Discussion:   EVALUATING OUR HYPOTHESIS/ PROCEDURAL ALTERATIONS   Our hypothesis stated: 1) If the number of phage present in a sample of raw sewage was amplified, then the bacteriophage could be isolated from its E. coli B host via centrifugation and filtration.  2) If this step is accomplished successfully, then upon plating the phage filtrate, distinct plaques would would be visible on a fresh lawn of  able to see distinct plaques on a fresh lawn of E. coli.  3) If new tubes of E. coli were then inoculated with an individual plaque, and the filtration process repeated, only plaques of this selected size should be visible on a new plate. The first two parts of this hypothesis were successfully supported. After enriching the original sewage sample using the deca-strength phage broth,  the aforementioned centrifugation and filtration steps of the procedure were performed.  One, three and six drops of our phage filtrate were plated, in combination with soft nutrient agar and E. coli on hard TSA plates.  However, when the incubated plates were observed, individual plaques were not seen.  In addition, no E. coli lawns grew, even on the control plate, which contained only E. coli  (no phage).  From these results, it was deduced that our phage filtrate was too concentrated, and that most likely, E. coli lawns had grown, but there were so many phage present that all of the bacterial cells were lysed out, which left no individual zones of clearing.  In effect, there were so many plaques present, that they overlapped one another, leaving only small specks of E. coli among large quantities of voracious phage.  In addition, it was suspected that contamination was a factor this first time around a) because the control plate did not grow on a lawn of E. coli and b) some of the experimental filtrate plates contained small off-white bacterial colonies, among other unidentified growth.  Hard nutrient agar plates for this initial attempt at isolating phage were prepared from scratch.  However, for all subsequent replications of the procedure, we utilized pre-made TSA plates, to reduce the potential for contamination.  At this point, the procedure was altered by diluting the remaining filtrate in a 5-tube serial dilution.  The result was four tubes, containing phage: 1/10, 1/100, 1/1000 and 1/10,000 of its original strength.  Mixtures of soft nutrient agar, E. coli and three drops of each of these dilutions of phage filtrate were placed into four tubes, along with three tubes containing 1, 3 and 6 drops of original-concentration phage filtrate.  This second time around, two types of control plates were used, one containing only E. coli and another just with soft nutrient agar overlaying the TSA plate.  After incubation, the presence of distinct plaques was observed on select plates.  The presence of these zones of clearing indicated that we the first part of the hypothesis was proven.  That is, amplifying bacteriophage from a raw sewage sample and inoculating the phage into a fresh E. coli host was an effective way of isolating (and visualizing) phage.    The plates with 1 drop of original concentration filtrate and with the 1/10 dilution filtrate bore the best results.  E. coli plates containing 3 and 6 drops of original concentration filtrate were completely lysed out again.  And, we found that the 1/100, 1/1000 and 1/10,000 filtrate dilutions were too dilute to support any sizeable phage growth (only a few tiny, pinpoint-size zones of clearing formed).  The controls worked as expected. To prove the second part of our hypothesis, four large, clear and distinct plaques were chosen from the 1/10 dilution plate and transferred  to fresh E. coli using a sterile needle.  However, in addition to the large plaques observed on the plates, there also appeared to be a second type of phage which produced much smaller plaques on each of the plates.  The presence of these smaller plaques indicated that a single species of bacteriophage had not been isolated.  In a final attempt to isolate a single species of phage, a 5-tube serial dilution from the filtrates of each of these plaques was prepared (a total of 20 experimental plates).  Unfortunately, plaques only formed on the 1/1000 and 1/10,000 dilutions from two of the filtrates, and they were not readily countable.  Also, these final plates still contained both tiny and large plaques, indicating that a single type of phage still had not been isolated.   There are three potential sources of this failure.  The first is due in part to the transfer process of plaques from the plate.  Despite using a sterilized needle, the larger plaques on the plate were in close proximity, if not surrounded by the tiny and more numerous  other plaques.  Secondly, there is the possibility that the tiny plaques produced by the alternate phage type actually formed within the larger plaques.  