logging in or signing up sterilisation and Disinfection By Prof. Satish Polshettiwar, MIP Pune satishmip Download Post to : URL : Related Presentations : Let's Connect Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Copy embed code: Embed: Flash iPad Dynamic Copy Does not support media & animations Automatically changes to Flash or non-Flash embed WordPress Embed Customize Embed URL: Copy Thumbnail: Copy The presentation is successfully added In Your Favorites. Views: 2013 Category: Education License: All Rights Reserved Like it (4) Dislike it (0) Added: March 18, 2011 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript Slide 1: MAEER’S, MAHARASHTRA INSTITUTE OF PHARMACY, PUNE-38. CONTROL OF MICROBIAL GROWTH Prof. S. A. Polshettiwar M. Pharm, D.I.T., PhD Slide 2: Control of Microbial Growth: Introduction Early civilizations practiced salting, smoking, pickling, drying, and exposure of food and clothing to sunlight to control microbial growth. Use of spices in cooking was to mask taste of spoiled food. Some spices prevented spoilage. In mid 1800s Semmelweiss and Lister helped developed aseptic techniques to prevent contamination of surgical wounds. Before then: Nosocomial infections caused death in 10% of surgeries. Up to 25% mothers delivering in hospitals died due to infection Reasons for controlling microorganism : Reasons for controlling microorganism To prevent contamination in sterile product To prevent transmission of microorganisms responsible to cause disease. To prevent decompostion,spoilage of food and food product. To prevent contamination of unwanted microbes in pure culture To prevent contamination in aseptic area. Sterilization : Sterilization Sterilization (or sterilisation, see spelling differences) is a term referring to any process that eliminates (removes) or kills all forms of life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media.Sterilization can be achieved by applying the proper combinations of heat, chemicals, irradiation, high pressure, and filtration. Slide 5: Control of Microbial Growth: D efinitions Sterilization: Killing or removing all forms of microbial life (including endospores) in a material or an object. Heating is the most commonly used method of sterilization. Commercial Sterilization: Heat treatment that kills endospores of Clostridium botulinum the causative agent of botulism, in canned food. Does not kill endospores of thermophiles, which are not pathogens and may grow at temperatures above 45oC. Slide 6: Control of Microbial Growth: D efinitions Disinfection: Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases. Usually involves the removal of vegetative or non-endospore forming pathogens. May use physical or chemical methods. Disinfectant: Applied to inanimate objects. Antiseptic: Applied to living tissue (antisepsis). Degerming: Mechanical removal of most microbes in a limited area. Example: Alcohol swab on skin. 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. Slide 7: Control of Microbial Growth: D efinitions Sepsis: Comes from Greek for decay or putrid. Indicates bacterial contamination. Asepsis: Absence of significant contamination. Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery. Aseptic techniques are also used to prevent bacterial contamination in food industry. Slide 8: Control of Microbial Growth: D efinitions Bacteriostatic Agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them. Suffix stasis: To stop or steady. Germicide: An agent that kills certain microorganisms. Bactericide: An agent that kills bacteria. Most do not kill endospores. Viricide: An agent that inactivates viruses. Fungicide: An agent that kills fungi. Sporicide: An agent that kills bacterial endospores of fungal spores. Slide 9: Control of Microbial Growth: Rate of Microbial Death When bacterial populations are heated or treated antimicrobial chemicals, they usually die at a constant rate. Slide 10: Control of Microbial Growth: Rate of Microbial Death Several factors influence the effectiveness of antimicrobial treatment. 1. Number of Microbes: The more microbes present, the more time it takes to eliminate population. 