[3dem] Seeking Advice on BSL-2 CryoEM Laboratory Setup and Safety Protocols

Thu Jun 19 18:23:24 PDT 2025

Dear all,

As there is interest in this topic within the community, I would like to share a summary of the replies I have received and in 3dem. Below are some highlights:

Important!! There is no international standard. All the requirements will depend on your country and university.

#1 Oxford Particle Imaging Centre, University of Oxford, UK (Thanks to Helen and especially OPIC for their contributions.)
Comments
Risk Assessment

  1.  Perform case-by-case risk assessments for each organism and share with the local health and safety team. Each institution tends to have a slightly different risk assessment format.
  2.  For some cases, you will need to get HSE approval in the UK e.g. GMO Class 2 samples. This is best determined by your local health and safety team, since your organisation might already have permission, for instance, to work with GMO Class 1 agents.
  3.  NOTE! Bacteria and prions are tricky and often not possible because they are difficult to inactivate without damaging the electron microscope.
  4.  NOTE! At CL3 it may be possible to avoid the requirement for a validated method for room fumigations for EM work.
  5.  It is also important to consider the use of sharps (tweezers) in your risk assessment.
Validation

  1.  In house validation tests are ONLY necessary for CL3 work. For this, you would need to perform validation tests to demonstrate that the infectious agent is inactivated after X time at room temperature. Additional validations are needed to demonstrate that the infectious agent is inactivated after X time with disinfectant (important to find the minimal time required to do this).
  2.  For CL2 samples, it is sufficient to use evidence from disinfectant companies or peer reviewed articles
(it is good to find the minimal time required for this).
Disinfection & Decontamination

  1.  Use 70% ethanol (make sure that this is in excess as evaporates quickly) or 10% Chemgene with thorough rinsing after validated contact time (avoid Virkon as this corrodes equipment).
  2.  Validated contact time (e.g., 10 minutes) is essential.
  3.  Fumigation can be used for entire microbiological safety cabinets containing equipment e.g. loading station, nanocab if available (likely overkill CL2 material) but avoid hydrogen peroxide for nanocab and cassette.
Laboratory Design & Safety

  1.  Number of air changes is important. Negative pressure is in place for CL3 laboratories.
  2.  Consult local health and safety regulations for specific requirements.
  3.  Keep laboratory as clear and clean as possible.
Others

  1.  Prior to work, it is important to notify the suppliers of instruments that we are performing work and that we have risk assessed/performed the validations such that the engineers are safe.
  2.  Keep a log of work performed with instruments at CL2.
  3.  Ensure that adequate PPE is available (for CL2, users should wear gloves, safety glasses and a laboratory coat).
  4.  Document stored samples.

OPIC Equipment
Level
Setup
Decontamination
GP2
CL2/CL3

  1.  Within a Class 1,2 or 3 microbiological safety cabinet (this might require customisation) if pathogen of interest is transmitted by aerosol.
  2.  For CL2 agents that are not aerosols, write a risk assessment to perform preparation outside of a cabinet.
GP2 can be baked out after use. All surfaces and tools must be wiped with the validated disinfectant (e.g 10 % chemgene) and left for the validated contact time after use.
Glacios
CL2

  1.  If transmitted by aerosol, samples are loaded into the nanocab within a microbiological safety cabinet. If not aerosol transmission, samples can be loaded in a  CL2 laboratory.   As before, users must wear appropriate PPE, which includes gloves.
After loading, nanocab must either be fumigated or heated (if validation tests show that can inactivate after x time at 37 degreesC for instance). All surfaces and tools must be wiped with the validated disinfectant (e.g 10 % chemgene) and left for the validated contact time after use.
Krios and Arctis
CL2/CL3

  1.  Ensure that grids are counted in/out pre and post loading. For CL3 work it is necessary to transport the nanocab in a special transport box such that the target is doubly contained without creating a bomb.
Validation tests for target indicate time that it takes to deactivate in a dry environment at room temperature. After experiments have completed, the system can be cryocycled and left at room temperature for the indicated time. In emergency cases, and/or where inactivation cannot be achieved after a sensible time at room temperature, microscopes have heating elements within the housing such that there is an option to heat cycle the entire instrument to 60 °C.
Ancillary (eg clipping/loading station)
CL2/CL3

  1.  As with everything, risk assess on a per target basis and depending on the target, it is possible to do outside of the cabinet e.g. no aerosols. Use of sharp tweezers should be risk assessed.
Both can be placed in an oven.
Laboratory preparation
CL2/CL3

  1.  When CL3 experiments are desired, the suite access is changed to only CL3 trained users and PPE appropriate for CL3 is adopted. All laboratories in which CL3 work is to take place are cleared and deep cleaned at the end of work to comply with CL3 laboratory practices.  A cold start where everything is treated as CL3 is scheduled before the actual experiments take place. The laboratory building is maintained to comply with CL3 legislation e.g. operates under negative pressure.

