Construction and organization of a BSL-3 cryo-electron microscopy laboratory at UTMB

Abstract

A unique cryo-electron microscopy facility has been designed and constructed at the University of Texas Medical Branch (UTMB) to study the three-dimensional organization of viruses and bacteria classified as select agents at biological safety level (BSL)-3, and their interactions with host cells. A 200 keV high-end cryo-electron microscope was installed inside a BSL-3 containment laboratory and standard operating procedures were developed and implemented to ensure its safe and efficient operation. We also developed a new microscope decontamination protocol based on chlorine dioxide gas with a continuous flow system, which allowed us to expand the facility capabilities to study bacterial agents including spore-forming species. The new unified protocol does not require agent-specific treatment in contrast to the previously used heat decontamination. To optimize the use of the cryo-electron microscope and to improve safety conditions, it can be remotely controlled from a room outside of containment, or through a computer network world-wide. Automated data collection is provided by using JADAS (single particle imaging) and SerialEM (tomography). The facility has successfully operated for more than a year without an incident and was certified as a select agent facility by the Centers for Disease Control.

Structures of enveloped virions determined by cryogenic electron microscopy and tomography

“…As many enveloped viruses are human and animal pathogens, their production, purification and preparation for cryo-EM requires suitable containment facilities and bio-safety protocols (Sherman et al, 2013). To circumvent the need for decontaminating cryo-EM equipment, which may be in some cases impractical, purified virions can be inactivated for example by chemical fixation (Bharat et al, 2011;Halldorsson et al, 2018;Li et al, 2016b) or ultraviolet radiation (Park et al, 2011;Ye et al, 2018;Zhong et al, 2016) prior to cryo-EM sample preparation.…”

Room, Suite Scale, Class III Biological Safety Cabinet, and Sensitive Equipment Decontamination and Validation Using Gaseous Chlorine Dioxide

“…4 Because of the increased concentrations, it has been documented to damage painted surfaces, epoxy surfaces, and electronics. [4][5][6][7][8][9] Additionally, for any decontamination to be successful, the agent must be able to distribute throughout the space and penetrate all nooks and crannies. VPHP vapors have been shown to have limited distribution and penetration abilities.…”

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

“…Since equipment for sample preparation and EM analysis is rarely available in high containment biosafety laboratories, samples must be inactivated before leaving these facilities, which is achieved by chemical fixation. Note, however, that few BSL laboratories in the world are equipped with freezing devices, as well as with cryo-microscopes (e.g., [ 52 ]), where examination of virus-infected cells can be performed in their most genuine state. Chemically inactivated samples can be also subsequently subjected to cryo-fixation outside the BSL laboratory.…”