Bacterial cultures were grown to mid-log phase in THB. 5 ml of OD600=0.4 culture was spun down and resuspended in 500 μl H2O. Serial dilutions of bacterial suspension were plated to confirm equivalent cfu. 400 μl of the bacterial suspension was placed in a 2 ml screw cap tube with 1 ml chloroform. Tubes were shaken for 5 min in a mini-beadbeater-8 (Biospec Products), then spun at ~13,000 x g for 10 min. Hyaluronic acid in aqueous phase was determined using a Hyaluronic Acid Test Kit (Corgenix) per manufacturer’s instructions.
Protocol detail
Bacterial Hyaluronic Acid Quantification
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Corresponding Organization : University of Montana
Other organizations : University of Wollongong, University of Technology Sydney
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Variable analysis
independent variables
- Bacterial culture growth to mid-log phase in THB
- Chloroform exposure
- Hyaluronic acid content in aqueous phase
- Culture volume (5 ml)
- Culture optical density (OD600=0.4)
- Resuspension volume (500 μl)
- Bacterial suspension volume (400 μl)
- Chloroform volume (1 ml)
- Shaking time (5 min)
- Centrifugation speed (~13,000 × g)
- Centrifugation time (10 min)
- Serial dilutions of bacterial suspension plated to confirm equivalent CFU
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