Laboratory Exercises in Virology

Phage Typing.....Phage Propagation

• Phage typing
  The object here is to determine which bacterial strain is being used. The determination is made by showing the existence of some exterior characteristic of a bacterial strain. Since phages are highly specific with regard to their attachment sites, and bacterial strain that possesses that "site-molecule" on its exterior will be sensitive to being killed by that phage.

  1. Obtain a petri plate filled with nutrient agar; sector the plate into sixths and label as shown in Figure 1.
     

  2. Swab onto each of these sections (with NO overlapping!) the appropriate strains from the stock bottles of bacterial cultures (Figure 2).

  3. Obtain a syringe containing one or the other of three phages. ØK1E, ØK5, or P1. Carefully drip ONE drop onto the middle of each swabbed sector - where the X's are in Figure 2.

  4. Incubate the plates for 8 or more hours. Look for any large plaques in the middle of your smears. An example is shown in Figure 3 of a phage that can only infect 'rough' E. coli (not having an LPS layer).
     

     • Phage Propagation

       1. Sterilize about 50 ml of L-broth (0.8% tryptone + 0.7% glucose) in 500 ml flasks with beakers set over the open ends.

       2. When cool, inoculate (note 1 "N" in the word!) with a strain that is sensitive to the virus you are to use.

       3. Place the flask in a waterbath at 37°C and be prepared to aerate the flask later.

       4. When you notice that there is just barely visible turbidity, note the time, and wait for 1 more hour.

       5. Add 1 ml of the virus suspension. Be careful to not transfer any of the CHCl₃ (chloroform) that may be in the bottom of the viral container. (CHCl₃ rapidly kills most bacteria.)

       6. Immediately begin aerating the culture by bubbling some air slowly into the container. Make sure that your container is far away from the cultures of other students since you do not want to cross infect.

       7. In about 2 to 3 hours, you should note a slight clearing of your culture as many cells have begun to lyse. You may notice some brown crud collecting near the meniscus - that's cell walls. Wait no longer! Chill in ice-bath, and then add some CHCl₃ and shake flask to lyse the cells.

       8. Centrifuge down the contents at 10,000 rpm for 5 minutes in the cold.

       9. Ultrafilter the supernatant to sterilize the viral (hope!) suspension.

       10. Test supernate for virulence on a lawn of the susceptible bacteria.
           1. as on the phage-typing protocol
           2. by determining pfu/ml

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