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