Caltech/Protocols/Titering

Titering

1. Grow fresh overnight cultures of the bacteria of interest (i.e. LE392, D1210, etc.)
   

   An example plate. Dark spots are cleared zones in a lawn of bacteria.
2. Incubate the cultures until they reach an OD600 of roughly 0.1
   • More specifically, when the culture is swirled, cloudiness is observed
3. Do NOT titer with bacterial cultures that have just been diluted from a culture past OD600 of 0.8 (complications will occur)
4. Place agar/agarose plates in the 42 C oven and ready the 48 C water bath
5. Prepare fresh 10 mM MgSO4 solution from the 1 M MgSO4 stock solution (100 ul MgSO4 in 10 mL sterile water works conveniently)
6. In 1.5 mL eppendorf tubes, add 1 mL of the 10 mM MgSO4 solution.
7. Prepare phage dilutions by adding appropriate amount of phage into MgSO4 solution (i.e. 1000-fold dilution is 1 ul phage in 1 ml MgSO4)
8. After addition of phage to solution, invert tube at least 12 times for sufficient mixing
9. For further dilutions, can simply dilute made phage dilutions (i.e. 1,000,000-fold can be obtained from taking 1 ul 1000-fold diluted phage and adding to another 1 mL of 10 mM MgSO4
10. Aliquot 0.1 mL cell solution (i.e. LE392, D1210, etc.) into new 1.5 mL eppendorf tubes
11. To begin infection, add appropriate amount of diluted phage to cell solution (10 ul usually works well for starters) and start the timer
12. During this 30 minute infection period, label the plates and place back in the 42 C oven
13. ~3 minutes before the end of infection, can take 3 mL aliquots of the top medium in 13 mL falcon tubes or 5 mL round-bottom tubes and microwave till complete liquification has occurred.
14. Right before the end of infection, take out the plates and place on the bench with the cover on top. Have a flame close-by to prevent contamination. Also, set the pippette to the right volume of infected cell solution and have sterile pipette non-filter tips ready
15. Very quickly with pipette in hand, pippette out infected cell solution, add to top medium, invert the tube at least 2 times, and pour the mixed contents onto the corresponding plate.
16. Rock the plate gently to allow the top medium to uniformly cover the plate and use the sterile tips to poke any bubbles.
17. Let the plate cool on the bench for at least 5 minutes (10 minutes is usually enough) with the cover completely on the plate.
18. Place the plate in the 37 C incubator.