Biosafety Unit, FHML,MUMC+
Procedure 5: Safe use of a microbiological safety Cabinet type II
1. Subject matter and scope:
A Microbiological Safety Cabinet type II (BSC-II) is a Safety Cabinet, which in good use
protects both the employee as well as the used material. In the Cabinet is a downwardoriented sterile air flow (Laminar Air Flow), created. At the front and back of the
workbench is the air sucked off. By opening the Cabinet, additional air is sucked in at the
front side. This will create an air curtain. The recirculating air passes through a HEPA
filter. Also the air that is blown out at the top, passes through a HEPA filter. A HEPA filter
extracts more than 99.97% of particles with a diameter smaller and larger than 0.3
microns from the air.
A BSC-II is used when working with microorganisms of risk class 2 and 3 or gmo’s of
containment level 2 or higher, with risk of infection through inhalation of aerosols. In
addition, the cabinet will be used for sterile work , for example cell cultures or when
working with non-volatile carcinogenic or reprotoxic substances.
To protect both the researcher as well as the experiment as much as possible, the BSC
must be used in a correct way and must be properly cleaned and maintained. Within the
University and hospital maintenance is conducted annually. This can be seen at a label,
on which the date of control is listed, on the front of the Cabinet.
2. Abbreviations and definitions
Aërosol
microscopic small liquid droplets that spread through the air
BSO
Biosafety Officer
GMO
Genetic Modified Organism
HEPA filter:
High Efficiency Particulate Air filter is a small dust filter that retrieve
submicronparticles according to the specifications.
ML-I/ML-II
Microbiology Laboratory Class I/II
BSC
Biosafety Cabinet
Biosafety Unit, FHML,MUMC+
Product protection
Protection of cells etc. against infection or contamination from
outside the Biosafety Cabinet
3. Where to place a BSC
For correct functioning of a BSC, it requires as little as possible disturbance of the airflow.
This can be promoted by the placement of the Cabinet already. This should comply with the
following conditions:
-as far as possible from the grids for the air supply into the room
-minimum 1.5 m to the walkway for the Cabinet and a door at right angles to the front
-at least 1 m from a door in a wall parallel to the front
-at least 3 m from a opposite lying Cabinet.
Keep at least 30 cm around and above the cabinet free to access in case of maintenance.
After the placement the downflow and inflow must be checked by a certified company, e.g.
the company Partical Measurement Validation (PMV), Woerden
Biosafety Unit, FHML,MUMC+
4. Work Instruction
Safe working method:
let the Cabinet run at least 10 minutes before you start, so that the laminar air flow at all is
set and only sterile air is blown in the Cabinet
wear a tightly closed lab coat with cuffs or sleeve caps
Don’t wear rings, watches and bracelets and wear gloves if necessary
check that there is sufficient air flow (green LED)
Disinfect the workbench with a suitable disinfectant (see work instruction disinfection),
please take care of the grids at the front and the back and watch the right contact time of
the disinfectant.
Collect the materials used for the work needed in advance.
Avoid disruptions to airflow (downflow):

do not place any material on the air grid’s.

do not place any objects other than the necessary materials in the Cabinet

use preferably no Bunsen burners, because that disrupt the downflow.

