Microbiology P 1 PDF

Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology Lab 1 : Orientation Microbiology lab Medical Microbiology classified into: 1- Bacteriology is Study of bacteria 2- Myc...

Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology Lab 1 : Orientation Microbiology lab Medical Microbiology classified into: 1- Bacteriology is Study of bacteria 2- Mycology is study of fungi 3- Virology is study of viruses 4- Parasitology is study of protozoan & helminthes 5- Immunology is study of resistance to disease Tools and Equipment 1  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology Microbiology Lab Practices and Safety Rules : 1. Hand Hygiene: Always wash your hands with disinfectant soap when entering and leaving the lab to reduce contamination risks. 2. No Eating or Smoking: Food, drinks, chewing gum, and smoking. Do not place anything in your mouth, and avoid storing food near microorganisms. 3. Protective Gear: Wear a lab coat and safety glasses or a long-sleeved shirt that can be easily removed. Keep protective clothing in the lab and avoid wearing it outside. 4. Dress Code: Avoid loose clothing and wear closed-toe shoes to ensure safety. 5. Organized Workspace: Keep your workspace clear of unnecessary items, and store personal belongings in designated areas to prevent accidents. 6. Disinfecting Work Areas: Clean your workspace with 70% ethanol or 10% bleach before and after each session to ensure a sterile environment. appropriate disinfectant on a routine basis, and especially after spills, splashes, or other contamination. 7. Labeling and closing reagents: Clearly label all containers. Always replace caps on reagent bottles and bacterial cultures. Open Petri dishes only when necessary. 8. Sterilizing equipment: Sterilize inoculating loops and needles by flaming them in a Bunsen burner before placing them down. Turn off the burner when not in use. Tie back long hair when using the Bunsen burner to prevent accidents. 9. Sterilizing with alcohol: Be cautious when using alcohol for sterilization—avoid placing papers or flammable materials nearby. 10. Handling microorganisms: Treat all microorganisms as potential pathogens. Do not take cultures outside the laboratory. Use proper techniques to prevent contamination. 11. Wearing protective gear: Wear disposable gloves when working with potentially infectious samples. Follow good bacteriological techniques to avoid exposure to pathogens. 12. Sterilization of materials: Sterilize equipment and materials after use. Do not pipette by mouth; always use a pipetting aid. 13. Handling biohazard materials: Treat everything as a biohazard. Autoclave liquids and cultures before disposal, and dispose of solid waste in a biohazard bag before autoclaving. 2  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology 14. Handling waste and accidents: Dispose of broken glass in designated containers, and sharp objects (e.g., needles) in sharps containers. Report any spills or accidents immediately, and clean small spills carefully. Report all injuries, regardless of severity. 3  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology The Equipments/Apparatus used in the microbiology labs include a bunch of different kinds of instruments required for a lot of different processes conducted within those laboratories. As shown table 1. No. Equipment/Apparatus Use 1. Loop (wire/plastic) Routine inoculation of agar slopes/deeps and small volumes of liquid media ; making streak plates 2. Pipette Transfer of measured volumes/drops of culture/sterile (calibrated/dropping; solutions (dry, non-absorbent cotton wool plug in neck glass/plastic) prevents contamination) 3. Conical flask Large volumes of liquid media for inoculation and liquid/media for short-term storage (non-absorbent cotton wool plug prevents contamination but does not reduce evaporation during long storage) 4. Petri dish Plastic: pre-sterilised for streak/spread/lawn/pour (plastic/glass) plates; Glass: only for materials for sterilisation by hot air oven, e.g. paper discs 5. Bunsen burner Sterilisation of wire loops and (with alcohol) metal forceps and glass spreaders 6. Autoclave Sterilisation of media, solutions and equipment before use and contaminated items afterwards; melting solidified agar media for use 7. Microwave oven Melting solidified agar media for use (but not in vessels with metal caps and not for sterilisation) 8. Incubator Incubation of cultures (but many cultures will grow at room temperature in the interval between lessons) 9. Water bath Suitable temperature for keeping melted agar media molten for use (ca 50 °C); accurate temperature control 10. Thermometer Checking incubator/water bath temperatures 11. Refrigerator Storage of heat-labile materials 12. Microscope, slides, Microscopical observations cover slips, stains, staining rack, immersion oil 13. Gas ring/hot plate Steam generation in autoclave 14. Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed. 15. Biological Safety A Biological Safety Cabinet (BSC), also known as a Cabinet Biosafety Cabinet is mainly used for handling pathogenic biological samples or for applications that require a sterile work zone. A biological safety cabinet creates inflow and downflow of air that provides operator protection. 4  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology Microbiology is the study of all living organisms that are too small to be visible with the naked eye. This includes bacteria, archaea, viruses, fungi, prions, protozoa and algae, collectively known as 'microbes'. These microbes play key roles in nutrient cycling, biodegradation/biodeterioration, climate change, food spoilage, the cause and control of disease, and biotechnology. Thanks to their versatility, microbes can be put to work in many ways: making life-saving drugs, the manufacture of biofuels, cleaning up pollution, and producing/processing food and drink. 5  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology Review of the tools used in the laboratory with the function of each tool Tools 1-Loop The loop is used in the cultivation of microbes on plates by Transferring inoculums for streaking. Touching a broth or a culture plate will gather enough microbes (0.01ml) for Inoculation. The inoculation loop is sterilized with flame or another heat source. 2-Pipette Used for transfer of cultured and uncultured broth from tube or flask to other and placed in can sterilized by autoclave inside the can. 3-Spreader (L-shape) Used for spreading bacterial cell on surface of solid medium in petriplate ,before using placed in alcohol and then sterilized by flame of burner. 6  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology 4-Petri-Dish(Petri-plate) Used for place the solid medium in it, glass petri-dish used for many times and sterilized by oven or autoclave ,while sterilized plastic plates used for one time. 5-Swab Used for swabbing bacterial cells on the surface of solid medium in Petri plate, must be placed in test tube and sterilized by autoclave, it used for one time. 6-Test tube Used to place the liquid or solid or semisolid medium for stabbing or placed as slant for culture of bacteria ,it sterilized by autoclave. 7-Needle Used for transfer of bacterial cells to a solid medium or semi-solid medium by stabbing, sterilized by the flame of burner before and after use. 8-Slide Used for examination of bacterial smear under microscope, it used for one time. 7  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology 9-Cover-Slips Placed on the slide, the bacterial smear may be between the cover and the slide, it used for one time. 10-Flask Used for place cultured and uncultured broth in it , sterilized after plugs with cotton by autoclave. 11-Beaker Used for graduated the volume of liquids. Sterilized by oven. 12-Cylinder(Graduated Cylinder) Used for graduated the volume of liquids, sterilized by oven. 8  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology 13-pH paper Used to know the pH of the medium or any liquids 14-Rack May be wooden, metallic or plastic used to stand and hold the tube. 15-Burner May be gaseous or alcoholic , used for sterilized the loop , needle and other metallic tools by flame (dry heat sterilization). Equipments 1-Autoclave Wet heat sterilization= death by protein denaturation It’s an equipment with: High temperature (121C˚) High pressure 1 atm(15 pound/inch2) used to sterilize media 9  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology 2-Oven Dry heat sterilization=death by oxidation Equipment with high temperature only (180C˚) for (90 min) used to sterilized some of metallic tools and glass wares 3-Incubator Is a device used to grow and maintain microbiological cultures or cell cultures. The incubator maintains optimal temperature ,humidity and other conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside 4-Refrigerator Used to maintain the sterilized media and broth when not used to avoid the contamination , and also to preserve the bacterial culture for long time by preventing the growth at 4C˚. 5-Biosafety cabinet (BSC) Also called a biological safety cabinet or microbiological safety cabinet is aenclosed,ventilated laboratory workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens 6-Waterbath 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 11  Mahmood Yaseen Mukhlif Practical Microbiology – 2nd stage Department of Medical Laboratory Technology 7-Centrifuge A machine with a rapidly rotating container that applies Centrifugal force to its contents. 11