As we discussed in class, bacteriophage are viruses that infect bacteria (bacteriophage means bacteria eater). Bacteriophage that infect E. coli sometimes are referred to coliphage. Generally, bacteriophage are referred to simply as phage. As is true for all viruses, phage can replicate only within host cells. In other words, coliphage can replicate only within E. coli. Phage must attach to a receptor on the surface of a bacterial cell in order to initiate an infection. This interaction between the phage and receptor is very specific - a given phage type only will bind to a specific receptor molecule. Thus, all phage are not alike.
As we have seen in the lab., not all E. coli are alike. Variability
exists in the growth curvesand antibiotic resistance of different E. coli
isolates. One could hypothesize that these isolates of E. coli
also possess some differences in their surface molecules and, as a result, differences
in the phage receptors present on their surfaces (remember - these surface molecules
serve some important function for the bacteria; they do not exist on the cell
surface merely as portals of entry for phage!). To examine if, in fact,
such receptor differences do exist, we will conduct a phage typing experiment.
Each person will isolate a phage that grows readily in her/his E. coli
isolate. Then, we will investigate whether or not each of these phage
isolates is capable of replicating within the other E. coli isolates.
At the end of this experiment, we will be able to produce a table that shows
the sensitivity or resistance of each person's E. coli to each of the
phage isolates. Each group also is encouraged to develop and conduct additional
experiments with their phage and E. coli isolates in an attempt to learn
more about the diversity of these isolates. For most of these experiments,
your group can work on it's own. Each person must, however, have an enriched
phage preparation by next week (April 2 or 3).
Bacteriophage Isolation
Each person will isolate a bacteriophage specific for his or her own isolate
of E. coli. Isolate the phage following the directions provided.
Because most of the steps are very short and require 1 to 2 day incubations
between them, we will not do the procedures together as a class. Rather,
we will go over the protocols today. You can carry out the phage isolation
according to your own schedule. The only requirement is that you complete
Day 4 in time for lab on April 2 or 3.
Obtaining bacteriophage
Not surprisingly, bacteriophage can be found in places where numerous
hosts exist. Because coliphage use E. coli as a host organism,
these phage can be found in places where E. coli are plentiful.
We saw earlier in the semester that all of us harbor numerous E. coli
(and other bacteria) in our lower intestines. Obviously, those E.
coli leave our bodies. Thus, sewage contains E. coli from
every one in town. And, as a result, sewage also contains numerous
coliphage.
I have obtained a sewage sample from the Rocky River waste treatment plant in Mooresville. From this material, we will isolate different strains of phage.
Isolation of bacteriophage
Day 1: Inoculate 5 mL tryptic soy broth with your E.
coli. As we did before, touch a sterile needle to a colony and
transfer it to the tube of broth. Incubate overnight at 37oC.
Day 2: Inoculate another tube with 4.5mL sewage, 0.5mL of your overnight E. coli culture, and 0.5mL of 10x tryptic soy broth (what do we mean by '10x tryptic soy broth'? Why do we use 10x TSB for this step?). Incubate 24-48 hours at 37oC. During this incubation, phage in the sewage sample capable of binding to your E. coli will replicate in and eventually lyse the bacteria. Thus, this step serves as a means of amplifying the phage that can infect your bacteria.
Centrifuge your tube in the table top centrifuge for 10 minutes at 2500rpm. Remember to balance the centrifuge! This centrifugation step should cause the bacteria and other cell debris to form a pellet at the bottom of the tube.
Take up 3mL of the supernatent into a 5 cc syringe. Attach a 0.22 micron filter to the base of the syringe and filter the supernatent into a sterile tube. This filter will prevent any remaining bacteria from passing through, but will allow phage to pass through. Label the filtrate "Enriched phage prep." Store in the refrigerator.
Set up another overnight culture of your E. coli.
Day 3: Transfer 100 microliters of an overnight E. coli growth onto the center of a 60 mm Petri plate containing tryptic soy agar (these plates will be in the refrigerator). With a bent glass rod, spread the bacteria over the plate. Next, place 10 microliters of your enriched phage prep in the center of the plate. Incubate at 37oC overnight.
Day 4: Examine your plate. Because we added thousands (or maybe millions!) of bacteria to the plate, we will not see isolated colonies. Rather a confluent lawn of bacteria will cover the agar. The phage, though, will infect and kill the bacteria in the center of the plate, creating a visible region of no bacteria - a plaque. This simple experiment demonstrates that you have isolated phage capable of infecting your strain of E. coli. Return the plate to the incubator and incubate it for several more days. Do you notice anything unusual?
On the day before next week's lab, inoculate a tube of tryptic soy broth
(approximately 5mL) with your E. coli isolate and incubate overnight
at 37oC.