Lecture 4 sterilization

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  • 1. Definition Sterilization: the elimination of all transmissible agents Sterilization (such as bacteria, prions and viruses) from a surface, a piece of equipment, food or biological culture medium. Prof. S.T. Yang Disinfection: the elimination of only the pathogenic Dept. Chemical & Biomolecular Eng. microorganism The Ohio State University Why Sterilization? Source of Contaminants “To reduce the amount of contaminants present in the culture medium” Raw materials Equipment and Instruments Manufacturing Process Containers and closure systems Manufacturing environment people 1
  • 2. Contaminants Prevention Cause loss of productivity and product Using pure inoculum to start the yield fermentation Contaminate and degrade final products Sterilizing medium, fermentor, air, etc Could outgrow the production organisms Maintaining aseptic conditions Could results in lysis of the culture (phage Use natural selection to resist the growth contaminants) of contaminants: pH, EtOH, high Temperature, etc. Control of Microbial Growth Control of Microbial Growth Early civilizations practiced salting, In mid 1800s Semmelweiss and Lister smoking, pickling, drying, and exposure of helped developed aseptic techniques to food and clothing to sunlight to control prevent contamination of surgical wounds. microbial growth. Before then: • Nosocomial infections caused death in 10% of Use of spices in cooking was to mask surgeries. taste of spoiled food. Some spices • Up to 25% mothers delivering in hospitals prevented spoilage. died due to infection 2
  • 3. Sterilization Disinfection Sterilization: Killing or removing all forms of Disinfection: Reducing the number of pathogenic microbial life (including endospores) in a microorganisms to the point where they no longer material or an object. cause diseases. Usually involves the removal of Heating is the most commonly used method of vegetative or non-endospore forming sterilization. pathogens. Commercial Sterilization: Heat treatment that May use physical or chemical methods. kills endospores of Clostridium botulinum the Disinfectant: Applied to inanimate objects. causative agent of botulism, in canned food. Antiseptic: Applied to living tissue (antisepsis). Does not kill endospores of thermophiles, which Degerming: Mechanical removal of most microbes in a are not pathogens and may grow at temperatures limited area. Example: Alcohol swab on skin. above 45oC. Sanitization: Use of chemical agent on food-handling equipment to meet public health standards and minimize chances of disease transmission. E.g: Hot soap & water. Biological Terms Biological Terms (cont’d) Sepsis: Comes from Greek for decay or putrid. Bacteriostatic Agent: An agent that inhibits the Indicates bacterial contamination. growth of bacteria, but does not necessarily kill them. Suffix stasis: To stop or steady. Asepsis: Absence of significant contamination. Germicide: An agent that kills certain Aseptic techniques are used to prevent microorganisms. contamination of surgical instruments, medical Bactericide: An agent that kills bacteria. Most do not personnel, and the patient during surgery. kill endospores. Aseptic techniques are also used to prevent Viricide: An agent that inactivates viruses. bacterial contamination in the food industry. Fungicide: An agent that kills fungi. Sporicide: An agent that kills bacterial endospores or fungal spores. 3
  • 4. Factors Affecting Sterilization Types of Sterilization Number of Microbes: The more microbes present, the Heat more time it takes to eliminate population. Type of Microbes: Endospores are very difficult to Chemicals destroy. Vegetative pathogens vary widely in susceptibility to different methods of microbial control. Filtration Environmental influences: Presence of organic Irradiation material (blood, feces, saliva) tends to inhibit antimicrobials, pH etc. Others – pressure, desiccation, freezing, Time of Exposure: Chemical antimicrobials and etc. radiation treatments are more effective at longer times. In heat treatments, longer exposure compensates for lower temperatures. Heat Sterilization Heat Sterilization Heat: Kills microorganisms by denaturing their enzymes When bacterial populations are heated or and other proteins. Heat resistance varies widely among treated with antimicrobial chemicals, they usually microbes. die at a constant rate. Thermal Death Point (TDP): Lowest temperature at which all of the microbes in a liquid suspension will be killed in ten minutes. Thermal Death Time (TDT): Minimal length of time in which all bacteria will be killed at a given temperature. Decimal Reduction Time (DRT): Time in minutes at which 90% of bacteria at a given temperature will be killed. Used in canning industry. 4
