Microbiological safety in diagnostic laboratoria!

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1 ! Microbiological safety in diagnostic laboratoria! 1. Introduction! 3! 2. Building and design! 4! 3. Hardware, purchase and maintenance.! 4! 3.1 Purchase of hardware! 4! 3.2 Maintenance and repair of hardware.! 4! 4. Personal hygiene! 5! 4.1 Handhygiene! 5! 4.2 Lab-coat, footwear and personal clothing! 5! 4.3 Personal protection gear.! 6! Eye protection and gloves.! 6! 5. Operation procedures! 6! 5.1 Aerosoles! 6! 5.2 Administration! 6! 5.3 Pipetting! 7! 5.4 Centrifugation! 7! 5.5 Homogenising! 7! 5.6 Cryopreservation! 8! Ultra-low freezer! 8! Fluid nitrogen.! 8! 5.7 Biological Safety Cabinet! 8! 5.8 Microtome and cryostat! 10! 5.9 Receptacles! 10! Receptacles for blood! 10! 5.10 Ultrasonic bath! 10! 5.11 Transport of receptacles with diagnostic patient material! 10! Internal transport.! 10! External transport! 11! 5.12 Vacuum-pumps! 11! 5.13 Vortexing! 12! 6. Cleaning and disinfection of items for reuse! 12! 6.1 Measures upon spillage! 12! 1 of 18

2 6.1.1 Clean and disinfect! 12! 6.2 Centrifuge! 13! 6.3 Refrigerator and freezer (-20 )! 13! 6.4 Lab-coat! 13! 6.5 Lab-table, wall and floor! 13! 6.6 Microbiological safety cabinet! 13! 6.7 Microtome and cryostat! 14! 6.8 Eye protection! 14! 6.9 Analysis equipment! 14! 6.10 Pipettes! 14! Manual and automatic pipettes! 14! Glass pipettes! 14! 6.11 Receptacles! 14! 6.12 Dry-stove! 14! 6.13 Water-bath and CO2-stove! 14! 7. Solid and fluid biological waste! 15! Appendix A! 16! APPENDIX B! 17! Literatuur! 18 2 of 18

3 ! 1. Introduction! In upcoming industrial countries, laboratories are catching up fast and the laboratories are being loaded with high tech equipment. As usual, quality assurance is the division that comes second. Most labs cannot meet the standards of the high income countries. Nonetheless, each patient not only deserves affordable diagnostics, it should also be reliable. The WHO-AFRO came up the Stepwise Laboratory Improvement Process Towards Accreditation (SLIPTA) to strengthen laboratory systems (1). It s more of a checklist and doesn t provide practical guidelines to improve safety and quality in a microbiology diagnostic laboratory. In the Netherlands, the Workgroup Infection Prevention (WIP) has a large collection of guidelines for the health-sector that is accessible to everyone, at least, if you understand Dutch. This document, Microbiological Safety in Diagnostic Laboratory has been translated by me from Dutch to English (hence, it might be a bit Dunglish ). The WIP stated that the guidelines are free to use, on condition that there is a reference to its source. Therefore, feel free to use these guidelines and please refer to the Workgroup Infection Prevention in The Netherlands (2). 3 of 18

