Lab Microbiology History & Safety PDF

[TRANS] LESSON 1: BRIEF HISTORY IN MICROBIOLOGY & LABORATORY SAFETY AND INFECTION CONTROL ▪ Causative agent: Bacillus anthracis (bioterrorism BRIEF HISTORY IN MICROBIOLOGY...

[TRANS] LESSON 1: BRIEF HISTORY IN MICROBIOLOGY & LABORATORY SAFETY AND INFECTION CONTROL ▪ Causative agent: Bacillus anthracis (bioterrorism BRIEF HISTORY IN MICROBIOLOGY agent) ROBERT HOOKE ▪ Bioterrorism agent - can be used to invade a An English scientist and architect, a polymath, recently specific place, to bring disease in a community. called "England's Leonardo", who, using a microscope, was ▪ Discovered in 1876 the first to visualize a microorganism. o Koch’s Postulates Discovered the law of elasticity. o He stated that the stretching of a solid object is proportional or equal. Cell History o Robert Hooke, an English scientist decided to examine thin slices of plant material and was struck by the appearance of regular box-like structures he names “cells” (1665). o Without realizing it, Hooke discovered the fundamental unit of all living things. ANTON VAN LEEUWENHOEK Dutch microscopist who was the first to observe bacteria and protozoa. His research on lower animals refuted the doctrine of LOUIS PASTEUR spontaneous generation, and his observations helped lay A French biologist, microbiologist, and chemist renowned the foundations for the sciences of bacteriology and for his discoveries of the principles of vaccination, microbia protozoology fermentation, and pasteurization. Discovered compound microscope He is remembered for his remarkable breakthroughs in the He extended the studies of Marcello Malpighi causes and prevention of diseases, and his discoveries He gave the first description of red blood cell have saved many lives ever since. Used the term “Animalcules” which means tiny living He reduced mortality from puerperal fever and created the organisms. first vaccines for rabies and anthrax. Compound Microscope created by Leeuwenhoek “Father of Modern Bacteriology” “Father of Ancient Bacteriology and Scientific Microscopy” Pasteur and Roux - proved that the attenuated type of o Thus, the Science Bacteriology was born. vaccine is effective Magnification lens of the compound microscope that o He demonstrated that microorganisms are present in Leeuwenhoek use: 50x - 300x magnification the air and can contaminate sterile solutions, but air itself does not create microbes. The air only carries the bacteria. o Note: Rabies is a virus. The pathologist can identify rabies by seeing the negri bodies. Negri bodies can only be seen in rabies. Example of Air bacteria: Bacillus cereus (Fried Rice Bacillus). The specimen of choice when examining this bacterium is the fried rice itself. ALEXANDER FLEMMING ROBERT KOCH A Scottish physician and microbiologist best known for A German physician and microbiologist. discovering the enzyme lysozyme and the world's first o As one of the main founders of modern bacteriology, broadly effective antibiotic substance which he named he identified the specific causative agents of penicillin tuberculosis, cholera, and anthrax, and gave o Lysozyme can be found in saliva. Saliva contains experimental support for the concept of infectious antimicrobial agent. disease, which included experiments on humans and Penicillin animals o Comes from the fungi Penicillium notatum (New name: Discovered the bacteria in 1882 Penicillium chrysogenum). o Tuberculosis o A broad-spectrum antibiotic. They are effective against ▪ Causative agent: Mycobacterium tuberculosis Gram-Positive and Gram-Negative bacteria. o Cholera Prescription is needed to buy this medicine ▪ Causative agent: Vibrio cholerae PAUL EHRLICH ▪ Can be caused by eating shellfish German physician and scientist who worked in the fields of ▪ Stool of patient with Cholera: Rice watery stool hematology, immunology, and antimicrobial chemotherapy. o Anthrax Among his foremost achievements were finding a cure for R.H. AGARRO, S.L. BOLUSAN, C.V. LUCERO | BSMT 1  TRANS: LESSON 1 syphilis in 1909 and inventing the precursor technique to o Gamma radiation: Gram staining bacteria. ▪ used for evacuated tubes o The methods he developed for staining tissue made it ▪ 1500 – 2000 radiation possible to distinguish between different types of blood ▪ Use of gamma radiation to bacteria cells, which led to the ability to diagnose numerous ▪ causes a mutation in DNA and generates blood diseases. peroxides (Agua oxinada / hydrogen peroxide – Syphilis used for cleaning the wound because it can kill o Syphilis is a bacterial infection usually spread by bacteria) sexual contact. The disease starts as a painless sore ▪ It has a shorter wavelength, higher energy — typically on your genitals, rectum or mouth. CHEMICAL METHODS (STERILIZATION) o Syphilis spreads from person to person