1 laboratory safety

1. 1 Biosafety of Microbiological Laboratory

1.2 departmental safety documents

(1) Biological hazard protection: refer to the national standard GB 19489-2004 "General Requirements for Laboratory Biosafety". It was released on 0 1-5, 2004 and implemented on 0101. )

(b) Laboratory safety: For principles and measures, see (General Guidelines for Biosafety of Microbiology and Biomedical Laboratories, China People's Congress and China Health Industry Standard WS 233-2002). Published in 2002- 12-03 and implemented in 2003-08-0 1. )

1.3 application:

The above procedures are applicable to all departments of microbiology department. Staff or visitors must abide by the rules.

1.4 amendments to new regulations or existing regulations:

Any new regulations or amendments to existing regulations must be notified to the person in charge of department security and related personnel, who will advise the department head to consider implementing these regulations.

1.5 biological hazard

1.5. 1 The biohazard level related to various operations depends on:

A. Disposal of infectious substances by themselves:

According to the potential danger of infectious substances to individuals and communities and the possibility of air transmission, they are divided into different grades. See "General Requirements for Laboratory Biosafety" for the classification adopted. Principles and measures (see general guidelines for biosafety in microbiology and biomedical laboratories).

B. Equipment and facilities used:

"Laboratory Safety" stipulates the requirements for handling infectious substances of different grades. These qualified equipment should undertake important clinical work, and the department head must ensure the pollutant level.

C. Training, especially safety training and safety experience accumulation of employees. All employees should receive certain training, be able to handle any substances that may be encountered in the laboratory safely, and avoid contact with infectious substances that cannot be handled in the experiment or exceed the laboratory regulations. Even if the exposure accident occurs in the laboratory, it should be handled correctly.

1.5.2 Classification of infectious substances

1.5.2. 1 classification system

Biological hazard protection: [see 1.2 (a)]. According to the harm degree of biological factors to individuals and groups, it is divided into four grades, and the harm grade ⅳ is the most harmful.

1.5.2.2 Hazard group/biosafety level: 1-4.

A. Risk group/biosafety level: 1- No pathogenicity to healthy adults and no harm to the community. Usually, general microbial operation is enough, no special safety equipment is needed, and there should be hand washing facilities near the work. The culture medium contains unknown substances, so proper treatment is very important for all levels of safety.

B risk group/biosafety level: 2- substances related to human diseases, which are moderately dangerous to laboratory staff and have a certain degree of community danger. The vast majority of substances involved in the hospital microbiology room belong to this safety level, and biological hazard warnings should be posted to restrict access. Eye-catching slogans should be used. If the first and second substances can spread or splash in the air, the corresponding operation console should be used. The laboratory should provide similar protective clothing and gloves, goggles, especially goggles with contact lenses, and hand washing facilities with similar safety level, and there should be autoclaving devices near the work area. There should be specific procedures for disinfection of all wastes and reusable equipment.

C. Risk population/biosafety level: 3- Substances related to human diseases, which are potentially airborne, can cause serious and fatal diseases, are highly dangerous to laboratory staff and have a certain degree of community danger. All specimens containing or suspected to contain biological cases must be labeled with "Pollution Precautions" and marked on the specimen container and application list. However, as a preventive protection, this is not enough. All blood, body fluids, separated human tissues, etc. It should be realized that general protection should be regarded as the source of infection. At this biosafety level, the laboratory should control access to it. All the substances involved should be operated by the first or second stage workbench, or all the wastes, including work clothes and reusable equipment, can be decontaminated according to the precautions of the second stage operation.

D. Risk group/biosafety level: 4-Danger to individuals, such as biosafety level 3, but highly dangerous to the community. These infectious substances include:

Tick encephalitis virus

Chilean Banya-Congo bovine hemorrhagic fever virus

Marburg virus

Ebola virus

Lagsa fever virus

Zhu Ning HF virus

Machupo HF virus

Guanaruto HF virus

Monkey herpesvirus (B virus)

All experimenters must observe the following precautions (1.6):

1.6. 1 work clothes

All laboratory members must always wear work clothes provided during work. When leaving the laboratory, the work clothes must be hung in the designated place, and it is not allowed to wash the work clothes outside the laboratory.

