Transcriptomics During One - Step Growth Curves for Cellulophaga Phages
Do a plaque assay to determine the PFU / ml of the lysate you plan to use .
Inoculate a new culture ; i . e . , pick a colony into a 125 ml flask containing MLB media .
The next day , transfer 10 ml of this culture to 500 ml of new media in a Fernbach flask ( short and fat 2800 ml ) .
Inoculate enough 500 ml cultures for your experiment .
Immediately after the transfer , take a 'time 0' growth reading .
Continue taking readings in this way periodically .
Graph the results as you go !
Determine the concentration of your culture at the time you want to start the infection .
Calculate the total number of cells in each of your 500 ml cultures .
Calculate how many phages you should add for MOI 3 .
Add phages to the experimental flask and an equal volume of MSM to the control flask and start your timer .
Immediately , dilute the infection 1 : 1 in MLB media ; i . e . , add 500 ml fresh media to each flask and swirl to mix .
Take a sample immediately after dilution .
Continue sampling in this way for 8 hours .
The next day , count the plaques on all plates that have a countable number of them .
The next day , count any new plaques that have appeared ; 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
