1 Laboratory Safety
1.1 Biosafety in Microbiology Laboratory
1.2 Section Safety Documentation
(a) Biohazard Protection: The actual operating procedures are shown in (General Requirements for Laboratory Biosafety, National Standard of the People's Republic of China*** and GB 19489-2004. 2004-01-01. 05 issued, 2004-10-01 implementation.)
(b) Laboratory safety: principles and measures see (General Guidelines for Biosafety in Microbiological and Biomedical Laboratories, People's Republic of China *** and the national standard for the health industry WS 233-2002. 2002-12-03 release, 2003-08-01 implementation.)
1.3 Application:
The above operating procedures should be used in all departments of the Microbiology Department. Staff or visitors must comply with the regulations.
1.4 Regarding new regulations or modifications to existing regulations:
Any new regulations or modifications to existing regulations must be communicated to the person responsible for the safety of the department and to the relevant staff, who will advise the Head of Department to consider their implementation.
1.5 Biological Hazards
1.5.1 The level of biological hazards associated with various operations is determined by:
a. The infectious substances handled per se:
Infectious substances are classified into levels according to their potential danger to individuals and communities and their potential for airborne transmission. The classification levels used are detailed in "General Laboratory Biosafety Requirements"; principles and measures (see "General Guidelines for Biosafety in Microbiological and Biomedical Laboratories").
b. Equipment and facilities used:
The requirements for handling different classes of infectious substances are set out in Laboratory Safety. These compliant equipment are required to perform important clinical work, and the head of the department must ensure that the level of contaminants is kept at the appropriate level.
c. Training, especially staff safety training and safety experience. All employees should receive some training to be able to safely handle any laboratory substances that may be encountered, to avoid exposure to infectious substances that can not be handled by the experiment or outside the regulations in the laboratory, even if the exposure accident in the laboratory, even if the exposure accident occurs to be able to handle it correctly.
1.5.2 Classification of infectious substances
1.5.2.1 Classification system
Biohazard protection: [see 1.2 (a)], according to the degree of biological factors on the individual and the group will be divided into 4 levels, Hazard Class Ⅳ most hazardous.
1.5.2.2 Hazard Groups/Biosafety Levels: 1-4
a. Hazard Groups/Biosafety Levels: 1 - Not pathogenic to healthy adults and not hazardous to the community. General microbiological operations are usually sufficient, no special safety equipment is needed, and hand washing facilities should be available in the vicinity of the work. Culture media contain unidentified material, so proper handling is important at all levels of safety.
b. Hazard Groups/Biosafety Levels: 2 - Substances associated with human disease, which pose a moderate risk to laboratory workers and a degree of community hazard. The vast majority of substances involved in hospital microbiology laboratories are at this level of safety. Biohazard warnings should be posted and access restricted, with conspicuous signs, and appropriate workstations should be used if airborne or splashing Class I or II substances can be generated. Laboratories should provide similar protective clothing and gloves, eye protection, especially for contact lens wearers, hand washing facilities at a similar level of safety, and autoclave sterilization units should be available in the immediate work area. There should be specific procedures for decontamination of all waste and reusable equipment.
c. Hazard Groups/Biosafety Levels: 3 - Human Disease-Related Substances that are potentially airborne, capable of causing serious, fatal disease, highly hazardous to laboratory workers, and have some degree of community hazard. All specimens that contain or are suspected of containing biological material must be labeled with a "Caution Contamination" label and included in both the specimen container and the checklist. However, as a precautionary protection this is not sufficient. There should be a general awareness of the need to protect all blood, body fluids, isolated human tissues, etc., which should be treated as infectious agents. At this biosafety level, the laboratory should control access to them. Anyone working with such substances should use a Class I or II bench, and Class II precautions can be followed to decontaminate all waste, including work clothes and reusable equipment.
d. Hazard Groups/Biosafety Levels: 4 - Dangerous to individuals as in Biosafety Level 3, but highly dangerous to the community. These infectious substances include:
Tick-borne Encephalitis Virus
Jiribia-Congo Bovine Hemorrhagic Fever Virus
Marburg Virus
Ebola Virus
Lagsa fever Virus
Junin HF Virus
Machupo HF Virus
Guanaruto HF Virus
Herpesvirus simiae (B Virus)
1.6 The following precautions must be observed by all laboratory staff:
1.6.1 Work Uniforms
a. All laboratory members must wear the provided coveralls at all times while working. When leaving the laboratory, coveralls shall be hung in the designated area and shall not be worn outside the laboratory.
b. When handling specimens containing or suspected to contain Biosafety Level 3 substances, isolation gowns must be worn over the overalls.
c. Gloves must be worn when handling specimens and media in the biosafety bench.