  • 5. Heat Sterilization – Moist Heat Heat Sterilization – Moist Heat Moist Heat: Kills microorganisms by coagulating Moist Heat (Continued): their proteins. Reliable sterilization with moist heat requires In general, moist heat is much more effective than temperatures above that of boiling water. dry heat. Autoclave: Chamber which is filled with hot steam under Boiling: Heat to 100oC or more at sea level. Kills pressure. Preferred method of sterilization, unless material vegetative forms of bacterial pathogens, almost all viruses, is damaged by heat, moisture, or high pressure. and fungi and their spores within 10 minutes or less. Temperature of steam reaches 121oC at 15 psig (2× atmospheric pressure). Endospores and some viruses are not destroyed this Most effective when organisms contact steam directly or are contained in a quickly. However brief boiling will kill most pathogens. small volume of liquid. All organisms and endospores are killed within 15 minutes. Hepatitis virus: Can survive up to 30 minutes of boiling. Require more time to reach center of solid or large volumes of liquid. Endospores: Can survive up to 20 hours or more of boiling. Autoclave Moist Heat - Pasteurization Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc. Classic Method of Pasteurization: Milk was exposed to 65oC for 30 minutes. High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72oC for 15 seconds. Ultra High Temperature Pasteurization (UHT): Milk is treated at 140oC for 3 seconds and then cooled very quickly in a vacuum chamber. Advantage: Milk can be stored at room temperature for several months. 5
  • 6. Heat Sterilization – Dry Heat Filtration Sterilization Removal of microbes by passage of a liquid or gas Dry Heat: Kills by oxidation effects. through a screen-like material with small pores. Direct Flaming: Used to sterilize inoculating loops and Used to sterilize heat sensitive materials like needles. Heat metal until it has a red glow. vaccines, enzymes, antibiotics, and some culture Incineration: Effective way to sterilize disposable items media. (paper cups, dressings) and biological waste. High Efficiency Particulate Air Filters (HEPA): Used in Hot Air Sterilization: Place objects in an oven. operating rooms and burn units to remove bacteria from air. Require 2 hours at 170oC for sterilization. Dry heat Membrane Filters: Uniform pore size. Used in industry transfers heat less effectively to a cool body, than moist and research. Different sizes: heat. 0.22 and 0.45 μm Pores: Used to filter most bacteria. Don’t retain spirochetes, mycoplasmas and viruses. 0.01 μm Pores: Retain all viruses and some large proteins. Freezing Sterilization Desiccation Sterilization Low Temperature: Effect depends on microbe Dessication: In the absence of water, microbes and treatment applied. cannot grow or reproduce, but some may remain Refrigeration: Temperatures from 0 to 7oC. viable for years. After water becomes available, Bacteriostatic effect. Reduces metabolic rate of they start growing again. most microbes so they cannot reproduce or produce toxins. Susceptibility to dessication varies widely: Freezing: Temperatures below 0oC. Neisseria gonnorrhea: Only survives about one hour. Flash Freezing: Does not kill most microbes. Mycobacterium tuberculosis: May survive several months. Slow Freezing: More harmful because ice crystals Viruses are fairly resistant to dessication. disrupt cell structure. Over a third of vegetative bacteria may survive 1 year. Clostridium spp. and Bacillus spp.: May survive decades. Most parasites are killed by a few days of freezing. 6
  • 7. Pressure-driven Sterilization Radiation Sterilization Osmotic Pressure: The use of high Three types of radiation kill microbes: concentrations of salts and sugars in foods is - Ionizing Radiation used to increase the osmotic pressure and create a hypertonic environment. - Ultraviolet light (Nonionizing Radiation) Plasmolysis: As water leaves the cell, plasma - Microwave Radiation membrane shrinks away from cell wall. Cell may not die, but usually stops growing. Yeasts and molds: More resistant to high osmotic pressures. Staphylococci spp. that live on skin are fairly resistant to high osmotic pressure. Ionizing Radiation Ultraviolet Radiation Gamma rays, X rays, electron beams, or higher Nonionizing radiation energy rays. Have short wavelengths (less than 1 nanometer). Wavelength is longer than 1 nanometer. Dislodge electrons from atoms and form ions. Damages DNA by producing thymine dimers, Cause mutations in DNA and produce which cause mutations. peroxides. Used to disinfect operating rooms, nurseries, Used to sterilize pharmaceuticals and cafeterias. disposable medical supplies. Food industry is interested in using ionizing radiation. Disadvantages: Damages skin, eyes. Doesn’t Disadvantages: Penetrates human tissues. May penetrate paper, glass, and cloth. cause genetic mutations in humans. 7