via skin or Ethylene oxide mucous membrane contact with these sores. o Most common o His laboratory discovered arsphenamine (Salvarsan), o Used in gaseous form for sterilizing heat sensitive the first effective medicinal treatment for syphilis, objects Formaldehyde Vapor and Vapor Phase H2O2 LABORATORY SAFETY AND INFECTION CONTROL o Sterilized HEPA filters in BSCs (Biosafety Cabinets) - STERILIZATION designed to removed organisms larger than 0.3um A process that kills “all form” of microbial life including Glutaraldehyde bacterial spores”. o Sporicidal – can kill bacteria that contain spores o Spore causes pathogenicity. They are able to cause o Used for medical equipment infection. Peracetic acid Destruction of all forms of life o Effective in the presence of organic material, used for Example of Sterilization: Autoclave - 120°C, 15 PSI surgical instruments. (pressure) for 15 minutes o Effective against vegetative microorganisms and Bacteria that are spore forming fungal spores. 1. Bacillus species o Vegetative microorganism: bacteria that grow in culture 2. Clostridium species media, commonly isolated in culture media PHYSICAL METHODS (STERILIZATION) DISINFECTION INCINERATION Destroys pathogenic organisms, but not necessarily all microorganism or spores. Most common method of treating infectious waste. Sterilization - Removal or destruction of all forms of life, Safest method. including bacterial spores Temperature: 870°C to 980°C Alcohol – commonly used Note: PHYSICAL METHOD (DISINFECTION) o The Philippines is the first and thus far the only country in the world with a national ban on incineration, BOILING following the passage of two landmark laws nearly 20 100°C (boiling point of water) for 15 minutes years ago: the Philippine Clean Air Act of 1999 and the Kills vegetative bacteria Ecological Solid Waste Management Act of 2000. o Bacterial cells that are metabolizing and undergoing MOIST HEAT (AUTOCLAVE) binary fission Sterilize biohazardous trash and heat-stable objects. o "alive" and replicating Machine needed: “Autoclave” Binary fission: multiplication process of bacteria Biological indicator (Quality Control): Bacillus Prokaryotes: w/o nucleus stearothermophilus vials incubated at 56’C. Eukaryotes: Mitosis Autoclave for culture media, liquids, utensils, glass pipettes and other instrument - 121°C, 15 PSI for 15 minutes PASTEURIZATION Autoclave for decontamination of biohazardous waste - 63°C for 30 minutes or 72°C for 15 seconds 132°C, 15 PSI for 30-60 minutes. Encounters mostly in Milk DRY HEAT Kills food pathogen without damaging the nutritional value Longer exposure times and higher temperature than moist or flavor heat While pasteurization kills or inactivates many Sterilize items such as glass wares, oil, or powders microorganisms, it is not a form of sterilization, Biological indicator: Bacillus subtilis because bacterial spores are not destroyed. o Bacillus subtilis It extends shelf life via heat inactivation of enzymes that most common laboratory contaminant spoil food Bacillus subtilis var niger 3 Types: FILTRATION o Low temperature holding (LTH) ▪ Batch method o Method of choice for antibiotic solutions, toxic ▪ Destroys milk-borne pathogens chemicals, radioisotopes, vaccines and carbohydrates, ▪ Reduce the rate of spoilage of food without which are all “heat sensitive” affecting its taste Filtration of air: HEPA (High efficiency particulate air) filters ▪ 63 ̊C for 30 minutes o Standard Filter size of HEPA: 0.3 µm o High-temperature short-time (HTST) o Liquid Filtration: Membrane filters ▪ Flash pasteurization IONIZING RADIATION / COLD STERILIZATION ▪ 72 ̊C for 15 minutes (followed by immediate Sterilizing disposables (e.g. plastic syringes, catheters, or cooling) gloves before use) o Ultra-high temperature (UHT) Type of ionizing radiation used for sterilization: Gamma ▪ 140 ̊C for 3 secs (followed by immediate cooling in radiation a vacuum chamber) R.H. AGARRO, S.L. BOLUSAN, C.V. LUCERO | BSMT 2  TRANS: LESSON 1 Quaternary are strong enough to cause adverse health NON-IONIZING RADIATION effects in people who are exposed to them. Ultraviolet light (UV) The Problem: Biological indicator: Bacillus pumilus o Skin irritants Cause DNA base pair mismatching by producing thymine o Can irritate lungs dimers o Linked to asthma o Linked to fertility issues A – T: 2 hydrogen bonds o Linked to reproductive harm C – G: 3 hydrogen bonds o Promote superbugs (anti-bacterial resistant bacteria) o It has a longer wavelength, > 1 µm, low energy CHEMICAL HYGIENE PLAN CHEMICAL METHOD Guidelines: CHEMICAL STERILANT OR BIOCIDES 1. Proper labeling of chemical containers Chemicals used to destroy all form of life 2. Manufacturer’s Material Safety Data Sheet (MSDS) It is under disinfection, but they are able to kill all form of life - Include