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Determining the performance of BSL-3 laboratory HEPA air filtration systems for reducing airborne contamination
using model microorganisms
ZhanBo Wen
State key laboratory of pathogen and biosecurity
Beijing Institute of Microbiology and Epidemiology
Main contents
� Introduction
� Materials and Methods
� Results
� Discussions
� Acknowledgements
(Laboratory biosafety manual, WHO, Third edition, 2004)
Introduction
BSL-3 laboratory is designed and provided for work with risk group
3 microorganisms and with large volumes or high concentrations of
risk group 2 that pose an increased risk of aerosol spread.
���� BSL-3 and BSL-4 laboratory are designed and provided for
work with risk group 3 and group 4 microorganisms.
(Kaiser, 2007, Science, Vol317, 1852-1854)
Introduction
���� Emerging and re-emerging infectious diseases::::SARS, H5N1 avian influenza, H1N1 influenza.
���� China is a major contributor to the worldwide
infectious disease burden because of its population
size.
���� Following the 2003 SARS epidemic, the concept of
aerosol transmission was so well accepted. After that
China has been built several BSL-3 laboratories.
Introduction
���� High-efficiency particulate air (HEPA) filtration system of
BSL-3 lab was to reduce airborne contamination to avoid
microbial aerosol leak to the environment.
���� The attempts to evaluate the respiratory protection
equipments performance directly with biologic particles have
been done using airborne bacteria and virus. (Brosseau1997,
Reponen 1999, Balazy 2006, Eninger 2008, Wen 2010)
���� But there is a lack of direct measurement data on the
efficiency of BSL-3 laboratory HEPA filtration system
against aerosolized biological particles.
Introduction
Aim of this study
To ensure there was no environmental leak risk of
microbial aerosol through the BSL-3 laboratory air
filtration system actually in a working laboratory
using biological aerosol methodology .
Materials and Methods
���� Test organism
Serratia marcescens (0.7μm× 1.0μm) one of the
smallest bacteria was used as test organisms.
���� BSL-3 laboratory air filtration system
The BSL-3 laboratory HEPA filtration systems had two
HEPA filters. First HEPA filters were in BSL-3 laboratory
and distributed in each contaminated lab. The exhausted air
through the first HEPA filters were aggregated together and
filtrated through the second HEPA filter before exhausting
into the out air. So the contamination air was ventilated out
through two HEPA filter system to avoid the leak of the
microbial aerosol.
Materials and Methods
Materials and Methods���� Size of aerosol particles
A multistage Andersen sampler was used to determine the
size of S. marcescens aerosol particles . The size of particle
collected was determined from the presence of growth on
each of the plates used.
0.60~~~~1.0Sixth
1.1~~~~2.0Fifth
2.1~~~~3.3Fourth
3.4~~~~4.7Third
4.8~~~~7Second
>7First
Particles distribution
((((μμμμm))))Stage
Materials and Methods���� Aerosol challenge to test filters
Wind direction
Nebulizer
The first HEPA filter
Lab wall
Figure 2 Test rig of BSL-3 lab HEPA air filtration system filtration efficiency against bacterial aerosol
Outside labInside lab
Wind chanel
Upstream biosampler
Downstream biosampler
Materials and Methods���� Aerosol challenge to test filters
1) DV40 nebulizer with S. marcescens suspension was
aerosolized by applying compressed air to the nebulizer at
10L/min.
2) The bacterial aerosol was generated before the filter in the
BSL-3 lab. At the upstream and downstream of the first
HEPA filter and the second HEPA filter, air samplers were
used to sample the air to detect the model bacterial aerosol
concentration.
3) AGI-30 air sampler was used to collect the air before
filtration, the flow rate was 12.6 L/min, 10min.
4) Merck100 air sampler was used to collect the air after
filtration of the first HEPA and the exhaust of the second
HEPA filter , the flow rate was 100L/min, 5min.
