Safe operating procedures for microbiology laboratories

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1 Laboratory Safety

1.1 Biosafety of Microbiology Laboratory

1.2 Department Safety Document

(a) Biohazard protection: For practical operating procedures, see ("General Requirements for Laboratory Biosafety", National Standard of the People's Republic of China GB19489-2004. Released on 2004-01-05, 2004-10-01 Implementation. )

(b) Laboratory safety: For principles and measures, see ("General Guidelines for Biosafety in Microbiological and Biomedical Laboratories", National Health Industry Standard WS 233-2002. Released on 2002-12-03, implemented on 2003-08-01. )

1.3 Application:

The above operating procedures apply to all departments of the Microbiology Department. Staff or visitors must comply with regulations.

1.4 Regarding new regulations or amendments to existing regulations:

Any new regulations or amendments to existing regulations must be notified to the person responsible for department safety and relevant personnel, who will advise the department director Consider implementation.

1.5 Biological hazards

1.5.1 The level of biological hazards associated with various operations depends on:

a. The infectious material itself being handled:

Infectious substances are divided into different levels according to their potential risk to individuals and communities and the possibility of airborne transmission. For details of the grade classification adopted, see "General Requirements for Laboratory Biosafety"; principles and measures (see "General Guidelines for Biosafety in Microbiological and Biomedical Laboratories").

b. Equipment and facilities used:

Requirements for handling different levels of infectious materials are specified in "Laboratory Safety". These qualified equipment must undertake important clinical work, and department heads must ensure the appropriate levels of contaminants.

c. Training, especially employee safety training and safety experience accumulation. All employees should receive certain training and be able to safely handle any substances that may be encountered in the laboratory, and avoid exposing infectious substances in the laboratory that cannot be handled or are not regulated by the experiment, even if the exposure accident occurs in the laboratory, even if the exposure accident occurs will be handled correctly.

1.5.2 Classification of infectious substances

1.5.2.1 Classification system

Biological hazard protection: [See 1.2 (a)], based on biological factors The degree of harm to individuals and groups is divided into four levels, with harm level IV being the most harmful.

1.5.2.2 Risk group/biosafety level: 1-4

a. Risk group/biosafety level: 1 - not pathogenic to healthy adults, not harmful to the community Hazardous. Normal microbiological procedures are usually sufficient and no special safety equipment is required. Hand washing facilities should be available near the work area. The media contains unidentified substances, so proper handling is critical to all levels of safety.

b. Hazard group/biosafety level: 2 - Human disease-related substances, which are moderately dangerous to laboratory workers and pose a certain degree of community risk. Most of the substances involved in hospital microbiology laboratories belong to this safe level. Biohazard warnings should be posted and access should be restricted. Eye-catching slogans should be used. If Class I and II substances that can cause airborne transmission or splashing are used, corresponding operating stations should be used. . Laboratories should provide similar protective clothing and gloves, and eye protection, especially for contact lens wearers, should also provide hand washing facilities with similar safety levels, and there should also be autoclave sterilization devices adjacent to the work area. There should be specific procedures for the decontamination of all waste and reused equipment.

c. Risk group/biosafety level: 3 - Substances related to human disease, potentially airborne, can cause serious, fatal diseases, highly dangerous to laboratory workers, and have a certain degree of community Danger. All specimens that contain or are suspected of containing biological evidence must be marked with a "Beware of Contamination" label and must be marked on both the specimen container and the declaration form. However, preventive protection is not enough. General precautions should be taken with all blood, body fluids, isolated human tissues, etc., and they should be treated as sources of infection. At this biosafety level, laboratories should control access.

Any work involving this type of material should be operated using a Class I or II workstation, or you can follow Class II operating precautions and decontaminate all waste, including work clothes and reusable equipment.

