Sterilization, Disinfectant and Antiseptic Techniques PDF

Sterilization, disinfectant and antiseptic Sterilization:- is killing of all living forms of microbes including spores. Disinfectants:- are antimicrobial agents that are applied to non-living objects to destroy microorganisms. (Reducing the number of pathogenic microorganisms). Antisepti...

Sterilization, disinfectant and antiseptic Sterilization:- is killing of all living forms of microbes including spores. Disinfectants:- are antimicrobial agents that are applied to non-living objects to destroy microorganisms. (Reducing the number of pathogenic microorganisms). Antiseptics:- are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection , or rot. Note That not all disinfectants are antiseptics because an antiseptic additionally must not be so harsh that it damages living tissue. Sterilization methods Sterilization methods are divided into:- 1- Chemical sterilization method 2- Physical sterilization method I-Chemical sterilization method Chemical sterilization is typically used for devices that would be sensitive to the high heat used in steam sterilization, and for devices that may be damaged by irradiation (rubbers and plastics can become more brittle after irradiation). Chemical sterilization method Chemical sterilization method :- it is divided into 1- Chemical liquid sterilization 2- Chemical vapour sterilization. 1 1- Chemical liquid sterilization Disinfectants classification into: High level disinfectants. Use for large number of spores after prolonged exposure , Vegetative bacteria ,Tubercle bacilli ,Fungi ,Viruses example H2O2(3-6%) Intermediate level disinfectants. Use for Few number of spores ,Vegetative bacteria,Tubercle bacilli ,Fungi ,Enveloped viruses (HBV, HIV). Example 70% ethyl alcohol and isopropyl alcohol. Low level disinfectants. Use for Mainly vegetative bacteria, Some fungi , Narrow range of viruses eample Providon(Iodine 5 and 10%). 2- Chemical vapor sterilization includes 1-Chemical Vapor under pressure (Chemiclave):- This process uses a mixture of chemicals, including alcohol, formaldehyde, ketone, acetone, and water, that are heated under pressure to form a sterilizing gas. Sterilization requires 20 minutes at 131° C and 20 lbs pressure when instruments are either unwrapped or bagged. 2- Fumigation :- to produce the fumigant, potassium permanganate should be mixed with formalin in a ratio (w/v) of 2:3. When the correct ratio of formalin and potassium permanganate is used 2-Physical sterilization method Physical sterilization methods use physical agents or processes to eliminate or inactivate all forms of microorganisms, including bacteria, viruses, and spores, from surfaces, equipment, and materials. Here are some common physical sterilization methods: 2 1-Autoclaving (Moist Heat Sterilization) Uses pressurized steam at high temperatures, typically 121°C (250°F) for 15–20 minutes. effective for sterilizing media, lab instruments, surgical tools, and biohazard waste. kills most microorganisms, including spores, due to the combined effects of heat and moisture. 2- Dry Heat Sterilization Involves heating materials to high temperatures, usually around 160– 180°C (320–356°F) for 1–2 hours in a dry heat oven. suitable for sterilizing metal instruments, glassware, and powders.works by oxidizing microbial cells, effectively killing them without moisture. 3-Filtration Removes microorganisms from liquids or air by passing them through a filter with pore sizes small enough (typically 0.2 microns) to trap microbes. used for heat-sensitive liquids, such as vaccines or enzyme solutions, and in HEPA filters for air sterilization. effective for removing bacteria and larger viruses but may not retain smaller viruses or prions. 4-Radiation (Ionizing Radiation) Involves gamma rays or electron beams to sterilize materials by breaking down DNA in microorganisms. used for sterilizing medical equipment, disposable plastics, and some pharmaceuticals. penetrates deeply and is effective even for heat-sensitive materials. 5- Ultraviolet (UV) Radiation Uses UV-C light (wavelength around 254 nm) to damage the DNA of microorganisms, preventing their replication. commonly used to sterilize 3 air, water, and surfaces in healthcare and lab settings. limited penetration depth, effective only for surfaces or thin layers of water or air. 6-Boiling Boiling water (100°C or 212°F) for 10–15 minutes can kill most bacteria and viruses but may not be effective against all spores. often used as a basic disinfection method rather than true sterilization. useful for sterilizing certain household items, baby bottles, and non-heat-sensitive items in emergencies. 7- Incineration Destroys contaminated materials by burning them at very high temperatures. commonly used for medical waste disposal, such as contaminated dressings, pathological waste, and sharps. completely eradicates all microorganisms by combustion but is only suitable for items intended for disposal. Diagnosis of Bacteria Diagnosing bacterial infections involves identifying and characterizing bacteria present in a patient sample (e.g., blood, urine, sputum). Techniques include: A. Microscopy  Gram Staining: A basic staining technique to classify bacteria as Gram-positive or Gram-negative based on cell wall structure, which aids in initial identification.  