1.6.2 Handwashing Sink
The handwashing sink shall be supplied with detergent and paper towels.
1.6.3 Hand washing
All staff must wash their hands when leaving the work area and remove their overalls and gloves first.
1.6.4 Food, Drink, Smoking, Cosmetics
Food and drink are not permitted in any room where specimens are processed. Smoking is not permitted in any area where specimens are handled. Cosmetics are not permitted in any area where specimens are handled.
1.6.5 Cuts and abrasions
Cuts and abrasions on the hands must be covered with protection.
1.6.6 Personal Clothing
Personal clothing is not allowed in the laboratory and must be kept in a locked cabinet.
1.6.7 Mouth pipettes
This is prohibited. Mechanical pipetting devices are required.
1.7 Routine precautions should be used in the manipulation of all blood, body fluids, and tissue specimens:
1> The use of syringes, needles, or other sharp instruments should be avoided whenever possible.
2> Used needles and disposable cutters must be disposed of in special buckets with lids for safe disposal and to prevent overfilling of containers.
3> Used needles must not be reused or manipulated by hand.
4> Broken glass must be placed in a hard container (not by hand) and then placed in a black garbage bag, or a yellow garbage bag if contaminated.
5> In contact with potentially infectious specimens media, tissues, protective gloves must be worn to prevent direct skin contact. If there is visible contamination of the gloves, the contamination must be removed before replacing them. Staff with dermatitis or wounds on the hands of the need for indirect contact with infectious substances should wear protective gloves.
6> Hands should be routinely washed after handling specimens, at the end of the work, and even when gloves are worn as specified above.
7> When contamination of blood, blood products or other body fluids occurs, work should be stopped to wash hands and wear disposable gloves before removing the contaminated area. It is recommended to use sodium hypochlorite solution containing 1000ppm of effective chlorine. If the contaminated area is larger cover it with a damp cloth soaked in sodium hypochlorite solution for 10min to decontaminate it and label it with the appropriate warning, discard the waste and gloves into a biosafety grade physical container. Be aware that splashes and runoff can carry contamination beyond the main contaminated area.
8> All potentially contaminating substances used in the laboratory need to be decontaminated, preferably under high pressure, before handling.
9> Any operation that may generate splashes or aerosols should be performed in a biosafety bench. These operations include centrifugation, separation of serum, mixing, sonication, collection of tissue fertilized eggs, and handling of specimens containing concentrated infectious material.
1.8 Laboratory Material Handling
a> Black plastic bags are used to collect wastes that are either uncontaminated or have been autoclaved for disposal.
b> Waste paper is repeatedly used to collect uncontaminated waste paper, used paper towels, etc.
c> Waste paper is used to collect uncontaminated waste paper.
c> All potentially infectious materials must be pressurized or incinerated.
d> All discarded specimens, media and laboratory waste must be placed in special containers
e> White polypropylene canisters containing 2% freshly prepared hypochlorite solution are used for the temporary disposal of specimens, contaminated tips and swabs that are discarded at work, to be disposed of at the end of the day and then under high pressure.
f> Yellow plastic bags are used to dispose of contaminated laboratory waste. They should be sealed and then taken by the workers for incineration.
g> Lidded, labeled rigid containers are used to hold needle sharps.
1.9 Sterilization of Laboratory Infectious Substances
High Pressure
a. All infectious substances in media should be subjected to high pressure unless they are prepared for incineration, the great advantage of high pressure is that it keeps the infectious substance out of the laboratory in a safe manner.
b.High-pressure equipment used for this purpose should be operated by laboratory workers under the supervision of a medical technologist.
c. High-pressure equipment shall be tested and serviced periodically by the hospital equipment group. These tests shall include commercially available germ cell test strips. Multiple pieces of high-pressure equipment should have a record of use that is continuously recorded by the medical technologist from the date of use. Any discrepancies should be reported to safety personnel.