  • 8. Microwave Radiation Chemical Sterilization Wavelength ranges from 1 millimeter to 1 meter. Phenol and Phenolics Heat is absorbed by water molecules. Halogens May kill vegetative cells in moist foods. Alcohols Bacterial endospores, which do not contain Heavy Metals water, are not damaged by microwave radiation. Quaternary Ammonium Compounds Solid foods are unevenly penetrated by microwaves. Aldehydes Trichinosis outbreaks have been associated with Gaseous pork cooked in microwaves. Peroxygens Chemical - Phenolic Chemical - Halogens Phenol (carbolic acid) was first used by Lister as a disinfectant. Iodine: Rarely used today because it is a skin irritant and has strong Tincture of iodine (alcohol solution) was one of first odor. antiseptics used. Used in some throat sprays and lozenges. Combines with amino acid tyrosine in proteins and Acts as local anesthetic. denatures proteins. Phenolics are chemical derivatives of phenol Stains skin and clothes, somewhat irritating. Cresols: Derived from coal tar (Lysol). Iodophors: Compounds with iodine that are slow Biphenols (pHisoHex): Effective against gram-positive staphylococci and streptococci. Used in nurseries. Excessive releasing, take several minutes to act. Used as skin use in infants may cause neurological damage. antiseptic in surgery. Not effective against bacterial endospores. Destroy plasma membranes and denature proteins. Betadine Advantages: Stable, persist for long times after applied, Isodine and remain active in the presence of organic compounds. 8
  • 9. Chemical – Halogens (cont’d) Chemical - Alcohols Chlorine: Kill bacteria, fungi, but not endospores or naked viruses. When mixed in water forms hypochlorous acid: Act by denaturing proteins and disrupting cell membranes. Cl2 + H2O ------> H+ + Cl- + HOCl Evaporate, leaving no residue. Hypochlorous acid Used to mechanically wipe microbes off skin before Used to disinfect drinking water, pools, and sewage. injections or blood drawing. Chlorine is easily inactivated by organic materials. Not good for open wounds, because cause proteins to Sodium hypochlorite (NaOCl): Is active ingredient of coagulate. bleach. Ethanol: Drinking alcohol. Optimum concentration is 70%. Chloramines: Consist of chlorine and ammonia. Less Isopropanol: Rubbing alcohol. Better disinfectant than effective as germicides. ethanol. Also cheaper and less volatile. Chemical – Heavy Metals Chemical – Heavy Metals (cont’d) Oligodynamic action: Very tiny amounts are effective. Copper Copper sulfate is used to kill algae in pools and fish Silver tanks. 1% silver nitrate used to protect infants against gonorrheal eye infections until recently. Selenium Kills fungi and their spores. Used for fungal infections. Mercury Also used in dandruff shampoos. Organic mercury compounds like merthiolate and mercurochrome are used to disinfect skin wounds. Zinc Zinc chloride is used in mouthwashes. Zinc oxide is used as antifungal agent in paints. 9
  • 10. Chemical – Quats Chemical – Aldehydes Widely used surface active agents. Inactivate proteins by forming covalent crosslinks with Cationic (positively charge) detergents. several functional groups. Include some of the most effective antimicrobials. Effective against gram positive bacteria, less effective against gram-negative bacteria. Formaldehyde gas: Also destroy fungi, amoebas, and enveloped viruses. Excellent disinfectant. Zephiran, Cepacol, also found in lab spray bottles. Commonly used as formalin, a 37% aqueous solution. Pseudomonas strains that are resistant and can grow Formalin was used extensively to preserve biological in presence of Quats are a big concern in hospitals. specimens and inactivate viruses and bacteria in Advantages: Strong antimicrobial action, colorless, vaccines. odorless, tasteless, stable, and nontoxic. Irritates mucous membranes, strong odor. Disadvantages: Form foam. Organic matter interferes Also used in mortuaries for embalming. with effectiveness. Neutralized by soaps and anionic detergents. Chemical – Aldehydes (cont’d) Chemical – Gaseous Sterilizers Chemicals that sterilize in a chamber similar to an Glutaraldehyde: autoclave. Less irritating and more effective than formaldehyde. Denature proteins, by replacing functional groups with One of the few chemical disinfectants that is a alkyl groups. sterilizing agent. A 2% solution of glutaraldehyde (Cidex) is: Ethylene Oxide: Bactericidal, tuberculocidal, and viricidal in 10 Kills all microbes and endospores, but requires minutes. exposure of 4 to 18 hours. Sporicidal in 3 to 10 hours. Toxic and explosive in pure form. Commonly used to disinfect hospital instruments. Highly penetrating. Also used in mortuaries for embalming. Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment. 10
  • 11. Chemical – Peroxygens Chemical – Peroxygens (cont’d) Oxidizing agents Hydrogen Peroxide: Disrupt membranes and proteins. Used as an antiseptic. Oxidize cellular components of treated microbes. Not good for open wounds because quickly broken down by catalase present in human cells. Ozone: Effective in disinfection of inanimate objects. Used along with chlorine to disinfect water. Sporicidal at higher temperatures. Helps neutralize unpleasant tastes and odors. Used by the food industry and to disinfect contact More effective killing agent than chlorine, but less lenses. stable and more expensive. Highly reactive form of oxygen. Benzoyl Peroxide: Made by exposing oxygen to electricity or UV light. Used in acne medications. Efficiency of Different Chemical Chemical – Peroxygens (cont’d) Antimicrobial Agents Peracetic Acid: One of the most effective liquid sporicides available. Sterilant : Kills bacteria and fungi in less than 5 minutes. Kills endospores and viruses within 30 minutes. Used widely in disinfection of food and medical instruments because it does not leave toxic residues. 11
  • 12. Sterilization in fermentation Liquid medium Fermentors and other equipment Air 12