Biosafety Unit, CRISP, FHML, MUMC+
Working instruction for activities in a ML-I lab
I Introduction
This work instruction is intended as basic document for ML-I labs en needs to be
supplemented to the individual department situation. List in addition the possible rules
from the license (I&M). Besides this we refer to the Safe Working with GMO procedure.
All employees working in a ML-I lab need to be acquainted with this procedure and also
with the contents of the license to which they participate. Next to this work instruction a
procedure has been drawn up in which the organizational aspects regarding the GMOactivities have been written.
II Admittance
The admission is limited to those that are aware of the risks of the activities for man and
environment. Activities can start only after the instruction by the responsible Investigator
(RI) or the Departmental Biosafety Expert (DBE).
Cleaning personnel only has admittance for activities as laid down in the Cleaning
procedure for GMO-spaces.
Maintenance personnel only have admittance after consulting the DBE or the person
responsible for the laboratory room.
Independent activities with GMO’s can only be done by project employees, who have
been signed up and admitted by the BSO (see procedure for License Application).
Remaining personnel, like students, intern’s en guest employees only have admittance as
long as their supervisor is present.
III Clothing
While working in the ML-I lab a closed long-sleeved lab coat must be worn (white coat
with green collar or green line on collar or breast pocket).
After the activities the lab coat and gloves remain in the ML-I space.
In case of contamination of the lab coat, f.i. by spillage or accidents with GMO’s, the
coat will need to be autoclaved immediately.
All coats must be offered to CGSA for autoclaving in an autoclavable plastic bag.
If ones skin is not in optimal condition or when the experiment does so require,
disposable gloves should be worn.
Personal possessions, like coat, sweater, or bag must be put away outside the workspace.
Nov. 2011
Biosafety Unit, CRISP, FHML, MUMC+
IV Work techniques
1. General
Doors and windows must be kept closed during the activities.
Besides administration of the experimental data, no other administrative work is allowed.
Workspace must be kept clean and tidy.
No excessive furniture or instruments are allowed.
Vermin is not allowed.
Smoking, eating, drinking, saving of food, applying cosmetics or putting in contact lenses
is not allowed: long loose hair should be put up.
Avoid hand- face contact.
Pipetting with the mouth is not allowed, use a balloon or pipetman
To prevent contamination of mechanical parts of the pipette, filter tips can be used.
Use only paper tissue, if needed.
Before leaving the workspace ones hands need to be washed with water and soap.
2. Prevention of aerosol formation
Prevent aerosol formation by waiting several minutes with the opening of tubes until the aerosols
have been settled down, leaving the graft needles to dry before flaming, and draining the pipette
against the lining of a tube instead of using force by pressing the pipette.
3. Use of Biosafety cabinet
See Procedure voor het werken in een veiligheidskabinet typeII
4. Syringes and needles
To minimize prick accidents, aerosol forming or spillage, one must avoid using the
syringe as much as possible.
Use disposable syringes and needles only;
Fill the syringe carefully and prevent forming of air bubbles: keep the point of the needle
in the fluid, when squeezing the syringe to drain the fluid.
Dispose the needle in the dedicated container immediately.
5. Decontamination and disinfection
Sufficient disinfectants must be available before starting the experiment. See Instructie
voor desinfectie in laboratoria en proefdierruimten.
The work surfaces (table or safety cabinet) must disinfected at the end of the activities or
when not possible certainly at the end of each working day.
In case of spillage the spillage needs to be cleaned by covering with a tissue drenched in
70% ethanol, 0.1% active chloride or another suitable disinfectant, leave this to soak for 5
minutes and then clean up, tissues with spillage must be disposed of in a green WIVA
barrel. Then clean the surface with water and disinfect again.
6. Biological contaminated waste and contaminated material
With the exception of hand wash water no contaminated water from the lab is allowed to
be disposed of through the sewer, unless it has been disinfected.
Nov. 2011
Biosafety Unit, CRISP, FHML, MUMC+
Liquid waste can be autoclaved or decontaminated with 0.1% active chloride in water (f.i.
with one tablet of stafilex per liter, for 30 minutes).
GGO- and biological waste is collected in the green WIVA barrels. When the barrel is
full it is locked with a yellow lid. The outside of the barrel will be disinfected with an
appropriate disinfectant and a sticker will be put on the lid stating the GGO number. Full
barrels must not be put in the hallway (see Instructie voor afvoer van mogelijk
microbiologisch besmet afval).
Dirty glassware, which has been in contact with GGO’s will be disinfected with 0.1%
active chloride solution then rinsed and washed.
Glass ware polluted with GGO’s or microorganism is offered to CGSA in a shatterproof
and leak proof container. CGSA will then autoclave it straight away.
7. Centrifugation
Check whether the tubes are intact and close them carefully.
Use closed centrifuges (preferably with screw cap) and/or closed buckets.
Fill the centrifuge tubes till maximum 4/5 of its volume. If angle rotors are used the
centrifuge tube can only be filled till 2/3 of its volume. The fluid in the centrifuge tube
can not come into contact with the tubes lid.
Wait for 30 minutes after centrifugation before opening the buckets/tubes to let the
aerosols be knocked down. An alternative would be to open the buckets/tubes in a safety
cabinet.
Rotors contaminated as a result of leakage or breakage need to be disinfected straight
after centrifugation (see Instructie voor desinfctie in laboratoria en proefdierruimten).
8. Use of fridges, freezers and nitrogen tank
If GGO material is stored outside the ML-I lab, the location needs to be mentioned in the
logbook. Provide all the material which is kept outside the ML-I lab with a label so that
it’s recognizable as GGO material. Register this storage always.
9. Water baths
Refresh the water regularly and add disinfectants (f.i. Prothemal or Aquaclean). Don’t
use Natriumazide this because of explosive properties.
10. CO2 stifling
To prevent fungus growth in the water pan a copper water pan can be used or use a piece
of copper (5 eurocents). Refresh the water of the water pan in the CO2-stifle regularly
and add disinfectants (f.i. Prothemal or Aquaclean).
11. Ultrasonic- and mixing equipment, magnetic stirrers and such
This equipment can only be used inside a safety cabinet, unless it’s a closed barrel. In that
case the barrels need to be opened in a safety cabinet. Wait several minutes before
opening the barrels so the aerosols will have been knocked down
12. Shipping of GGO materials
For shipping GGO material see procedure vervoer van biologisch material.
Nov. 2011
Biosafety Unit, CRISP, FHML, MUMC+
13. Transport of GGO materials inside the building
To transport GGO materials to another location (also inside the building) a closed, break
proof and leak proof container need to be used at all times.
Nov. 2011