Viruses: The Bacteriophages

Introduction
Viruses are infectious agents with both living and nonliving characteristics.

1. Living characteristics of viruses
   
   1. They reproduce at a fantastic rate, but only in living host cells.
   2. They can mutate.
2. Nonliving characteristics of viruses
   
   1. They are acellular, that is, they contain no cytoplasm or cellular organelles.
   2. They carry out no metabolism on their own and must replicate using the host cell’s metabolic machinery. In other words, viruses don’t grow and divide. Instead, new viral components are synthesized and assembled within the infected host cell.
   3. They possess DNA or RNA, but never both.

Viruses are usually much smaller than bacteria. Most are submicroscopic, ranging in size from 10–250 nanometers.

Structurally, viruses are much more simple than bacteria. Every virus contains a genome of single-stranded or double-stranded DNA or RNA that functions as its genetic material. This is surrounded by a protein shell called a “capsid” or “core”, which is composed of protein subunits called capsomeres. Many viruses consist of no more than nucleic acid and a capsid, in which case they are referred to as “nucleocapsid” or “naked” viruses.

Most animal viruses have an envelope surrounding the nucleocapsid and are called enveloped viruses. The envelope usually comes from the host cell’s membranes by a process called budding, although the virus does incorporate glycoprotein of its own into the envelope.

Bacteriophages are viruses that infect only bacteria. In addition to the nucleocapsid or head, some have a rather complex tail structure used in adsorption to the cell wall of the host bacterium.

Since viruses lack organelles and are totally dependent on the host cell’s metabolic machinery for replication, they cannot be grown in synthetic media. In the laboratory, animal viruses are grown in animals, in embryonated eggs, or in cell culture. (In cell culture, the host animal cells are grown in synthetic medium and then infected with viruses.) Plant viruses are grown in plants or in plant cell culture. Bacteriophages are grown in susceptible bacteria.

Today we will be working with bacteriophages, since they are the easiest viruses to study in the lab. Most bacteriophages, such as the Coliphage T4 that we are using today, replicate by the lytic life cycle and are called lytic bacteriophages.

The lytic life cycle of Coliphage T4 consists of the following steps:

1. Adsorption. Attachment sites on the bacteriophage tail adsorb to receptor sites on the cell wall of a susceptible host bacterium.
2. Penetration. A bacteriophage enzyme “drills” a hole in the bacterial cell wall and the bacteriophage injects its genome into the bacterium. This begins the eclipse period, the period in which no intact bacteriophages are seen within the bacterium.
3. Replication. Enzymes coded by the bacteriophage genome shut down the bacterium’s macromolecular (protein, RNA, DNA) synthesis. The bacteriophage genome replicates and the bacterium’s metabolic machinery is used to synthesize bacteriophage enzymes and bacteriophage structural components.
4. Maturation. The bacteriophage parts assemble around the genome.
5. Release. A bacteriophage-coded lysozyme breaks down the bacterial peptidoglycan,
causing osmotic lysis of the bacterium and release of the intact bacteriophages.
6. Reinfection. 50–200 bacteriophages may be produced per infected bacterium, and they now infect surrounding bacteria.

Some bacteriophages replicate by the lysogenic life cycle and are called temperate bacteriophages. When a temperate bacteriophage infects a bacterium, it can either (1) replicate by the lytic life cycle and cause lysis of the host bacterium, or it can (2) incorporate its DNA into the bacterium’s DNA and assume a noninfectious state. In the latter case, the cycle begins by the bacteriophage adsorbing to the host bacterium and injecting its genome, as in the lytic cycle. However, the bacteriophage does not shut down the host bacterium. Instead, the bacteriophage DNA inserts or integrates into the host bacterium’s DNA. At this stage, the virus is called a prophage. Expression of the bacteriophage genes controlling bacteriophage replication is repressed by a repressor protein and the bacteriophage DNA replicates as a part of the bacterial nucleoid. However, in approximately 1 in every million to 1 in every billion bacteria containing a prophage, spontaneous induction occurs. The bacteriophage genes are activated and bacteriophages are produced, as in the lytic life cycle.

In this exercise, you will infect the bacterium Escherichia coli B with its specific bacteriophage, Coliphage T4.

In the first part of the lab, you will perform a plaque count. A plaque is a small, clear area on an agar plate where the host bacteria have been lysed as a result of the lytic life cycle of the infecting bacteriophages. As the bacteria replicate on the plate, they form a “lawn” of confluent growth. Meanwhile, each bacteriophage that adsorbs to a bacterium will reproduce and cause lysis of that bacterium. The released bacteriophages then infect neighboring bacteria, causing their lysis. Eventually a visible self-limiting area of lysis, a plaque, is observed on the plate.

The second part of the lab will demonstrate viral specificity. Viral specificity means that a specific strain of bacteriophage will only adsorb to a specific strain of susceptible host bacterium. In fact, viral specificity is just as specific as an enzyme-substrate reaction or an antigen-antibody reaction. Therefore, viral specificity can be used sometimes as a tool for identifying unknown bacteria. Known bacteriophages are used to identify unknown bacteria by observing whether or not the bacteria are lysed. This is called Phage typing.