Acid-Fast Staining: Used primarily for detecting Mycobacterium species (e.g., tuberculosis), which have unique cell walls. 4   Phase-Contrast and Dark-Field Microscopy: Enhance visualization of bacterial morphology and motility, useful for bacteria that are difficult to stain. B. Culture Methods  Agar Plates: Samples are cultured on selective or differential media to promote bacterial growth. Blood agar and MacConkey agar are common types used to identify pathogens based on colony morphology and color changes.  Broth Cultures: Bacteria can be cultured in liquid media to detect growth, typically indicated by turbidity. This method is useful for anaerobic bacteria and bacteria that don’t grow well on solid media. C. Biochemical Testing  Catalase and Coagulase Tests: Used to differentiate types of Staphylococcus species, with coagulase-positive indicating Staphylococcus aureus.  Oxidase and Urease Tests: Help identify bacteria based on enzyme presence, useful for differentiating among Gram-negative bacteria.  Automated Systems: Systems like VITEK and BD Phoenix automate biochemical testing for faster bacterial identification. D. Molecular Methods  Polymerase Chain Reaction (PCR): Detects bacterial DNA or RNA, enabling rapid identification of specific pathogens. PCR is highly sensitive and used for pathogens difficult to culture. 5   DNA Sequencing: Sequencing bacterial genomes can provide highly specific identification and information on antibiotic resistance. 2. Blood Analysis Blood analysis evaluates the components and characteristics of blood to detect infections, blood disorders, and other health conditions. A. Complete Blood Count (CBC)  Automated Hematology Analyzers: Machines that count red blood cells (RBCs), white blood cells (WBCs), and platelets and measure hemoglobin, hematocrit, and other parameters.  Blood Smear Microscopy: A manual examination of blood smears under a microscope to observe cell morphology. It is essential for diagnosing infections like malaria and conditions such as leukemia or anemia. B. Hemoglobin and Hematocrit Tests Measures hemoglobin concentration and the proportion of blood that is RBCs. Low levels can indicate anemia, while high levels may indicate polycythemia. C. Blood Coagulation Tests  Prothrombin Time (PT) and Partial Thromboplastin Time (PTT): Assess blood clotting function, essential for diagnosing bleeding disorders and monitoring anticoagulant therapy. 6   Platelet Function Tests: Evaluate platelet adhesion and aggregation, important for detecting platelet disorders and bleeding risks. D. Flow Cytometry Used for detailed analysis of blood cell populations by tagging cells with fluorescent antibodies. Flow cytometry is especially useful for diagnosing leukemias and lymphomas and monitoring immune function. E. Bone Marrow Analysis Bone marrow biopsies or aspirations provide information about blood cell production, important for diagnosing hematologic cancers, anemias, and bone marrow disorders. Clinical Chemistry Clinical chemistry involves measuring various chemical components in blood and other bodily fluids, which can indicate the function of organs, metabolic state, and presence of diseases. A. Spectrophotometry  Measures the absorbance of light by substances in blood, such as glucose, cholesterol, and proteins, by analyzing color changes that correlate with concentration levels.  Enzymatic Reactions: Many tests (e.g., liver enzyme tests) use specific enzyme reactions that produce color changes detectable by spectrophotometry. 7 B. Electrolyte Analysis Measure electrolytes (e.g., sodium, potassium, chloride) in blood. Imbalances can indicate kidney disease, dehydration, or heart issues. C. Immunoassays Enzyme-Linked Immunosorbent Assay (ELISA): Uses antibodies to detect specific proteins or hormones, such as insulin, TSH, or troponin. ELISAs are essential in diagnosing infections, hormone imbalances, and cardiac events. D. Chromatography Gas Chromatography (GC) and High-Performance Liquid Chromatography (HPLC): Separate and identify compounds in blood or urine, useful for drug testing, toxicology, and monitoring therapeutic drug levels. E. Electrophoresis Separates proteins based on their charge and size, used in protein analysis to detect abnormal protein levels or patterns, as seen in multiple myeloma and other blood disorders. F. Automated Analyzers Modern labs use automated analyzers to measure various biochemical markers like glucose, lipids, liver enzymes, and kidney function markers, enabling high-throughput and rapid diagnostics. 8 Laboratory Safety and Accident Prevention 1. First Aid: Keep first aid kits, eyewash stations, and emergency showers accessible. Train staff in handling injuries like burns, cuts, or chemical exposure. 2. Biochemical Hazards: Use proper storage, labeling, and PPE for chemicals. Work in fume hoods and dispose of waste safely. 3. Biological Hazards: Follow biosafety protocols, use biological safety cabinets, and disinfect surfaces. Wear PPE and handle pathogens with care. 4. General Safety: Follow Standard Operating Procedures (SOPs), conduct regular safety checks, and maintain safety equipment. Always report hazards. 9