information on the nature of the chemical, because they are able to kill disk performing bacteria that is the precautions to take if the chemical is spilled difficult to kill due to their characteristics and disposal recommendations. Used for shorter periods – disinfectants 3. Written chemical safety training and retraining Used in living tissue (skin) – antiseptics program. National Protection Association Diamond DISINFECTANTS Applied to inanimate objects ex. Sodium Hypochlorite (NaOCl) – bleach solution (zonrox) – Ratio: 1:10, Cresols, Chlorine (used for pools) ANTISEPTICS Applied topically on the skin and inhibits sepsis infection o ex. Iodophor and Hexachlorophene o Alcohols o Non-sporicidal o 70% ethyl alcohol is more effective as a disinfectant that 95% alcohol. o 70% solutions – most effective because it penetrate the cell wall more completely which permeates the entire cell, coagulates all proteins, and then cell lysis o Apoptosis: program cell death o Cell lysis: Destruction of cells HEAVY METALS 1% silver nitrate newborns infected with gonorrhoeae infection. Ophthalmia Neonatorum (conjunctivitis’ of the newborn) is an eye infection. As the newborn passes the vaginal area of the infected mother, the newborn is also infected o Gonorrhea “Tulo” ▪ Causative Agent: Neisseria gonorrheae ▪ Most common sexually transmitted infection ▪ Found in the vagina HALOGENS Iodine o A tincture with alcohol or as an “iodophor” with neutral polymer. 70% ethyl alcohol o Iodophor, most common compound used for skin disinfection before drawing blood specimen for “culture” or surgery. CHLORINE Most often used in the form of sodium hypochlorite household bleach (1:10 ratio) dilution of bleach after blood spills. QUATS (QUATERNARY COMPOUNDS) Detergents, inactivated by organic materials such as blood. R.H. AGARRO, S.L. BOLUSAN, C.V. LUCERO | BSMT 3  TRANS: LESSON 1 TYPES OF FIRES indigenous or exotic agents that may cause serious or Class A - Ordinary combustibles potentially lethal disease through the inhalation route of Class B - Flammable liquids & gases exposure. Class C - Electrical, Energized Electrical Equipment Agents are primary transmitted by aerosols. Class D - Combustible Metals Observe level 2 procedures with additional lab clothing Class K - Cooking oils and Greases and controlled access to the laboratory. Ex. Mycobacterium tuberculosis, Francisella tularensis, ELECTRICAL SAFETY and Brucella All plugs should be three-prong, grounded type. BIOSAFETY LEVEL 4 STANDARD PRECAUTIONS Required for work with dangerous and exotic agents that Blood and body fluids from every patient be treated as pose a high individual risk of aerosol-transmitted potentially “infectious”. laboratory infections and life-threatening disease. Applies to blood and all body fluids except sweat. All procedures are performed under maximum containment. BIOSAFETY Use mostly for research studies. Frequently acquired infection (shigellosis, salmonellosis, Ex: Arbovirus, Filovirus, Arenavirus tuberculosis, brucellosis, and hepatitis) Biosafety is to keep harmful agents from people. Biosecurity is to keep people from harmful agents. Biosafety cabinets- remove particles that may be harmful REFERENCES to the employee who is working with infective biologic specimens. Notes from the discussion by Prof. Roel Guinto, RMT, MSMT Biosafety Cabinet Types: o Class I, open front National University PowerPoint presentation: Prof Roel o Class II Type A1 o Class II Type A2 Guinto, RMT, MSMT o Class II Type B1 o Class II Type B2 o Class III BIOSAFETY LEVEL 1 Suitable for wok involving well-characterized agents known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment. Ex: Bacillus subtilis, Mycobacterium gordonae Precautions includes standard good laboratory techniques. BIOSAFETY LEVEL 2 Suitable for work involving agents that pose moderate hazards to personnel and the environment. Most organisms can cause laboratory acquired infection. Ex. HIV, HBV, Staphylococcus, Salmonella, and Shigella. Observe good laboratory techniques, limiting access to the laboratory, training laboratory personnel, and utilizing Class I and II BSCs. CLASS II BSC SUBTYPES Class II Type A1 o 70% recirculated and 30% exhausted o It is utilized for biological samples. Class II Type A2 o 70% recirculated and 30% exhausted o Most commonly used BSC. o Most used for both biological and clinical samples. o It is utilized for specimens treated with minimal concentration of volatile chemical. Class II Type B1 o 30% recirculated and 70% exhausted Class II Type B2 o 0% recirculated and 100 exhausted BIOSAFETY LEVEL 3 Applicable to clinical, diagnostic, teaching, research, or production facilities where work is performed with R.H. AGARRO, S.L. BOLUSAN, C.V. LUCERO | BSMT 4  TRANS: LESSON 1 R.H. AGARRO, S.L. BOLUSAN, C.V. LUCERO | BSMT 5