- Practice serial dilutions
- Perform a plaque assay
- Determine PFUs
Knowing how to determine the number of microorganisms in a sample is extremely important in microbiology, and requires accurate pipetting, aseptic technique, and calculation skills. In this exercise, you will again prepare dilutions and enumerate the microorganisms in a sample. Although the principles are the same, you will be enumerating viruses instead of bacteria and conserving on materials by using micropipettes and smaller volumes in your dilutions.
The number of viruses in a sample can be determined by direct count using electron microscopy or by determining the number of infectious virus particles using a plaque assay. Viruses are obligate intracellular parasites. Therefore, they require a host cell in which to grow. Some viruses lyse the cells in which they have replicated while others appear to cause little cell damage. A plaque assay can be used to enumerate viruses that lyse their host cells. In a plaque assay the host cells and virus are incubated together for a short time to allow the virus to attach to and enter the host cell. Then the mixture in plated within a semi-solid agar. This semi-solid agar is poured onto a "bottom agar" that serves to supply adequate nutrients for the host cell. At the end of one cycle of virus replication a cell infected with a single virus particle will lyse, releasing hundreds of new viruses. In the semi-solid medium these newly released viruses can only infect neighboring cells; after a second cycle of replication these neighboring cells will be lysed. Those cells that escape infection will continue to grow. After 24 to 48 hours plates that were not infected with a virus will contain a confluent layer of cells (they will resemble the TNTC plates of the preceding exercise). Those plates that were infected with several hundred viruses may actually appear clear- the viruses will have infected and lysed all of the host cells. Those plates that contain an intermediate number of viruses will have plaques, clear or partially clear circular areas in an otherwise turbid background of cellular growth. Each plaque represents the result of one infectious virus, called a plaque forming unit, or PFU. Many animal and bacterial viruses can be enumerated using a plaque assay. Bacterial viruses are also referred to as phage; in this exercise you will be using a well-characterized bacterial phage, T4, and its host cell, Escherichia coli.
In this exercise you will also be setting up your own dilution tubes and using micropipetters in this laboratory. Your laboratory instructor will go over the proper use of these pipetters and maintenance of aseptic technique.
Cultures Escherichia coli strain B culture
Phage sample T4
Media 4 petri plates containing "bottom agar"
4 tubes containing 5 mL of "top agar" - these tubes will be in the 50°C water bath
Supplies Sterile Tryptic Soy broth diluent and 10 sterile microcentrifuge tubes
Micropipetters and sterile tips
A. Sample Dilution and Plating
1. Prepare 10-fold serial dilutions of the virus through 10-6
a. To do this, first prepare 6 dilution tubes by aseptically pipetting 900 µL of
Tryptic Soy broth diluent to each tube - use one pipette tip.
b. Add 100 µL of the T4 phage sample to the first dilution tube, close the
cap, and vortex to mix thoroughly. This is a 10-1 dilution
(100 µL/100 µL + 900 µL = 100 µL/1,000 µL = 1/10 = 10-1).
c. Use a new pipette tip to transfer 100 µL from the 10-1 dilution to the next
dilution tube, mix thoroughly, this is your 10-2 dilution.
d. Continue making dilutions of the phage until you have used all 6 dilution
tubes and have a 10-6 dilution of the phage in the 6th tube.
2. Label your remaining 4 sterile tubes 10-4, 10-5, 10-6, and 10-7, and pipette 100 µL from the appropriate phage dilutions into these labeled tubes (use a new pipette tip for each transfer).
3. Add 100 µL of the Escherichia coli culture to each of the 4 labeled tubes. (Use the same pipette tip; add Escherichia coli to the tube with the most diluted phage first and work back.)
4. Incubate bacteria with virus for 10 minutes to allow for adsorption (attachment) of the virus to the bacteria.
5. While you are waiting, label the agar plates with your name, lab section, and the virus dilution that will be plated on it: one plate for each 10-4, 10-5, 10-6, and 10-7. Reset the micropipette for 200 µL.
6. After the 10 minutes, get the tubes of top agar from the 50°C water bath; working quickly, wipe off the tubes. You may want to get one top agar tube at a time so you won't have to rush; top agar solidifies within a few minutes once it is removed from the 50°C bath.
7. Pipette 200 µL from a labeled tube into a top agar tube, vortex for about 2 seconds, pour onto the appropriately labeled bottom agar plate. Do this for all four labeled tubes.
8. Allow the top agar to solidify (about 5 minutes) before moving the plates. Invert the plates and incubate at 30°C.