Phage typing is useful in identifying strains of such bacteria as Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species. For example, by using a series of known staphylococcal bacteriophages against the Staphylococcus aureus isolated from a given environment, one can determine if it is identical to or different from the strain of Staphylococcus aureus isolated from a lesion or food. This can be useful in tracing the route of transmission.

Plaque Count
Materials
Bottle of sterile saline, sterile 10.0-mL and 1.0-mL pipettes and pipette fillers, sterile empty dilution tubes (7), Trypticase soy agar plates (3), bottle of melted motility test medium from a water bath held at 47°C.

Cultures
Trypticase soy broth culture of Escherichia coli B, suspension of Coliphage T4.

Procedure

1. Take 2 tubes containing 9.9 mL of sterile saline, 2 tubes containing 9.0 mL of sterile saline, and 3 sterile empty dilution tubes, and label the tubes as shown in.
2. Dilute the Coliphage T4 stock as described below.
   
   1. Remove a sterile 1.0-mL pipette from the bag. Do not touch the portion of the pipette that will go into the tubes and do not lay the pipette down. From the tip of the pipette to the “0” line is 1 mL; each numbered division (0.1, 0.2, etc.) represents 0.1 mL; each division between 2 numbers represents 0.01 mL.
   2. Insert the cotton-tipped end of the pipette into a blue 2-mL pipette filler.
   3. Flame the sample of Coliphage T4, insert the pipette to the bottom of the tube, and withdraw 0.1 mL of the sample by turning the filler knob towards you. Reflame and cap the tube.
   4. Flame the first (10^–2) dilution tube and dispense the 0.1 mL of sample into the tube by turning the filler knob away from you. Draw the liquid up and down in the pipette several times to rinse the pipette and help mix. Reflame and cap the tube.
   5. Mix the tube thoroughly by holding the tube in one hand and vigorosly tapping the bottom with the other hand. This is to assure an even distribution of the bacteriophage throughout the liquid.
   6. Using the same pipette and procedure, aseptically withdraw 0.1 mL from the first (10^–2) dilution tube and dispense into the second (10^–4) dilution tube and mix.
   7. Using the same pipette and procedure, aseptically withdraw 1.0 mL (up to the “0” line); from the second (10^–4) dilution tube and dispense into the third (10^–5) dilution tube. Mix as described above.
   8. Using the same pipette and procedure, aseptically withdraw 1.0 mL from the third (10^–5) dilution tube and dispense into the fourth (10^–6) dilution tube. Mix as described above. Discard the pipette.
3. Take the 3 remaining empty, sterile tubes and treat as described below.
   
   1. Using a new 1.0-mL pipette and the procedure described above, aseptically remove 0.1 mL of the 10–6 bacteriophage dilution and dispense into the third (10^–7) empty tube.
   2. Using the same pipette and procedure, aseptically remove 0.1 mL of the 10^–5 bacteriophage dilution and dispense into the second (10^–6) empty tube.
   3. Using the same pipette and procedure, remove 0.1 mL of the 10–4 bacteriophage dilution and dispense into the first (10^–5) empty tube. Discard the pipette.
4. Using a new 1.0-mL pipette, add 0.5 mL of E. coli B to the 0.1 mL of bacteriophage in each of the 3 tubes from step 4 and mix.
5. Using a new 10.0-mL pipette, add 2.5-mL of sterile, melted motility test medium to the bacteria-bacteriophage mixture in each of the 3 tubes from step 3, and mix.
6. Quickly pour the motility medium-bacteria-bacteriophage mixtures onto separate plates of trypticase soy agar and swirl to distribute the contents over the entire agar surface.
7. Incubate the 3 plates rightside-up at 37°C until the next lab period.

Viral Specificity
Materials
Trypticase soy agar plates.
Cultures
Trypticase soy broth cultures of 4 unknown bacteria labeled #1, #2, #3, and #4; suspension of Coliphage T4.

Procedure

1. Using a wax marker, draw a line on the bottom of both trypticase soy agar plates, dividing them in half. Number the 4 sectors 1, 2, 3, and 4, to correspond to the 4 unknown bacteria.
2. Draw a circle about the size of a dime in the center of each of the 4 sectors.
3. Using a sterile inoculating loop, streak unknown bacterium #1 on sector 1 of the first trypticase soy agar plate by streaking the loop through the circle you drew. Be careful not to streak into the other half of the plate.
4. Using the same procedure, streak the 3 remaining sectors with their corresponding unknown bacteria.
5. Using a sterile Pasteur pipette and rubber bulb, add 1 drop of Coliphage T4 to each sector in the area outlined by the circle.
6. Incubate the 2 plates rightside-up at 37°C until the next lab period.

Results
Plaque Count
Observe the 3 plates for plaque formation and make a drawing.

• 1/100,000 (10^–5) dilution
• 1/1,000,000 (10^–6) dilution
• 1/10,000,000 (10^–7) dilution

Viral Specificity
Make a drawing of your results and show which of the unknowns (#1, #2, #3, or #4) was E. coli.

Performance Objectives
Discussion

1. Define the following: bacteriophage, plaque, and phage typing.
2. Describe the structure of the bacteriophage coliphage T4.
3. Describe the lytic life cycle of bacteriophages.
4. Define viral specificity.

Results

1. Recognize plaques and state their cause.
2. Interpret the results of a viral specificity test using Coliphage T4.