ISOLATION AND TITRATION OF COLIPHAGES FROM SEWAGE SAMPLE

 ISOLATION AND TITRATION OF COLIPHAGES FROM SEWAGE SAMPLE

 

AIM:

 To isolate and titration of coliphages from the given sewage sample.

PRINCIPLE:

 Phages are viruses that infect bacteria. Coliphage infects bacteria like Escherichia coli. Sewage is a rich source of coli phages, because sewage contains faecal matter and is rich in organic matter. Faecal bacteria grow enormously by utilizing organic matter present in sewage. Isolating phage from sewage is not an easy process. It requires stepwise careful isolation and enrichment process.

 Bacteriophages can be isolated from different environments. Since they grow and reproduce within bacteria, one would expect to find them wherever a large population bcteria is present. For example, large numbers of E.coli grow in the intestinal tract of warm-blooded animals. Therefore, animal manure and untreated sewage are excellent sources of coliphages. Concentration of bacteriophages specific for a particular host may be relatively low in natural environmnet, the first step in the isolation procedure is an enrichment step. When the sample is incubated with a population of the proper host, phages specific for that bacterium will greatly multiply in number. The phges then are much easier to isolate since they are present in greater numbers. Membrane filtration serves to remove cell debris and most bacteria. Any bacteria that may remain in the enriched culture can be killed and lysed by treatement with chloroform. The filtrate containing bacterial viruses may now be stored in the refrigerator for months. Individual viruses in the filtrate can be isolated by mixing a small amount of filtrate with a young culture of the host bacterium and then spreading the mixture out on the surface of a petriplate containing nutrient agar. This is called the double-layered culture technique. In practice, the viruses and the bacteria are mixed with a dilute agar medium (the top agar) and then poured in a thin layer on the surface of harder bottom, or base agar. When the top agar solidifies, the viruses and bacteria are immobilized. Whenever a virus particle is present in the agar, it will infect an adjacent bacterial cell, reproduce, and lyse its host cell. The virus particle will then give rise to millions of virions, and a clear area of lysed bacteria will develop in the bacterial lawn. This clear area is called a plaque. Ideally, each virus will produce one plaque containing enormous2 / 2 Numbers of its progeny. Samples of each plaque can then be removed and used to culture large quantities of a single type of virus for further study. Because each plaque arises from a single virus particle, a count of the number of plaques will enable one to calculate the concentration of viruses in the original undiluted sample.

 

MATERIALS REQUIRED:

 

 Overnight culture of E.coli and fresh sewage collected in screw-capped bottles, filtration unit, test tubes, centrifuge, conical flask. Flask of raw sewage, 0.22 and 0.45 µm membrane filters,  filter  apparatus,  luminium  foil,  500-ml  flask,  graduated  cylinder,  1  tube  broth, concentrated broth, 24 hours E.coli broth culture, incubator, bunsen burner, chloroform, sterile screw-cap tubes, sterile saline (0.85% NaCl), petriplates, hard agar, water bath, thermometer, wax pencil.

 

PROCEDURE:

 

 1.   ISOLATION OF SAMPLE:

     Add 5 ml of nutrient  broth and 5ml of E.coli culture to 45ml of sewage sample available in 250ml conical flask aseptically

     Incubate the flask for 24hours at 37 degree Celsius

 

 2. FILTERATION AND SEEDING:

      Centrifuge the enriched sample at 2500rpm for 20 mins

      Decant the supernatant into a 125ml flask

      Filter the supernatant through a sterile membrane filter apparatus to collect bacteria free phage, phage containing filterate in the vaccum flask.

     Prepare the hard nutrient agar and pour it in the sterile petriplate and allow it to solidify.

      Prepare soft agar in test tube of 5ml quantities and maintain in liquid conditions.

      Inoculate the 0.1ml of E.coli culture to all molten soft agar tubes using sterile 1ml pipette.

      Add 1 to 5 drops of sewage filterate to labelled molten soft agar tubes using a sterile pipette and mix properly.

     Pour properly mixed soft agar into appropriately labelled hard agar plate.

     Allow soft agar media to harden.

      Incubate all plates in a inverted position for 24 hours at 37 degree Celsius and observe plates after incubation

 

3. TITRATION:

      Obtain about 50 ml of raw sewage. Centrifuge it for 10mins using clinical centrifuge.

      Add 20ml of supernatant to 500ml containing 20ml of sterile,twofold concentrated broth.

      Inoculate the mixture with 1ml of an overnight E.coli culture.inoculate for 24 hours at 35 degree Celsius.

     Inoculate a tube broth culture tube with 1 ml of E.coli and incubate it at 35 degree Celsius overnight or for 24 hours. This will serve as a stock culture.

 

4. ISOLATION OF PHAGES:

      Filter the phage enriched sewage through  another 0.45µm membrane filter. Transfer

 8.0ml portions of the filtrate into sterile, Screw cap tubes.

      Add 0.2ml of chloroform to each screw-cap tube with a sterile pipette. Mix thoroughly.

      The virus stock can be used immediately or kept in the refrigerator until it can be analyzed.

     Prepare a serial dilution of the enriched bacteriophage sample. Withdraw a sample and transfer an aliquot to the next tube and mix properly. With a new pipette, transfer an aliquot to the next tube in the series (Use a new, sterile pipette for each transfer).

     After all dilutions have been prepared, add 2 drops of the overnight E.coli culture to the melted, cooled 10⁻⁴to 10⁻⁸ dilution tubes of soft agar. Keep the tubes in the 48° to 50°C water bath as much of the time as possible during this process.

     Transfer 0.5ml of 10⁻⁴  dilution to a 45°C soft agar top tube. Mix quick and thoroughly, and pour the soft agar aseptically into a petri plate containing sterile bottom agar. Immediately spread the top agar over the surface of the base nutrient agar by tilting the plate. Set the plates aside to harden. repeat this procedure with each of the other four dilution.

     Incubate the plates in an inverted position at 35°C for 8 to 24 hours.

RESULT:

 

 Clear lytic areas are seen on the plate. E.coli has grown inro a lawn. Clear lytic area indicates the presence of phages.