WEEK 1.pdf

LABORATORY QUALITY 1 SYSTEM BIOSAFETY & WASTE MANAGEMENT necessary part of molecular biology laboratory 1. Provide us knowledge needed to solve key problems in human health 2. One of major components of laboratory quality system 3. The application of the knowledge and techniques is very important to...

LABORATORY QUALITY 1 SYSTEM BIOSAFETY & WASTE MANAGEMENT necessary part of molecular biology laboratory 1. Provide us knowledge needed to solve key problems in human health 2. One of major components of laboratory quality system 3. The application of the knowledge and techniques is very important to prevent the Laboratory and personnel exposure to potentially infectious agents or biohazard 4. In any laboratory, safety is very important. We must break the chain of infection and break the transmission of diseases through proper hand hygiene. PRIMARY GOAL OF LQMS ensure that lab personnel is safe from potentially harmful of infectious agents SAFETY Coming in contact with human blood or blood products (plasma, serum, etc.), or with certain chemicals used in the laboratory, is potentially hazardous. Fundamental objective of any biosafety in molecular biology lab is to contain potentially harmful agents. Safety involves taking precautions to protect you and coworkers against infection, injury or poisoning. HAND HYGIENE Persons must wash their hands after working potentially hazardous materials and before leaving the Laboratory. 20 SECONDS LATHER & SCRUB 10 SECONDS RINSE DO NOT USE HAND DRYERS 40-60 SECONDS HANDWASHING PROCEDURE ALCOHOL & SANITIZERS ALTERNATIVES ALCOHOL-BASED CLEANSERS not recommended after contact with spore- forming bacteria, including Clostridium difficile and Bacillus sp. Hands should be washed regularly before and after touching the specimens before and after putting your gloves A number of infectious diseases can be spread through contaminated hands respiratory infection (influenza, coronavirus) G.I.T infection (salmonella) Handwash with soap & water if hands are visibly soiled Liquid soap is better than bar soaps to prevent higher chances of transmission If hands are not visibly soiled, use alcohol-based sanitizers DECONTAMINATION/DISINFECTION DECONTAMINATION Process of removing or neutralizing chemical or Biological Agents so that they no longer pose a hazard. Reduce level of microbial contamination Free from the risk of infection transmission 1. STERILIZATION Removal or destruction of all forms of life, including bacterial spores killing all forms of microorganisms from vegetative to spore-forming organisms autoclave: most common gamma radiation: deep penetrating destroying dna and rna short wavelength but high energy used only for heat sensitive materials (ex. syringe) 2. disinfection removal, inhibition or killing of microorganisms including potential pathogens by using chemical agents usually or inanimate objects does not remove all bacterial spores eliminate most pathogen but not all microbes reduce level of microbial contamination chemical disinfectants sodium hypochlorite ethyl alcohol used in surfaces, inanimate objects 3. antisepsis antimicrobial substances typically applied in the skin to reduce possibility of sepsis formation or putrefaction similar with disinfectant (can’t kill bacterial spores) solutions used for cleaning wounds, can be applied directly to skin (human & animals) most commonly used antiseptic: iodine solution hydrogen peroxide 10% sodium hypochlorite 70% ethyl alcohol benzalkonium chloride (found in lysol) standard precaution Minimum infection prevention practices that apply to all patient care, regardless of suspected or confirmed infection status of the patient, in any setting where health care is delivered (CDC) 1. wear proper ppe, avoid physical hazards avoid touching eyes, nose or mouth with gloved or unwashed hands 2. all specimens shall be treated as infectious (even if specimen is free from disease; regardless if blood is present 3. avoid wearing jewelry 4. refrain from using mobile electronic devices inside the laboratory 5. keep work area tidy, clean, and free of cluster and materials not necessary for the work being done. decontamination solutions FORMS OF DECONTAMINATION 70% ETHYL ALCOHOL SODIUM HYPOCHLORITE SPILLS: 1:10 OR 10% GENERAL: 1:100 OR 1% CONTACT TIME: 10-15MINS MORE SPILLS REQUIRES HIGHER CONTACT TIME ALL SURFACES AND EQUIPMENT MUST BE DECONTAMINATED BEFORE AND AFTER USAGE. 