limit the actions to perform to a minimum

make quiet movements
check after spilling whether the space under the worktop is polluted and clean if necessary.
Replace the filter, when this also has become contaminated (see for the procedure: under
maintenance and management)
Biosafety Unit, FHML,MUMC+
Cleaning
After finishing, the work bench and grids have to be cleaned with a tissue, which is
moistened with a suitable disinfectant for the work, which is not aggressive for the stainless
steel workbench, e.g. 70% ethanol or 1% Virkon S). Note the incubation time of the
disinfectant (min 30 sec for 70% ethanol and 10 minutes for VirkonS). Clean afterwards
with water in case of Virkon S.
Clean the outside of the Cabinet (also the top) regularly with a damp cloth.
Disturbances and incidents
At alarm signal/lamp (too low airflow):
• Stop work immediately and close open and sterile materials!
• Fix the cause of the failure
• clean workbench with 70% ethanol solution or another suitable disinfectant.
If the issue cannot be resolved: take the material out of the Cabinet.
Create worksheet, etc. that clean with a tissue dampened with a 70% ethanol solution or
other suitable disinfectant.
Close the work opening (with the corresponding attachment) and put a label on the
outside. Warn the IDEE (tel. 81295) or TD (azM) Tel. 75505, azM)
Biosafety Unit, FHML,MUMC+
Maintenance and Management
Maintenance of the Biosafety Cabinet class II is yearly validated by a certified company, e.g.
PMV to Woerden.
• Prior to the annual maintenance, the BSC must be cleaned and the workbench upper part
as well as the lower part, must be disinfected.
Replace at least once a year, for example prior to the annual maintenance the pre-filter.
This can be peformed by the IDEE or by the labmanager. Ask the IDEE (UM) or TD (azM)
proceed as follows:
• use gloves, wear a lab coat and a P2-closed mouth mask.
• Disinfect the work surface of the Cabinet with a suitable disinfectant
• lift the sheet out of the closet and clean the bottom with a tissue with a suitable
disinfectant
• take a plastic bag; take the prefilter by rolling it and drop the prefilter in the plastc bag, in
a way (inside out) so that the outside of the plastic bag cannot be infected. Binds the plastic
bag and put it in the supplied biological waste bin.
Clean the drip tray, walls and window. Place the new filter, cut it on the good length and
make sure that it fits well. Place worksheet, and evt armrest and the window in the normal
working position.
• Fill out the release form, in accordance with the procedure release ML-I/ML-II laboratories
and ask the signature of the BSO in case of a ML-II lab.
The maintenance work on a Biosafety Cabinet class II are administered in accordance with
the quality management system of the IDEE/TD. The HEPA filters are replaced by PMV. This
is determined according to air speed, temperature measurements with prolonged use,
circulation etc. and is listed in the maintenance report.
The little HEPA filter in the Exhaust can be taken out without the filter actually touching.
This is encased in plastic and features a Biohazard sign.
If the HEPA filter needs to be replaced:-PMV brings the disinfection device in the BSC.
Biosafety Unit, FHML,MUMC+
-The space must be closed by the space administrator.
On the entrance door to the workspace shows that it is not allowed to go inside.
-
Disinfection will be perfomed overnight
The next day, the Cabinet has to be put on for one hour, in order to get rid of the rest
fumes through the room ventilation.
-
Slide the windshield up, turn off the acoustic signal
Lift the inside bench of the BSC, clean the bottom and disinfect this with a suitable
disinfectant. Remove the front filter as shown above and place a new well fitting filter.
The HEPA filter is by PMV encased in plastic. This can be offered through the route of
the solid waste: please call the RNL: tel: 81164
Place workbench back and check if ereything is working correctly and leave the Cabinet
for a run of 15 minutes.
Check the replacement of the maintenance sticker (with new date)
-
IDEE/TD archives the control report and sents a copy to the Labmanager of the Cabinet
-
IDEE/TD sents an overall reporting annually to the Biosafety Officer.
-
Apply if FD (UM) or TD (azM) space supply tubes and drains and the fan, which is located
in the in the technical compartment of a biohazard sign, or the symbol for dangerous
substances
5. Responsibilities
Employee IDEE(UM) of TD(azM)
* all maintenance activities to the BSC

Check that the maintenance will meet the yearly maintenancecontract,

Archive the testreports
Labmanager, responsible for the BSC
Biosafety Unit, FHML,MUMC+

contact IDEE or TD

Change the prefilter in case of contamination

archive copy testreport
BSO

yearly check that resposibilities will be taken

keep this procedure up-to-date
6. Comments

De Biosafety Officer has a Dutch and English language Instruction video on Safe working in
a Biosafety Cabinet.

In the Movie “Exactly as it should be” is the operation of a BSC further explained and
demonstrated. See References.

At het front of each BSC, you will find a detailed description of the Safety Control of the
Cabinet and the safe working procedure.

“Bock Advies” courses “working in an BSC”, is organised often by Clean Air te Woerden.

The operation of a Biosafety Cabinet according to the European standard EN 12469 d.d.
2000 (performance requirements for microbiological safety cabinets) relates to the
following qualifications:
o the downflow should average between 0.25 and 0.50 m/s.
o In Annex H represents the opinion, that the ideal value 0.4 m/s and that none
of the 8 measurements on a number of defined places in the BSC differs more
than 20% of this value.
o the average inflow speed must be at least 0.4 m/s and up to 1.0 m/s. above 1.0
m/s will occur unwanted turbulence.
o Also recommends an annual check to the standard of the performance
requirements. The sound pressure level at 1 m from the middle of the work
Biosafety Unit, FHML,MUMC+
opening would not be more than 65 dB (A) if the background noise is less than
55 dB
Referenties

P. Herman en K. Pauwels. Microbiologische veiligheidswerkkasten. Wetenschappelijk Instituut
Volksgezondheid, afd. Bioveiligheid en Biotechnologie, Brussel, 2006. Te vinden onder de
volgende link: Biosafety Cabinets

.
MBL-TNO. Microbiologische veiligheidskabinetten.
o
Eigenschappen en werking. Videoband van 17 minuten. Rijswijk, 1990,
o
Beïnvloeding van de werking, videoband van 17 minuten, Rijswijk 1990 (beide te
lenen bij de BVF; Zowel Nederlands- als Engelstalig beschikbaar.

Workinstruction disinfection

website www.crispmaastricht.nl

Instructie DVD” Exactly as it should be”; link:
http://www.youtube.com/watch?v=xbdi95IxUw8&feature=youtu.be
Information
Biosafety Officers:
Natasja Kisters, tel: 81137
e-mail: n.kisters@maastrichtuniversity.nl
Marjanne Markerink, tel: 82986
e-mail: m.markerink@maastrichtuniversity.nl