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

Tick-borne encephalitis virus

Trinidad and Tobago-Congo bovine fever virus

Marburg Virus

Lagsa fever Virus

Junin HF Virus

Machupo HF Virus

Guanaruto HF Virus

Herpesvirus simiae (B Virus)

1.6 The following precautions must be followed by all experimental workers:

1.6.1 Work clothes

a. All experiments Room members must wear the provided work clothes at all times while working. When leaving the laboratory, work clothes must be hung in a designated place, and work clothes must not be worn outside the laboratory.

b. When handling specimens that contain or are suspected of containing biosafety level 3 substances, isolation gowns must be worn over work clothes.

c. Gloves must be worn when handling specimens and media in the biosafety operating station.

1.6.2 Hand sink

Detergent and paper towels must be provided at the hand sink.

1.6.3 Washing hands

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

1.6.4 Food, drinking water, smoking, cosmetics

Food and drinks are not allowed into any room where specimens are processed. Smoking is not allowed anywhere where specimens are processed. Smoking is not allowed in any room where specimens are processed. Use cosmetics wherever specimens are handled.

1.6.5 Wounds and abrasions

Wounds and abrasions on the hands must be covered with protective coverings.

1.6.6 Personal Clothing

Personal clothing is not allowed into the laboratory and must be kept in a locked cabinet.

1.6.7 Oral pipette

This is prohibited. Requires use of mechanical pipetting devices.

1.7 Routine precautions should be used in all operations with 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 thrown into special lids and buckets for safe disposal and to prevent overfilling of containers.

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

4> Broken glass must be placed in a hard container (do not use your hands), and then placed in a black garbage bag. If it is contaminated, use a yellow garbage bag.

5> When in contact with potentially infectious specimen culture media and tissues, protective gloves must be worn to prevent direct skin contact. If the gloves are visibly contaminated, they must be removed before changing. Protective gloves should be worn by workers with dermatitis or wounds on their hands who are required to come into indirect contact with infectious materials.

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

7> When contamination of blood, blood products or other body fluids occurs, you should stop working, wash your hands, wear disposable gloves and then clear the contaminated area. It is recommended to use sodium hypochlorite solution containing 1000ppm available chlorine. If the contaminated area is large, cover it with a damp cloth soaked in sodium hypochlorite solution for 10 minutes to decontaminate it, and mark the corresponding warning. Discard the waste and gloves into a biosafety-level physical container. Be aware that splashes and runoff can carry contamination beyond the main contamination zone.

8> All potentially polluting substances used in the laboratory need to be decontaminated, preferably by high pressure first and then processed.

9> Any operation that may generate splash or aerosol should be performed in a biosafety operating station. These operations include centrifugation, separation of serum, mixing, sonication, collection of tissue fertilized eggs, and processing of specimens containing concentrated infectious material.

1.8 Laboratory Material Disposal

a> Black plastic bags are used to collect uncontaminated or high-pressure waste that needs to be processed.

b> Waste paper is often used to collect uncontaminated waste paper, used paper towels, etc.

c> All potentially infectious materials must be autoclaved or incinerated.

d> All discarded specimens, culture media and laboratory waste must be placed in special containers

e> Use white polypropylene tanks to hold 2% freshly prepared hypochlorous acid solution. It is used for temporary disposal of specimens, contaminated tips and cotton swabs during work, and then high-pressure processing at the end of the work day.

f> Yellow plastic bags are used to dispose of contaminated laboratory waste. They should be sealed and then burned by workers.

g> A lidded, labeled rigid container for holding needle sharps.

1.9 Disinfection of laboratory infectious substances

High pressure

a. All infectious substances in the culture medium must be under high pressure, unless they are to be incinerated. The biggest benefit is that keeping infectious material out of the laboratory is safer.

b. High-voltage equipment used for this purpose should be operated by laboratory workers under the supervision of a medical technician.

c. High-voltage equipment should be regularly tested and inspected by the hospital equipment team. These tests include commercially available spore test strips. Multiple high-voltage equipment should have usage records and be recorded continuously by a medical technician from the date of use. Any discrepancies must be reported to security personnel.

1.10 Decontamination

Decontamination agent:

1> Hypochlorous acid solution: freshly prepared 2 containing 1000ppm available chlorine in a white polypropylene can % hypochlorous acid solution, placed in multiple studios.