B. When handling specimens containing or suspected of containing biosafety level 3 substances, isolation gown must be worn outside the work clothes.

C gloves must be worn when handling specimens and culture media in the biosafety console.

1.6.2 sink

The sink must be equipped with detergent and paper towels.

1.6.3 Wash your hands

All staff must wash their hands before leaving the work area and first take off their work clothes and gloves.

1.6.4 food, drinking water, smoking and cosmetics

Food and drinks are not allowed to be brought into any room where specimens are processed. Smoking is not allowed anywhere where specimens are handled. Cosmetics are not allowed in any place where specimens are handled.

1.6.5 wounds and abrasions

Wounds and abrasions on hands must be covered with protective materials.

1.6.6 Personal clothes

Personal clothes are not allowed to be brought into the laboratory and must be stored in a locked cabinet.

1.6.7 oral pipette

This is forbidden. Mechanical pipetting devices are required.

1.7 routine precautions are applicable to all operations of blood, body fluids and tissue samples:

1 & gt； Avoid using syringes, needles or other sharp tools as much as possible.

2> Used needles and disposable knives must be thrown into special containers with lids and barrels for safe disposal and to prevent the containers from being overloaded.

3> Used needles are not allowed to be reused or operated by hand.

4> broken glass must be put into a hard container (don't use your hands) and then put into a black garbage bag. If it is polluted, use yellow garbage bags.

5> Wear protective gloves when contacting the culture medium and tissues of potentially infectious specimens to prevent direct contact with the skin. If gloves are obviously contaminated, they must be removed before replacement. Workers with dermatitis or wounds on their hands should wear protective gloves if they need indirect contact with infectious substances.

6> Wash your hands regularly after handling specimens, finishing work and even wearing gloves specified above.

7> When blood, blood products or other body fluids are contaminated, you should stop working and wash your hands, and wear disposable gloves to clean the contaminated area. It is suggested to use sodium hypochlorite solution containing available chlorine 1000ppm. If the polluted area is large, cover 10min with a wet cloth soaked with sodium hypochlorite solution to remove the pollution, and mark the corresponding warning, and discard the waste and gloves into the biosafety level physical container. Pay attention to splashing water droplets and flow will bring pollution outside the main pollution area.

All potential pollutants used in the 8> laboratory need to be decontaminated, and it is best to treat them at high pressure first.

9> Any operation that may generate splash or aerosol should be carried out on the biosafety console. These operations include centrifugation, serum separation, mixing, ultrasound, collecting the tissue of fertilized eggs and processing samples containing concentrated infectious substances.

1.8 laboratory material handling

A> Black plastic bags are used to collect unpolluted or high-pressure wastes that need to be treated.

B> waste paper is often used to collect unpolluted waste paper, used paper towels and so on.

C> All potentially infectious substances must be autoclaved or incinerated.

D> All discarded specimens, culture media and laboratory wastes must be put in special containers.

E & gt2% freshly prepared hypochlorous acid solution is packed in a white polypropylene tank, which can be used for the temporary treatment of discarded specimens, contaminated tips and cotton swabs at work, and then subjected to high pressure treatment after one day's work.

F> yellow plastic bags are used to treat contaminated laboratory wastes. Workers should seal them up and burn them.

G> A hard container with a lid and label is used to hold sharp needles.

1.9 disinfection of infectious substances in the laboratory

high-pressure

A all infectious substances in the culture medium should be pressurized unless they are prepared for incineration. The biggest advantage of high pressure is that infectious substances can leave the laboratory to ensure safety.

The high-voltage equipment used for this purpose should be operated by laboratory staff under the supervision of medical technicians.

C. High-voltage equipment shall be regularly tested and overhauled by the hospital equipment group. These tests include commercially available spore test paper experiments. A number of high-voltage equipment should have use records, which should be continuously recorded by medical technicians from the date of use. Any discrepancy should be reported to the security personnel.

1. 10 is used for decontamination.

