Phage-spreading plate assay for phage spot production
Phage-spreading plate assay for phage spot production
Source of content: compact guide to molecular biology 5th ed.
Operation method
serial dilution method
Principle
This method enables the isolation of pure phage tribes from individual phage spots, and it allows the concentration of phage matrices to be determined.
Materials and Instruments
Escherichia coli Move 1. Cultivate E. coli in λ broth medium containing 0.2 % maltose and 10 mmol/L MgSO4 until saturation. Add 0.3 ml of E. coli culture solution to five 8 mmX80 mm test tubes. Caveat 1. all materials in contact with E. coli should be sterile. 2. be sure to loosen the cap of the bottle with the top agar medium before placing it in the microwave!3. It is best to prepare control plates that meet both of the following conditions: uninfected E. coli moss and cell-free top agar medium. For more product details, please visit Aladdin Scientific website.
Maltose Medium
Plate Microwave Test tube Heater Capillary tube
Be sure to loosen the caps of the bottles with the top agar medium before placing them in the microwave!
2. Thaw the top layer of agar medium in the microwave oven or boil in a water bath for 15 min. cool the bottles at room temperature for 5 min, then place the bottles with melted agar medium in a water bath at 45-50°C.
3. Make serial dilutions (e.g., 100-fold dilutions) of the phage lysate with SM. Add 0.1 ml of the first dilution to one E. coli test tube, then 0.1 ml of the second dilution to another test tube, and the other dilutions in turn. Label the tubes with the E. coli/phage mixtures so that they are not confused with each other. Place the test tubes at room temperature and incubate for 20 min.
4. Transfer the test tubes to a water bath or heater at 37 °C for 10 min. remove the test tubes from the water bath.
5. Add 2.5 ml of top agar medium to each tube, shake gently to mix, and pour the contents onto the plate. Gently tilt the plate to spread the agarose medium over the entire surface of the plate. Place the plate in a 37°C incubator for 6-12 hours.
6. Select individual phage spots from a dilution plate with less dense phage spots using a sterile capillary tube or toothpick. For future use, cut a piece of agar containing phage spots with a capillary tube and pop it into a test tube containing 1 ml of SM (or put the tip of a toothpick into the liquid and swirl it gently). If needed, count the phage spots on a dilution plate and estimate the number of infectious phages in the initial reservoir.