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

Begin with a selective nutrient agar plate supplemented with blood containing RBCs.

Streak Staphylococcus aureus (S.aureus), a Gram-positive pathogenic bacterium, onto the agar and incubate.

During incubation, bacteria utilize nutrients, grow, and form colonies.

S. aureus secretes hemolysin, a pore-forming toxin that lyses the RBCs in the agar and produces clear, transparent zones around colonies, indicative of hemolysis.

Select a hemolytic colony, inoculate it into a nutrient broth, and incubate under rotating conditions to support bacterial growth.

Using a spectrophotometer, measure optical density (OD) to determine bacterial concentration.

Based on the OD reading, transfer an aliquot into a fresh nutrient broth to dilute the cells, and incubate in a water bath with shaking.

Measure the OD at regular intervals.

An OD near 0.8 indicates that the bacteria have reached the exponential growth phase with uniform physiology.

This hemolytic S. aureus is ready for host-pathogen interaction studies.

Begin by streaking S.aureus strains of interest on blood agar plates from a frozen glycerol stock and incubating the plates at 37 degrees Celsius for 16 to 24 hours to confirm their hemolytic phenotypes based on their ability to lyse red blood cells and to form clear transparent zones.

Inoculate single colony isolates of each strain in 4 milliliters of tryptic soy broth, or TSB, in sterile glass tubes and rotate the tubes at 37 degrees Celsius for 16 to 24 hours in a tube roller at an approximately 70-degree angle and 70 rotations per minute. The next morning, measure the optical density at 600 nanometers, or OD₆₀₀, of the cultures on a spectrophotometer and dilute the cells to an OD₆₀₀ of 0.05 in 25 milliliters of sterile TSB at a 5 to 1 flask to volume ratio in 125 milliliter DeLong flasks. Incubate the cultures in a 37 degree Celsius water bath at 280 rotations per minute.