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Appendix 2: Laboratory Rules for Working with
Biological Material
Director’s Directive 3/2019
RECETOX Operating Regulations
Preliminary Provisions
1. The rules described in this Appendix to the RECETOX Operating Regulations apply to all
RECETOX laboratories.
2. Any changes to this Appendix are subject to approval by the Brno Regional Hygiene Station.
General Information
Description and Focus of the Laboratories
The RECETOX laboratories analyse environmental and biological samples and study the
pollutants in the environment and in biological matrices.
Location and layout of the laboratories
Location: The laboratories are located within the University Campus in BrnoBohunice
in building A29, street address Kamenice 753/5, and in
building INBIT, street address Kamenice 771/34.
Layout: In building A29 (commissioned in 2013), the laboratories are located across four
floors. The environmental processes and toxicology laboratories are located on the 1st basement
floor and 1st (ground-level) floor. The Trace Analysis Laboratories are located on the 2nd floor.
These laboratories are separated by a glass partition and are subject to restricted access rules. The
ecotoxicology laboratories are located on the 3rd floor. The building provides appropriate sanitary
facilities.
In building INBIT (commissioned in 2008), the laboratories are
located on the 1st basement floor and on the 3rd floor. The
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laboratory for processing biological material and the isolation
laboratory are located on the 1st basement floor. The metabolomics
laboratory is located on the 3rd floor along with the MELISA
laboratory, two microbiome laboratories (pre- and post-PCR) and
a room with deep freezers for storing samples.
The laboratory working hours are from 6 a.m. to 10 p.m. on working days.
Description of Biological Material Samples
Biological material is material of environmental and biological origin that comes from humans,
other organisms or parts of the environment (the “biological material”). Since biological material
could contain pathogens, it is a potentially infectious material and must be treated as such. The
laboratories primarily analyse the following biological material:
• Human and animal samples (fat, muscle, brain, skin and liver tissue; blood and blood
derivatives; hair; buccal swabs etc.);
• Human and animal secretions and excretions (breast milk, urine, stool, sperm etc.);
• Environmental samples (biological waste, wastewater, samples taken from polluted
watercourses, soils and sediments etc.).
Monitored and Analysed Groups of Substances
Persistent Organic Substances
Organochlorine pesticides, cyclodiene pesticides, polychlorinated biphenyls, polychlorinated
biphenyls with dioxin-like toxic effects, polychlorinated dibenzo-p-dioxins and dibenzofurans,
polybrominated diphenyl ethers, new brominated flame retardants, hexabromocyclododecanes,
organophosphorus flame retardants, polyaromatic hydrocarbons etc.
Polar Organic Substances
Polar pesticides, perfluorinated substances, pharmaceuticals, personal care products, sweeteners,
alkylphenols, steroids etc.
Metals
Metals. The laboratories also conduct speciation analyses.
Biomarkers and Biological Macromolecules
Nucleic acids (DNA and RNA), proteins, metabolites, low molecular weight biomarkers etc.
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Division of Laboratories Working with Biological Material According
to the Tasks Performed
Sample Receipt and Inventory Records
Building A29
• Chemical laboratories – laboratory no. 226 and 1S09
• Toxicology laboratories – laboratory no. 325
Building INBIT
• Samples for microbiome analysis – laboratory no. 0.21
• Samples for metabolome analysis – laboratory no. 3.24
• Samples for further processing and storage in the biobank – laboratory no. 0.22
Sample Storage
Building A29
• Temperature controlled freezers – room no. 1S29 and 213
• Cryogenic storage dewars (-196°C) – room no. 1S29
Building INBIT
• Freezers (-20°C) – laboratory no. 0.21, 0.22 and 3.24
• Freezers (-80°C) – laboratory no. 3.21, 3.24
Building A1
• Freezers (-20 °C) – laboratory no. S217a
• Freezers (-80 °C) – laboratory no. S217a
Sample Preparation for Analysis
Building A29
• Drying, freeze-drying, homogenisation, fat determination etc. – laboratory no. 226
• Extraction, gel chromatography, column adsorption chromatography – laboratory no. 216,
222, 224, 225, 228 and 1S09
• Ecotoxicological testing – laboratory no. 325
• Student laboratories – laboratory no. 114, 115, 118
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Building INBIT
• Isolation of biological macromolecules (DNA, RNA) – laboratory no. 0.21 and 0.22
• Separation of blood fractions and elements – laboratory no. 0.22 and 3.25
• Processing, fractionation and aliquoting of biological material (venous and cord blood,
urine, buccal swabs, stool, saliva, dried blood spot) – laboratory no. 0.22
• Quantification of proteins – inflammation markers, gangliosides and metabolites
(homogenisation, extraction, trypsinisation, alkylation, vacuum drying, weighing etc.) –
laboratory no. 3.24
• Quantification of proteins – inflammation markers, gangliosides and metabolites (protein
reduction, spectrophotometric quantification of proteins etc.) – laboratory no. 0.21
Sample Analysis
Building A29
• Instrumental methods of chemical analysis – GC-MS, GC-MS/MS, GC-HRMS, HPLCMS,
HPLC-MS/MS, ICP-MS, (GC/HPLC)-ICP-MS, AAS, UHPLC-MS – laboratory no.
