Lab 1 Safety in Microbiology Lab PDF

Basic Microbiology Practical No.: 1 (Online) Safety in Microbiology Lab and Microbiology Lab Instrumentations Part A: Microbiology Lab Practices and Safety Rules Intended Learning Objectives (ILOs): By the end of this practical the...

Basic Microbiology Practical No.: 1 (Online) Safety in Microbiology Lab and Microbiology Lab Instrumentations Part A: Microbiology Lab Practices and Safety Rules Intended Learning Objectives (ILOs): By the end of this practical the student will be able to understand: 1. Safety rules and behavior inside the laboratory. 2. Different Biosafety levels 3. Safety equipment and how to use it. 4. How to deal in emergency cases. 5. Good laboratory practices and techniques. Introduction/background The laboratory environment is to be regarded at all times as a restricted area. All lab users, staff or students, are likely to be exposed to many different hazards. This include Fire, electrical, chemical and Bio-hazards. Therefore, safety in the laboratory is the primary concern and it is extremely important that the laboratory rules are followed at all times. A microbiology laboratory is a unique environment that requires special practices and containment facilities. The three main elements of safe containment of microorganisms are: 1. Good laboratory practices and technique 2. Safety equipment and 3. Facility design. Page 1 **If you are not sure about anything, ALWAYS consult one of the academic staff** Laboratory Biosafety levels: According to Centre of Diseases Control and prevention (CDC), there are 4 different levels of biosafety labs, where each one of them is designated and equipped for the isolation of specific microorganisms according to their infectious hazards: Page 2 Precautions: General behavior/ safety measures (rule). v Upon entering the laboratory, coats, books, and other personal belongings should be placed in specified locations and never on bench tops (except for your lab manual). v No running or “horse play” Report any injuries & accident immediately. Do not clean them yourself, contact the instructor immediately! In case further treatment is necessary. v No food and drink consumption and storage in laboratories, laboratory-like areas and offices where microorganisms are stored v There will be — No Smoking, No eating or Drinking — No pencils, fingers, hair, labels etc. in the mouth, eye, nose v At the beginning and the end of each laboratory session, bench tops should be wiped with a disinfectant solution. v Observe strict personal hygiene. Wash your hands with antimicrobial soap before leaving the laboratory at the end of the session. v Do not wash glassware. All the glassware used in the Microbiology Lab must be sterilized before it can be washed. v Removal of media, equipment, and cultures from the laboratory is prohibited unless so directed by the instructor v Make sure the gas valve is turned off before you leave the laboratory. v You must report to your supervisor (checkout) prior to leaving the laboratory setting. Page 3 Why is Lab Safety Important? Lab safety is a major aspect of every lab based biomedical class. Lab safety rules and symbols are needed so that students do not injure themselves or their classmates Laboratory Clothing Laboratory Gowns or Lab Coats: v Lab coats must be worn at all times in the laboratory. v Anyone wearing inappropriate lab coat will not be allowed to work in the laboratory. v Lab coats must be of sufficient length to protect the worker (i.e., down to about the wearer's knees), have long sleeves, and elastic or restricted cuffs. Foot wears: v Closed shoes must be worn at all times (Absolutely no flip-flops allowed) in the laboratory. Anyone wearing inappropriate footwear will not be allowed to work in the laboratory v Students MUST not wear sandals or open toed or canvas shoes because of the constant danger of cuts and infections from broken glass found on the lab floors and the possibility of chemical spills. Eye wears: v Safety glasses must be worn for all activities in the laboratory except when using the microscope or computer or at other times as designated by the Instructor. Page 4 Hand wears: v Laboratory gloves are NOT to be worn in communal areas. v Door handles, telephones, computer keyboards and mice, lift buttons, etc. are not to be touched with gloves v Rubber or disposable gloves should be worn when handling / working with: Human blood or other body fluids Dangerous chemicals Infectious, or potentially infectious materials Lab Safety Equipment: All students and staff working in the lab must be aware of safety equipments and their locations: Common laboratory equipments: 1. Safety Shower. 2. Eye Wash. 3. Safety Goggles. 4. Fire Extinguisher. 5. Fire Blanket. 6. Fume Hood or Safety Cabinet. Page 5 Lab Safety Symbols Found in textbook: Page 6 Chemical Hazard Symbols and Definitions Page 7 Waste disposal: v All samples must be considered to be infectious. v Use of “Universal Precautions” handling v Never assume any sample is “safe” Where to dispose the waste in lab: Reasons for proper waste disposal: Hazardous chemicals down the drain can lead to pollution of ground water, lakes, rivers, etc. Plants and animals will die if they are exposed to hazardous chemical waste. Serious health problems will become present in people if hazardous waste finds its way into drinking water. Page 8  Part B: Instrumentations used in microbiology lab Intended Learning Objectives (ILOs): 1. Equipment's and apparatus uses / handled in microbiology laboratory. 