Question about MOI of S. flexneri

[Sc]

Hey all,

 

Im hung up on how to calculate MOI for the bacteria Im currently studying. My procedure is to innoculate a single colony into 4mL of BHI for my overnight culture, then for activation I am following Hanajima-Ozawa et al by aliquoting 120uL of overnight into 3mL of BHI for 3 hours to achieve mid-exponential growth. This is where it gets tricky, Im forced to spin this down and resuspend in less media because ridiculous concentrations of S. flex are required for infection. However, I dont know where to go from here since Im unsure of the concentration before spinning them down and how much to resuspend in/ infect with.

 

I know that I need 10^7 to 10^8 bugs/mL for infection. Im also using HeLa, MDCK and CaCo2 cells at a confluency of about 80% on a 13mm slide in a 24 well plate.

 

Any insight? Thanks in advance

 

Steve