. This is your 10-8 dilution.
13. Aseptically transfer 100 μL of SALINE into tube E. coli #1, close the lid and vortex. This will become your negative control.
14. Aseptically transfer 100 μL of dilution tube #2 to E. coli #2, close the lid and vortex.
15. Aseptically transfer 100 μL of dilution tube #3 to E. coli #3, close the lid and vortex.
16. Aseptically transfer 100 μL of dilution tube #4 to E. coli #4, close the lid and vortex.
17. Aseptically transfer 100 μL of dilution tube #5 to E. coli #5, close the lid and vortex.
18. Aseptically transfer 100 μL of dilution tube #6 to E. coli #6, close the lid and vortex.
19. Aseptically transfer 100 μL of dilution tube #7 to E. coli #7, close the lid and vortex.
20. Let all seven tubes stand for 15 minutes. We are allowing time for the virus to infect the bacteria.
21. Remove one soft agar tube (containing 2.5 ml of agar) from the hot water bath and add the entire contents of E. coli #1 to it. Using a sterile plastic transfer pipet, mix the contents by pipetting up and down twice. (DO NOT USE YOUR MICROPIPETTER FOR THIS OR YOU WILL LOSE POINTS!!!)
22. Immediately pour the mixture onto plate A. Then, gently tilt the plate back and forth until the soft agar mixture is spread evenly across the solid medium.
Remember that you need to take the soft agar plates and nutrient agar plates out one at a time. Otherwise, the agar will solidify before you have a chance to pour.
23. Allow the agar to solidify completely with the lid on at your table (15-20 minutes). Then, invert and place in the 37°C incubator to grow overnight.