BACTERIAL INOCULUM

A) STOCK CULTURE

    • Acclimate bacterial vial
      • Remove bacterial vial from the fridge
      • Place vial inside the hood and let equilibrate at room temperature
    • Rehydrate bacterial pellet
      • Grab 1 bacterial pellet from the vial using forceps
      • Place the vial in a tube with 0.5 ml of DD water
      • Crush the pellet with a cotton swab to get a homogenous suspension
    • Streak on a TSA dish
      • Roll the cotton swab other one third of a TSA petri dish
      • Streak using an inoculating loop to cover the rest of the dish
    • Incubate
      • Close the lid and turn the plate upside down
      • Place on the incubator for 24 hs

B) SINGLE COLONY GROWTH

    • Grab one CFU from the stock culture
      • Remove stock culture from the incubator
      • Using an inoculating loop, grab one CFU
    • Mix CFU with TSB
      • Place the CFU in a bottle containing TSB
      • Mix thoroughly
    • Incubate
      • Close the media bottle lid
      • Place the bottle in the Incubator shaker for 18 hs

C) BACTERIAL INOCULUM

    •  Prepare Suspension Medium
      • Combine TSB, DD water, and Triton-X100
      • Adjust pH value
      • Autoclave
    • Measure Single Colony Growth (SCG) concentration
      • Remove Single Colony Growth from the incubator
      • Estimate the concentration by using either microscopic or spectrophotometry methods (*this step has to be done rapidly since the SCG can only be used within a two hour period)
    • Dilute Single Colony Growth
      • Dilute the SCG using suspension medium to achieve a concentration of 6 x 105