Detergent:

1 & gt； Hypochloric acid solution: 2% hypochlorous acid solution containing available chlorine 1000ppm is freshly prepared in white polypropylene tanks and placed in multiple studios.

2 & gt5% Printol: Its 50% aqueous solution is used to treat the pollution splashed on the floor and furniture.

3> Glutaraldehyde: freshly prepared to a certain concentration, mainly used for decontamination of instruments such as centrifuges that cannot use sodium hypochlorite.

4 & gt75% ethanol: mainly used for quick decontamination of clean surfaces.

1. 1 1 centrifuge

1.11.1Note:

The test tube used in a> centrifuge should be made of thick tube or plastic, and it should be carefully checked for defects before centrifugation.

B> centrifuge should be placed in a proper position, and the centrifuge cylinder should be visible during operation, so that the centrifuge frame can be correctly placed on the rotor.

C> centrifuge barrels and centrifuge frames are used in pairs and should be carefully balanced after loading.

D> spilled the solution in the test tube in order to avoid falling off the shelf. At first, the rotation speed was very low, and then it gradually increased.

The inner cylinder of e> centrifuge should be searched regularly by senior personnel, and the inner wall must be cleaned regularly with glutaraldehyde to remove pollution.

When f> centrifuge level 3 specimens containing or suspected of containing biosafety operation, it must be sealed, and the sealing cover can only be opened in the 1 level biosafety operation console.

1. 1 1.2 centrifuge, the test tube is broken.

A> If the test tube breaks or is suspected during centrifugation, the motor should be turned off immediately, and the centrifuge cover cannot be opened until 30 minutes later.

B> If rupture is found after centrifugation, the machine cover shall be closed immediately and kept for at least 30 minutes.

C> immediately notify the security officer.

D> put on thick gloves and use tweezers or cotton needles to remove broken glass fragments.

E> All broken test tubes, glass fragments, centrifugal barrels and rotors must be put into a special disinfectant to remove pollution. The disinfectant is required to be non-corrosive and effective for soaking biosafety substances. Then, either soak for 24 hours or under high pressure.

F> undamaged test tubes with lids should also be put into another container filled with disinfectant, and then their contents should be treated.

My> centrifugal cylinder must be cleaned with glutaraldehyde cotton swab overnight, then wiped repeatedly, then washed with water and dried.

G> sealed centrifuge barrels filled with biosafety level 3 substances must be taken out in a sealed state and opened in the biosafety 1 level operation console. If the test tube is broken, the sealing cover of the centrifuge barrel should be loosely covered to apply high pressure to the centrifuge barrel.

1. 12 Rupture and overflow

Security personnel should be informed of any major rupture spillage. When dealing with spills containing or suspected of containing biological substances, the safety personnel should be consulted first.

1. 12. 1

A> Cover the spilled container fragments with a paper towel soaked with 20% sodium hypochlorite.

B> coverage 10- 15 minutes.

C> put on disposable gloves, clean paper towels and debris with suitable containers (such as plastic bags, but excluding broken glass or other sharp objects), and clean them with a piece of cardboard. Do not use brushes or dust collectors unless they are suitable for high pressure. Keep your fingers away from the broken glass.

D> puts debris and used garbage into the laboratory dustbin.

E> Wipe the contaminated area and the possible splash area around it with routine working disinfectant.

1. 13 leaked specimen

The samples that have not leaked in the laboratory should be put in plastic sealed bags and sent back to the ward.

1. 14 ultraviolet disinfection: the culture medium, reagent repackaging and patient serum should be kept in refrigerators at -70℃ and -30℃. Wear gloves when handling materials stored in a refrigerator at-30 C or below, for example, when taking out samples or putting them in a low-temperature refrigerator.

1. 15 Biosafety Cabinet (Taiwan Province)

The laboratory should be equipped with a secondary biosafety cabinet.

1. 15. 1 level

1. Basic requirements: directional flow protection, which requires airflow to flow into the safety cabinet from the outside, away from the operator and pointing to infectious substances. After being filtered and discharged by HEPA, it is tightly guided to the outside by a conduit to eliminate all kinds of steam after decontamination.

Basic requirements: the filtered gas flows vertically, which not only protects the operator but also protects the work from pollution.