1S05, 1S18, 1S21, 1S31, 214, 229, 231, 232, 233 and 325
• Other types of analysis (biomarkers) – laboratory no. 315, 316, 321, 323, 324 and 325
Building INBIT
• Analysis of biological macromolecules (DNA, RNA) – laboratory no. 0.21, 3.22, and 3.23
• Biochemical and immunochemical quantification of biomarkers in full blood, plasma,
serum and urine – laboratory no. 0.21
• MELISA – detecting hypersensitivity to heavy metals and other allergens (chemicals,
toxins, moulds etc.) – memory lymphocyte immunostimulation – laboratory no. 3.25
Process Experiments
Building A29
• Distribution balance – laboratory no. 1S09 and 1S11
Statutory Requirements
According to Government Regulation No. 361/2007 Coll., the minimum measures for ensuring
occupational health and safety, detailed hygienic requirements and signage at the workplace,
detailed requirements for work practices and health and safety information concerning tasks that
could expose employees to biological agents and endanger their health must include the following:
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• No eating, drinking and smoking in a workplace where there is a risk of contamination by
a biological agent. Staff must not leave the designated workplace while wearing personal
protective equipment.
• The sanitary facilities provided must correspond to the nature of the work.
• Personal protective equipment must be provided.
• Personal protective equipment must be stored in a designated area, checked, cleaned and
disinfected, preferably before each use but always after each use. Defective personal
protective equipment must be repaired or substituted before it is used again.
• An authorised employee must prepare guidelines for safe collection, handling and
processing of human or animal samples, which are then approved by their superior.
• Personal protective equipment that may be contaminated with a biological agent must be
removed. Prior to decontamination, cleaning or disposal, personal protective equipment
must be stored separated from street clothes.
• Vaccination, when advisable, particularly for employees who are not immune to the
biological agent that they are, or could be, exposed to at work.
• Employees must be informed of any incidents involving the handling of biological agents.
If the processing and/or analysis of biological material listed above in the “Description of
Biological Material Samples” section of this Appendix to the Operating Regulations could involve
any risk of the exposure of employees to biological agents classified as risk group 2, 3 or 4, the
measures taken to protect the health and safety of employees must also include the following:
• Minimising the number of employees exposed or probably exposed.
• Adapting the work practices and technical protective measures designed to prevent or
minimise the release of the biological agent into the environment at the workplace.
• Using personal protective equipment, if there is no other way of preventing the exposure
of the employee to the biological agent.
• Maintaining hygiene practices designed to prevent or minimise accidental transmission or
the release of the biological agent outside the workplace.
• Conducting tests to detect any presence of the biological agent used at work outside the
closed system, if necessary and practicable.
• Providing resources for easy collection, storage and disposal of waste in a safe and
identifiable bin, which can also be specially adapted for the purpose.
• Adjustments required for the safe handling and transport of the biological agent at the
workplace.
General Measures
• Access to laboratories processing biological material is only permitted for authorised and
duly trained persons.
• Performing tasks that involve biological material is only permitted for duly trained persons
authorised to work with such material.