1.10 Decontamination
Decontaminants:
1> Hypochlorite solution: freshly configured 2% hypochlorite solution containing 1000 ppm of active chlorine in white polypropylene canisters placed in multiple studios.
2> 5% Printol: its 50% aqueous solution is used to treat contamination from spills on floors and furniture.
3> Glutaraldehyde: freshly prepared to a certain concentration, it is mainly used to decontaminate instruments that cannot use sodium hypochlorite, such as centrifuges.
4> 75% ethanol: mainly used for rapid decontamination of clean surfaces.
1.11 Centrifuges
1.11.1 Precautions:
a> The tubes used to make the centrifuge should be thick or plastic, and should be carefully inspected for defects before centrifugation.
b> The centrifuge should be placed in a suitable position, and the centrifuge cylinder should be visible to all operators so that the centrifuge rack can be correctly placed on the rotor.
c> The centrifugal drum and rack are paired and need to be carefully balanced after loading.
d> To avoid dislodging the rack and spilling the solution in the test tube, start with a low speed and gradually increase it.
e> The inner cylinder of the centrifuge should be regularly retrieved by a senior person and the inner wall must be cleaned regularly with glutaraldehyde to decontaminate.
f> When centrifuging specimens that contain or are suspected to contain Biosafety Operation Level 3 specimens, they must be sealed with a lid, which can only be opened inside a Level 1 Biosafety Station.
1.11.2 Tube rupture during centrifugation
a> If a tube rupture occurs or is suspected while centrifugation is in progress, the motor shall be immediately de-energized and the lid of the centrifuge shall not be opened until 30 min have elapsed.
b> If a rupture is detected after centrifugation, close the lid immediately and keep it closed for at least 30 min.
c> Notify the safety officer immediately.
d> Wear thick gloves and use tweezers or cotton identification with tweezers to remove broken glass fragments.
e> All broken tubes, glass fragments, centrifugal buckets and rotors must be decontaminated by placing them in a special disinfectant that is non-corrosive and effective in immersing biosafety substances. Then, either soak up to 24 hours, or high pressure.
f> Unbroken, capped test tubes should also be placed in another container containing a disinfectant1 before their contents are treated.
i> Centrifugal cylinders must be cleaned with a glutaraldehyde cotton swab, overnight, and then repeatedly wiped, then cleaned and washed with water and dried.
g> Sealed centrifugation vats containing biosafety level 3 substances must be removed in a sealed state and opened in a level 1 biosafety console. If a test tube is ruptured, the centrifuge drum sealing lid should be loosely closed and high pressure applied to the centrifuge drum.
1.12 Ruptures and Spills
Safety personnel should be notified of any significant ruptures of spills. Advice should be obtained from safety personnel before handling spills that contain, or are suspected of containing, biological cases.
1.12.1 Spills of viral media
a> Cover spilled contaminants with container debris using paper towels soaked in 20% sodium hypochlorite.
b> Cover for 10-15 min.
c> Wearing disposable gloves, sweep paper towels and debris from suitable containers (e.g., plastic bags, but not from containers with broken glass or other sharp objects) with a piece of cardboard, not with a brush or duster unless they are suitable for high pressure. Keep fingers away from broken glass.
d> Place debris and post-use waste in the laboratory waste bin.
e> Wipe down the contaminated area and its surrounding potential splash zone with a routine work disinfectant.
1.13 Leaking specimens
Specimens that do not leak in the laboratory but are sealed in plastic and returned to the ward.
1.14 Ultraviolet Sterilization: Media, reagent dispensing and patient sera should be stored in -70°C and -30°C refrigerators. Gloves should be worn when handling substances stored in refrigerators at -30°C or below, e.g., when removing or inserting specimens from or into cold refrigerators.
1.15 Biosafety Cabinet (Bench)
The laboratory should be equipped with a Class II biosafety cabinet.
1.15.1 Level
I: Basic requirements: Directed flow protection, requiring airflow to flow into the cabinet from the outside and away from the operator, pointing toward infectious agents. After HEPA filtration exhaust, it should be directed to the outside by conduit in order to remove all kinds of vapors after decontamination.