IIA: 70% of the filtered gas is circulated to the working area, and the waste gas is led outside and filtered by HEPA. The average air velocity in the internal flow working area is 0.4m/s or slightly higher.

IIB: The average inflow gas velocity is 0.5 m/s or more. According to different types (IIB 1, IIB2, IIB3), the gas emission ratio ranges from 70% to 100%. Choose different types according to the job. For example, toxic chemicals and radioisotopes cannot be recycled to the work area.

Basic requirements: Operators should safely isolate infectious diseases, and the whole operation process should be improved. The environment where the safety cabinet is located should also be isolated to a certain extent to prevent gloves from breaking.

1. 15.2 installation of biosafety cabinet

In order to play a certain role in safety protection, the safety cabinet must pay attention to the installation position, and the exhaust gas should pass through the pipeline to protect the environment from pollution. If you need to purchase this kind of equipment, you should contact the security office of CUHK for guidance on the type, brand, style, installation location and installation, and the adjacent relationship with other facilities.

1. 15.3 routine inspection and safe operation, and disinfection of the work area.

There are operating instructions detailing the daily use of the safety cabinet, which should also include the actions that should be taken when the safety cabinet has an accident. Every user must read the regulations before signing.

1. 15.4 Note:

Safety cabinets should be kept as unsafe as possible, but sometimes users are forced because of accidents in safety cabinets. At this time, a notice should be posted to warn others not to use it again.

1. 15.5 Purification and recertification

By trained personnel, wearing personal protective equipment, to do pollution work. Requirements: The safety cabinet is steamed with paraformaldehyde dry steam under closed conditions, and the concentration is 8500ppm. (The volume of the safety cabinet should include peripheral power supply and waste gas filtering equipment. ) at a temperature of 20-25℃ and a relative temperature of not less than 60% for 4h. If allowed, formaldehyde vapor can be directly taken out of the room; If not, it can be absorbed by amino bicarbonate in the safety cabinet.

Because of the limited penetration of air, it is best to use HEPA filter when dealing with potentially infectious substances, and then discard them after high pressure or incineration.

1. 17.5.2 Re-certification

This work is carried out by specialized personnel. After decontamination, the performance of the safety cabinet is re-certified, the filter screen is replaced, whether there is air leakage, whether the air flow meets the requirements, and the performance of the filter screen is tested. Re-certification should be carried out at least once a year, or after the equipment is moved and the filter is repaired. Records of decontamination, maintenance and recertification shall be kept for easy reference by users.

2. Experimental safety operation procedures:

2. 1 The staff should prevent the spread and infection of pathogenic microorganisms in the process of collecting specimens, and make detailed records on the source, collection process and methods of specimens.

2.3 Smoking, eating, etc. It is absolutely forbidden during the experiment. Don't touch your head and face with your hands.

2.4 When highly dangerous aerosols are easily generated during sample processing, appropriate personal protective equipment, biosafety cabinets and/or other physical protective equipment should be used at the same time. If aerosol containing biological factors may be generated, it should be operated in a suitable biosafety cabinet. Bacteria separation and culture, strain unsealing, seed transfer, grinding, dilution and other operations should be carried out in the biosafety cabinet.

2.5 When using the inoculation ring, the inoculation ring should burn in the internal flame of the working lamp to avoid splashing of bacterial liquid or bacterial blocks.

2.6 When diluting the bacterial liquid, slowly insert the straw and needle into the bottom of the test tube or flask, and pay attention to avoid bubbles or aerosols.

2.7 When using a syringe to add samples, the used needle should not be put back into the sleeve or pulled off from the syringe and needle, but should be directly put into the sharp instrument collector to avoid cutting the skin and causing inoculation infection.

2.8 The strain bank shall be managed by designated personnel, and shall be implemented in accordance with the national measures for the administration of microbial strains. 2.9 Don't wear contaminated protective gloves to touch doorknobs, instruments or areas outside the poisonous bacteria area during poisonous bacteria operations, so as to avoid inadvertently expanding the scope of pollution.

