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!