Lab 1 Orientation to the Laboratory PDF

Lab 1 Orientation to the Laboratory By Dr. Mohammed Ali Eid Assistant Professor of Microbiology 06/10/2024  Sample Receipt and Storage Laboratory Microscopy Layout Culturing: incubators, autoclaves, and other necessar...

Lab 1 Orientation to the Laboratory By Dr. Mohammed Ali Eid Assistant Professor of Microbiology 06/10/2024  Sample Receipt and Storage Laboratory Microscopy Layout Culturing: incubators, autoclaves, and other necessary equipment to grow and isolate microorganisms. Laboratory Layout Biohazard Disposal: ‫اﻟﺗﺧﻠص ﻣن اﻟﻣﺧﺎطر‬ ‫اﻟﺑﯾوﻟوﺟﯾﺔ‬ Hand washing Stations Emergency Equipment: Locations of fire extinguishers, first Aid Kits, safety showers, eye wash stations, Emergency Exits.  Familiarization with Common Lab Equipment Microscopes: The primary tool for visualizing microorganisms. Autoclaves: Used for sterilizing lab equipment and waste. Bunsen Burners: Used for heat-related procedures. Centrifuges: Used to separate different components of a sample.  Familiarization with Common Lab Equipment Incubators: Provide controlled environments for growing microorganisms. Pipettes: For precise measurement and transfer of liquids. Personal Protective Equipment (PPE): Includes lab coats, gloves, safety glasses, and other equipment used for personal protection.  Personal Protective Equipment (PPE) No Eating or Drinking Handle Chemicals and Biological Materials Safely Dispose of Waste Properly General Know Emergency Procedures: Laboratory Use Equipment Correctly Safety Rules Maintain Cleanliness No Unauthorized Experiments Report Accidents and Unsafe Conditions Do Not Work Alone Microscopic Techniques Light Microscopy: provide magnification up to 1000-2000 times. routine tasks like identifying bacteria and observing cell structures Parts of a Microscope and Their Functions Eyepiece(Ocular Lens): The lens you look through at the top of the microscope, usually providing 10x or 15x magnification. Objective Lenses: These are the lenses closest to the sample. Most microscopes have several objective lenses of different magnifications (e.g., 4x, 10x, 40x, 100x), mounted on a rotating tower for easy switching. Stage: The flat platform where the slide with the sample is placed. Parts of a Microscope and Their Functions Stage Clips: These hold the slide in place on the stage. Light Source (Illuminator): Provides the light necessary to view the sample. It can be a mirror reflecting ambient light or a built-in light. Condenser: Controls the amount of light reaching the sample from the light source. It can also help focus the light for better illumination.  Parts of a Microscope and Their Functions Coarse and Fine Focus Knobs: These are used to move the stage up and down to bring the sample into focus. Body Tube: Holds the eyepiece and objective lenses at the correct distance from each other. Base and Arm: The base supports the microscope, while the arm connects the base to the body tube and lenses. The arm is also where the microscope should be held when being carried. Lab 2 Sterilization and Disinfection By Prof. Dr. Mohammed Ali Ahmed Eid 12/10/2024  Sterilization: It is the killing or removal of all microorganisms, including bacterial spores, which are highly resistant. Disinfection: Sterilization It is the killing of most, but not all microorganisms, mainly the pathogenic ones. For adequate disinfection, and pathogens must be killed but some organisms and bacterial spores may survive. Disinfection Disinfectants vary in their tissue-damaging from the corrosive phenol-containing compounds, which should be used only on inanimate objects, to less toxic materials such as ethanol and iodine which can be used on skin surface. Chemicals used to kill microorganisms on the surface of skin and mucous membrane are called antiseptics.  1-physical agents: heat Classification Filtration of sterilizing Radiation. agents: 2- Chemical agents. Chemical disinfection methods are widely used for sanitizing surfaces, water treatment, and other applications  Physical sterilization. I. Heat: Glassware and medium are routinely sterilized by heat. A. Dry Heat: Direct flame: whenever rapid and repeated sterilization is required; the simplest method is direct flaming. This Type of sterilization is used for metal instruments such as platinum wire loop, forceps and scissors etc. Bunsen burner is commonly used for sterilization by dry heat (direct flame). Incineration for disposable of infection material. B. Hot air: All dry glassware and metal instrument are usually sterilized by this method. Generally, the instruments are left in a Hot Air Oven on a temperature of 160- 180ºС for one hour.  Physical sterilization. C. moist heat:- 1. pasteurization: uses heat 63ºС at 30 sec. The organisms such as Brucella or Salmonella and tubercle bacilli which contribute to milk born-disease are readily killed by this process. The alternative method raises the temperature of milk to 72 ºС (161 º F) for 15- 20 seconds and is referred to as the flash process. N.T: bacterial spores are not killed by this method. Physical sterilization. 2. Steam under pressure (high pressure steam): The autoclave or pressure cooker is the instruments used for high pressure steam sterilization. Then steam is placed under pressure in an autoclave. Bacteriological media, surgical instruments are sterilized in the autoclave at 121ºС (15 Ibs) for 15 minutes. Physical sterilization. II. Filtration: The principle of this method is to pass the material to be sterilized through special bacterial filters which hold back any bacteria present; the filtrate is thus obtained bacteria free. This method is used for the sterilization of fluids that do not withstand heating, e.g. sera, Plasma, Vitamins and antibiotic solutions. Either positive pressure on the liquid to be filtered should be exerted or negative pressure by sucking from the filtrate container on the filtration in order to enhance the process. Physical sterilization. III. Radiation: It is employed commercially for the sterilization of large amount of pre-packed disposable items such as plastic syringes and Catheters that are unable to withstand heat. It is done by applying ultra-violet rays, this process induces thymine dimmer of DNA and this interferes with replication of Micro-organism.  