Bacteriophage Enrichment from Water PDF

Bacteriophage Enrichment from Water Section 8  Bacteriophages are the most abundant life forms on earth. Bacteriophages are common in soils and direct counting methods have found 107– 109 per gram of soil. In this chapter we will describe simple enrichment techniques which hav...

Bacteriophage Enrichment from Water Section 8  Bacteriophages are the most abundant life forms on earth. Bacteriophages are common in soils and direct counting methods have found 107– 109 per gram of soil. In this chapter we will describe simple enrichment techniques which have been used successfully in both postgraduate research and undergraduate laboratory exercises. Sewage treatment plants are ideal sources from which bacteriophages are isolated.  Enrichment of Phage from Aqueous Materials Materials:- 1. Collecting Samples of pond water, pond sediment, raw sewage etc., collected in sterilized Erlenmeyer flasks or screw-capped bottles 2. Centrifuge. 3. Sterile Luria broth (LB) or Tryptic Soy broth (TSB) supplemented with 2mM CaCl2, stored in 100 or 500 ml sterile Pyrex screw-capped bottles. 4. Sterile 125 ml Erlenmeyer flasks. 5. Overnight 5–10 ml broth cultures of the host bacterium grown in LB or TSB. 6. Agar plates containing LB or TSB supplemented with 1mM CaCl2.  Method of Enrichment 1. Centrifuge the sewage suspensions at 10, 000 × g for 10min to remove particulates. The supernatant can be used directly in enrichments involving rapidly dividing bacterial cultures, it can be filter sterilized by passage through 0. 2μm low protein binding membrane filters. 2. The supernatant can be stored at 4◦C in the presence of several ml of chloroform in a glass or solvent-resistant plastic bottle. 3. For aerobic hosts, pipette 10 ml of sterile broth containing 2mM CaCl2 into a 125 ml Erlenmeyer flask, and add 10 ml of clarified (and filtered) sewage. 4. Inoculate the flask with 0.1ml of an overnight broth culture of the desired host bacterium and incubate at the appropriate growth temperature with gentle mixing (50 rpm). 5. After 24–48 h incubation, centrifuge the contents of the flask at 10, 000 × g for 10min. decant the supernatant into a small screw-capped bottle or a series of capped test tubes. 6. As a general protocol add approximately 0.5ml of chloroform to the clarified crude lysate, shake and store at 4◦C.  Detection and Quantification of Bacteriophage 1. Testing Enrichment for Phage:- At this stage of the enrichment it is recommended that you spotted your lysate on the host cells to see whether it contains any phage active on your host bacterium. This can be accomplished in the following manner: 1. Spread loopfuls of host bacteria down the surface of agar plates prepared with the same medium supplemented with Ca2+. 2. Allow the fluid to dry and then place 5μl of the clarified enrichments on the streaks, incubate overnight and observe for zones of cell killing 2-Quantification of Viruses:- The plaque assay is one of the most accurate ways to measure virus infectivity. Plaques are clear zones that develop on lawns of host cells. Theoretically, each plaque results from infection by a single virus particle. The virus plaque is analogous to the bacterial colony.  Ouchterlony Double Diffusion - Patterns Principle: - The key reaction of immunology and immune defense is the interaction of antibodies and antigens. This interaction is responsible for the body’s defense against viral and bacterial infections and other toxins. The body’s defense mechanism recognizes foreign substances, or antigens, and raises specific antibodies against them. - An antigen reacts with a specific antibody to form an antigen-antibody complex, the composition of which depends on the nature, concentration and proportion of the initial reactants.  Materials:- 1. Agarose 2. 1X Assay buffer 3. Antiserum 4. Test Antigens (Ag1 and Ag2) 5. Glass slide 6. Gel punch with syringe 7. Template 8. Incubator (37˚C) 9. Conical flask 10. Measuring cylinder 11. Alcohol 12. Distilled water 13. Micropipette and pipette tip 14. Petri plate 15. Cotton  Procedure:- 1. Prepare 25 ml of 1.2% agarose (0.3 g /25 ml) in 1X assay buffer by boiling to dissolve the agarose completely. 2. Cool the solution to 55-60°C and pour 4 ml/slide on to grease free glass slide placed on a horizontal surface. Allow the gel to set for 30 minutes. 3. Punch wells by keeping the glass plate on the template. 4. Fill the lower well with 10µl of antiserum and the upper two wells with 10 µl each of Antigen 1 and 2. 5. Keep the glass plate in a moist chamber overnight at 37°C. 6. After incubation, observe for opaque precipitin lines between the antigen and antisera wells.  Observations and Results Observe for the presence of precipitin lines between the antigen and antisera wells. If pattern A or ‘pattern of identity’ is observed between the antigens and the antiserum, it indicates that the antigens are immunologically identical. If pattern B or ‘pattern of partial identity’ is observed, it indicates that the antigens are partially similar or cross-reactive. If pattern C or ‘pattern of non-identity’ is observed, it indicates that there is no cross- reaction between the antigens,i.e the two antigens are immunologically unrelated.  Pattern of Identity: A The antibodies in the antiserum react with both the antigens resulting in a smooth line of precipitate. The antibodies cannot distinguish between the two antigens. i.e., the two antigens are immunologically identical. Pattern of Partial Identity: B In the ‘pattern of partial identity’, the antibodies in the antiserum react more with one of the antigens than the other. The ‘spur’ is thought to result from the determinants present in one antigen but lacking in the other antigen Pattern of Non-Identity: C In the ‘pattern of non-identity’, none of the antibodies in the antiserum react with antigenic determinants that may be present in both the antigens, i.e., the two antigens are immunologically unrelated as far as that antiserum is concerned.