1. CHEMICAL AGENTS EFFICACY IS BASED ON DIFFERENT FACTORS: ORGANIC LOAD COMPOSITION OF CHEMICAL AGENTS MICROBIAL LOAD Number of bacteria or microorganism present in the specimen Higher viral load= higher conc. of reagents used Type of Organism Spore forming, capsulated, enveloped, vegetative (bacteria) enveloped, or non-enveloped(virus) Condition of surface to be decontaminated Disinfectant concentration, pH, temperature, contact time, environmental humidity RESISTANCE OF MICROORGANISMS TO DISINFECTANTS Prions Bovine spongiform encephalopathy (Mad cow) Creutzfeldt- Jakob disease BACTERIAL SPORES Bacillus subtilis Clostridium sporogenes MYCOBACTERIA Mycobacterium tuberculosis; Mycobacterium bovis HYDROPHILIC VIRUSES (NON-LIPID, NONENVELOPED) Rhinovirus; Adenovirus Fungi Cryptococcus sp. Candida sp. VEGETATIVE BACTERIA Streptococcus pneumoniae; Staphylococcus aureus LIPOPHILIC BACTERIA (LIPID, ENVELOPED) Herpes Simplex; Cytomegalovirus; HIV PRION most resistant to disinfection also known as Infective proteins. They don't have nucleic acids. The patient who has CJD undergoes brain surgery, all the equipment and instrument must be disinfected. CJD / Creutzfeldt-Jakob disease is an example of human prion. DISINFECTANTS: destroys nucleic acids (DNA and RNA) Bacterial spores can survive in higher temperature. Mycobacteria has different cell membrane so it is harder for the disinfectants to penetrate. Hydrophilic Viruses are the least resistant due to their lipid in the membrane. The disinfectant can easily melt the lipid. TYPES OF CHEMICAL DISINFECTANTS 1. SODIUM HYPOCHLORITE Contact Time: 10-15 Minutes Bought in concentrations of 5-8.25%, most common: 5.25% PREPARATION 1:10 or 1:100 At least 5000 ppm, no more than 10,000 ppm Chlorine for decontamination CORROSIVE (highly reactive): Needs to be washed after (Steel) DO NOT USE CONCENTRATED SOLUTION (produces toxic gas) DO NOT AUTOCLAVE BLEACH SOLUTIONS Effect: Oxidative effect of Hypochlorous acid Avoid the following: Exposure to air (↓ free Chlorine CONC. due to Chlorine evaporation) do not store in direct sunlight and varying temperatures (NaOCl solutions can be stored up to 6 months if this is followed!) Bleach loses 20-50% of its concentration/efficacy after 6mos Household bleach active ingredient (Ex: Zonrox) SPILLS (Chemical/Blood/Specimen spills) GENERAL DISINFECTION 10% (1PART + 9PARTS H2O) 1% (1PART + 99PARTS H2O) 2. ALCOHOL Best preparation: 60-90% Alcohol ↑ Water conc while maintaining high Alcohol concentrations ↑ contact time of Alcohol to microorganism best prepartion 60-90% alcohol Disinfection: Ethyl Alcohol ANTISEPSIS ethyl and isopropyl best concentration to achieve contact time requirement 70% more volatile easily evaporates so it does not meet the contact time requirement If used as a disinfecting agent: Without Bleach:2-5minutes With Bleach: No need for contact time Bactericidal, Fungicidal, Virucidal, non-Sporicidal Allowed to evaporate from the surface to which they Were applied to achieve maximum effectivity. Effect: Cause denaturation of protein and dissolution of lipid membrane Should not be used as a lone decontaminant for MOLECULAR TESTING – causes aggregation of Nucleic Acids not eradicating them 70% Ethyl Alcohol: Disinfectant & antiseptic at the same time can be consumed by human since it is found in liquors Isopropyl Alcohol: also known as rubbing alcohol which is commonly used as antiseptic only 3. uv Room disinfectant A type of non-ionizing radiation that causes damage to cellular DNA by producing Thymine dimers Wavelengths: 250nm: Most lethal wavelength: >1 nm: Longer wavelength and low energy Microorganisms are destroyed when water is passed under the UV Lamps Quality Control Indicator: Bacillus pumilus disadvantages: Can only destroy those under its direct contact. Eye and skin irritation, skin cancer: Basal cell carcinoma, squamous cell carcinoma, malignant melanoma Wears out overtime. Has little to no effect on RNA. 4. LYSOL Active Ingredients: Benzalkonium Chloride or Hydrogen Peroxide Capable of oxidative burst Formerly has PHENOL (Carcinogenic) Phenolic Compounds- Earliest germicide KNOWN EFFECTIVE AGAINST MANY MICROORGANISMS Effects: Benzalkonium Chloride: Disruption of cell membrane, resulting in leakage of cell contents. Hydrogen Peroxide: Degrades Organic compounds by the highly reactive Hydroxyl radical (-OH). SPILL CONTROL GENERAL STEPS 1. Add concentration bleach (>5.25% available Chlorine) to a final concentration of 10% Bleach 2. Let the solution sit for 30-60 minutes. Larger volumes may require longer contact times. 