MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #17: VIRUSES

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REVISED: 08/04/99

INTRODUCTION

Viruses are basically a GENOME surrounded by a PROTEIN COAT. Viruses are OBLIGATE INTRACELLULAR PARASITES that must live and develop inside of living cells. Viruses have the ability to BIND specifically to their target or host cell and to either enter the host cell or to inject their genome into the host cell. Once the viral genetic material is in the host cell it TAKES OVER the metabolism of the host cell and converts it to the process of manufacturing NEW viral entities (VIRIONS). When complete virions are produced within the host cell they leave the cell in a #variety of ways. Some viruses cause the host cell to BURST or LYSE, thereby instantly releasing all the enclosed virions. Other viruses leave their host cells by a complex process of BUDDING OFF the host's cytoplasmic membrane. In these cases the mature virus usually takes some of the host's cytoplasmic membrane with it when it leaves. Even though the host cells of budding viruses do not burst, they eventually die.

In order to study viruses they must be cultivated in a CULTURED CELL. In the case of bacteria this is easily accomplished as you will see in this experiment. However, with animal and plant viruses it is more difficult because not all eukaryotic host cells can be cultivated. Thus any viruses that use cells that can not yet be cultivated as their hosts are difficult to study. One example of such a virus is the HUMAN PAPILLOMA VIRUS that causes #genital warts. When a host cell is available, they are cultivated as a SINGLE LAYER OF CELLS on a solid surface prior to their infection by the virus. This single layer is called a LAWN.

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Figure 1. Phage dilution series. What is the number of phage/ml in the first tube?

The general means of cultivating a virus on a host lawn involves DILUTING the viral stock to a point where there are only a few hundred virions or less in a ml of solution. Then a portion of the diluted virus is added to the host cells and allowed to infect them. In the case of phage the infected cells are plated on media in a petri dish, whereas eukaryotic viruses are added directly to a lawn of eukaryotic cells. Following introduction into the host-culture the viruses proceed to reproduce in the cell lawn. Released virions subsequently infect fresh ADJACENT host cells in the lawn. As the infected host cells die the RELEASED VIRIONS diffuse away from the infected cell and spread the infection to the surrounding host cells. As the infection spreads in an ever-enlarging circle of death it leave a HOLE or empty space in the host-cell lawn. This hole is called a PLAQUE. It has been shown that only a single virion is required to produce a plaque. In this way the number of virions in a sample can be quantitated by counting the number of plaques formed on a host lawn.

Phage have two life styles. LYTIC or VIRULENT phage take over a host cell and produce new virions and quickly lyse the host cell (20 to 40 min). A second type of phage, called a TEMPERATE phage, can integrate (fuse) its DNA into the host's genome as a PROPHAGE or PROVIRUS. In this state, called the LYSOGENIC STATE, the phage genes are NOT EXPRESSED. Rather, they remain quiescent for many generations, until the lytic cycle is INDUCED. Following induction the new phage virions are rapidly made and the host cell is lysed. Some eukaryotic viruses, called #RETROVIRUSES can also enter a provirus state. Retroviruses, like the HIV virus, are RNA viruses that are converted into DNA once they enter the host cell. The viral DNA genome is then integrated into the host's genome and becomes a PROVIRUS. See #Chap. XVII for details of this life cycle.

In this exercise you will observe the plaques produced by #bacteriophage.

 

PURPOSE OF LABORATORY:

  1. To demonstrate phage replication and plaque formation.
  2. To demonstrate plaque formation in tissue cultures

RELATIONSHIP TO LECTURE MATERIAL

GENERAL INSTRUCTIONS:

  1. Use aseptic technique throughout the procedure.
  2. Examine the virus infected tissue cultures if they are available.

PROCEDURE:

  1. Read pg. 87, 115-116 in A Photographic Atlas for the Microbiology Laboratory.
  2. Working as pairs, obtain a medium plate and label it appropriately with your NAME, DATE and the DILUTION assigned to your table.
  3. Locate the following:
    1. The melted TOP AGAR in a warm water bath.
    2. The PHAGE SAMPLE at the dilution for your table.
    3. The BACTERIAL HOST culture.
  4. Working as a team carry out the following steps within about 30 seconds.
    1. Using a sterile pipette, add 100 microliters (0.1 ml) of the PHAGE sample to the melted TOP AGAR and mix by swirling the solution.
    2. Quickly add 100 microliters of the BACTERIAL HOST to the same TOP AGAR tube, mix and IMMEDIATELY pour the mixture over the surface of the plate-medium. Tilt the liquid agar so that it covers the entire surface of the bottom agar.
  5. Allow the top agar to solidify for about 15 min. Invert the plate and incubate it at 37oC.
  6. At the next period examine it for plaques as seen on pg. 87 in the Atlas. Count the plaques and calculate the original phage concentration from the dilution as given you by the instructor.
  7. Also examine the tissue culture with viral plaques in them.

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

Copyright © Dr. R. E. Hurlbert, 1999.
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