4 ! 2. Building and design! Essential requirements voor Biosafety Laboratory class II:! Airflow from outside into the workspace of the laboratory.! Setup a work-place voor administrative activities outside the working environment of the lab. Or separate the administrative workplace from the wet- and dry laboratory activities.! Make sure there are sufficient protective gear and materials to be able to perform safely all activities in the lab.! For the structure of the laboratory:! - Work-places, floors, walls and doors are covered with non-absorbing material.! - Work-places are resistent to water, acids, bases, solvents, disinfectants and decontamination reagents. Preferably, this also applies to walls, floors and doors.! - Work-places can easily be cleaned.! Provide the lab with:! - Sink, soap and alcohol dispenser, of which the tap of the sink and dispenser can be operated without using hands.! - hooks to hang up lab-coats.! The door of the laboratory has a window with plain glass.! The floor has a mounting skirting board.! Do not place the microbiological safety cabinet close to a door or at a place where there are many movements.! Drainage where leakages, splashes or forming of aerosols are minimised.! 3. Hardware, purchase and maintenance.! Use equipment of which the manufacturer specifically comments that the equipment meet the requirements for cleaning and/or disinfection. Preferably use equipment that prevents the forming and spreading of aerosols.! Preferably use automated analysers, which only work when the system is physically closed and in which patients material from the receptacle is taken without having to opening them beforehand.! Motivation: A closed-system analyser will prevent the propagation of aerosols in the environment.! Use a microbiological safety cabinet that meets or exceeds Class IIA of the EN standard.! Motivation: Class II and Class III provide product-, environment- as personal protection. Class I does not provide product-protection.! 3.1 Purchase of hardware! When deciding on the purchase of new laboratory equipment, it is important to take into account that the equipment can be disinfected and has self containment of aerosols! Note: Disinfectants need to be effective for micro-organisms that occur in patient material. Consider also the potential for cleaning and disinfecting control panels.! Preferably purchase equipment with automatic data processing.! Motivation: Automatic dataprocessing by, for example barcodes separates technical- from administrative procedures so that keyboards and such are not contaminated.! 3.2 Maintenance and repair of hardware.! 4 of 18

5 Take preventive measures before offering equipment for installation or repair to prevent infection and contamination.! 4. Personal hygiene! In diagnostic laboratories Good Laboratory Practice (GLP) associated rules of conducts should be applied! During laboratory work avoid any external contact with the workers own mucous membranes:! - Do not lick the labels;! - Do not eat, drink or chew gum in the workspace;! - Do not put writing utensils in the mouth or behind the ear;! - Do not touch the face (especially mouth, eyes, nose);! - Do not put on cosmetics;! - Do not put in or remove contact lenses;! - Tie up long hair;! - No smelling on materials.! Store personal belongings (mobile phones, mp3 players, bags, jewellery, personal clothing, etc.) outside the workspace.! Keep doors and windows of the work area closed during work.! Do not keep food and drink in the work area of the laboratory.! Note: These rules also apply to areas designated for accepting, storing or processing of patient material.! 4.1 Handhygiene! Apply hand hygiene:! - Before and after working in the microbiological safety cabinet;! - After removing gloves;! - When leaving the work area of the laboratory.! Note: When hands are visibly dirty or feel sticky, wash hands with water and soap.! 4.2 Lab-coat, footwear and personal clothing! Wear in the laboratory workspace closed and space-bound lab coats.! Note: The lab coat has long sleeves and covers all personal clothing. Preferably, the sleeves are provided with an elastic cuff that encloses the wrist. To prevent contamination of the examination material through the sleeves of the lab coat, the employee may consider to pull the gloves over the (over-)sleeves.! Exception: For time-consuming activities at which the risk is that material is contaminated through the sleeves of the lab coat of the laboratory technician, preference can be given to wear a lab coat with short sleeves. To prevent infections to the arms, hand hygiene is applied to both the hands and forearms to beyond the elbows. The technician can also wear a lab coat with short sleeves but with over-sleeves to prevent possible contamination on the skin. The employee must remove the sleeves on the correctly.! When working in the biological safety cabinet, preferably use a lab coat with a back closure and sleeves with elastic cuffs around the wrists.! Wear closed shoes.! Hang personal clothes you are not wearing, like jacket or sweater, in a designated area outside the working area of the laboratory.! When leaving the working area of the laboratory, hang up the lab coat on the coat-hook inside the workspace.! 5 of 18

