9-1 Defining and counting bacteriophage

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Viruses are obligate parasites of organisms and almost every life-form known is victimized by these pests. Viruses that attack bacteria are termed bacteriophage or phage. Bacteriophage consist of at least two components, an outer protein coat and inside the coat, a nucleic acid. The nucleic acid (either DNA or RNA) encodes for proteins involved in taking over the host cell's machinery and perverting it to form new virus.

There are three phases in the life cycle of a bacteriophage; attachment, replication, and release. During attachment, the virus non-covalently binds to its host at a phage receptor and injects its viral nucleic acid. Of course the host bacteria does not have special structures just for phage attachment. These viral receptors are proteins (pili, flagella, transport enzymes, or other membrane components) involved in various host functions. Once the nucleic acid is inside, the phage replication cycle takes place. The phage must then be released from the bacteria to repeat their life cycle. This is accomplished either by lysing the bacterium or by budding out of the bacterial cytoplasmic membrane. For more details about bacteriophage, a very interesting subject, read the section on bacteriophage in the microbiology textbook.

Figure 9-1 Bacteriophage life cycle

The bacteriophage life cycle involves attachment, replication and release. Bacteriophage recognize a protein on the outside surface of the microbe, such as the flagella, a pilus, or a receptor for transport of a sugar. They bind this protein and inject their nucleic acid inside. Replication then begins inside the host cell. After replication, the virus leaves the host cell either by budding out of the host membrane, or by lysing the host cell.

Bacteriophage Enumeration

Since phage are obligate parasites, a host must be provided in order to enumerate them. Providing a host and counting the number of bacteriophage is most easily accomplished by using an agar overlay. In this method, a small number of phage particles, obtained by serial dilution in appropriate buffer, is mixed with an excess of host bacteria. The phage-bacteria mixture is added to molten agar and then poured into a plate containing solidified agar. (The extra agar in the plate is needed to prevent the thin layer containing the phage and bacteria from drying out.) The bacteria multiply in the medium forming a confluent lawn on the plate, causing the Petri plate to be mostly opaque. In places where bacteriophage are present, they multiply and lyse the bacteria, causing a zone of clearing (a plaque) on the plate. Theoretically, each plaque is formed by one virus and the number of plaques multiplied by the dilution factor is equal to the total number of viruses in a test suspension. This is analogous to bacterial cell enumeration and the same guidelines for CFU's apply to plaque forming units (PFU's)

In this chapter we will examine three experiments dealing with phage. First, phage strain JL-1 will be enumerated using its host E. coli to familiarize you with the procedures necessary for counting phage. Second, the process of phage-typing, a method for discovering the host ranges of viruses, which is also useful for bacterial identification will be introduced. Third, an attempt will be made to isolate viruses from nature.

Period 1

Materials

Broth culture of Escherichia coli strain B

Diluted suspension of phage strain JL-1 (a 10-5 dilution)

3 dilution blanks (9 ml)

3 tubes of melted Top Agar (4 ml/tube) - in 55°C water bath

3 plates of Bottom Agar

Micropipettes and sterile tips

  1. Label the dilution blanks 10-6, 10-7 and 10-8. Label the Bottom Agar plates 10-7, 10-8 and 10-9.
  2. Prepare three serial, decimal (1/10) dilutions of the phage suspension provided which is already a 10-5 dilution of the original (undiluted) suspension. Note that you are making cumulative dilutions of 10-6, 10-7 and 10-8 of the original suspension.
  3. Obtain three tubes of Top Agar from the 55°C water bath. Be sure to work quickly, as the medium will solidify before too long if left at room temperature. (The tubes may be placed in a cup of hot water-50-55°C-at your desk.) Label the tubes 10-7, 10-8 and 10-9, each representing the respective plated dilution to be made.
  4. Pipette 0.1 ml from the 10-8 dilution into the Top Agar tube labeled 10-9. The same pipette tip can be used to inoculate the remaining Top Agar tubes in similar fashion as long as you are going from the more dilute to the more concentrated dilutions.
  5. Using a fresh pipette tip, inoculate 0.1 ml of the host culture into each tube of Top Agar.
  6. Gently mix each tube of Top Agar and pour the entire contents onto the surface of the same-labeled plate of Bottom Agar. Rotate the plate to distribute the agar layer evenly.
  7. After the medium has solidified in each plate, incubate at 37°C for 1-2 days (or 30°C if the incubation is more than 2 days). Plaques will begin to appear in the growing lawn within several hours!

Period 2

Figure 9-2 Phage plates

Results of the phage titration experiment showing Petri plates with phage plaques (the clear circular areas) on them.

  1. Count the plaques on the plate containing between 30 and 300 plaques. Determine the concentration of plaque-forming units (PFUs/ml) in the original, undiluted suspension. Record results in the table below.

Plated dilution of phage JL-1

Number of plaques (if countable)

10-7

10-8

10-9

Therefore, the number of PFUs of phage JL-1 per ml of the undiluted suspension =

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