#2 A facility in the USA
Comments
Risk Assessment & Validation

  1.  The risk studying human derived cell lines is generally low and setting up freezing equipment in a BSL2 space may be fine. Pathogens however are a different story.
  2.  When working with pathogenic viruses, always perform work inside a biosafety cabinet (BSC) due to aerosol generation during freezing.
Disinfection & Decontamination

  1.  Proper decontamination methods include autoclaving, bleach, aerosolized hydrogen peroxide, or UV exposure according to the US EPA.
  2.  Ethanol is not approved by EPA for disinfecting pathogens and it was used only with follow-up bleach washes.
  3.  Post-imaging, store grids in liquid nitrogen or disinfect (10% bleach solution) immediately to prevent contamination.
  4.  The vacuum system in TEM would be pretty harsh on any pathogen. UV light on exhausted materials from the backing pump or TFS heating element to 60°C could assuage such concerns.
Laboratory Design & Safety

  1.  BSL2 samples would be segregated in separate dewars and kept in secure areas
Others

  1.  Safety measures should be able to protect everyone in all health status, including immunocompromised person.
  2.  Electron beam imaging is likely to destroy pathogen due to radiation damage.

Equipment
Level
Setup
High Pressure Freezer
BSL2

  1.  Had extra security and the lab was under negative pressure
  2.  Heard other facilities may have some extra ventilation hoods being put over HPF devices
Krios
BSL2

  1.  Not under negative pressure as anything that entered the Krios room was cryofixed
Krios
BSL3

  1.  Any sample that came out of the BSL3 either had to be UV exposed prior to cryo-fixation or chemically inactivated.
  2.  All inactivation protocols had to show that the pathogen was rendered non-infectious through either plaque assay or some PCR based assay.

#3-8
#
From
Comments
3
Switzerland

  1.  During sample preparation, all the tools and devices are BSL2 and reserved only for that.
  2.  After fixation or frozen, it is considered just any other sample.
4
USA

  1.  All equipment can be operated under normal conditions - i.e. in the open on a lab bench, with special considerations for the evacuation of N2 gas or Ethane.
  2.  Issues regarding BSL-2 have only arisen during disposal of equipment.
5
France

  1.  Insufficient liquid nitrogen or a vacuum pump failure in TEMs should not be an issue as the temperature and pressure are far bellow triple point.
  2.  Electron beam that will heat up the sample will destroy the sample.
  3.  With BSL-2 ventilation setting and personal protection equipment, the risk for service engineer in TEM rooms is low.
6
USA

  1.  The risks regarding pathogens at the BSL-2 level is rather minimal. Having a cryo-TEM in a BSL-3 environment is a completely different story.
7
N/A

  1.  BSL2 grids discard into a tube of bleach, and eventually the tube is discarded via the regular BSL2 biohazard waste stream.
  2.  Wipe down inside the GP2 with 70% ethanol, as well as the freezing/clipping tools, and use a slide warmer at a fairly high temperature just inside the range for heat inactivation for good measure (>45˚C).
  3.  An Aclar "anti-contamination" ring can be placed on Vitrobot foam pads to prevent media soaking into the pads.
8
A lab in Switzerland

  1.  All freezing instruments, equipment and storage are in a dedicated BSL-2 lab. Freezing instruments are not in BSC but pipet under the cabinet, bring it to the grid in the chamber. Use mask and gloves to reduce the risks.
  2.  Work under the assumption that the risk of potential aerosol creation is negligeable as long as you are not milling and as long as your microscope remains cold. Extra cautious when putting BSL-2 grids and avoid having them in the microscope over the weekend without close supervision.

Thank you again to everyone who contributed. If you have further comments or experiences to share, please feel free to continue the discussion.

Regards,
Jason

From: Jason NG (CPOS)
Sent: 16 June 2025 15:53
To: 3dem at ncmir.ucsd.edu
Subject: RE: [3dem] Seeking Advice on BSL-2 CryoEM Laboratory Setup and Safety Protocols

Dear all,

Thank you very much for your replies to my email and to those who messaged me privately regarding the BSL-2 lab setup. I sincerely appreciate the detailed information and experiences you have shared-they are extremely helpful and valuable.

I am currently summarizing all your comments and suggestions to present to my senior. Please feel free to continue the discussion and share any further opinions or advice you may have.

Regards,
Jason

From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> On Behalf Of Jason NG (CPOS) via 3dem
Sent: 12 June 2025 17:00
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: [3dem] Seeking Advice on BSL-2 CryoEM Laboratory Setup and Safety Protocols

Dear CryoEM Community,

We are currently evaluating the risks of becoming BSL-2 CryoEM laboratory and would greatly appreciate your guidance on several safety and setup considerations.

Specifically, we are interested in understanding:

  *   Given that the electron beam can potentially generate aerosols or cause warming within TEMs, what disinfection protocols or measures have you found effective for maintaining biosafety?
  *   Is implementing negative room pressure necessary in a CryoEM lab at BSL-2?
  *   For sample preparation instruments such as the TFS Vitrobot and Leica GP2, is it recommended to place them inside a biosafety cabinet? How about large instruments like the Leica ICE-are there specific safety measures we should consider?

Your insights and experiences would be extremely helpful as we evaluate the best practices for creating a safe and effective CryoEM environment. Thank you very much for your time and guidance.

Best Regards,
Jason NG
Senior Technical Assistant
LKS Cryo-EM Laboratory

[cid:image001.jpg at 01DBE1C4.F71045C0]

Centre for PanorOmic Sciences
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