2. Proper uses and care of instruments. 3. Standard operation setting of each equipments. Introduction: All Microbiology Laboratory require specific equipment in order to isolate and identify the unknown microorganism. A modern microbiology laboratory should be furnished with the following equipment: 1. MICROBIOLOGICAL SAFETY CABINET or HOODS or LAMINAR AIR FLOW CHAMBERS: v Microbiological safety cabinet is a piece of laboratory equipment that provides a safe working area for people handling material potentially contaminated with pathogens as well as protecting the sample from any external contamination. v Classification of Biosafety cabinet Class I A ventilated cabinet with an inward airflow and outlet HEPA filters. It was biosafety previously referred to as the CDC Hood and served a valuable function in its cabinet time by protecting personnel and the environment. Because it offers no product protection, it has been essentially obsolete for the past several decades. Class II (types Laminar Flow Biological Safety Cabinets that protect personnel, product and A and B) environment. They provide inward airflow to protect personnel, downflow HEPA filtered air to the work area to protect the product and exhaust HEPA filtered air to protect the environment from particulate and aerosol hazards. Page 9  Class III Essentially a ventilated glovebox. This is a gas-tight chamber operated cabinet through sealed gloves which provide a complete barrier between the worker and hazardous material. The glovebox is maintained under negative pressure with HEPA filtered supply air and double HEPA filtered exhaust air. v Laminar Air Flow is based on the flow of air current to create uniform velocity, along parallel lines, which helps in transforming microbial culture in aseptic conditions. Uses: It is used in microbial inoculation and isolation studies. In addition, it is utilized for protection of user, samples and the environment from hazardous contamination. 2. INCUBATORS An apparatus in which environmental conditions, such as temperature and humidity, can be controlled, often used for growing bacterial cultures or providing suitable conditions for a chemical or biological reaction. As for temperature, most commonly used at 37°C. Most bacteria grow well under such conditions. Page 10 Uses: It is used in cultivating, multiplying and in the characterization tests of microorganisms. This device provides the heat necessary for the growth of microorganisms. 3. HOT AIR OVEN OR LAB OVEN Hot air ovens are electrical devices used in sterilization. In sterilizing instruments for various aseptic procedures, especially if that cannot be autoclaved (Dry heat sterilizer) like powders. Generally, they can be operated from 50 to 300 °C. There is a thermostat controlling the temperature. USES: For sterilization v These are digitally controlled to maintain the temperature. The standard settings for a hot air oven are: ü 1.5 to 2 hours at 160 °C ü 6 to 12 minutes at 190 °C Page 11 4. AUTOCLAVE: An autoclave (Moist Heat Sterilizer) is a device to sterilize equipment and supplies by subjecting them to high pressure steam (≈15lbs) at 121° C or more, typically for 15 to 20 minutes depending on the size of the load and the contents. It was invented by Charles Chamberland in 1879. Uses: The main purpose of this device is to sterilize materials and media under pressure and steam. 5. LBORATORY REFRIGERATOR and DEEP FREEZER (-86oC) Refrigerators are used to cool samples or specimens and maintain temperature for preservation. Uses: The device is used for the storage of the stock solutions, chemicals, kits and nutrient media that should be maintained at certain temperatures. Page 12 Uses of Deep Freezer: It is used to store stock cultures in microbiology. It is a device used to store materials which should be kept at low temperatures (cells, tissues, enzymes, proteins, etc.) 6. BUNSEN BURNER: The Bunsen burner consisting of a vertical metal tube connected to a gas source and producing a very hot flame from a mixture of gas and air let in through adjustable holes at the base. The heat generated from the flame help obtaining the required aseptic condition to culture some microorganisms. Named after the German chemist Robert Wilhelm Bunsen, who contributed to its development, the Bunsen burner was already known to Michael Faraday, who may have created the first design in 1855. Uses: Heating, Sterilization & Combustion 7. INOCULATION LOOP / NEEDLE Its tip is a wire made of platinum or nichrome (Nickel + Chromium). The wire forms a small loop with a diameter of about 5 mm. The inoculation loop is always sterilized in a flame until it becomes red hot before and after each use. After flame sterilization, the loop must be cooled so that the next cells to touch Needle - Inoculation of agar slopes/dips. (Disposable plastic loops and needles) Page 13 Uses: To inoculate/dips test samples into culture media for bacterial or fungal cultures, antibiograms, etc. 8. CENTRIFUGE MACHINE: A laboratory centrifuge is a piece of laboratory equipment, driven by a motor, which spins liquid samples at high speed. There are various types of centrifuges, depending on the size and the sample capacity. Based on automatic rotor recognition, up to date centrifuges have a button for automatic conversion