3. Dispose of the solution down the sanitary sewer. INACTIVATION OF PRIONS INSTRUMENT: Immerse in 1N NaOH or 2.5% NaOCl for 1 hour; remove and rinse in water, and then transfer to open pan and treat in a gravity displacement (121C) or porous load (134 C) autoclave for 1 hour. Surfaces: Spray or pour 1N NaOH or 2.5% on surface and let sit on 1 hour. Ensure surfaces stay wet for entire period. Surfaces should be clean of any gross contamination as organic material can reduce the effectiveness of the solutions. Incineration- used for disposable lab equipment 800-1000°C INACTIVATION OF MICROORGANISMS AUTOCLAVE Principle: Steam under pressure Boiling point of water increases in high pressure Fastest and simplest method of sterilization all organisms (except Prions) and spores are killed within 15 minutes. Biological Indicator: Geobacillus stearothermophilus 121C, 15 psi, for 15 minutes Media, Liquids, Utensils, Glass Pipettes, and Instruments for Assay 132C, 15 psi, 30-60 minutes Decontaminating Medical Wastes QUALITY CONTROL FOR AUTOCLAVE BIOLOGICAL INDICATOR: Incubate for 24hours at 37°C No color change IF sterilization is complete Turns color YELLOW if growth Happens (ineffective) Autoclave Tape: Turns black if Sterilization is complete The tape has white diagonal lines with Geobacillus stearothermophilus BIOSAFETY CABINET Calibrated and certified before use (by supplier) Practice proper usage, placement, and decontamination The Biosafety Cabinet is a requirement to process infectious specimens. Enclosed container minimizing our exposure to hazardous materials TYPES OF BIOSAFETY CABINET PROTECTION FILTER 1. HEPA Filter Standard for Biosafety High Efficiency Particulate Air 99.97% efficiency at 0.3 microns (pore size) Made from Pleated borosilicate glass, arranged in random fibers These are exhaust system of the biosafety cabinet that effective traps all of the infectious agents. Hence, the air that will come out is free of microbes. 99.97% efficiency at 0.3 microns and up. 2. ULPA Filter Ultra-low Penetration Air 99.999% efficiency at 0.12 microns 3. SULPA Filter Super Ultra-low Penetration Air 99.9999% efficiency at 0.12 microns To achieve maximum cleanliness Used in reference laboratories & BSL4 "Curtain of Air" at the openingprotection of product Laminar flow of filtered air within the BSC-Protection of the personnel Filtration of exhausted airProtection of the environment BIOSAFETY MANAGEMENT Ensure the development and adoption of a biosafety management plan and a safety or operations manual. Ensure that regular training in laboratory safety is provided. Personnel should be advised of special hazards, and required to read the safety or operations manual and follow standard practices and procedures There should be an arthropod and rodent control program. Most infectious diseases are transmitted through these animals Appropriate medical evaluation, surveillance and treatment should be provided for all personnel in case of need, and adequate medical records should be maintained. HEALTH AND MEDICAL SURVEILLANCE Provision of active or passive immunization were indicated Ex: Passive- Hepa B Ig vaccine; Antitetanus Active- Ab produced Facilitation of the early detection of laboratory- acquired infections Exclusion of highly susceptible individuals (e.g., pregnant women or immuno-compromised individuals) from highly hazardous laboratory work Provision of effective personal protective equipment and procedures. safety practices 1. PRE-ANALYTICAL Specimen collection: use antiseptic technique Specimen preparation Specimen transport: use appropriate containers that IS properly sealed, with labels, and biohazard symbol 2. ANALYTICAL Testing: apply the safety rules 3. POST-ANALYTICAL Disposal DEVELOP PERSONAL SAFE WORK HABITS Wash hands before and after entering the lab Change gloves frequently to prevent cross contamination Wear lab coat or apron Dispose of contaminated sharps and waste immediately after testing Pipetting by mouth is strictly forbidden Never eat, drink or smoke at the test site Keep food out of the laboratory/testing site refrigerator MAINTAIN CLEAN AND ORDERLY WORK SPACE Keep work areas uncluttered and clean Disinfect work surfaces daily Restrict or limit access when working Keep supplies locked in a safe and secure area Keep emergency eye wash units in working order and within expiry date PROPER DISPOSAL OF BIOWASTES Good Safety Practices: Identify Hazards Implement safety strategies to