5) The collecting samples were incubated at 30℃℃℃℃ for 24h,
counted the colonies on the agar plates and then transferred
the result to the concentration of CFU (colony forming unit )
per liter. The filtration efficiency was determined by the
aerosol concentration upstream and downstream of the tested
HEPA filters.
Materials and Methods
Materials and Methods
���� How to calculate Filtration efficiency
The percentage efficiency of the HEPA filter was
calculated using the following formula, where A was
the concentration of bioaerosol challenging the
HEPA filter and B was after filtration.
S. marcescens was determined in terms of cfu/liter.
Efficiency (%)=1----B/A××××100%
���� Particle size distribution of S. marcescen aerosol
Fi gur e 3 Par t i cl es si ze of S. mar cescens aer osol
1%2%
8%24%19%
46%
>7μ m 4. 8- 7. 0μ m 3. 4- 4. 7μ m 2. 1- 3. 3μ m 1. 1- 2. 0μ m 0. 60- 1. 0μ m
Results
Count median diameter (CMD) of S. marcescen aerosol was 1.77μμμμm
>99.9999<0.00222808#
>99.9999<0.00236007#
99.99050.331716#
>99.9999<0.00223765#
>99.9999<0.00253244#
99.98360.636513#
>99.9999<0.00252072#
>99.9999<0.00240991#
Filtration efficiency
of the first
HEPA filter
(%)
Concentration of
bacterial aerosol
after the first HEPA
filters (cfu/L)
Challenge
concentration of
bacterial aerosol
(cfu/L)
HEPA
filter
Table 2 Filtration efficiency of the first HEPA filter of BSL-3 filtration system
Note: After replaced 3# filter and airproofed 6# filter fixed bracket no
Serratia marcescens was detected in the filtration air, the filtration
efficiency were >99.9999%.
Results
���� Efficiency of second HEPA filter
S. marcescens aerosol was not detected after filtrating
through the second HEPA filter at each testing and
filtration efficiency was >99.9999%.
Results
Discussion
���� Besides safe microbiological techniques, primary barriers
(safety equipment and personal protective equipment) and
secondary barriers (facility safeguards) are now regarded as
vital elements of containment measures.
���� Air filtration system with HEPA filters is one of the most
important secondary barriers to prevent the escape of
infectious agents from the working place of the BSL-3 lab to
the environment.
���� There are no reports to evaluate the air filtration system of
BSL-3 lab with the deliberate release of bacterial aerosol
actually in a working laboratory setting.
���� Due to aerosol safety issues involved with the generation of high
bacterial aerosol concentrations, the method uses non-
pathogenic microorganisms. In this study, a bacterial model (S.
marcescens) was used to test the efficiency of air purification
system of BSL-3 lab.
���� S. marcescens has been used previously as model bacterial
aerosol because of its aerosol stability. (Furuhashi 1981, Holton
1994, Heidelberg 1997, Barker 2005, Ko 2007, Patel 2008)
Discussion
���� In our past study Merck 100 sampler collection efficiency was
higher than AGI-30 when the concentration of the aerosol was
at low level (Yang 2009). So Merck 100 air sampler was used
to collect the air after filtration of the HEPA, the flow rate was
100L/min, 5min, so the testing limit was 0.002 cfu/L(2 cfu/m3).
���� In the BSL-3 lab the Serratia marcescens aerosol concentration
was very high and mimicked the serious contaminated status
and no Serratia marcescens was detected in the exhaust air.
Discussion
���� This test method not only evaluated the HEPA filters but also
the installation process such as: airproof of the filter fixed
bracket, airproof glue, fixed screw and whether the HEPA
filter was damaged during the installation.
���� In the future research when evaluating BSL-3 lab doing viral
research we will try to use viral model aerosol.
Discussion
Acknowledgements
���� The authors thank Dr Sun Zhenhai for technical
assistance for this work.
����This work was supported by the National science and
technology support program of China (No. 2008BAI62B05)
and National importance infectious disease program of
China (No. 2009ZX10004-501).
Thank you for your attention!