rpm (rotation per minute) Uses: Separation/Isolating suspensions like serum & plasma from blood. Page 14 9. MAGNETIC STIRRER Or HOTPLATE STIRRER: A magnetic stirrer or magnetic mixer / Hotplate stirrer is a laboratory device that employs a rotating magnetic field to cause a stir bar (also called "flea") immersed in a liquid to spin very quickly, thus stirring it. Magnetic stirrers often include a hot plate or some other means for heating the liquid. Uses: For mixing and keeping the chemical solutions and mixtures at a certain time and temperature by the help of a magnetic bar. 10. VORTEX MIXER: Vortex mixers designed for mixing solutions in test tubes or small flasks with a circular, oscillating movement Page 15 Uses: It is used for mixing. Vortex agitates the solutions in the tube, flask and so on in certain speed and duration. 11. COLONY COUNTER: Colony counter is an instrument used to count colonies of bacteria or other microorganisms growing on an agar plate. Method: Uses a pencil-like rod with a metal tip, which is connected by an electrical connection to the gelled growth medium; when touched to a colony on the plate, the completion of the electrical circuit causes an increment of 1 unit on the readout of the colony counter. (Automated Colony Counter) Uses: For Counting of bacterial colony 12. WATER BATH: A water bath is laboratory equipment made from a container filled with heated water. It is used to incubate samples in water at a constant temperature over a long period of time. All water baths have a digital or an analogue interface to allow users to set a desired temperature. Page 16 Uses: Warming of reagents, melting of substrates or incubation of cell cultures. It is also used to enable certain chemical reactions to occur at high temperature. 13. SENSITIVE BALANCE: They are used in precise weighing of small amounts (up to milligrams) of samples and chemicals used for preparing media and stock solutions. Uses: For weighing media and chemicals. Page 17 14. WATER DISTILLER: Distillation is a process of separating the component substances from a liquid mixture by selective evaporation and condensation. Uses: For liquid (water) distillation 15. LABORATORY THERMOMETER: A laboratory thermometer is a tool used in laboratories to measure temperature with a high level of accuracy. Uses: For measuring temperature. Page 18 16. ANAEROBIC JAR: It is provide anaerobic condition for growth of anaerobic bacteria. Uses: For anaerobic cultivation of bacteria. 17. MICROSCOPE: It is an instrument used to see objects that are too small for the naked eye. (Greek, Mikros = small, Skopein= To look or see) There are many types of microscopes. The most common microscope is light or compound microscope. Page 19 MICROSCOPE MAGNIFYING OIL: OIL IMMERSION: It is a technique used to increase the resolving power of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens. Cedar wood oil or immersion oil: Immersion Oil contributes to two characteristics of the image viewed through the microscope: finer resolution and brightness. Applying immersion oil: The applicator rods with a taper-point or ball- end make it easy to apply oil to the positioned slide The magnifications of the image in a compound microscope are as follows: Objective lens Eye piece Total magnification 10x 10x 100x 40x 10x 400x 100x 10x 1000x Uses: For visualizing minute structures including microbes. Page 20 GENERAL INFORMATION ON USE OF THE MICROSCOPE 1. Take the microscope out of the cabinet and use both hands to lift it to your work area. 2. Note the three objectives: low power (10X), “high dry” (40X), and oil immersion (100X). Adjust the eyepieces in order to use both eyes when viewing specimens 3. Place a drop of immersion oil on the subject before examining it with the oil immersion objective (100X). Swing that objective into place and use the fine adjustment knob to bring the subject into focus. Use only the fine adjustment with oil immersion lenses. 4. Higher magnification requires more light so you may need to increase the amount of light 5. Turn off the light source and set the intensity control to zero. 6. Always clean the microscope before returning it to its cabinet, especially the lenses and stage. Wipe any dust or oil off the instrument. Use only lens paper and the cleaning fluid to clean the lenses. Rotate the low power objective into place before putting the microscope back into the cabinet. 7. Always examine your microscope carefully at the start of each lab period; report any problems with the microscope to the Instructor immediately. 8. Check your microscope before the end of each lab period (during which you have used the microscope) to ensure that it has been cleaned and prepared for storage properly. Page 21 References: 1. Turner, B. and K.K.Shamsid-Dean. 2005. Good, Messy, Frothing Fun. Teaching Problem-Based Lab Safety. Science Scope. April/May: 10-13 2. Medical Laboratory Manual for Tropical Countries by Monica Cheesbrough. 3. Color Atlas of microbiology by Luis M 4. http://nobel.scas.bcit.ca/debeck_pt/science/safety.htm 5. Textbook of Microbiology by Ananthanarayan and Panikar, ISBN 81-250-2808-0 6. Textbook of Medical Laboratory Technology by Praful B. Godkar, Darshan P. Godkar ISBN 9381496196 7. Bharti Arora, D. R. Arora (2015): Practical Microbiology. ISBN: 81-239-14095-9. 8. Steven Obenauf, Susan Finazzo (2016): Microbiology Fundamentals. A Clinical Approach. Page 22