Overview of Methods

 

These biological methods are actually very simple and require only a few materials!  However, since it is difficult to acquire any new technique without an experienced person nearby to train you, seeing the protocols in two different ways is very helpful.  In this first way, all the protocols are looped together, to illustrate how they are used to isolate a new phage.  In the second way, wherein the protocols are fully described, the incidentals of the procedure are made evident.  These incidentals are hyperlinked to the outline. 

adsorption

This is an outline for all phage-purification procedures. 

I.  Sample collection and preparation.

1.    Collect environmental samples in 50 mL conical tubes.

2.    If the samples are solid, add 3-5 mL MP phage buffer.

3.    Spin to pellet debris, unless the supernatant is clear enough to filter.

4.    Filter sterilize supernatant with 0.22 uM filters.

 

II.    First round of infection to screen for plaques

1.    Infect 0.1 mL Mycobacterium smegmatis mc2155 grown without tween with 0.1 mL of your filtered sample.

2.    Add 4 mL Middlebrook Noble Top Agar

3.    Plate on 7H9 media.

4.    Incubate at 37C overnight.

 

III.    Second round of infection (plaque purification).

1.    Use a pipette tip to pick a single plaque into 100 uL phage buffer plus calcium.

2.    Serially dilute it 10-2, 10-3, 10-4.

3.    Infect 0.1 mL mc2155 with 0.1 mL of dilutions

4.    Add 4 mL Middlebrook Noble Top Agar

5.    Plate on 7H9 media.

6.    Incubate at 37C overnight.

 

IV.    Final Plaque purification (Isolating a phage from a plaque).

1.    Use a pipette tip to pick a well isolated plaque from one of the above dilution plates into MP phage buffer.

2.    Serially dilute it 10-1, 10-2, 10-3

3.    Infect 0.1 mL mc2155 with 0.1 mL of phage (undiluted) and dilutions

4.    Add 4 mL Middlebrook Noble Top Agar

5.    Plate on 7H9 media.

6.    Incubate at 37C overnight.

 

V.    Extracting Phage from Plates.

1.    To a nearly cleared plate, add 4 mL MP phage buffer,  swirl gently.

2.    Let sit at room temperature a couple of hours.

3.    Siphon up liquid and filter it through a 0.22 uM filter.

 

VI.    Spot tests.

1.    Mix 0.5 mL mc2155 and 4.5 mL MBTA+Ca.

2.    Plate on 7H9 media (As for this protocol, except omit any phage at this time)

3.    Serially dilute lysate 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9, 10-10 using MP phage buffer.

4.    Spot 5 uL of these onto the plate after it has cooled enough to gel.

5.    After the spots evaporate, incubate at 37C overnight.