Bacteriophage Enrichment Techniques

Bacteriophage Enrichment from Water

• Bacteriophages are the most abundant life forms on Earth.
• Bacteriophages are common in soil, with direct counting methods finding 10⁷-10⁹ per gram of soil.
• This chapter describes simple enrichment techniques used successfully in postgraduate and undergraduate research and laboratory exercises.
• Sewage treatment plants are ideal sources for bacteriophage isolation.

Enrichment of Phage from Aqueous Materials - Materials

• Collecting: Samples of pond water, pond sediment, or raw sewage collected in sterilized Erlenmeyer flasks or screw-capped bottles.
• Centrifuge: Equipment for separating liquid from solid parts.
• Broth: Sterile Luria broth (LB) or Tryptic Soy broth (TSB) supplemented with 2mM CaCl₂, stored in 100 ml or 500 ml sterile Pyrex screw-capped bottles.
• Flasks: Sterile 125 ml Erlenmeyer flasks.
• Bacterial Cultures: Overnight 5-10ml broth cultures of the host bacterium grown in LB or TSB.
• Agar Plates: Agar plates containing LB or TSB supplemented with 1mM CaCl₂

Method of Enrichment

• Centrifuge sewage suspensions: At 10,000 x g for 10 minutes to remove particulates; supernatant can be used directly for enrichments using rapidly dividing bacterial cultures. It can be filter sterilized using 0.2µm low protein binding membrane filters.
• Store supernatant: At 4°C in the presence of several ml of chloroform in a glass or solvent-resistant plastic bottle.
• 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. Inoculate with 0.1 ml of an overnight broth culture of the desired host bacterium and incubate at the appropriate growth temperature with gentle mixing (50 rpm).
• Incubation and Centrifugation: After 24-48 hours, centrifuge contents at 10,000 x g for 10 minutes. Decant the supernatant into a small screw-capped bottle or a series of capped test tubes.
• Add Chloroform: As a general protocol, add approximately 0.5 ml of chloroform to the clarified crude lysate, shake, and store at 4°C.

Detection and Quantification of Bacteriophage - Testing Enrichment for Phage

• Spot the lysate (the solution containing the bacteriophages) onto host cells to test if phage is present.
• Spread: Spread loopfuls of host bacteria on agar plates prepared using the same broth as the host cells but supplemented with Ca2+.
• Dry and Spot: Allow the fluid to dry, then place 5 µl of the clarified enrichments onto the streaks.
• Incubate and Observe: Incubate overnight and observe for zones of cell killing (plaques).

2- Quantification of Viruses - Plaque Assay

• Plaques are clear zones that develop on lawns of host cells, each resulting from infection by a single virus particle.
• The plaque assay measures virus infectivity.
• The figures in the document show a detailed method, but it cannot be represented in this format.

Enrichment Protocol

• The method, using molten top agar, adds a mixture of the bacterial solution containing phage suspension onto a layer of solidified nutrient agar on an agar plate, resulting in a phage lawn and distinct plaques.

Ouchterlony Double Diffusion - Patterns

• Principle: The key reaction in immunology and immune defense is the interaction of antibodies and antigens to create antigen-antibody complexes. The type of interaction determines immunological identity and is related to the nature, concentration, and proportion of initial reactants.

Ouchterlony Double Diffusion - Materials

• Agarose
• 1X Assay buffer
• Antiserum
• Test Antigens (Ag1 and Ag2)
• Glass slide
• Gel punch with syringe
• Template
• Incubator (37°C)
• Conical flask
• Measuring cylinder
• Alcohol
• Distilled water
• Micropipette and pipette tip
• Petri plate
• Cotton

Ouchterlony Double Diffusion - Procedure

• Prepare agarose solution, cool to 55-60°C, and pour onto a glass slide.
• Allow the gel to set.
• Use a template to punch wells in the agar.
• Fill the wells with antiserum and antigens.
• Incubate overnight at 37°C in a moist chamber.
• Observe for precipitin lines between antigen and antisera wells.

Ouchterlony - Observations and Results

• Observe precipitin lines between antigen and antiserum.
• Pattern A (Identity): Indicates immunologically identical antigens (a single line joining the antigen and antiserum wells).
• Pattern B (Partial Identity): Indicates partially similar or cross-reactive antigens (a line joining the wells, but with a spur).
• Pattern C (Non-Identity): Indicates unrelated antigens (no cross-reaction).

Patterns of Identity, Partial Identity, and Non-Identity

• The patterns of identity, partial identity, and non-identity in Ouchterlony double diffusion are clearly described in the diagrams.