2> 5% Printol: Its 50% aqueous solution is used to treat spills on floors and furniture.

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

4> 75% ethanol: mainly used for rapid decontamination of clean surfaces.

1.11 Centrifuge

1.11.1 Notes:

a> The test tubes used in the centrifuge should be thick or plastic and should be Check carefully for any defects.

b> The centrifuge needs to be placed in a suitable position, and the centrifuge cylinder should be visible during operation so that the centrifuge rack can be correctly placed on the rotor.

c> The centrifuge bucket and centrifuge rack are used in pairs and need to be carefully balanced after loading.

d> In order to avoid the rack falling off and the solution in the test tube spilling, start with a low speed and then gradually increase it.

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

f> When centrifuging specimens that contain or are suspected to contain biosafety level 3 specimens, they must be sealed and the sealing cover can only be opened in a level 1 biosafety operation station.

1.11.2 The test tube ruptures during centrifugation

a> If the test tube ruptures or is suspected to occur during centrifugation, the motor should be powered off immediately and the centrifuge should be turned on after 30 minutes. build.

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

c> Notify the security officer immediately.

d> Wear thick gloves and use tweezers or tweezers to remove broken glass fragments.

e> All broken test tubes, glass fragments, centrifuge barrels and rotors must be placed in special disinfectants for decontamination. The disinfectants are required to be non-corrosive and effective against biosafety substances. Then, either soak for 24 hours, or high pressure.

f> Unbroken, capped test tubes should also be placed in another container containing disinfectant1 before disposing of their contents.

i> The centrifuge cylinder must be cleaned with a glutaraldehyde cotton swab, left overnight, wiped again, washed with water and dried.

g> Sealed centrifuge barrels containing biosafety level 3 substances must be taken out in a sealed state and opened in a level 1 biosafety operating station. If the test tube ruptures, the centrifuge barrel sealing cap should be tightly closed and high pressure applied to the centrifuge barrel.

1.12 Ruptures and Spills

Safety personnel should be notified of any significant rupture and spillage. Before handling a spill that contains or is suspected to contain biological cases, advice should be obtained from safety personnel.

1.12.1 Leakage of virus culture medium

a> Cover the spilled contaminant container fragments with paper towels soaked in 20% sodium hypochlorite.

b> Cover 10-15min.

c> Wear disposable gloves, sweep paper towels and debris into a suitable container (such as a plastic bag, but not containing broken glass or other sharp objects), use a piece of cardboard to clean, do not use a brush or Dust dusters unless they are suitable for high pressure. Keep your fingers away from broken glass.

d> Place debris and post-use waste in the laboratory waste bin.

e> Wipe the contaminated area and possible splash areas around it with regular work disinfectant.

1.13 Leaked specimens

Specimens that are not leaked in the laboratory must be sealed in plastic and returned to the ward.

1.14 Ultraviolet disinfection: Culture media, reagent packaging and patient serum should be stored in -70°C and -30°C refrigerators. Gloves should be worn when handling materials stored in refrigerators at temperatures of -30°C or below, such as when taking specimens out of or placing specimens in low-temperature refrigerators.

1.15 Biological safety cabinet (station)

The laboratory should be equipped with a Class II biological safety cabinet.

1.15.1 Level

I: Basic requirements: Directional flow protection requires airflow to flow into the safety cabinet from the outside and away from the operator, directed toward infectious agents. After being filtered and discharged by HEPA, it should be led to the outdoors through a duct to remove all kinds of decontaminated steam.

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

IIA: 70% of the filtered gas is recirculated to the work area, and the exhaust gas is directed outdoors for HEPA filtration. The average airflow velocity in the flow-through work area is 0.4m/s or slightly higher.

IIB: The average inflow gas flow rate is 0.5m/s or better. According to different types (IIB1, IIB2, IIB3), the gas discharge ratio ranges from 70% to 100%. Choose different types according to the job. For example, toxic chemicals and radioactive isotopes cannot be recycled to the work area.