contain hazards Audit existing practices to determine whether new ones are needed PROPER DISPOSAL OF BIOWASTES Anatomical clinical infectious waste which requires disposal by incineration Hazardous waste to be collected by a licensed waste handler and incinerated in a suitably permitted or licensed facility Waste which may need to be “treated". Hazardous waste to be collected by a infectious waste and licensed waste handler and potentially infectious incinerated in a suitably permitted waste known to contain or licensed facility pathogens Cytotoxic and cytostatic waste Hazardous waste to be collected by a licensed waste handler and incinerated in a suitably permitted or licensed facility RECYCLED CONTAINERS Offensive/hygiene waste To be collected by a suitable waste handler and disposed of in a suitably permitted or licensed landfill site Domestic (municipal) waste To be disposed of in a suitably permitted or licensed landfill site Amalgam waste For recovery Recycling wastes such as glass, plastic, paper, etc Collected by an authorised recycling operator LABORATORY WASTE MANAGEMENT 1. BIOLOGICAL WASTE Exposure: Ingestion, Inoculation, Tactile Contamination, Aerosolization, Inhalation of Infectious materials Swabs Respiratory infections/ Inactivated (Heat, Chemical) and autoclaved before disposal Blood HBV, HCV, HIV Blood-borne pathogens Inactivated and Autoclaved TISSUES Should be fixed with a fixative Unfixed tissues must be inactivated and autoclaved Other body fluids Sputum: Tuberculosis, other respiratory infections Inactivated and autoclaved PREVENTION: Gloves Primary barrier protection Not reused or washed for reusing Masks/Respirators, Protective Eyewear, Face Shield for exposure from splashes to the mouth, eyes,nose Decontaminate work area regularly CHEMICAL HAZARDS: Stored in certain sections of the Laboratory Not in use, packaging with leakage or corrosion: Replaced Water reactive chemicals: No contact with water MATERIAL SAFETY DATA SHEET Document that gives detailed information about a material and about any hazards associated with the material CHEMICAL HAZARDS IN THE GENOTYPING LABORATORY 1. Guanidinium thiocyanate commonly used in lysis buffers denaturing agent for RNA extraction Carcinogenic DO NOT MIX WITH BLEACH 2. Ethidium Bromide DNA staining in agarose gels, buffers For Agarose Gel Electrophoresis Intercalating agent, Nucleic Acid stain Fluorescence: Orange Not directly mutagenic: metabolites are mutagenic For visualization of result for PCR or hybridization techniques Mutagenic- causes mutation Used for research purposes only 3. Carcinogens Any substance that can cause cancer. Changes the metabolic processes by damaging the genomes Different types: Decontaminating Agents Dyes Probes and Labels DYES, PROBES AND LABELS 1. SYBR Green Replacement for Ethidium Bromide Can still bind to DNA with high affinity potential Carcinogen 2. Acrylamide (neurotoxin) Used in PAGE (Polyacrylamide Gel Electrophoresis) Cross-linking agent for gel chromatography and electrophoresis Can cause peripheral neuropathy, possible carcinogen 3. Phenol (organic solvent) Used in Phenol-Acetic Acid-Urea Polyacrylamide Gel Electrophoresis (PAU-PAGE) 4. Chloroform (organic solvent) Extraction of Proteins Eye damage (sclera), fainting, lifethreatening DEFINITIONS Antimicrobial An agent that kills microorganisms or suppresses their growth and multiplication. Antiseptic A substance that inhibits the growth and development of microorganisms without necessarily killing them. Antiseptics are usually applied to body surfaces. Biocide A general term for any agent that kills organisms. Chemical germicide A chemical or a mixture of chemicals used to kill micro-organisms. Any process for removing and/or killing microorganisms. The same term is also used Decontamination for removing or neutralizing hazardous chemicals and radio- active materials. Disinfectant A chemical or mixture of chemicals used to kill microorganisms, but not necessarily spores. Disinfectants are usually applied to inanimate surfaces or objects. Disinfection A physical or chemical means of killing microorganisms, but not necessarily spores. Microbicide A chemical or mixture of chemicals that kills microorganisms. The term is often used in place of “biocide”, “chemical germicide” or “antimicrobial”. Sporocide A chemical or mixture of chemicals used to kill microorganisms and spores. Sterilization A process that kills and/or removes all classes of microorganisms and spores.