Titering T7:
1. Grow the appropriate host strain (BL21 for wildtype or T7-415) overnight in M9TB media. Next morning, inoculate around 100 uL of the host strain in 4mL of M9TB and grow to an OD of 1.0 at 37 degrees (for BL21 this takes between 2-3 hrs). The cells can be stored at 4 degrees until ready for use. Do not store for more than 2 days.
2. Create a series of dilutions (in TB or LB) of the phage to be titered. This depends on what you estimate the phage concentration to be. Generally you want to make sure to cover a range of concentrations that includes concentrations of 100-1000 pfu/mL. This way you will get some plates with 10 to 100 plaques that will be easy to count.
3. Pipette 100 uL of the dilutions onto pre-warmed LB plates with the appropriate antibiotic resistance (for BL21, just use plain LB plates). Then pipette 100 uL of the host strain (see step 1) onto each plate. Then pour melted top agarose (around 3 mL) onto each plate and swish it all around so that the top agarose spreads evenly.
Note: I typically don’t do this step with more than 3 plates at a time. It is best to melt the top agarose a little while before hand so that it cools down a bit (to avoid killing cells/phage) before you use it. I recommend melting it in the microwave before you do the dilutions in step 2. Or just keep it in a 60 degree water bath.
4. When the plates solidify, invert them. Plaques will grow at 37 degrees in several hours (at least 3) or at room temperature overnight. I find that keeping the plates at room temperature overnight usually yields plaques of good size.
5. Example calculation. Say you get 5 plaques in the 1E-5 dilution plate. Then that means that the 1E-5 dilution has 50 pfu/mL (since I only plated .1 mL). Which means that the original concentration is 50E5 pfu/mL (just multiplied by 1E5).
Titering fd-tet
Fd-tet has a replication defect and this makes it impractical to titer by pfu. Rather, the titer can be measured in terms of transducing units (TU). Simply, cells infected with fd-tet will gain tet resistance and therefore will form colonies on tetracycline plates. The number of colonies formed gives the number of TU. This number is typically 20x lower than the number of phage particles (which can be measured with nanodrop if high enough concentration).
This protocol uses host cells grown in rich media. Another method is to prepare starved cells as host cells, which apparently gives a more reliable titer, but is more complicated.
1. Grow overnight culture of K91 in NZY media.
2. Next morning, incoculate around 300 uL of this culture in 20 mL LB (use 125 mL flask). Grow to an OD of 1.0 at 37 degrees (around 3 hours)
3. Slow shake the cells (90-100 rpm) at 37 degrees for 10-15 minutes. This allows them to regenerate F-pili.
4. Store the cells at 4 degrees (or on Ice). Try to use within the hour.
5. Dilute the phage (in TBS) to an estimated titer of around 1E7 TU/mL (though this is the ideal phage concentration, it is better to use less than it is to use more, since you don’t want phage competing for cells). If you are very unsure of the titer, then you can use a range of dilutions that covers 1E7 TU/mL.
6. Mix 20 uL of your diluted phage (from step 5) with 20 uL of your cells (from step 4) in a 1.5 mL eppindorf tube. Incubate at room temperature for 15 minutes.
7. Add 1 mL of NZY with .2ug/mL tet. Then shake the tube (upside down at 250 rpm) at 37 degrees for 30 minutes. This will allow infected cells to express the tet-resistance gene.
8. Serial dilute the cells (from step 7) up to 1E-3 in NZY with 15 ug/mL tet. Spread 100 uL of each dilution (up to 1E-3) onto a pre-warmed NZY plate with 40 ug/mL NZY. Invert the plate and put it in the 37 degree incubator overnight. Count the colonies the next day.
9. Example calculation. Say you get 10 colonies in the 1E-3 dilution plate. This means that the 1E-3 dilution solution has 100 infected cells/mL. Which means that your undiluted solution of 20 uL phage, 20 uL cells, and 1 mL NZY/.2ug tet has 100E3 (or 1E5) infected cells/mL. Which means that there are 1E5 infected cells/mL times 1.04 mL (volume of the solution) = 1.04E5 TU in the solution. Since those TU came from the 20 uL of phage, we conclude that that the phage titer was 1.04E5 TU/.02 mL = 5.2E6 TU/mL right before we used them to infect the host. Now if this was a dilution of some higher up phage stock, then the backward calculation is simple (ie. if we used a 1E-2 stock dilution to infect the cells, then the titer of the stock would be 5.2E8 TU/mL).