1-Alcohols Etyhl alcohol, Isoporopylalcohol (70% aqueous solution): Antiseptic to sterilize the thermometer, the skin before injection vein puncture. 2-Phenols: Uses: sterilization of surgical instruments, Chemical bathroom, hospital floor. Chlorohexidin ; as skin disinfectants. sterilization. 3-Heavy metal ions (metallic salt); (Mercury, siliver nitrate): Mercuric salt e.g Methiolate used as preservation for sera, bacterial and viral vaccine. Silver salt e.g AgNO3 (1%) used as eye drop for newborn infants to prevent infections by Neisseria gonorrhea. Lab 3 Isolation and Purification of Bacteria BY: Prof. Dr. Mohammed Ali Ahmed Eid 18/10/2024 Isolation Techniques SERIAL DILUTION METHOD This method involves diluting a sample through a series of steps, which reduces cell concentration. It's essential in quantifying bacteria for scientific investigations. Follow Isolation technique Filtration method: Membrane filter (MF) Separates bacteria based on size using filters with specific pore sizes. In this process, a liquid sample containing bacteria is passed through a filter membrane with very small pore sizes (0.2-0.45 μm) to remove bacteria from liquids. This technique is useful for isolating bacteria from mixed populations, purifying cultures, and sterilizing heat-sensitive solutions. Follow Isolation technique STREAK PLATE METHOD A technique to isolate pure bacterial colonies. By spreading diluted samples on agar plates, individual cells grow into separate colonies, allowing for analysis and study.  Enrichment culture Enrichment culture involves creating selective growth conditions Follow that favor the target microorganism Isolation while inhibiting or slowing the growth of others. technique This is achieved by manipulating factors such as nutrients, pH, temperature, oxygen levels, or adding selective agents. Purification Methods COLONY PICKING After isolation, specific colonies can be picked and cultured separately using streaking method to ensure purity. This step is crucial to avoid contamination.  Table of Techniques Technique Description Application Streak Plate Isolate colonies Pure cultures Serial Dilution Reduce concentration Quantitative studies Filtration Separate based on size Water treatment Enrichment Culture Enhance specific bacteria Research studies  Isolation form different source Soil: Dilute soil sample in sterile water Plate dilutions on appropriate media Use selective media for specific bacteria Water: Filter water through membrane filters Place filter on agar plate For low concentrations, use enrichment techniques Air: Expose agar plates to air for set time periods  Food: Homogenize food sample Follow Dilute and plate on selective media Incubate at appropriate temperatures Isolation Clinical samples: Use sterile swabs for collection form Streak directly on selective media May require enrichment for some pathogens different Plant surfaces: Wash plant parts in buffer solution source Plate wash solution on appropriate media Animal sources: Collect fecal samples or swabs Use selective enrichment broths Plate on differential media Lab 4 Colony Description, Selection, and Subculturing Prepared by Prof. Dr. Mohammed Eid 1/11/2024 Purpose of Colony Description Bacterial species often form colonies with unique sizes and appearances. These characteristics aid microbiologists in identifying and distinguishing different types of bacteria. Identified colonies can be used to obtain pure cultures of specific bacterial species. Factors Affecting Colony Morphology 1. Nutrient Composition and Availability: Bacteria may exhibit different colony morphology, and different growth patterns based on nutrients composition and availability. Different media provide varying levels of nutrients, and some media may contain specific compounds that influence colony morphology. 2. Temperature: Different temperatures can influence growth rates and the overall structure of bacterial colonies. Factors Affecting Colony Morphology 3. Oxygen Availability: Aerobic and anaerobic bacteria may form different types of colonies based on their oxygen requirements. 4. pH Level: The acidity or alkalinity of the growth medium (pH level) can impact bacterial growth and, consequently, colony morphology. Some bacteria thrive in acidic conditions, while others prefer alkaline environments. 5. Moisture Content: Some bacteria may produce more mucoid or slimy colonies under conditions of higher moisture. Factors Affecting Colony Morphology 6. Incubation Time: The duration of incubation can affect colony development. Longer incubation periods may lead to larger colonies, and the growth pattern over time can contribute to variations in morphology. 7. Genetic Factors: The genetic makeup of the bacteria plays a crucial role in determining colony morphology. Different strains of the same species may exhibit distinct colony characteristics. 8. Cooperative Behavior and Quorum Sensing: Bacterial communication through quorum sensing and cooperative behavior can result in specific colony structures. This is particularly evident when bacteria respond collectively to environmental conditions. Factors Affecting Colony Morphology 9. External Influences: Factors such as physical barriers, the presence of other microorganisms, or exposure to antimicrobial agents can influence colony morphology. Different factors could affect the A colony morphology of the same bacterial Isolates A. Bacillus subtilis growing on B nutrient-poor agar B. Bacillus subtilis growing on nutrient-rich agar. Bacterial Colony Morphology The general form of the colony and the shape of the edge or margin can be determined by looking down at the top of the colony. The nature of colony elevation is apparent when viewed from the side as the plate is held at eye level.