6 4.3 Personal protection gear.! Eye protection and gloves. Wear eye protection when risk there is risk of splashing and at indicated places such as:! - the cutting- and carving room;! - when working with liquid nitrogen;! - when working with corrosive, toxic or carcinogenic liquids or chemicals;! - when aerosols are formed.! Wear well-fitting gloves (preferably allergen-free) when working with patients and when indicated.! Note: Choose the correct type of gloves for the work, and clean them regularly.! Motivation: All patient material are potentially infectious. Gloves can also be indicated to protect against the harmful effects of chemical solutions (disinfectants, solvents, fixative, cytostatics) or as protection against cold instead of for microbiological reasons.! 5. Operation procedures! Consider all commercial and non-commercial biological products as potentially contaminated with pathogenic microorganisms.! Note: This also applies to biological products that may or may not have undergone a chemical or physical treatment to inactivate microorganisms or which have been frozen or fixated.! Motivation: All commercial and non-commercial biological products should be considered as potentially contaminated with pathogenic micro-organisms. Inactivation by chemical or physical treatment is sometimes possible. However, these inactivate methods may not be equally effective for all micro-organisms, often bacterial spores survive the treatment.! None of the freezing methods (cryostat, liquid nitrogen or 'ultra-low' freezer) can disinfectant material. Although fixative is a disinfectant, it still must be considered as potential contaminated material, because it can not be established whether all pathogenic micro-organisms have been inactivated.! 5.1 Aerosoles! Limit the formation of aerosols.! Note: Aerosols arise on the friction surface between air and liquid. Even when working cautiously, the formation of aerosols can not be prevented, only reduced. Aerosols can move over long distances while carrying pathogenic microorganisms. Infection can occur through inhalation but also after contact with descended aerosols on working surfaces.! Work careful and cautiously when performing lab procedures where aerosols are released. Pipette gently with an (automatic) pipette and let the fluid drain along the wall and do not blow out the remaining fluid; remove caps / lids gently from tubes and centrifuge preferably sealed receptacles (tubes) in enclosed holders. See Appendix A for a summary of aerosol-forming operations.! Motivation: often, the route of laboratory infection can not be determined, but it is likely that a large portion of laboratory infections caused by aerosols (3-5).! Perform operations where aerosol formation cannot entirely be prevented, such as homogenisation, vortexing open receptacles and sonification, preferably in a microbiological safety cabinet class IA, IIA (EN standard 12469) or in a properly functioning fume hood.! 5.2 Administration! Process data preferably automatically.! Motivation: Automatic data processing for example through barcodes, separates technical from administrative procedures so that keyboards do not become infected.! 6 of 18

7 Laminate paper procedures. Motivation: Plastic folders can be cleaned and disinfected.! 5.3 Pipetting! Pipettes are used for transferring or distributing fluids.! Types of pipettes are:! - Graduated and volumetric pipets;! - Air-displacement pipettes;! - Positive-displacement pipettes.! For the graduated and volumetric pipets, automatic and manual propipetters are available.!! Use for transferring or distributing liquid patient material:! - a propipetter with graduated- or volumetric pipet with a filter;! - an air-displacement pipet with a sterilisable tip-holder and tip-shooter in combination with a filter-tip; or! - a positive-displacement pipette.! Note: The filter prevents that the inside of the propipetter gets infected. In "air-displacement" pipettes, the inside of the tip holder may be contaminated when filter tips are not used.! The "positive-displacement" pipette does not get infected on the inside. The liquid and air coming from the patient material does not come into contact with the tip holder.! Let the liquid flow gently (not blowing) along a surface of the pipette.! 5.4 Centrifugation! Centrifuge patient material in closed unbreakable receptacles in screw-capped sealed container or in a closed rotor.! Motivation: In case of breakage or leakage of the receptacles, aerosols are not distributed outside the closed centrifuge.! Fill and close the receptacles while restricting the forming of aerosol. See section 5.1. how aerosol-forming can be limited.! Motivation: Prevent formation to aerosols.! Enter the receptacles up to two-thirds. Motivation: Receptacles filled up for two-third prevents the fluid cap to become infected during centrifugation, which lowers the risk of contaminating hands when opening the receptacles.! Wear gloves when inserting and removing receptacles. Preferably use centrifuges that lock automatically. Open the sealed receptacle containers or closed rotor taking aerosol precautions (eg in a microbiological safety cabinet or after the centrifuge has stood still for ten minutes).! Motivation: Aerosols can escape by releasing the locked receptacle aerosol containers or locked rotor. directly after centrifugation. The aerosols sink down after ten minutes.! Check for leakage of the receptacles in the receptacle holder or rotor.! 5.5 Homogenising! Homogenisation of diagnostic tissues can take placebo using for example a blender, a grinder, a potter and by ultrasonic vibration.! Homogenise tissue preferably in a microbiological safety cabinet class IA, IIA, or in a working fume hood.! Motivation: This protects the employee against aerosols.! Clean and disinfect the 'homogeniser' after use! 7 of 18