II: basic requirements: the filtered gas flows vertically, both to protect the operator and to protect the work from contamination.
IIA: 70% of the filtered gas recirculation to the work area, the exhaust gas is directed to the outdoor by HEPA filtration. And within the flow through the type of work area of the airflow velocity average 0.4m / s or slightly higher.
IIB: the flow into the gas flow rate average 0.5m/s or better. According to different types (IIB1, IIB2, IIB3), the gas discharge ratio from 70% to 100%. Select different types according to the work. Such as toxic chemicals and radioisotopes can not be recirculated to the work area.
III: basic requirements: the operator and the infectious material safety isolation, and the entire operation process should be raised. The environment in which the safety cabinet is located must also be isolated to some degree to prevent glove breakage.
1.15.2 Installation of biological safety cabinets
Safety cabinets to play a certain safety protection must pay attention to the location of the installation, the exhaust gas should be piped to protect the environment from contamination. If this type of equipment is required, the CUHK Safety Office should be contacted for guidance on type, brand, style, location and installation, and proximity to other facilities.
1.15.3 Routine Inspection and Safe Operation and Disinfection of Work Areas
An operating procedure detailing the routine use of the cabinet should also include actions to be taken in the event of an accident with the cabinet. Each user must read and sign the protocol.
1.15.4 CAUTION:
Safety cabinets should be kept in an unsafe condition whenever possible, but sometimes users are forced to do so because of an accident with the cabinet. In this case, a caution should be posted to warn others not to use it again.
1.15.5 Decontamination and Recertification
Contamination is done by trained personnel wearing personal protective equipment. Requirements: Safety cabinets are vaporized with dry paraformaldehyde vapor under airtight conditions using a concentration of 8500 ppm. (The volume of the safety cabinet should include peripheral supply and exhaust gas filtration equipment.) At a temperature of 20-25 ° c, relative temperature is not less than 60% of the environment to maintain 4h, formaldehyde vapor, if allowed, can be pumped directly out of the outdoors, if not allowed can be absorbed in the safety cabinet with amino bicarbonate salt.
Because of the limited penetration of air, it is best to use HEPA filters when dealing with potentially infectious substances, as well as high pressure or incineration before disposal.
1.17.5.2 Recertification
This is performed by specialized personnel to recertify the performance of the cabinet after decontamination and filter replacement, to test for air leaks, to check for proper air flow rates, and to test the performance of the filter. Recertification should be done at least once a year, or after the unit is moved and the filter is repaired. There should be decontamination, maintenance and recertification records, and should be easy for users to access.
2. Laboratory safety procedures:
2.1 Staff should prevent the spread of pathogenic microorganisms and infections in the process of collecting specimens, and the source of the specimen, the collection process and method of detailed records.
2.3 Smoking, eating and drinking are absolutely prohibited in the course of the experiment, and do not stroke the head and face with your hands.
2.4 The use of appropriate personal protective equipment (PPE), biosafety cabinets, and/or other physical protection equipment is required when handling samples that are susceptible to the generation of highly hazardous aerosols. If aerosols containing biological factors can be generated, they should be operated in appropriate biological safety cabinets. Operations such as isolation and culture of bacteria, strain opening, transferring, grinding, and dilution should be performed in a biological safety cabinet.
2.5 When operating with inoculation rings, the rings should be burned in the inner flame of the work lamp to avoid splashing of bacterial fluids or clumps.
2.6 When diluting the bacterial solution, the pipette and syringe should be inserted slowly into the bottom of the test tube or flask, and carefully operated to avoid bubbles or aerosols.
2.7 When using a syringe to add samples, the used needle should not be re-inserted into the set or pulled out of the syringe and needle, it should be put directly into the sharps collector to avoid scratching the skin and causing inoculation infection.
2.8 The strain library should be managed by a special person, and in accordance with the national microbial strain management methods. 2.9 In the process of sterilization operations, do not wear protective gloves that have been contaminated to touch the door handle, instruments or areas outside of the sterilization area, in order to avoid expanding the scope of contamination due to carelessness.
