One - step growth curves for Cellulophaga phages
Inoculate a new culture ; ie , pick a colony into a new flask containing 10 ml of MLB media .
Immediately after the transfer , take a ‘time 0’ growth reading .
Continue taking readings as performed in step 2 periodically .
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 concentration of your culture at the time you want to start the infection .
Calculate the volume of host culture needed for 108 cells .
Pipet this amount into a 1.5 ml tube .
Add 107 phages to the tube and start your timer for 15 minutes to allow the phages to adsorb to the host cells .
After 15 minutes , dilute the infection 1 : 1000 in MLB media in a 250 ml flask .
Take a sample immediately after dilution – this is ‘time 0’ .
Steps for centrifuged sample :
Steps for samples that are not centrifuged :
Continue sampling in this way for 8 hours .
Store the filtered samples at 4°C .
The next day , count the plaques on all plates that have a countable number of them .
Decide which dilution gives the best count at each time point for the next time you do this same phage - host pair .
The next day , count any new plaques that have appeared and add these to your original count .
Count again on the third day .
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 .