8 5.6 Cryopreservation! Patients materials can be stored for long periods, by means of storage at temperatures of -150 or less. These low temperatures can be reached by making use of an ultra-low freezer or liquid nitrogen! Ultra-low freezer Use special insulating gloves when there is a chance that skin can come into direct contact with the cold parts of the plant.! Note: The insulating gloves should at least be able to withstand temperatures of -180 and should be impermeable to micro-organisms.! Fluid nitrogen. Place the tank with liquid nitrogen in a space with an oxygen meter in which an audio and a visual alarm will go off when the oxygen content is dropping below 19.5%.! Note: An oxygen meter is also obligatory for a room in which containers with liquid nitrogen are stored. If the oxygen level is too low, one must immediately leave the room and not allowed to enter until the oxygen level is back normal.! Preferably do not work in an enclosed space in which a tank of liquid nitrogen is located.! Motivation: In a confined area nitrogen vapours can easier remain in the room, making the oxygen level in this space below acceptable the threshold.! Wear a face shield when working liquid nitrogen and when inserting and removing samples in/ from a liquid nitrogen container.! Use special insulating gloves when there is a risk that the hands come into contact with liquid nitrogen or with cold parts of the installation.! Note: The insulating gloves need at least to be able to withstand temperatures of -180 and should be impermeable to micro-organisms. Make sure the gloves are not too tight so that they can be removed quickly when they get in contact with liquid nitrogen.! Motivation: Preventing frostbite on the hands.! Use cold-resistant receptacles for storage of patient material in a storage tank containing liquid nitrogen. Caution! Upon opening of the receptacle after removing from the liquid nitrogen, there is a risk of explosion from pressure that has been build-up in the receptacle. This is caused by liquid nitrogen warming up and becoming gaseous. It is necessary to remove carefully the pressure from the receptacle by opening the cap a little without taking it off. Always do this in the microbiological safety cabinet and hold the bottom of the receptacle toward you and the opening towards the rear wall of the microbiological safety cabinet. Use as an extra safety measure a tissue. If the content still escapes the receptacle, the contamination should be removed as indicated in Section 6.! Save the patient material in the gas phase of the liquid nitrogen. Motivation: Storing the patient material in the gas phase of the liquid nitrogen will prevent possible transmission of micro-organisms through the liquid phase of the liquid nitrogen.! 5.7 Biological Safety Cabinet! Allow the microbiological safety cabinet to run for at least 10 minutes or the amount of time specified by the manufacturer before the beginning of the experiment.! Motivation: After turning on the microbiological safety cabinet, it takes at least 10 minutes before the flow stabilises. Maintaining a period of 10 minutes, or a period of time specified by the manufacturer between two experiments prevents cross-contamination via the air.! Disturb the airflow in the microbiological safety cabinet class as little as possible by: - placing only the necessary objects next to each other in the microbiological safety cabinet;! - not blocking the air vents on the front and back of the working-area;! 8 of 18