• Regular annual re-training and training of new employees are provided by an employee
authorised by a RECETOX manager, who is qualified for working with potentially
infectious material.
• Only persons vaccinated against hepatitis B can be authorised to work with biological
material. New employees/students must submit a vaccination certificate (of at least two
doses of the vaccine), a recent hepatitis B antibody screening test (HBsAg antibody titre
over 10 IU/l) or a medical certificate for proven previous infection with hepatitis B.
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Employees/students not vaccinated against hepatitis B will not be allowed to work with
biological material until they complete the vaccination (after the second dose of the vaccine
at the earliest).
When working with biological material, the persons authorised to work with biological material
must follow the guidelines below to prevent contamination and the emergence and spread of
infections:
• No eating, drinking and smoking in the laboratories.
• Sterile supplies and instruments must be handled aseptically.
• The instruments and the environment must be decontaminated in accordance with the
Laboratory Operating Procedures.
• The recommended personal protective equipment according to the nature of the task must
be worn at work: trousers, shirt, laboratory coat, shoes, single-use gloves, face masks.
• Staff must not leave the designated workplace while wearing personal protective
equipment.
• Samples of biological material can only be received for analysis in standardised containers
and decontaminated crates.
• Samples of biological material can only be received if accompanied by a duly completed
protocol or dispatch note that includes, but is not limited to, the following information:
description of the material (type of the sample), amount, origin, place and date of
collection, mode of transport and storage, method of disposal and potential infection risks
and other hazards.
• Samples of biological material must be moved between different laboratories so that they
do not degrade due to physical influences and so that there is no threat to people.
• All waste (including gloves) produced in the laboratories while working with biological
material is considered infectious and is placed in suitable labelled containers. The waste is
separated according to the type: general infectious solid waste, infectious liquid waste,
infectious sharp waste – glass and infectious sharp waste – metal (scalpels and needles).
• Single-use gloves must be used when handling biological material. After removing the
gloves, staff must wash their hands with soap, dry them thoroughly with paper towels and
disinfect them using the recommended no-rinse hand sanitiser.
• After finishing work in the laboratory, all the work surfaces (on benches and boxes) must
be wiped with a disinfectant (ethanol-petrol blend, ethanol, commercial disinfection etc.).
• Only supplies that are designated for working with biological material may be used when
preparing samples. Work surfaces must be designated according to the nature of the task.
• If necessary, the benches that would otherwise come in contact with biological material
must be covered only with single-use supplies (aluminium foil and paper towels) and the
cover changed after each series of experiments.
• Centrifugation: When centrifuge rotors or individual rotor adapters are fitted with a closing
lid, the lid must always be in place during centrifugation. If there is no lid, the centrifuges
must be placed in a box/fume hood or in a separate room. The tubes must always be
closed properly before being placed in the centrifuge. If a tube is damaged in the
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centrifuge, this can produce a large amount of aerosol. Sudden stopping of the centrifuge
can also produce aerosols. In these situations, the necessary steps must be taken to prevent
exposure of employees to the aerosol by using suitable personal protective equipment to
protect the respiratory system. This personal protective equipment must also be used while
the centrifuge and surrounding area are decontaminated.
• Only single-use plastic supplies (pipettes, tips, Pasteur pipettes, tubes etc.) may be used
when working with biological material. These supplies are placed in their original
packaging after use and then in strong waste bags or suitable plastic containers. After
finishing the task, the bag or container is securely closed. Full bags and containers are
placed in the appropriate waste bins.
• Glassware and laboratory instruments used for processing biological samples must be
decontaminated immediately after use, then sterilised and stored separately from other
laboratory glassware and instruments.
• Single-use instruments must never be used repeatedly, even if sterilised.
• It is recommended to remove any jewellery from your hands.
• In the case of infectious disease, the necessary steps must be taken to prevent an epidemic.
If required by legislation, the presence of an infectious disease must be reported to the
public health protection authority (Regional Hygiene Station of the South Moravian
Region in Brno).
• In the case of an incident in the laboratory (serious injury, skin or mucous membrane
contamination etc.), the employees follow the instructions outlined in the OHS fact sheet,
which is available in each laboratory.
