Heat-killed bacteria

Protocol for heat-killing Pseudomonas or MRSA:
  • Grow an overnight culture in LB broth with shaking
  • Wash the bacteria (centrifuge) 2 times with PBS to remove the conditioned-LB.  Save and filter sterilize the LB if you want to look at secreted compounds as well  
  • Adjust the OD to 1 in PBS and aliquot bacteria into 1.5 ml Eppendorf tubes.
  • Heat 10 min at 95°C on pre-warmed heat block    
  • Freeze aliquots of the the OD 1 PBS stocks at -80.  Streak some of the heat-killed bacteria on an LB plate and incubate at 37°C for 24-48 hours to ensure there is no growth.  Do not use for cell culture experiments until you have verified no growth.  Alternatively, inoculate a liquid culture and incubate ≥24 hours
  • When you are ready to use the heat killed bacteria, dilute to OD 0.01 (1:100) with media and stimulate your cells with that for inflammation studies.  You may have to dilute further depending on the cells you are using.  
  • This protocol was adapted from Seiler, et al. 2013 J Immunol. 190:1603-1613