III: Basic requirements: Operators are safely isolated from infectious agents, and the entire operation process must be improved. The environment where the safety cabinet is located must also be isolated to a certain extent to prevent gloves from breaking.

1.15.2 Installation of biological safety cabinets

To perform a certain safety protection function, the safety cabinet must pay attention to the installation location. The exhaust gas should pass through the pipeline to protect the environment from pollution. If you need to purchase this type of equipment, you should contact the CUHK Safety Office for guidance on type, brand, style, installation location and installation, and proximity to other facilities.

1.15.3 Routine inspections and safe operations as well as disinfection of the work area

There is an operating procedure that describes in detail the routine use of the safety cabinet and should also include the actions that should be taken in the event of an accident. action. Each user must read the regulations and sign them.

1.15.4 Note:

Safety cabinets should be kept in an unsafe condition as much as possible, but sometimes users are forced to do so due to safety cabinet accidents. At this time, a notice should be posted to warn others against further use.

1.15.5 Decontamination and Recertification

Contamination work should be done by trained personnel wearing personal protective equipment. Requirements: The safety cabinet is steamed with dry paraformaldehyde steam under closed conditions, with a concentration of 8500ppm. (The volume of the safety cabinet should include peripheral supplies and exhaust filtration equipment.

) At a temperature of 20-25°C and a relative temperature of not less than 60% for 4 hours, formaldehyde vapor, if allowed, can be directly extracted outdoors. If not, it can be absorbed with amino heavy carbon salt in the safety cabinet.

Because of the limited penetration of air, it is best to use HEPA filters when handling potentially infectious materials, and high pressure or incineration before disposal.

1.17.5.2 Recertification

This work is carried out by specialized personnel. After decontamination, the performance of the safety cabinet is recertified, the filter is replaced, and there is testing for air leakage and debugging. Check whether the air flow rate complies with regulations and test the performance of the filter. Recertification should be done at least once a year or after the unit is moved or the filter is repaired. Records of decontamination, maintenance and recertification shall be maintained and shall be readily accessible to users.

2. Experimental safety operating procedures:

2.1 Staff should prevent the spread and infection of pathogenic microorganisms during the collection of specimens, and provide detailed information on the source, collection process and methods of the specimens. Record.

2.3 Smoking, eating, etc. are absolutely prohibited during the experiment, and do not touch the head or face with your hands.

2.4 When handling samples where highly hazardous aerosols are likely to be generated, appropriate personal protective equipment, biosafety cabinets and/or other physical protective equipment are required to be used at the same time. If aerosols containing biological agents can be generated, operations should be performed in an appropriate biological safety cabinet. Operations such as isolation and culture of bacteria, unsealing of bacterial strains, transfer, grinding, and dilution should all be performed in a biological safety cabinet.

2.5 When operating with an inoculating loop, the inoculating loop should be burned in the inner flame of the work lamp to avoid splashing of bacterial liquid or bacterial clumps.

2.6 When diluting the bacterial solution, the straw or needle should be slowly inserted into the bottom of the test tube or flask and be careful to avoid the generation of bubbles or aerosols.

2.7 When using a syringe to add samples, do not re-insert the used needle or pull out the syringe and needle. It should be placed directly into the sharps collector to avoid scratching the skin and causing inoculation infection.

2.8 The strain library must be managed by dedicated personnel and implemented in accordance with the national management regulations for microbial strains and viruses. 2.9 During the operation of germs, do not wear contaminated protective gloves and touch door handles, instruments or areas outside the germ area to avoid expanding the scope of contamination due to carelessness.

2.10 After the test, all test instruments and items that may be in contact with biohazardous substances or contaminated during the operation must be sterilized by high pressure. Equipment and instruments that cannot be sterilized by high pressure must be disinfected by high pressure. Use an effective disinfectant to scrub and then use ultraviolet light to disinfect at close range and for a long time.

