User:Vijay/Protocol/saureus-harvest

Overview
I do Staphylococcus aureus cell harvest at different time points (OD 0.3, 1.5, 4.0 at 560nm) corresponding to mid-exponential, late-exponential and post-exponential growth phase.

Materials

 * Tryptic Soy Agar (with appropriate antibiotics)
 * Tryptic Soy Broth (200ml broth in 500ml conical flask)
 * 560 nm spectrophotometer

Procedure

 * Preparation


 * 1) Streak the culture (wild type and the mutant) in TSA plates with corresponding antibiotics, incubate at 370C overnight.
 * 2) From the overnight plates, inoculate in to 5ml TSB, with appropriate antibiotics, incubate at 370C overnight, at 185 rpm


 * Procedure


 * 1) In a sterile 200ml TSB (in 500ml conical flask), inoculate 500ul of overnight culture (NO ANTIBIOTICS).
 * 2) Incubate at 370C, 150rpm
 * 3) Read OD at 560nm to collect cells at OD 0.3, 1.5 and 4.0.
 * 4) When the OD reaches 0.3 collect 10ml culture in a 15ml sterile falcon tube (3-5 tubes).Spin the tube at 4C, 10000rpm for 10minutes. Transfer the cells to a small eppendorf tube (1.5ml), spin it again and freeze
 * 5) When the OD reaches 1.5 and 4.0 collect just 1.5ml of the culture in a small eppendorf tube, spin it at 4C, 10000rpm for 10mintues and freeze