9 - making calm controlled movements;! - working deep in the microbiological safety cabinet and not at the front;! - not placing large equipment in the microbiological safety cabinet such as centrifuges;! - not using a Bunsen burner;! - not walking at short distance along the microbiological safety cabinet;! - keeping the doors of the room closed as much as possible during the work.! Motivation: Objects and arm movements cause air swirls in the air flow of the microbiological safety cabinet. By limiting the number of objects in the microbiological safety cabinet and making calm arm movements, the risk of contamination of the employee (s) and blowing micro-organisms to outside the cabinet can be prevented. Walking close by the microbiological safety cabinet causes a disturbance in the air flow.! Work in the microbiological safety cabinet always from 'clean' side to dirty' side. Note: Never move with a dirty pipette tip over clean utensils.! See Figure 1 for how to work from clean to dirty.! Figure 1: Work arrangement in a microbiological safety cabinet for right-handed person. Clean items are put on the left (a). The patient material is placed in the middle. In the middle of the working-zone the patient material is pipetted into clean materials and subsequently placed on the right side in the working zone (b). On the right, dirty side (c), contaminated pipettes are disposed in a moisture-proof container and other contaminated material in a waste bag. Left-handed persons carry out the process from the right to the left.! Keep contaminated consumables during work in the microbiological safety cabinet.! Motivation: This reduces the risk of spreading micro-organisms in the environment during work. See Figure 1 for the correct working method.! Before removal from the microbiological safety cabinet, first disinfect the exterior of all items inside the microbiological safety cabinet with 70% alcohol. Apply a contact time of 30 seconds (or the prescribed time from the manufacturer) for the alcohol with the surface and allow it to air dry.! Turn the microbiological safety cabinet off or put in standby mode, at least 10 minutes (or time specified by manufacturer) after finishing work.! 9 of 18

10 Close the window of the microbiological safety cabinet after work.! Immediately stop the work when there is an unexpected failure of the microbiological safety cabinet or when the alarm goes off because of an disturbance of the downflow; close open receptacles, clean if necessary the worksheet and close the window.! Resume operations when the microbiological safety cabinet has been running for at least ten minutes or by the company specified recovery time.! 5.8 Microtome and cryostat! Cut a frozen section always with the hood closed.! Note: Use personal protective gear when working with the hood opened.! Remove waste from the cryostat and microtome daily or after use. Use a disposable knife for cutting the tissue in a cryostat and microtome.! Caution! Wearing gloves for the removal of the knife does not protect against cutting incidents.! 5.9 Receptacles! Preferably use plastic receptacles for storage of patient material.! Motivation: With plastic the risk of breakage is less compared to glass.! Preferably use receptacles with a screw cap. Motivation: When when push-caps are removed, material is easily spilled and aerosol can be formed. When receptacles with a push caps or "safe lock" are reversed, fluid may come between the tube and the cap. Due to the centrifugal forces, the fluid can get outside the receptacle into the tube holder of the centrifuge.! Fill, close and open receptacles with aerosol restrictive measures. See section 5.1. on Aerosols.! Motivation: Preventing exposure to aerosols.! Remove cap or lid from a receptacle between finger and thumb in a circular motion. Caution! Upon opening vacuum tubes content can spill out. First aerate the vacuum tubes before removing the cap.! Motivation: The method described above prevents contamination and formation of aerosols. When opening receptacles "with the thumbs," there is a greater risk of infection and formation of aerosols.! Receptacles for blood Preferably use receptacles with gels or beads for the separation of blood components.! Motivation: The use of receptacles with gels or beads, a second centrifugation step is not required and it reduces pipetting so there is less chance of contamination.! 5.10 Ultrasonic bath! Use an ultrasonic bath preferably in a microbiological safety cabinet class IA, IIA (EN standard 12469) or in a properly functioning fume hood.! Motivation: During the ultrasonic vibration aerosol formation can occur.! 5.11 Transport of receptacles with diagnostic patient material! Clean and disinfect, if necessary, the outside of the receptacle for transport.! Motivation: Employees from internal transport must be protected from getting contaminated.! Internal transport. Use leakproof plastic packaging. Transport patient material preferably in unbreakable and leak-proof receptacles. 10 of 18

