Phage Testing Assay

This assay uses a lawn of phage-susceptible E.coli embedded in a layer of agarose. This top agarose lays on a bed of standard agar. If phage is present in a clone streaked onto the top agarose lawn, the lawn will be lysed on incubation.

Method for preparing top agarose

(i.e. Phage-susceptible E.coli clone)

The phage-susceptible E.coli clone culture (at Geneservice we have successfully used E.coli strains TG1, DH10B or JM101) must be freshly grown the day before making bacterial lawns.
If a negative control lawn is also going to be tested, we recommend using Q358 which is phage-resistant.

Method for preparing bacterial lawn

1. Pour the required number of PBA trays (11 x 7cm) or Q-trays (22 x 22cm) with LB agar and NO antibiotic and allow to set.
2. Place into an incubator at 37°C for at least one hour. This facilitates pouring of top agarose.
3. Make up top agarose by adding 4g agarose to 500ml LB broth without glycerol (0.8% agarose). Microwave until agarose is dissolved and place in a waterbath set to 45°C. The top agarose needs to sit in the waterbath for at least 30 minutes so that it has fully equilibrated to 45°C (if it is too hot the bacteria will be killed).
4. When the top agarose reaches 45°C add 11.25ml lawn culture (see above) and mix well.
5. Aliquot 40ml top agarose + bacteria (if using Q-tray) or 10ml top agarose + bacteria (if using PBA tray) into Falcon tubes keeping them at 45°C.
6. Take one Q-tray or PBA tray out of the incubator at a time, and in a safety cabinet quickly pour the top agarose from the Falcon tube onto the top of the LB agar base ensuring an even covering of top agarose. NB: Top agarose will set very quickly. Leave the plate open in the hood for at least 10 minutes to dry.
7. If plates are not to be used immediately, wrap them in clingfilm and leave them at 4°C until ready for use (use that day).
8. Streak each clone onto the lawn using a sterile tip.
9. Seal the lawn plate with parafilm and then wrap with clingfilm. Incubate overnight at 37°C. Lawn plates should never be opened after incubation if lysis is observed in order to prevent the spread of the phage throughout the laboratory.
   

Diagrammatic Representation of a Positive Phage Assay

Materials

For clean clone culture

Sterile 100ml conical flask
LB broth without glycerol
Glycerol stock of E.coli to be used as the lawn: Phage susceptible clone

E.coli strain e.g. TG1, DH10B or JM101 (Stratagene)

Q358 (DSMZ)

For bottom agar

PBA culture plates (CONS1001)or Genetix Q-trays (X602) depending on number of clones to be tested
LB agar without antibiotic

For top agarose

LB broth without glycerol
Ultrapure Electrophoresis Grade agarose (GIBCOBRL, 540-551OUB)
Microwave (Matsui M183BT)
Waterbath
Falcon 15ml conical tubes (2097) or Falcon 50ml conical tubes (2098)
Clingfilm