2.11 If accidental contamination occurs during the experiment, the person in charge should be notified immediately and prepared to deal with the contaminants and the corresponding area. Other prohibited methods should not be used for disinfection without authorization.

2.12 Any microbial samples and waste in the laboratory must be sterilized by high temperature and high pressure before they can be disposed of as general garbage.

2.13 Any personal protective equipment used in the laboratory should comply with the requirements of relevant national standards. Laboratories should ensure that adequate clean protective clothing with an appropriate level of protection is available for use. Other personal protective equipment should also be worn, such as gloves, protective goggles, masks, head and face protection, etc.

3. Laboratory waste treatment and disinfection operating procedures:

3.1 Clinical specimens and contaminated disposable items containing biohazardous substances in the laboratory should be removed after the test is completed. The operating table or experimental area should be disinfected with ultraviolet light at close range for more than 2 hours, and then subjected to high-pressure disinfection before discarding or incineration.

3.2 Reusable experimental supplies and equipment should be disinfected by close-range ultraviolet light irradiation for more than 2 hours on the operating table or experimental area after completion of the experiment, and then handed over to relevant personnel for high-pressure disinfection and boiling and cleaning .

3.3 Test tubes, pipettes, and syringes used for experiments must be placed in leak-proof containers and taken out after autoclaving.

3.4 Cultures or laboratory waste must be sterilized by high pressure and irradiated with ultraviolet light at close range for a long time before being discarded. The accumulation of garbage and laboratory waste is not permitted. Filled containers should be removed regularly. It should be stored in a designated safe place, usually within the laboratory area, until decontamination or final disposal.

3.5 Laboratory waste should be safely transported out of the laboratory in appropriate sealed and leak-proof containers.

Hazardous gases, aerosols, sewage, and waste liquids should be discharged after appropriate harmless treatment and should comply with relevant national requirements.

3.6 During the experiment, if the specimen or the pre-digestion solution containing the specimen is knocked over and contaminates the operating table or floor, the contaminated area should be covered with toilet paper filled with 70% alcohol. The toilet paper should be removed after 15 minutes. Can be removed.

3.7 Unsterilized sewage in the laboratory is prohibited from being discharged directly into the public drainage system, nor is it allowed to be mixed with residents' domestic waste.

4. Accident handling procedures:

4.1 If an accident occurs, the laboratory supervisor and the hospital biosafety office must be notified immediately, and the scene of the accident must be inspected under the guidance and supervision of relevant personnel. It is absolutely forbidden to give non-standard treatment to the accident scene without reporting it.

4.2 During the experiment, if contaminants splash onto the surface of the body, or cuts, stab wounds, burns, scalds, etc. occur, the experimental work should be stopped immediately for emergency treatment and the contaminated experimental clothes should be replaced. , clean the skin surface with disinfectant, disinfect the wound with iodine or alcohol, and flush the eyes with sterile saline.

4.3 If the bacterial liquid overflows, the culture tube containing the bacteria is broken, etc., causing medium or small area contamination, the contaminated area can be covered with gauze that is more than 25% larger than the contaminated area, and the edges are surrounded by absorbent cotton. Pour 5% phenol solution or 70% alcohol into the gauze, soak it for more than 2 hours (add an appropriate amount of solution during this period to prevent drying), and then irradiate it with a UV lamp at close range (within 1 meter) for more than 2 hours; soak contaminated instruments, containers, etc. immediately Soak in 70% alcohol for more than 2 hours, then enter the room again wearing protective clothing, put the contaminated spill and the gauze used for cleaning into a double-layer plastic bag that can withstand high pressure, and pressurize as soon as possible.

4.4 If aerosol pollution or large-scale pollution occurs, the experiment should be stopped immediately and the laboratory should be closed. The contaminated area should be disinfected by ultraviolet light overnight; the contaminated area should be fumigated and disinfected with 24-hour closed air the next day. (Aldehyde disinfection method: 5ml acetaldehyde + 2g potassium permanganate/m3 space).

4.5 Clinical medical staff should conduct medical follow-up on accident victims and disinfection personnel.