Finally, six drops of each dilution of filtrate were used, which proved to be too much, even at the greatest dilution of phage filtrate.   ECOLOGICAL ROLE OF BACTERIOPHAGE/CHARACTERISTICS/SIGNIFICANCE OF ISOLATION Bacteriophage have important roles in the ecosystem as a form of exerting natural selection pressures on populations of organisms. Host organisms like bacteria would evolve to resist viral infection or develop mechanisms with which to maintain a viable and reproducing population. Also, phages play a role in transduction, the transfer of genetic material between bacterial cells during infection. By exerting selective pressure and creating variety in bacterial gene pools (affecting both phenotype and genotype), viruses have a major impact directly on their host organism populations. Also, by lysing bacterial cells, the population of bacteria is kept in check, which affects the ecology of the bacteria's environment. Some viruses can infect other organisms that greatly impact ecosystems, such as cyanobacteria in marine ecosystems. By limiting the growth of cyanobacteria, marine ecosystems will not become eutrophic, where cyanobacteria and algae can choke the ecosystem of sunlight and oxygen. 2   INTERESTING CHARACTERISTICS Bacteriophage can exist outside of living cells as inert virions, but once they enter susceptible bacterial host cells, they begin one of a few potential replicating lifestyles.  Virulent, lytic phages completely overtake the bacterial metabolism machinery, which results in cell lysis.  Other types of phage leave the infected, but still living, host cell by extrusion.  Lysogenic temperate phages produce latent infections.  For this assignment, a lytic bacteriophage was targeted, so that macroscopic changes in the E. coli lawn (plaques) would be easily detectable.  Typically, each lysed E. coli cell releases 200 T4 phage (its burst size), and the entire lytic phage cycle, from attachment to release, takes about 30 minutes.  However, it is possible, that the single-stranded RNA  MS2 or QBeta phage, or the single-stranded DNA phage, PhiX174, (two additional groups of lytic phages), caused the lysis of E. coli cells that was observed.  The MS2 and QBeta phage have a burst size of 10,000, which could have produced the larger-sized plaques.  Phages are also instrumental in facilitating the transfer of bacterial genes via transduction.  General transducing phages can introduce any donor cell gene into a new bacterial recipient if bacterial DNA is accidentally packaged in the virus head.  In this manner, phage play a primary role in the evolution of bacterial cells.             One of the invaluable benefits of studying bacteriophage is that much of the information gleaned from these easy-replicable viruses can be applied to other eukaryotic-infecting viruses, especially those about which little is known because of the slow reproduction of the host organism (plants and animals).  This is one of the reasons why it is so important for researchers to have a clear-cut and effective procedure to isolate phages from their bacterial hosts.    MEDICAL (CLINICAL) SIGNIFICANCE/APPLICATION From a medical standpoint, having and utilizing an accurate step-by-step method to isolate phage might become even more important in the very near future because of bacterial cells’ rapid and growing resistance to antibiotics.  It is conceivable that at some point, the evolution of resistant bacteria could outpace the introduction of novel, effective antibiotic medications.  If this situation occurs, doctors might resort to using phage to treat bacterial infections.  Currently, the Research Institute in the USSR’s Georgia is conducting extensive research on the use of phage as therapeutic organisms.  However, to produce feasible phage medicines, the virus’s specific host range limitations must be overcome.  In a treatment of intestinal diseases, a mixture of 17 phages are concurrently used to combat the intended pathogens. In addition, researchers are trying to get around the fact that phage particles are identified as foreign bodies by our immune system. Furthermore, researchers warn that an especially effective protocol to purify phage must be devised, because it would be very dangerous if pieces of lysed E. coli cells got into the bloodstream.  In our experiment, our back-to-back triple centrifugation/filtration processes seemed to efficiently separate the virus from the E. coli.  However, there was no way of detecting how much debris from lysed E. coli cells was present in our filtrate (nor would it have affected our results in any significant way).  If we were to carry out this procedure in the future, we might concentrate our efforts on improving the accuracy of this isolation step, if such a method could be employed in a medical setting.  If these kinks can be ironed out of the existing protocol for isolating phage, these organisms could become a powerful tool in the fight against bacterial infections. 3