Isolation of Bacteriophages LECTURE 10: Isolation of Bacteriophages Viro102: Bacteriophages & Phage Therapy 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB)

Bacteriophage Isolation Bacterial viruses are generally present in natural habitats where bacteria are present. In this lecture we will discuss how to isolate and detect bacteriophage specific for E. coli in a wastewater sample.

Phage isolation The Subsequently Procedure below uses a we pre-incubation of the wastewater sample with an E.coli host strain to enrich, or amplify, the population of phage Subsequently we use a plating technique to detect and count specific phages from the enriched sample Phage isolation The plating technique is referred to as "plaque assay” and involves seeding a “lawn” of host bacteria with a small volume of sample containing phage When phage seeded on the lawn infect and lyse the host cells, they produce a plaque within the confluent opaque lawn of bacteria.

Setting Up Enrichment In a sterile 125 ml flask, combine 5 mL of wastewater with 5.0 mL of 10Xphage broth media and 1.0 mL of E. coli strain culture. Incubate overnight in a 37°C shaker with vigorous agitation. 10 mL of wastewater + 1.0 mL of media 1.0 mL of E. coli Plug it and incubate At 37 °C

Culture Incubator, 37°C

Dilution and Plating 3. Transfer 1 mL of the overnight enrichment culture to a 1.5mL microcentrifuge tube. 4. Add one drop of chloroform to the microcentrifuge tube and mix by finger vortexing or inversion. The chloroform disrupts cell membranes to facilitate release of free phage from infected cells that have not yet lysed.

1ml of Over night culture Add 1 drop of chloroform Vortex it

5. Centrifuge the micro-centrifuge tube at maximum RPM for 5-10 min. The goal is to collect insoluble cell debris into a pellet. Phage are too small to sediment at the low gravitational force generated in a micro-centrifuge and will remain suspended in the supernatant. 6. Repeat the centrifugation step by transferring the uppermost layer (supernatant) into a fresh micro-centrifuge tube. centrifuge again at maximum rpm for 5-10 min. (We are trying to remove all bacteria and cell debris from the supernatant.)

Centrifuge Transfer supernatant to fresh tube

The first microcentrifuge tube is contaminated with chloroform, and should be discarded in the appropriate hazardous waste container. 7. Transfer the supernatant of the second spin to yet another fresh microcentrifuge tube. Discard the second microcentrifuge tube as regular waste.

Serial Dilution of Phage Enrichment 8. Using autoclaved distilled water as the diluent, perform a dilution series of the supernatant (in steps of 10-2) to a total dilution of 10-6. The phage idealy should be diluted in TMG (Tris Mg Gelatin) Buffer, not the usual 0.9% NaCl that you use for E. coli. TMG Buffer contains Mg++ that stabilizes some phage virions and facilitates attachment to the host cell outer membrane. Save the remaining sample at 4°C.

Serial dilution

Plaque Assay for Phage Detection 10-1 dilution of phage sample + host bacteria 10-2 dilution of phage sample + host bacteria 10-4 dilution of phage sample + host bacteria 10-6 dilution of phage sample + host bacteria Add 0.1 ml of E. coli host culture to all the plating tubes Add 0.1 ml of the appropriate phage dilution to their respective tubes.

Gently mix the tubes and incubate for 10-15 minutes at 370C. This pre-incubation allows the phage to attach to the host cells more effectively than in the agar. Add 2-3 mL melted top agar to each of the tube.

Add 2-3 ml of melted Top agar Incubate for 10-15 Min. 0.1 ml E.COLI+ 0.1 ml of Phage dilution

Plaque Assay for Phage Detection Mix the tubes and immediately pour the contents of each tube onto the appropriate plate. Immediately rotate the plate gently so that the melted agar completely and uniformly covers the surface. Let the plates be allowed to solidify . Incubate the plates upside down at 37°C overnight before observing plaques next day.

Incubate at 37 °C for 24 hours and observe the results

Plaque formation, Results Examine plates (a dissecting microscope is helpful) for evidence of zones of clearing, or plaques, within the lawn of bacteria. The size and appearance of plaques may vary, at least in part because there are different types of phage present in the sample. Note particularly the difference between clear and turbid (murky) plaques. Turbid plaques may result when temperate phage establish lysogeny in some of the infected host cells. Clear plaques, on the other hand, indicate that the phage is lytic, or virulent (i.e. non-lysogenic).

B3 plaques LK1 plaques

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