2. Type of Microbes: Endospores are very difficult to destroy. Vegetative pathogens vary widely in susceptibility to different methods of microbial control. 3. Environmental influences: Presence of organic material (blood, feces, saliva) tends to inhibit antimicrobials, pH etc. 4. Time of Exposure: Chemical antimicrobials and radiation treatments are more effective at longer times. In heat treatments, longer exposure compensates for lower temperatures. Slide 11: Physical Methods of Microbial Control: Heat: Kills microorganisms by denaturing their enzymes and other proteins. Heat resistance varies widely among microbes. 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. Slide 12: Physical Methods of Microbial Control: Moist Heat: Kills microorganisms by coagulating their proteins. In general, moist heat is much more effective than dry heat. Boiling: Heat to 100oC or more at sea level. Kills vegetative forms of bacterial pathogens, almost all viruses, and fungi and their spores within 10 minutes or less. Endospores and some viruses are not destroyed this quickly. However brief boiling will kill most pathogens. Hepatitis virus: Can survive up to 30 minutes of boiling. Endospores: Can survive up to 20 hours or more of boiling. STERILIZATION METHODS : STERILIZATION METHODS Physical Methods: 1. Moist Heat Sterilization a. Autoclave b. Boiling at 100 deg. C c. Pasteurization d. heating with bactericide e. Tydallization 2. Dry heat sterilization a. Hot air oven b. Flaming c. Red hot technique d. Incineration 3. Filtration or Mechanical Method: Membrane filter 4. Chemical method Gaseous sterilization or by using disinfectant 5. Irradiation : UV., Gamma etc NOTE: End products must pass sterility tests. Autoclave: Closed Chamber with High Temperature and Pressure : Autoclave: Closed Chamber with High Temperature and Pressure Slide 16: Laboratory autoclave Industrial autoclave Autoclave: : Autoclave: An autoclave is a device to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C or more, typically for 15-20 minutes depending on the size of the load and the contents. It was invented by Charles Chamberland in 1879 Mechanism: : Mechanism: Saturated steam condensate on the material or microbes and gives up heat and the material or microbes get heated to 121oC or more depending upon the pressure. While it condenses, it hydrate the microbes by proving latent heat from vapor thereby making them vulnerable to killing. Moreover, when the steam condensate it contract and vacuum is created. Fresh store of steam takes its place and pushes I under pressure, penetrating the load and killing the microorganism. Killing is done by : Coagulation of protein Denaturation of protien Sterilization cycle or steps: Loading and packing of the autoclave Raising temp and pressure Holding the load at specific temp and pressure Cooling and unloading Slide 19: Physical Methods of Microbial Control: Moist Heat (Continued): Reliable sterilization with moist heat requires temperatures above that of boiling water. Autoclave: Chamber which is filled with hot steam under pressure. Preferred method of sterilization, unless material is damaged by heat, moisture, or high pressure. Temperature of steam reaches 121oC at twice atmospheric pressure. Most effective when organisms contact steam directly or are contained in a small volume of liquid. All organisms and endospores are killed within 15 minutes. Require more time to reach center of solid or large volumes of liquid. STERILIZATION : STERILIZATION 10 lb Pressure (115.5 deg. C)...30 minutes 15 lb Pressure (121.5 deg. C)...20 minutes 20 lb Pressure (126.5 deg. C)... 