Hand Hygiene Guidelines When Working With Biological Material
Hands are washed with soap and hot water. No-rinse hand sanitisers are used for hand
disinfection.
Hand Washing
When:
• Before and after any contact with biological material, including accidental contact.
• After contact with surfaces and objects (that are in direct contact with the biological
material).
• After removing gloves of any kind.
Steps:
1. Wet hands with water.
2. Apply soap and rub hands to spread the soap for at least fifteen seconds.
3. Thoroughly wash the palms and backs of the hands up to the wrist including the areas
between the fingers.
4. Rinse hands thoroughly with water.
5. Use your elbow to turn off the water faucet.
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6. Dry hands with a paper towel.
Total time: 40–60 seconds
Hand Disinfection
While working with biological material, the hands are disinfected whenever necessary. The hands
are always disinfected after completing a task that involved handling potentially infectious
material and removing your gloves. Hand sanitisers are always applied to dry hands.
Steps:
1. Apply a sufficient amount of the sanitiser (according to the manufacturer’s instructions)
in a cupped hand.
2. Keep the hands wet for the whole duration of the sanitising process.
3. Repeat each movement five times in no fixed order:
o Rub the hands palm to palm.
o Place the right palm over the left dorsum with intertwined fingers and rub.
Repeat for the other hand.
o Rub the hands palm to palm with the fingers intertwined.
o Rub the backs of the clasped fingers against the opposing palm with the fingers
intertwined.
o Rotationally rub the left thumb clasped in the right palm and vice versa.
o Rotationally rub backwards and forwards with the clasped fingers of the right
hand in the left palm and vice versa.
4. Wait until the sanitiser dries.
Do not rinse or wipe hands when the sanitiser has dried.
Total time: 20-30 seconds
Guide to Using Gloves
• Gloves are only donned once the hand sanitiser has completely dried.
• One pair of gloves may only be used for handling one series of samples.
• Single-use gloves must be removed immediately after completing the task they were used
for.
• Used gloves are disposed of as infectious waste.
• Damaged gloves must not be used.
• As gloves do not completely protect users against hand contamination, the hands must be
washed and disinfected after removing the gloves.
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Sterilising Instruments and Tools
Instruments and tools for repeated use are sterilised to remove any microorganisms so that they
can be used again with no risk of contaminating the samples. Sterilisation also protects the health
and safety of employees who handle biological material.
Pre-sterilisation Steps
All instruments and tools that are used are considered to be contaminated. If they are designed
for repeated use, they must be decontaminated immediately after use.
Decontamination guide:
1. Disinfect the tools with a suitable agent and remove any impurities.
2. Rinse the tools thoroughly with water.
3. Place the tools in a washer-disinfector for chemical decontamination. If required, they
may be also pre-decontaminated using a decontamination agent.
4. Dry the tools and inspect them to make sure they are fully functional and in good
condition.
5. Place the tools in suitable packaging for sterilisation.
Hot Air Sterilisation and Decontamination
Glassware, porcelain and earthenware are sterilised by a flow of hot air. Stainless steel tools are
sterilised in a designated hot air sterilisation oven in building A29.
The hot air sterilisation requirements are listed below:
Temperature Time Packaging
material
Sterilised material expiration
Glass, porcelain
400°C
8 hours No packaging No time limit
Stainless steel tools
200°C
120 min
Single-use
bags
No time limit
After finishing the sterilisation cycle, both the high-temperature hot air ovens and the lowtemperature
sterilisation ovens may only be opened once they have cooled down to 150°C.
Autoclave Sterilisation
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Sterilisation by wet heat and increased pressure is used to treat heat-stable solid and liquid
materials. The sterilisation programme is selected according to the sterilised materials (glass,
plastic, metal, liquids, objects with or without packaging). Successful sterilisation is indicated by a
change in colour of the autoclave tape, which must be applied to the items before they are placed
in the autoclave.
Autoclave sterilisation takes place in designated autoclaves and may only be performed by duly
trained persons.
Sterilisation Packaging
Stainless steel tools sterilised in the sterilisation oven are placed in sterilisation bags with forced
air circulation even after the bags are heat-sealed. Sterilisation bags, containers and aluminium foil
are used for autoclave sterilisation. Liquids are sterilised in suitable closable containers.
