Preparing a Hemolytic Staphylococcus aureus Culture for Host-Pathogen Interaction Studies

Abstract

Source: Behera, R.K. et. al., A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues. J. Vis. Exp. (2019)

This video demonstrates the preparation of hemolytic Staphylococcus aureus cultures to standardize bacterial growth and achieve metabolic synchronization for host-pathogen interaction studies.

Protocol

1. Preparation of bacterial inoculum
   1. Streak Staphylococcus aureus strains of interest for isolation on blood agar plates from a frozen glycerol stock. Incubate at 37 °C for 16–24 h to confirm hemolytic phenotypes based on their ability to lyse red blood cells (RBCs) and form clear transparent zones.
   2. Initiate overnight cultures by inoculating single colonies of each strain to 4 mL of tryptic soy broth (TSB) in sterile glass tubes. Rotate the tubes at 37 °C for 16–24 h in a tube roller set at approximately a 70° angle and 70 rotations per minute (RPM).
   3. Use a spectrophotometer to measure the optical density at 600 nm (OD₆₀₀) of the cultures from step 2.1.2. Use sterile medium as an optical reference (blank). Dilute these cells to an OD₆₀₀ of 0.05 in 25 mL of sterile Tryptic Soy Broth (TSB) in separate, 125 mL DeLong flasks (5:1 flask to volume ratio) and incubate cultures in a water bath (for proper heat transfer to the cultures), shaking at 280 RPM and set to 37 °C.
      NOTE: Growth of the inoculum can be performed in various ways; one method for growing cells to exponential phase is presented. Regardless, it is important to consistently use the same method for preparing the cells for inoculation.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5% Sheep blood	Hardy Diagnostics (Santa Maria, CA)	A10
Orbital shaking water bath	New Brunswick Innova	NA
Tryptic Soy Broth	VWR	90000-376
UV-visible spectrophotometer	Beckman Coulter-DU350	NA