15 minutes STERILIZATION : STERILIZATION Application: 1. Solutions sealed in containers ampuls, vials 2. Bulk Solutions 3. Glassware 4. Surgical Dressing 5. Instruments 6. Culture media STERILIZATION : STERILIZATION Advantages: Rapid, Inexpensive, Effective, Large volumes, Steam has Better penetrating power and applicable to wide variety of materials Shorter exposure of sterilization Disadvantages: 1. Cannot use for oily preparation (oil base ointment) 2. Cannot use for moisture sensitive preparations Slide 23: Physical Methods of Microbial Control: Moist Heat (Continued): 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. Slide 24: Physical Methods of Microbial Control: Dry Heat: Kills by oxidation effects. Direct Flaming: Used to sterilize inoculating loops and needles. Heat metal until it has a red glow. Incineration: Effective way to sterilize disposable items (paper cups, dressings) and biological waste. Hot Air Sterilization: Place objects in an oven. Require 2 hours at 170oC for sterilization. Dry heat is transfers heat less effectively to a cool body, than moist heat. Hot air Oven : Hot air Oven Hot air Oven : Hot air Oven It works on the principle of killing of microorganism by dry heat at 150 to 170oC for 1 hrs Killes microorgnaism by oxidation of protie. Slide 27: Physical Methods of Microbial Control: Filtration: Removal of microbes by passage of a liquid or gas through a screen like material with small pores. Used to sterilize heat sensitive materials like vaccines, enzymes, antibiotics, and some culture media. High Efficiency Particulate Air Filters (HEPA): Used in operating rooms and burn units to remove bacteria from air. Membrane Filters: Uniform pore size. Used in industry and research. Different sizes: 0.22 and 0.45um Pores: Used to filter most bacteria. Don’t retain spirochetes, mycoplasmas and viruses. 0.01 um Pores: Retain all viruses and some large proteins. Slide 28: Nitrocellulose filter Slide 30: A complete filtering set up Slide 31: Physical Methods of Microbial Control: Low Temperature: Effect depends on microbe and treatment applied. Refrigeration: Temperatures from 0 to 7oC. Bacteriostatic effect. Reduces metabolic rate of most microbes so they cannot reproduce or produce toxins. Freezing: Temperatures below 0oC. Flash Freezing: Does not kill most microbes. Slow Freezing: More harmful because ice crystals disrupt cell structure. Over a third of vegetative bacteria may survive 1 year. Most parasites are killed by a few days of freezing. Slide 33: Physical Methods of Microbial Control: Dessication: In the absence of water, microbes cannot grow or reproduce, but some may remain viable for years. After water becomes available, they start growing again. Susceptibility to dessication varies widely: Neisseria gonnorrhea: Only survives about one hour. Mycobacterium tuberculosis: May survive several months. Viruses are fairly resistant to dessication. Clostridium spp. and Bacillus spp.: May survive decades. Slide 34: Physical Methods of Microbial Control: Osmotic Pressure: The use of high concentrations of salts and sugars in foods is used to increase the osmotic pressure and create a hypertonic environment. Plasmolysis: As water leaves the cell, plasma 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. Slide 35: Physical Methods of Microbial Control: Radiation: Three types of radiation kill microbes: 1. Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy rays. Have short wavelengths (less than 1 nanometer). Dislodge electrons from atoms and form ions. Cause mutations in DNA and produce peroxides. Used to sterilize pharmaceuticals and disposable medical supplies. Food industry is interested in using ionizing radiation. Disadvantages: Penetrates human tissues. May cause genetic mutations in humans. Forms of Radiation : Forms of Radiation Radiation : Radiation Non- ionizing radiation (UV light) Ultraviolet light Damages DNA Adjacent thymines (pyrimidine base) form bonds Forms thymine dimers Inhibits correct replication of DNA UV lamps Germicidal lamps Disadvantage Rays do not penetrate, microbes on surfaces Cannot penetrate paper Prolonged exposure Eyes damage, burns, and skin cancer Slide 38: Physical Methods of Microbial Control: Radiation: Three types of radiation kill microbes: 2. Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations. Used to disinfect operating rooms, nurseries, cafeterias. Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and cloth. Slide 39: Physical Methods of Microbial Control: Radiation: Three types of radiation kill microbes: 3. Microwave Radiation: Wavelength ranges from 1 millimeter to 1 meter. Heat is absorbed by water molecules. May kill vegetative cells in moist foods. Bacterial endospores, which do not contain water, are not damaged by microwave radiation. Solid foods are unevenly penetrated by microwaves. Trichinosis outbreaks have been associated with pork cooked in microwaves Comparison of Methods : Comparison of Methods Slide 41: Chemical Methods of Microbial Control Types of Disinfectants 1. Phenols and Phenolics: Phenol (carbolic acid) was first used by Lister as a disinfectant. Rarely used today because it is a skin irritant and has strong odor. Used in some throat sprays and lozenges. Acts as local anesthetic. Phenolics are chemical derivatives of phenol Cresols: Derived from coal tar (Lysol). Biphenols (pHisoHex): Effective against gram-positive staphylococci and streptococci. Used in nurseries. Excessive use in infants may cause neurological damage. Destroy plasma membranes and denature proteins. Advantages: Stable, persist for long times after applied, and remain active in the presence of organic compounds. Slide 42: Chemical Methods of Microbial Control Types of Disinfectants 2. Halogens: Effective alone or in compounds. A. Iodine: Tincture of iodine (alcohol solution) was one of first antiseptics used. Combines with amino acid tyrosine in proteins and denatures proteins. Stains skin and clothes, somewhat irritating. Iodophors: Compounds with iodine that are slow releasing, take several minutes to act. Used as skin antiseptic in surgery. Not effective against bacterial endospores. Betadine Isodine Slide 43: Chemical Methods of Microbial Control Types of Disinfectants 2. Halogens: Effective alone or in compounds. B. Chlorine: When mixed in water forms hypochlorous acid: Cl2 + H2O ------> H+ + Cl- + HOCl Hypochlorous acid Used to disinfect drinking water, pools, and sewage. Chlorine is easily inactivated by organic materials. Sodium hypochlorite (NaOCl): Is active ingredient of bleach. Chloramines: Consist of chlorine and ammonia. Less effective as germicides. Slide 44: Chemical Methods of Control Types of Disinfectants 3. Alcohols: Kill bacteria, fungi, but not endospores or naked viruses. Act by denaturing proteins and disrupting cell membranes. Evaporate, leaving no residue. Used to mechanically wipe microbes off skin before injections or blood drawing. Not good for open wounds, because cause proteins to coagulate. Ethanol: Drinking alcohol. Optimum concentration is 70%. Isopropanol: Rubbing alcohol. Better disinfectant than ethanol. Also cheaper and less volatile. Slide 45: Chemical Methods of Control Types of Disinfectants 4. Heavy Metals: Include copper, selenium, mercury, silver, and zinc. Oligodynamic action: Very tiny amounts are effective. A. Silver: 1% silver nitrate used to protect infants against gonorrheal eye infections until recently. B. Mercury Organic mercury compounds like merthiolate and mercurochrome are used to disinfect skin wounds. C. Copper Copper sulfate is used to kill algae in pools and fish tanks. Slide 46: Chemical Methods of Control Types of Disinfectants 4. Heavy Metals: D. Selenium Kills fungi and their spores. Used for fungal infections. Also used in dandruff shampoos. E. Zinc Zinc chloride is used in mouthwashes. Zinc oxide is used as antifungal agent in paints. Slide 47: Chemical Methods of Control Types of Disinfectants 5. Quaternary Ammonium Compounds (Quats): Widely used surface active agents. Cationic (positively charge) detergents. Effective against gram positive bacteria, less effective against gram-negative bacteria. Also destroy fungi, amoebas, and enveloped viruses. Zephiran, Cepacol, also found in our lab spray bottles. Pseudomonas strains that are resistant and can grow in presence of Quats are a big concern in hospitals. Advantages: Strong antimicrobial action, colorless, odorless, tasteless, stable, and nontoxic. Diasadvantages: Form foam. Organic matter interferes with effectiveness. Neutralized by soaps and anionic detergents. Slide 48: Chemical Methods of Control Types of Disinfectants 6. Aldehydes: Include some of the most effective antimicrobials. Inactivate proteins by forming covalent crosslinks with several functional groups. A. Formaldehyde gas: Excellent disinfectant. Commonly used as formalin, a 37% aqueous solution. Formalin was used extensively to preserve biological