11 Transport receptacles preferably upright in racks. In air-tube mail distribution use shock- and spill-proof unbreakable plastic patterns.! Unload the received receptacles with patients material on a dedicated workstation.! Refer to internal SOP Specimen Transportation (6).! External transport The guidelines for external transport of diagnostic materials described within a country can vary between countries. For Ethiopia consult DRAFT guidelines FMHACA Guidance on regulations for the transport of Clinical Specimens for Diagnostic purpose (7).! Airlines may still impose additional requirements. These are described in the IATA Dangerous Goods Regulations (8).! 5.12 Vacuum-pumps! When applying vacuum, use only vacuum-pumps or -systems, not water-flow-pumps.! Motivation: aerosols are produced when using a water flow pump for the creation of a vacuum, which could cause a laboratory contamination.! For freeze-drying, use vacuum-pumps or -systems provided with hydrophobic absolute filters. Note: Place the hydrophobic absolute filter between the vacuum pump or central vacuum system and the collection bottle.! Motivation: The hydrophobic absolute filter prevents contamination of the vacuum pump or central vacuum system.! Replace the hydrophobic absolute filter at least every 6 months, and directly after a malfunction. See Figure 2 for the most ideal setup.!! 11 of 18

12 ! Figure 1: Setup of a vacuum pump.! The set-up includes a collection or suction vessel (A), an overflow collection vessel (B), and a vacuum protection in-line hydrophobic absolute filter or HEPA filter (C) placed immediately before the valve (D) to protect the vacuum system from contamination (9).! 5.13 Vortexing! Vortex open receptacles in a microbiological safety cabinet class IA, IIA (EN standard 12469) or in a properly functioning fume hood. 6. Cleaning and disinfection of items for reuse! For laboratory the disinfectants described in Appendix B are eligible. The different disinfectants are divided for disinfection of objects and surfaces of less than 0.5m 2 and of surfaces greater than 0.5m 2. When working with specific pathogens such as norovirus and Echinococcus eggs, it must be taken into account disinfectants are is less effective.! 6.1 Measures upon spillage! Visible contamination with blood or other body fluids on the floor and other objects are cleaned first and then disinfected with 70% alcohol or, if the surface is greater than 0.5m 2 with 1000 ppm chlorine. Make sure the object is dry prior to disinfection. Allow a minimum contact time of five minutes for the chlorine solution and a contact time of 30 seconds for 70% alcohol.! See Appendix B for an overview of the recommendations.! Note: Chlorine affected by stainless steel. When using chlorine, the disinfected surfaces should be wiped afterwards with water to remove chlorine residues.! Clean and disinfect Clean and disinfect contaminated surfaces, furniture or objects as follows:! 1. Pull on non-sterile gloves;! 2. Lay down absorbent paper surrounding the contamination;! 3. Clean the contaminated area with disposable cleaning materials. If necessary, first soak up fluid with a tissue;! 4. Remove the absorbent paper;! 5. Disinfect the contaminated area with a disinfectant (see Appendix B);! 6. Dispose the used paper and cleaning material. See Chapter 7;! 7. Pull off the gloves and apply hand hygiene.! Note: Intuitively, one would rather fit first disinfection instead of clean first. Cleaning beforehand is necessary because disinfectants are made partially ineffective by organic matter, such as blood 12 of 18

