Bacteriophage isolation by spotting on target host cells
The host cells are grown overnight in liquid cultures containing an appropriate growth medium for the organism .
Measure optical density spectrophotometrically at 525 nm ( OD525 ) and adjust OD to 0.3–0.5 with growth medium .
Soft agar ( 0.5 to 0.6 % agar in media of choice ) is melted in a water bath or a microwave oven .
Aliquot 4mL of soft agar into sterile tubes and keep just above solidification temperature until use .
200–300 µL bacterial culture is added to the 4 mL tubes with melted soft agar .
The bacteria - soft agar mixture is then vortexed and immediately poured onto an agar plate with an agar that supports growth of the host bacterium .
Distribute bacteria - soft agar mixture evenly on the plate , which is placed on a flat surface .
When the soft agar containing the target bacteria has solidified , triplicate aliquots of 5–10 µL of each of the environmental water samples from which phages should be isolated are spotted on top of the soft agar .
As a negative control 5–10 µL phage buffer ( e . g . , SM buffer : 450 mM NaCl , 50 mM MgSO4 , 50 mM Tris , 0.01 % Gelatin , pH = 8 ) or 0.02 µm filtered sample water is spotted in triplicate on the soft agar .
The plates are incubated for 1–3 d depending on the growth rate of the bacteria , and the presence of lytic phages in the sample is detected as a clearing zone ( plaque ) in the spotted area of the lawn of bacteria that develops over time on the plate .
If clearing zones appear in the spotted area , this indicates the presence of lytic phages , which can be isolated and purified Once detected as clearings in the spotting zone , phages are further isolated and purified from the plates .