2.10 After the end of the test, the operation of all possible contact with biohazardous substances or contaminated test instruments and items, can be high-pressure sterilization must be high-pressure disinfection, can not be high-pressure disinfection of equipment, instruments, should be used to effectively disinfectant scrubbing, and then to the ultraviolet lamp for a long time in close proximity to the disinfection.
2.11 accidental contamination occurs in the experiment, should immediately notify the supervisor and make preparations to deal with the pollutants and the corresponding area, shall not be unauthorized use of other prohibited methods of disinfection.
2.12 Any sample of microorganisms in the laboratory, the waste must be sterilized by autoclaving before disposal as general waste.
2.13 Any personal protective equipment (PPE) used in the Laboratory should meet the requirements of the relevant national standards. The Laboratory should ensure that sufficient clean protective clothing with appropriate levels of protection is available for use. Other PPE such as gloves, goggles, masks, head and face protection should also be worn.
3. Operational procedures for handling and disinfection of laboratory contaminants:
3.1 Clinical specimens containing biohazardous materials and contaminated disposables in the laboratory should be disinfected by close proximity irradiation with UV lamps for more than 2 hours on the operating table or in the experimental area after completion of the test and then disinfected with autoclave before being discarded or incinerated.
3.2 Reusable laboratory supplies and equipment, after the completion of the experiment, should be disinfected by ultraviolet lamp near irradiation on the operating table or in the experimental area for more than 2 hours, and then handed over to the relevant personnel to carry out high-pressure disinfection and boiling and scrubbing.
3.3 Test tubes, pipettes and syringes for experiments shall be packed in containers with lids that do not leak and removed after autoclaving.
3.4 Cultures or laboratory wastes shall be treated with autoclaving and UV lamps in close proximity for a long period of time before disposal. Accumulation of garbage and laboratory waste is not allowed. Filled containers should be removed periodically. Until decontaminated or finally disposed of, they should be stored in a designated safe place, usually within the laboratory area.
3.5 Laboratory waste shall be transported safely out of the Laboratory in appropriately sealed and leak-proof containers. Hazardous gases, aerosols, effluents, and waste liquids shall be discharged after appropriate and harmless treatment, and shall comply with relevant national requirements.
3.6 If a specimen or pre-digestive treatment solution containing a specimen is knocked over and contaminates the operating table or the floor during the course of an experiment, the contaminated area should be covered with tissue paper soaked with 70% alcohol, and the tissue paper should not be removed until 15 minutes later.
3.7 Undisinfected sewage in the laboratory is prohibited from being discharged directly into the public **** drainage system, and is not allowed to be mixed with residential waste.
4. Accident Handling Procedures:
4.1 If an accident occurs, the laboratory supervisor and the hospital biosafety office must be notified immediately, and under the supervision of the guidance of the relevant personnel to deal with the scene of the accident, it is absolutely prohibited to give the scene of the accident without a report of non-standardized treatment privately.
4.2 During the experiment, if the pollutant splashes onto the body surface, or there are cuts, puncture wounds, burns, scalds, etc., the laboratory work should be stopped immediately to carry out emergency treatment, replacement of contaminated laboratory clothing, skin surface with disinfectant to clean, wounds disinfected with iodine or alcohol, and the eyes rinsed with sterile saline.
4.3 If the bacterial liquid overflow, containing strains of culture tubes broken, resulting in medium and small area contamination, can be larger than the contaminated area of more than 25% of the gauze covering the contaminated area, the edge of the skimmed cotton surrounded by the gauze pouring 5% phenol solution or 70% of the alcohol, soak for more than 2 hours (during which the appropriate amount of added solution to prevent drying), and then irradiated by ultraviolet light at close range (within 1 meter) for 2 hours. Above; contaminated instruments, containers, etc. Immediately immersed in 70% alcohol for more than 2 hours, again with protective clothing into the room, the contaminated spills and gauze used to clean into a high-pressure-resistant double-layer plastic bag and as soon as possible high pressure.
4.4 If aerosol contamination or large area contamination occurs, the experiment should be stopped immediately and the laboratory closed, the contaminated area of the UV light irradiation disinfection overnight; the next day of the contaminated area 24 hours closed air fumigation disinfection (acetaldehyde disinfection method: 5ml acetaldehyde + 2g potassium permanganate / m3 space).
4.5 Clinical staff should conduct medical follow-up of accident victims and sterilizers.