Rickettsiae are small obligate intracellular gram-negative bacteria that are grown in a Biological Safety Level 3 (BSL-3) laboratory setting.This protocol uses antibiotic-free medium, and therefore cultures are susceptible to contamination by undesirable bacteria or fungi.This procedure provides guidance for the inoculation and growth of primary isolates derived from small volumes of clinical samples including blood and blood products in a small volume culture and includes topics of growth monitoring, changing media, freezing primary isolates, and troubleshooting in the event of contamination. This technique utilizes Vero E6 cells growth in small conical culture flasks.These flasks have a growth area of 10cm2 and can accommodate up to 5 mL of media.All techniques are to be performed in a biological safety cabinet (BSC) in a BSL-3 laboratory.Proper training and extreme caution is of the utmost importance when working with live rickettsial cultures.
Preliminary Authorization Basis Document For the Proposed Biological Safety Level 3 (BSL-3) Facility (B368) at Lawrence Livermore National Laboratory Revision 2