Chapter 8 - Bacteriophages, the Viruses of Bacteria

8 - -1 Enrichment of phage from nature

Bacteriophages can be recovered consistently from various environmental niches where these viruses may play a role in the natural control of bacteria. In the laboratory, an aqueous suspension of the source material (if not already liquid) is passed thorough a 0.45 µm membrane filter in order to produce a suspension of viruses free from contaminating bacteria and other cells. Procedures utilized in the phage enumeration experiment are then used to detect the viruses by their formation of plaques with one or more suitable bacterial host cultures.

Following is a procedure which utilizes direct plating for bacteriophages in the environment. This procedure is extended to include phage isolation (the picking of plaques and the culturing of phages in broth cultures of the host organism) and determination of the host range of the phage isolates (as done in the phage typing experiment).

Period 1

Materials

Young culture of Escherichia coli strain B or other host organism

Sewage or soil suspension, filtered through a 0.45µm membrane filter. ("Activated sludge filters easily and usually has abundant E. coli phage.)

1 dilution blank (9 ml)

3 tubes melted Top Agar (4 ml/tube; in 50°C water bath)

3 plates Bottom Agar

Micropipettes and sterile tips

Safety note: Gloves should be used if analyzing the sewage sample, as human viruses may be present in the filtered raw sewage!

1. Prepare a 1/10 dilution of the filtrate (i.e., the filtered sample). Inoculate the tubes of melted Top Agar (work as quickly as possible!) from the sample or dilution as follows:
• 0.5 ml of the undiluted sample.
• 0.1 ml of the undiluted sample.
• 0.1 ml of the diluted sample.
2. With a fresh pipette tip, inoculate 0.1 ml of the host culture into each tube of Top Agar.
3. Pour the contents of each Top Agar tube onto a separate plate of Bottom Agar and allow the plates to solidify before incubation at 30°C. If the next period is more than two days away, place the plates into the tray on stage for appropriate incubation.

Period 2

Materials

Young culture of host organism

4 tubes of Tryptone Yeast Extract (TYE) Broth (5 ml)

Micropipettes and sterile tips

1. Observe the plates for plaques. From the results of a countable plate, knowing the actual amount of original inoculum, the number of plaque-forming units (PFUs) per ml of the original sample can be calculated. (Keep in mind that this result will only apply to the phages able to lyse the particular host culture used.)
2. Look for variety in the different types of plaques present. Choose three well-isolated plaques.
3. Inoculate each of four tubes of TYE Broth with the appropriate host organism such that you see a slight turbidity in the broth.
4. With a sterile needle, inoculate each of the three chosen plaques into a separate tube of broth. The fourth broth tube will serve as a control to show normal growth of the uninfected host organism.
5. Place the tubes into the rack on stage such that they can be incubated for the short time needed to effect lysis by the phage of the developing broth cultures of host organisms. This is normally less than 24 hours for phage that grow on E. coli

Period 3

Materials

Dropper bottle of chloroform

Young broth cultures of 6-8 potential host strains

3-4 plates of Bottom Agar (well-dried)

6-8 sterile swabs

Micropipettes (preferably P20) and sterile tips

Do not inhale chloroform. Only use in a well-ventilated area

1. Observe each tube of TYE Broth, comparing the three tubes inoculated with plaques to the control tube. What we expect to work with now are lysates. For each plaque-inoculated culture that shows less turbidity than the control, proceed as follows.
2. Add a few drops of chloroform to each lysate. (Careful! See note above.) Vortex vigorously and frequently, and allow the chloroform to dissipate. What you are doing is killing off any remaining viable cells (including phage-resistant cells) in the lysate; the bacteriophages should remain active.
3. Following the procedure in the phage typing experiment, test your lysates for the ability to lyse the various strains of potential host cultures available. Make sure that the streaks of the test organisms are at least one inch wide and completely dried before you apply the drops of the lysates! Incubate the plates at 30°C until the next period.

Period 4

1. Observe the plates for plaques and tabulate your results.

8 - 1 Defining and counting bacteriophage

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.

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

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.

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

8 - 4 Summary of bacteriophage

Bacteriophage are viral parasites of bacteria and are some of the simplest self-replicating systems that contain an outer shell. Much of the early work deciphering the nature of genetics was done using bacterial viruses, and they still find utility today in molecular biology. This chapter has hopefully introduced you to these interesting creatures.