Sterilisation and Decontamination Records
Every sterilisation must be recorded in the sterilisation log. The sterilisation or decontamination
must be recorded by the employee who performed it.
A successfully performed sterilisation is documented by:
a) A record in the sterilisation log (type of material sterilised, parameters, date, name and
signature of the person who performed the sterilisation);
b) Written dated results of the sterilisation test for every batch.
Performance Check of the Sterilisation and Decontamination Device
The performance of the sterilisation and decontamination device is checked by its manager.
Check schedule:
1. New devices and devices that have been repaired must be checked before
commissioning.
2. Each device must be checked immediately if there is any doubt about the effectiveness
of the sterilisation and decontamination process.
3. Sterilisers older than 10 years must be checked after every 100 sterilisation or
decontamination cycles or once every six months, whichever comes first.
4. Sterilisers no older than 10 years from the date of production must be checked after
every 200 sterilisation cycles or once a year, whichever comes first.
5. Chemical sterilisation tests are used to prove that all the requirements of the
sterilisation and decontamination cycle have been met.
Disinfection
All laboratory instruments for repeated use that come into contact with potentially infectious
material must be disinfected and decontaminated by the laboratory staff immediately after use.
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Glassware and ceramics are first decontaminated chemically using a decontamination agent and
then placed in a decontamination washer and a high-temperature decontamination oven or
autoclave.
Recommended biocidal agents or disinfectants declared as medical devices or agents registered as
medicinal products for use in healthcare are used for chemical disinfection.
• Disinfection solutions are prepared according to the manufacturer’s instructions by
carefully measuring the required amount of water and disinfectant immediately before use.
Multi-day disinfectants may only be used for two-stage disinfection according to the
manufacturer’s instructions. Ready-made disinfectants may also be used.
• There is a regular rotation of disinfectants with different active components in the
disinfection regime (once a month) to prevent natural selection and antimicrobial
resistance and reduce allergic reactions in the staff. The Technical Laboratory Manager
keeps records of the disinfectants used.
• The disinfectants used must not contain the organic substances that are analysed
in the RECETOX laboratories.
• After using all the disinfectant in a dispenser, the dispenser is washed manually, refilled
and labelled with the name of the disinfectant, the date of refilling and the expiration date.
Alternatively, the empty package is disposed of as hazardous waste.
• Disinfecting solutions are stored in the designated containers correctly labelled with the
disinfectant name, concentration, preparation date and expiration date. Open containers
with disinfecting solutions must be covered.
• When handling disinfectants, staff must use suitable protective equipment and comply
with the manufacturer’s instructions for handling and storage.
• Areas and surfaces contaminated with a potentially infectious material are covered with
cellulose wadding soaked in disinfectant, which must be left on the surface for the
recommended exposure period of time (indicated on the disinfectant packaging).
Afterwards, the area is cleaned as usual.
Laundry Handling Guidelines
The laundry handling guidelines are subject to Decree no. 306/2012 Coll. The staff handling used
laundry must use personal protective equipment. After completing the task, the hands must be
disinfected. Used laundry is placed in packaging (either washable and disinfectable or single-use)
to prevent contaminating the surrounding area. Each RECETOX organisational unit with
laboratories that process biological material must designate a room for used laundry storage. The
room must allow ventilation and the floor and walls up to 150 cm must be washable and
disinfectable. The laundry is washed at the University Hospital Brno based on a contract with the
hospital. Laundry is collected according to the schedule available on the INTRANET. On the
collection day, the laundry must be brought down to the garage on the 2nd basement floor by 8
a.m. The cleaned laundry is returned on the following collection day. The Technical Laboratory
Managers are responsible for compliance with the laundry handling guidelines in their laboratory
section.
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Waste
Waste Handling Guidelines
The waste handling guidelines are subject to Act no. 185/2001 Coll. regulating waste and the
alterations to certain related acts, as amended. The provisions of Decree no. 306/2012 Coll.
regulating the conditions to prevent the emergence and spread of infectious diseases and the
hygiene requirements for healthcare facilities and social care institutions are applied accordingly.
There is a centralised waste collection system at the laboratories.
