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 .