13 (proteins). One must realise that a disinfectant works faster and better when the surface to be disinfected is cleaned up.! 6.2 Centrifuge! Clean and disinfect chemically the inside and the outside of the rotor and its matching lid, the tube holders with the lids and the rotor chamber of the centrifuge once a week or immediately after leakage as follows:! 1. Pull on sturdy gloves;! 2. Clean all parts with soap and water and dry them;! 3. Immerse the rotor tubes or containers with matching cover(s) into a disinfectant recommended by the supplier;! 4. Employ the contact time prescribed by the manufacturer. Beware of corrosive disinfectants;! 5. Remove the disinfectant after the correct contact time and rinse if necessary the disinfected parts with tap water;! 6. Beware of splashes.! Note: Consider thermal disinfection or sterilisation of centrifuges that are used daily, after fracture of a receptacle in a bucket or contamination of the rotor.! Check the rubber parts of the centrifuge prior to centrifugation and replace the rubber parts if necessary or treat them according to the manufacturer's specifications if they are dried up. Motivation: Good closure of the bucket prevents leaks and distribution of aerosols.! 6.3 Refrigerator and freezer (-20 )! Clean the refrigerator monthly or sooner when visibly dirty and dry the refrigerator before use.! Clean the freezer (-20 ) annually or sooner when visibly dirty and dry the freezer before use. Clean, dry and disinfect the refrigerator and freezer immediately when visibly contaminated with patient material.! 6.4 Lab-coat! Replace the lab coat daily or immediately after spilling or splashing with patient material on the lab coat.! 6.5 Lab-table, wall and floor! Disinfect the bench at start and after completion of work, and clean and disinfect the laboratory bench immediately when visibly dirty.! Motivation: Disinfect after work to leave a clean table that poses no risk to others. Clean before work because you never know what it happened on the table before.! Clean and disinfect the floor or wall immediately when contamination with patient material.! 6.6 Microbiological safety cabinet! Disinfect the work surface and the air vents of the microbiological safety cabinet with 70% alcohol before and after completion of work. Handle a contact time of the alcohol with the surface of 30 seconds, or the contact time prescribed by the manufacturer, and allow it to air dry.! Note: Disinfect the microbiological safety cabinet with formaldehyde or as directed by the manufacturer, after an contamination of parts which cannot be reached by hand, such as the grid. This should be carried out by the biological safety experts are means of a validated method.! Disinfect all objects with 70% alcohol before placement in the microbiological safety cabinet. Handle a 30 seconds contact time of the alcohol with the surface, or a contact-time prescribed by the manufacturer, and allow it to air dry.! Clean, dry and disinfect the microbiological safety cabinet when visibly contaminated with patient material. 13 of 18

14 Leave the microbiological safety cabinet to run for 10 minutes (or time prescribed by manufacturer) before continuing with the work.! 6.7 Microtome and cryostat! Clean the microtome and cryostat weekly with warm soapy water and immediately when visibly dirty. Note: Thaw the cryostat prior to cleaning.! Disinfect the cleaned and fully air-dried microtome or cryostat with 70% alcohol. Keep a contact time of at least 30 second or time prescribed by manufactuere.! 6.8 Eye protection! Clean the eye protection every day with warm water and soap or immediately when visibly dirty.! Clean and disinfect the eye protector immediately after contamination with patient material.! 6.9 Analysis equipment! Clean at the end of the day the keyboard or touch-screen of the analyser according to the manufacturer documentation.! Clean and disinfect the analysers (internal and external) as directed by the manufacturer.! 6.10 Pipettes! Manual and automatic pipettes Take the pipette (pipette holder including tip-shooter and -holder) of air-displacement pipettes and pipet-pump after work with a manufacturer's prescribed disinfectant or 70% alcohol and leave the pipette to air dry. Apply the prescribed contact time of the disinfectant.! Caution! Make sure the pipette holder or pipet holder withstand disinfection with 70% alcohol.! Note: Clean off visible contamination on the pipette holder and pipet-pump prior to disinfection. The tip-shooter and -holder of air-displacement pipettes can easily become infected. There are pipettes of which components can be autoclaved. Check this with the manufacturer. The tip holder can also become infected on the inside filter tips are not used.! Glass pipettes Insert glass pipettes in a disinfecting storage fluid immediately after use Clean the glass pipettes and let them dry and continue with thermal disinfection or autoclaving, as directed by the manufacturer.! 6.11 Receptacles! Directly clean and disinfect a on the outside contaminated receptacle.! 6.12 Dry-stove! Laboratories use different stoves: incubator (shaking incubator) and stoves for drying and curing (oven).! Clean, dry and disinfect the incubator monthly or sooner when visibly dirty.! 6.13 Water-bath and CO2-stove! Prevent growth of micro-organisms in a water bath:! - Add to water bath an agent that prevents the growth of bacteria. When using a copper water tank in the CO2-incubator, the addition of a bacterial growth inhibiting agent is redundant.! Note: A bacterial growth inhibitory agent must be permitted by law. The agent often contains a color indicator reacting to bacterial growth.! 14 of 18

