One-Step Growth Curves for Cyanophages
From an already growing culture, split cells at the ratio you will be splitting for the one step experiment.
Immediately after the split, take a 'time 0' growth reading using a plate reader.
Continue taking readings in this way at approximately the same time every day for 21 days.
Graph the results as you go and determine when exponential growth occurs!
Repeat 2 more times to ensure the host growth is consistent.
During your 3rd growth curve when exponential growth is occuring, fix 1 ml of cells with 5 µl 25% glutaraldehyde.
Flash freeze in liquid nitrogen daily.
Store in -80°C freezer.
Determine the cell count (using FCM or DAPI) that is associated with the culture fluorescence level during exponential growth.
It is best to infect the host in late-exponential (log linear) phase.
Do a plaque assay to determine the PFU/ml of the lysate you plan to use.
Calculate the volume needed for 107 phages.
Determine the length of time to sample during the one-step experiement.
Prepare dilutions for free phage and total phage by following the table found in guidelines.
Turn on water bath to 35°C Label a 1L flask with the phage, host, and your initials.
Add 396 ml SN media (or however much to q. to 400 ml) to the 1L flask.
Label 1 15ml snap cap tube the "Infection Tube".
Label 230 15ml snap cap tubes with the red, bolded text ids.
Add 1ml host to each tube.
Days in advance, pour 240 SN bottom agar plates (2ml SN bottom agar per plate).
Lay out the SN bottom agar plates.
Label 230 SN bottom agar plates with the red, bolded text ids.
Label 2 plates "Host (-)" Label 1.5µl centrifuge tubes with ALL THE IDS (See table 1 guidelines).
Add 900µl SN media to the ids highlighted in yellow.
Add 990µl SN media to the ids highlighted in blue.
Microwave SN top agar.
Aliquot heated SN media top agar into 50ml conical tubes.
Keep warm in the 35°C water bath.
Determine the concentration of your culture at the time you want to start the infection.
Calculate the volume of host culture needed for 108 cells (q. to 2ml with SN media).
Pipet this amount into the 15 ml snap cap tube labeled "Infection Tube".
Add 107 phages (q. to 2ml with SN media) to the tube.
Allow the phages to adsorb to the host cells for 1 hour.
Put the 4ml infection into 396ml SN media in a 1L flask to dilute the infection to 1:100.
Take a sample immediately after dilution - this is time 0.
Remove the plunger of 1ml luer-lock syringe.
Add a 0.2 µm 25mm diameter syringe filter to the syringe end.
Pipet 1 ml from the flask into the syringe using 1 ml serological pipet.
Add the plunger back to the syringe and 0.2µm syringe filter the 1 ml into a 1.5 ml centrifuge tube.
Perform a serial dilution to dilute the sample to 102 per ml (10-3 dilution).
Add 250µl of the 10-3 dilution and 250µl of the 10-2 dilution to the corresponding 15ml snap cap tube with 1ml host.
Allow phage and host to incubate for 2 hours.
Plate using 4ml SN top agar per plate.
Pipette 1ml from the flask into a 1.5ml centrifuge tube using the same 1ml serological pipette.
Perform a serial dilution to dilute the sample to 102 per ml (10-3).
Add 250µl of the 10-3 dilution and 250µl of the 10-2 dilution to the corresponding 15ml snap cap tube with 1ml host.
Allow phage and host to incubate for 2 hours and then plate using 4ml SN top agar per plate.
Continue sampling in this way for 16 hours (and sample one more time at 24 hrs).
At later time points more dilutions will need to be plated.
Be generous with what you plate (i.e., plate 10-2, 10-3, 10-4, 10-5).
The serial dilutions should be performed as shown below: 
In seven days, count the plaques on all plates that have a countable number of them.
Count again on day 14 and 21.
Calculate PFU/ml at each time point for both the centrifuged (free phage only) and not centrifuged (total phage) samples.
Graph the results.
Calculate burst size.
Take the FREE phage average of the time points on the plateau before the burst (A).
Take the FREE phage average of the time points on the plateau after the burst (B).
Subtract A and B.
This the total burst or new phages released (C).
Divide C by the number of infecting phage (TOTAL phages at T0 minus FREE at T0).
This is the burst size.