Waste is separated at the site of production and dangerous waste is placed in labelled, separated,
covered, closable, impermeable and mechanically resilient containers.
All waste produced while working with human biological materials is considered infectious.
Waste categories
• Halogenated waste;
• Non-halogenated waste;
• Waste containing heavy metals;
• Glass waste;
• Other sharp waste (scalpels, injection needles etc.);
• Infectious waste (biological material, waste contaminated with biological material);
• Other waste.
The persons authorised to work with biological material follow the principles below when working
with waste:
• Single-use instruments (serological pipettes, plastic pipettes etc.) are always returned to
their original packaging after use and separated according to the resulting type of waste.
• Liquid infectious waste (left after processing blood samples etc. in suction bottles) is
disposed of by decontaminating with a suitable agent (Savo, Chloramine) and placed in
labelled impermeable rigid containers for disposal. Alternatively, it may be collected in
collection bags suitable for autoclaving and placed in suction bottles, then decontaminated
in autoclaves and disposed of as general liquid waste.
• Analysed samples of biological material are decontaminated by autoclaving and then
disposed of as general waste. All waste produced while working with human biological
materials is considered potentially infectious.
• Separated waste is placed into labelled containers and removed from the workplace daily
to designated areas (building A29). Further waste handling, including the handover to the
authorised waste-management contractor, is arranged by the waste department of the
Management of the University Campus Bohunice.
• Infectious waste produced in building INBIT is removed by the authorised laboratory staff
at least two times a week and placed in the waste storage room (1S44 – 1st basement floor
corridor; the key is available from laboratory 0.21 in building INBIT).
• All hazardous waste is visibly labelled as such on the container. The label must include the
statutory waste type number, date and place of origin.
• Glass waste and other sharp waste (180101 N*), such as used needles and complete
syringes without caps, glass etc., must be placed in labelled, burnable, rigid, non-pierceable
and impermeable containers.
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• Solid burnable waste (180103 N*) is placed in labelled, separated, impermeable and
mechanically resilient containers that can be closed (such as a plastic bin with a lid and a
polythene bag inside). Liquid infectious waste is collected in single-use white plastic barrels
or in single-use suction bags with bacterial and hydrophobic filters (Medela).
• Prior to final removal, infectious waste may be stored in a designated enclosed space for
no longer than 3 days in the winter and 2 days in the summer.
• Infectious waste may be stored in a freezer or cooler area at 8°C or less for no longer than
1 month.
• All other waste is disposed of in accordance with the Operating Regulations of the
University Campus Bohunice – Appendix no. 6 (Provozní řád odpadového hospodářství UKB)
https://is.muni.cz/do/sukb/spolecne/provozni_rad_odpadoveho_hospodarstvi_ukb/P
rovozni_rad_odpadoveho_hospodarstvi_UKB_2018-02-26.pdf.
Cleaning
Laboratories are cleaned centrally according to the cleaning standards (see INTRANET).
• Laboratory staff clean and disinfect laboratory benches and fume hoods as required and
always after finishing work.
• Cleaning tools are always disinfected after use.
• Disinsection and pest control are provided by a specialised firm.
Water Supply and Wastewater Disposal
Drinking water is supplied by the water supply system of the city of Brno. Distilled and double
distilled water is produced using the distillation and filtration apparatuses in buildings A29 and
INBIT. Wastewater pipes are connected to the city sewer system. Selected laboratories have drains
fitted that can be used for the disposal of weak acids and bases (but not organic solvent). These
drains lead to a tank that collects chemical wastewater, which is then disposed of by a specialised
firm. The laboratory bench drains leading to the chemical wastewater tank can only be used for
disposing of the remaining quantities of chemical solutions miscible with water, in amounts that
do not represent a hazard for the water environment, water-soluble solvents (up to 0.5 l, diluted
at least ten times), and acids and hydroxides (diluted 30 times) if such waste cannot be collected
in the waste bins for operational reasons.
Final Provisions
The person authorised to interpret the provisions of this Appendix and ensure that they are
complied with is the OHS and FS Officer.
Brno, 9 September 2019
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……………………………….
prof. RNDr. Jana Klánová, Ph.D.
Director of RECETOX