15 - Heat the water once a week to at least 80 for ten minutes! Empty the water bath monthly or sooner if necessary (observe the color indicator), clean, dry and fill the bath with clean water.! 7. Solid and fluid biological waste! According to the National Waste Management Regulations [REF. needed]!! 15 of 18

16 Appendix A! Procedures that cause formation of aerosols.!! Draining;! Centrifugation with open receptacles;! Over-inoculation and smearing;! Flaming the inoculation loop;! Homogenising lyophilised biopsies (mortar and pestle);! Homogenising tissue in liquid (grinder);! Inoculating eggs;! Cooling inoculation loop;! Removing air;! Mixing;! Spilling;! Taking out needle from vacuum tube;! Removing needle from syringe;! Opening receptacles;! Pipetting;! Using spatula;! Vortexing open receptacles;! Sonification.! 16 of 18

17 APPENDIX B! Use of disinfectants in laboratoria for surfaces and objects.! Indication Disinfectant Exposure time Disinfection of small surfaces (less than 0.5 m Disinfection of large areas (larger than 0.5 m # 70% ethanol is usual! Ethanol 60-90% 1000 ppm of active chlorine (0.1%) * The surface should remain wet for at least 30 seconds and then air-dried When immersed in alcohol, exposure time is at least 5 minutes. The surface should remain wet for at least 30 seconds and then air-dried&.! When immersed in alcohol, exposure time is at least 5 minutes. & The use of alcohol in a spray bottle does not guarantee that the entire surface is disinfected. With spraying the contact time of 30 seconds is not reached for the entire surface. Rubbing the surface with 70% alcohol is will ensure the proper contact-time. In some countries the spraying of alcohol is associated with health risks and therefore prohibited by law.! * A chlorine concentration of less than 1000 ppm has limited effectiveness against viruses. Caution! Chlorine affects stainless steel! 17 of 18

18 ! Literatuur! 1. WHO Guide for the Stepwise Laboratory Improvement Process Towards Accreditation in the African Region Werkgroep Infectieziektepreventie in The Netherlands. objectid=rivmp:192923&type=org&disposition=inline! 3. Sewell DL. Laboratory-associated infections and biosafety. Clin. Microbiol. Rev. 1995; 8(3): ! 4. Pike RM. Laboratory-associated infections: incidence, fatalities, causes, and prevention. Annu. Rev. Microbiol. 1979; 33:41-66.! 5. Wedum AG. Laboratory safety in research with infectious aerosols. Public Health Rep. 1964; 79: ! 6. International Clinical Laboratories SOP. Specimen Transportation.! 7. FMHACA Guidance on Regulations of Biological/clinical Specimens Transport for Diagnostic Purpose. Adopted from the Recommendations on the Transport of Dangerous Goods, Model Regulations, 17th revised edition, New York and Geneva, United Nations, 2011, and WHO/ HSE/GCR/ Guidance on regulations for the transport of infectious substances ! 8. website: infectious_substances.aspx.! 9. General Biosafety Practices and Procedures. Laboratory Vacuum Lines. ehs.research.uiowa.edu/545-laboratory-vacuum-lines! 18 of 18

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