2. After the10 test, the testing instruments and articles that may come into contact with biological hazards or be contaminated during the operation must be disinfected under high pressure, and the equipment and instruments that cannot be disinfected under high pressure should be scrubbed with an effective disinfectant, and then disinfected for a short distance and a long time with an ultraviolet lamp.

2. 1 1 In case of accidental pollution in the experiment, the person in charge shall be informed immediately, and preparations shall be made for handling pollutants and corresponding areas, and other prohibited methods shall not be used for disinfection without authorization.

2. 12 Any microbial samples and wastes in the laboratory must be sterilized at high temperature and high pressure before they can be treated as general garbage.

2. 13 Any personal protective equipment used in the laboratory shall meet the requirements of relevant national standards. The laboratory should ensure that there are enough clean protective clothing with appropriate protection level available. You should also wear other personal protective equipment, such as gloves, goggles, masks, head and face shields, etc.

3. Laboratory sewage treatment and disinfection procedures:

3. 1 clinical specimens containing biological hazards and contaminated disposable goods in the laboratory should be disinfected with ultraviolet lamp at close range for more than 2 hours at the operation desk or experimental area after inspection, and can only be discarded or burned after high-pressure disinfection.

3.2 Reusable experimental supplies and equipment should be disinfected at close range for more than 2 hours on the operating table or experimental area after the experiment is completed, and then handed over to relevant personnel for high-pressure disinfection and boiling washing.

3.3 Test tubes, straws and syringes used in the experiment should be packed in closed containers and taken out after autoclaving.

3.4 Cultures or laboratory wastes must be autoclaved and exposed to ultraviolet light for a long time before being discarded. It is not allowed to pile up garbage and laboratory waste. Full containers should be transported away regularly. Before decontamination or final disposal, it should be stored in a designated safe place, usually in the laboratory area.

3.5 Laboratory wastes shall be safely transported out of the laboratory in proper sealed and leak-proof containers. Harmful gases, aerosols, sewage and waste liquid should be discharged after proper harmless treatment and should meet the relevant national requirements.

3.6 During the experiment, if the specimen or pretreatment liquid containing the specimen is knocked over and pollutes the console or the ground, the polluted area should be covered with toilet paper saturated with 70% alcohol, and the toilet paper can be removed after 15 minutes.

3.7 Laboratory sewage without high temperature disinfection is prohibited from being directly discharged into the public drainage system and mixed with domestic garbage.

4. Accident handling procedures:

4. 1 In case of an accident, the laboratory director and the hospital biosafety office must be informed immediately, and the scene of the accident should be handled under the guidance and supervision of relevant personnel. It is absolutely forbidden to give irregular treatment to the scene of the accident without reporting.

4.2 During the experiment, if pollutants splash on the body surface, or there are cuts, stab wounds, burns, scalds, etc. , should immediately stop the experimental work for emergency treatment, and replace the contaminated experimental clothing. The skin surface should be cleaned with disinfectant, the wound should be disinfected with iodine or alcohol, and the eyes should be washed with sterile saline.

4.3 If the medium or small area is polluted due to the overflow of bacterial liquid or the rupture of the culture tube containing strains, it can be covered with gauze that is more than 25% of the polluted area, and the edge is surrounded by absorbent cotton. Pour 5% phenol solution or 70% alcohol into gauze, soak it for more than 2 hours (add a proper amount of solution to prevent drying), and then irradiate it with ultraviolet lamp for more than 2 hours (within 1 meter); Contaminated instruments, containers, etc. Immediately soak in 70% alcohol for more than 2 hours, then put on protective clothing and enter the room again, and put the contaminated spill and cleaning gauze into a double-layer plastic bag that can withstand and apply high pressure as soon as possible.

4.4 In case of aerosol pollution or large-scale pollution, the experiment should be stopped immediately and the laboratory should be closed, and the polluted area should be disinfected with ultraviolet lamp overnight; The next day, the polluted area was fumigated with closed air for 24 hours (acetaldehyde disinfection method: 5ml acetaldehyde +2g potassium permanganate /m3 space).

4.5 Clinical medical personnel shall conduct medical follow-up for accident victims and disinfection personnel.