ISOLATION OF BACTERIOPHAGE CLERIGO, GEHAN ALYANNA V. DIMAANO, PETER BOB Z. DECIO, JOHN LAWRENCE GOCO, AMELIA BERNADETTE O.

INTRODUCTION  VIRUSES – ultramicroscopic entities that’s consist of nucleic acid surrounded by protein coat.  BACTERIOPHAGE - are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria.). - are used in the diagnostic laboratory for the identification of pathogenic bacteria (phage typing).

COMPOSITION OF BACTERIOPHAGE  - Although different bacteriophages may contain different materials they all contain nucleic acid and protein.  nucleic acid can be either DNA or RNA but not both and it can exist in various forms. The nucleic acids of phages often contain unusual or modified bases. These modified bases protect phage nucleic acid from nucleases that break down host nucleic acids during phage infection.

 The number of different kinds of protein and the amount of each kind of protein in the phage particle will vary depending upon the phage.  The simplest phage have many copies of only one or two different proteins while more complex phages may have many different kinds.  The proteins also function in infection and to protect the nucleic acid from nucleases in the environment.

STRUCTURE OF BACTERIOPHAGE  SIZE - T4 is among the largest phages; it is approximately 200 nm long and nm wide. Other phages are smaller. Most phages range in size from nm in length.  HEAD OR CAPSID- All phages contain a head structure which can vary in size and shape. Some are icosahedral (20 sides) others are filamentous. - is composed of many copies of one or more different proteins.

- acts as the protective covering for the nucleic acid.  TAIL - Many but not all phages have tails attached to the phage head. - is a hollow tube through which the nucleic acid passes during infection. - complex phages like T4 the tail is surrounded by a contractile sheath which contracts during infection of the bacterium. It also T4 phages also have a base plate where tail fibers are attached.

 NOTE: The base plate and tail fibers are involved in the binding of the phage to the bacterial cell. But not all phages have base plates and tail fibers. T4 Bacteriophage (TEM x390,000) © Dennis Kunkel Microscopy, Inc.Dennis Kunkel Microscopy, Inc Used with permission T4 bacteriophage Negative stain electron micrograph © ICTVICTV

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OBJECTIVES At the end of the activity, the students are expected to:  Isolate bacteriophage from raw sewage.  Explain the importance of enriching the samples before isolating becteriophages.  Calculate the plaque forming units per millimeter of the sample.  Recognize viral growth in bacteria.

MATERIALS

PROCEDURE A. ENRICHMENT METHOD  Aseptically mix together 5 ml TSB, 5 ml E.coli, and 25 ml raw sewage in a 250 ml E. flask.  Incubate at 37°C for 48 hrs.

B. ISOLATION METHOD Inoculate E.coli in 20 ml liquefied TSA. Maintain at 40°C Set aside (part B1) ‏ Centrifuge enriched suspension at 2500 rpm for 20 mins. Decant supernatant into sterile E. flask. Heat for 15 mins. Pour 1 ml of supernatant into sterile plate Add 20 ml of part B1 Swirl contents Incubate at 37°C for 24 hrs.

After 24 hrs. Observe presence of clearing (plaques) ‏ Count plaque forming units on each plate Compute for pfu/ml Pfu/ml = (number of plaques)(dilution factor)/volume plated Decontaminate materials used Answer items 1-5 in worksheet

DATA AND RESULTS  Table 17.1 Plaque Forming Units Per Millmeter of Water Sample. SAMPLESNUMBER OF PLAQUES Pfu/ml E.coli474.7 x 10 ¯ ¹

PFU – Plaque forming units Measures infectious particles in a given sample. Count the number of PFUs on a series of plates:

 Of the four samples, which has the greatest number? Account for such observation. - Having just one result, E.coli has 47 plaque formations.

 Does a phage plaque grow to a specific size? Why? - Yes, because phage cannot move. So when the originally infected bacteria lyse (burst) only its neighboring cells become infected. This process results to a round shaped spread of phages thus a perfectly round plaque.

 Is it possible for a phage to clear out the whole plate? Explain. - Yes it is possible for the phage to clear out the whole plate because the mechanism of the phage is to insert their DNA into the bacterial cell and make it their own.

 The presence of plaques indicates viral growth and thus they are considered living entities. Are these bacteriophages considered to be microorganisms? Why?  No, because bacteriophages are viruses, and viruses are not considered to be living microorganisms although they are capable of making a copy of themselves.

REFERENCES  pathmicro.med.sc.edu/mayer/phage.htm  Tortora,G.J.,B.R.Funke, and C.L.Case.Microbiology:An Introduction (7 th ed.) USA: Addison Wesley Longman Inc.,2001.