<ol><li>Preparation of bacterial inoculum<ol style='list-style-type:decimal;'><li>Streak Staphylococcus aureus strains of interest for isolation on blood agar plates from a frozen glycerol stock. Incubate at 37 &#176;C for 16&#8211;24 h to confirm hemolytic phenotypes based on their ability to lyse red blood cells (RBCs) and form clear transparent zones.</li><li>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 &#176;C for 16&#8211;24 h in a tube roller set at approximately a 70&#176; angle and 70 rotations per minute (RPM).</li><li>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 &#176;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.</li></ol></li></ol>

<table class='ck-table-resized'><tbody><tr><td>5% Sheep blood</td><td>Hardy Diagnostics (Santa Maria, CA)</td><td>A10</td><td>&#160;</td></tr><tr><td>Orbital shaking water bath</td><td>New Brunswick Innova</td><td>NA</td><td>&#160;</td></tr><tr><td>Tryptic Soy Broth</td><td>VWR</td><td>90000-376</td><td>&#160;</td></tr><tr><td>UV-visible spectrophotometer</td><td>Beckman Coulter-DU350</td><td>NA</td><td>&#160;</td></tr></tbody></table>

preparing a hemolytic staphylococcus aureus culture for host-pathogen interaction studies

Source: <a href='https://app.jove.com/v/59055/a-fluorescence-based-method-to-study-bacterial-gene-regulation'>Behera, R.K. et. al., A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues. J. Vis. Exp. (2019)</a>This video demonstrates the preparation of hemolytic Staphylococcus aureus cultures to standardize bacterial growth and achieve metabolic synchronization for host-pathogen interaction studies.

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

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

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.

preparing-hemolytic-staphylococcus-aureus-culture-for-host-pathogen

Research

JoVE Encyclopedia of Experiments

Microbiology

Microbiologie

Bacterial Pathogenesis and Host Interactions

Bacteriële pathogenese en interacties met de gastheer

bacterial-pathogenesis-and-host-interactions

Infection Models

Infectiemodellen

Een hemolytische Staphylococcus aureus -cultuur voorbereiden voor studies naar gastheer-pathogeen interacties

368 Views. Begin met een selectieve voedingsbodemplaat met bloedagar, aangevuld met RBC's.Streek Staphylococcus aureus (S.aureus), een grampositieve pathogene bacterie, op de agar en incubeer.Tijdens de incubatie gebruiken bacteriën voedingsstoffen, groeien en vormen kolonies.S. aureus scheidt hemolysine af, een porie-vormende toxine dat de RBC's in de agar lyseerd en heldere, transparante zones rond kolonies produceert, wat duidt op hemolyse.Selecteer e...

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

In dit artikel

Samenvatting

Protocol

Materialen

Herprints en machtigingen

Trefwoorden

Gerelateerde artikelen

Naam	Bedrijf	Catalogusnummer
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