specimens and inactivate viruses and bacteria in vaccines. Irritates mucous membranes, strong odor. Also used in mortuaries for embalming. Slide 49: Chemical Methods of Control Types of Disinfectants 6. Aldehydes: B. Glutaraldehyde: Less irritating and more effective than formaldehyde. One of the few chemical disinfectants that is a sterilizing agent. A 2% solution of glutaraldehyde (Cidex) is: Bactericidal, tuberculocidal, and viricidal in 10 minutes. Sporicidal in 3 to 10 hours. Commonly used to disinfect hospital instruments. Also used in mortuaries for embalming. Slide 50: Chemical Methods of Control Types of Disinfectants 7. Gaseous Sterilizers: Chemicals that sterilize in a chamber similar to an autoclave. Denature proteins, by replacing functional groups with alkyl groups. A. Ethylene Oxide: Kills all microbes and endospores, but requires exposure of 4 to 18 hours. Toxic and explosive in pure form. Highly penetrating. Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment. Slide 51: Chemical Methods of Control Types of Disinfectants 8. Peroxygens (Oxidizing Agents): Oxidize cellular components of treated microbes. Disrupt membranes and proteins. A. Ozone: Used along with chlorine to disinfect water. Helps neutralize unpleasant tastes and odors. More effective killing agent than chlorine, but less stable and more expensive. Highly reactive form of oxygen. Made by exposing oxygen to electricity or UV light. Slide 52: Chemical Methods of Control Types of Disinfectants 8. Peroxygens (Oxidizing Agents): B. Hydrogen Peroxide: Used as an antiseptic. Not good for open wounds because quickly broken down by catalase present in human cells. Effective in disinfection of inanimate objects. Sporicidal at higher temperatures. Used by food industry and to disinfect contact lenses. C. Benzoyl Peroxide: Used in acne medications. Slide 53: Chemical Methods of Control Types of Disinfectants 8. Peroxygens (Oxidizing Agents): D. 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. Efficiency of Different Chemical Antimicrobial Agents : Efficiency of Different Chemical Antimicrobial Agents The Bioburden : The Bioburden Initial number of microorganisms present in a given product. Sensitivity of Microorganism : Sensitivity of Microorganism Ideally , the sterilizing treatment to be used should be decided for each specific load with prior knowledge of the type and number of contaminating M.O. present. Sensitivity of Microorganism : Sensitivity of Microorganism Microorganism show their varying degrees of resistance to heat, radiation and chemicals. The vegetative forms of bacteria and fungi are most sensitive. The thermophilic bacteria, smaller viruses and mold spores are killed at temperature between 70 to 90 deg., while bacterial spores may be destroyed at 90 t o 120 deg. Temperature. Slide 58: SITES OF ANTIMICROBIAL ATTACK DNA replication Cytoplasm Death curves or Survivor curve : Death curves or Survivor curve Death in a microbial population is determined by assessing the reduction in the number of viable microorganism resulting from contact with a given destructive force. This can be represented graphically with a “survivor curve” drawn from the exposure time or dose. The death of a population of cells exposed to heat, radiation or toxic chemicals is often found to follow first order kinetics. Slide 60: Heating time or radiation dose Survivor Curve Fraction of survivors 10-1 10-2 10-3 10-4 10-8 A C B linear Slide 61: dN/dt = -kN where: N = number of viable organisms at time t k = thermal inactivation constant, or specific death rate Slide 62: Nt / No = e-kt Where: No = the number of original organisms present Nt = number of organisms remaining after time t Advantages : Advantages 1. The determination of death rates provides the facility to compare the resistance of the same microorganisms at different temperatures or to compare the resistance of different microorganism to the same lethal agent. E.g. temperature, radiation, chemical etc. 2. It may also be used to give quantitative measure of the effect of environmental factors such as pH, osmolarity and presence of various chemicals on the sterilization process. Physical Methods: Moist Heat : Physical Methods: Moist Heat Calculations using D and z values Given: For Clostridium botulinum spores suspended in phosphate buffer, D121 = 0.204 min How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 1012 spores to 100 spores (1 spore) at 121°C?Answer: Since 1012 to 100 is 12 decimal reductions, then the time required is 12 x 0.204 min = 2.45 min Physical Methods: Moist Heat : Physical Methods: Moist Heat Calculations using D and z values (cont.) Given the D value at one temperature and the z value, we can derive an equation to predict the D value at a different temperature: Physical Methods: Moist Heat : Physical Methods: Moist Heat Calculations using D and z values (cont.) First, write an equation for this line. Since the y axis is on a log scale, then y = log (D). The slope of the line is -1/z; we’ll let the y intercept be equal to c. Therefore: At a given temperature Ta, D = Da, so we can eliminate the “c” term Physical Methods: Moist Heat : Physical Methods: Moist Heat Calculations using D and z values (cont.) We can explicitly refer to the second temperature and D value as Tb and Db, so: Physical Methods: Moist Heat : Physical Methods: Moist Heat Calculations using D and z values (cont.) Given: For Clostridium botulinum spores suspended in phosphate buffer, D121 = 0.204 min and z = 10°C How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 1012 spores to 100 spores (1 spore) at 111°C?Answer: To answer the question we need to know D111, which we can calculate from the formula:log(D111/0.204) = (121-111) /10D111 = 0.204(10) = 2.04 min12D111= 24.5 min Physical Methods: Moist Heat : Physical Methods: Moist Heat Calculations using D and z values (cont.) Given: For Staph. aureus in turkey stuffing, D60 = 15.4 min and z = 6.8°C How long would it take to reduce a population of Staph. aureus in turkey stuffing from 105 cells to 100 cells at 55°C, 60°C, and 65°C?Answers: Work it out for yourself. Here are the answers.At 55°C: 419 minAt 60°C: 77 minAt 65°C: 14.2 min (((! : (((! The time taken under defined conditions to reduce a population by 90% or --------------- for the survivor curve to traverse 1 log cycle Written as: Dtemp: x min D121 : 2 min Slide 71: Determination of D value (thermal death rate) DECIMAL REDUCTION TIME (D value) : DECIMAL REDUCTION TIME (D value) log10No Time (mins) 1log cycle D value (mins) Thermal Death Time D value as a function of temperature for Clostridium sporogenes PA 3679 : D value as a function of temperature for Clostridium sporogenes PA 3679 Temp (oC) D (min) 110 19.65 113 10.46 115 4.92 118 2.56 121 1.23 Values for k and D for various bacterial spores at 121oC : Values for k and D for various bacterial spores at 121oC B.subtilis 2.6 0.9 B.stearothermophilus 0.77 3 Clostridium sporogenes 1.8 1.8 K min-1 D min Z value : Z value The temperature change that will bring about a 10-fold change in D value written as: z = yoC Bacterial spores have a Z-value in the range 10 to 15 deg. while most Non-sporing organism have Z-values of 4 to 6 deg. Z value : Z value log10D Temp (oC) 1 log cycle Z value (oC) Inactivation Factor (IF) : Inactivation Factor (IF) IF = 10t/D F - value : F - value F = D121(log a - log b) a = initial population b = final population Factors affecting heat resistance of microbial population : Factors affecting heat resistance of microbial population Type & Species Nature of cell Age Number Physico-chemical environment Heat : Heat DRY MOISTPRESSURE BATCH CONTINUOUS Heat resistance of a variety of microorganisms : Heat resistance of a variety of microorganisms D value z value B.stearothermophilus (115) 22.6 7.1 B.megaterium (100) 1 8.8 Salmonella typhimurium 0.5 4.2 (55.5) Yeast spores (55) 0.9 Adenovirus (55) 17 2.5 Batch Sterilizations curve : Batch Sterilizations curve time temp Heating Holding Cooling STERILIZATION : STERILIZATION Different organisms for different methods of sterilization. The organisms that are resistant to a particular sterilization method should be chosen as the marker organism STERILIZATION MONITORS : STERILIZATION MONITORS STERILIZATION MONITORS : STERILIZATION MONITORS It is essential that strict controls are carried out on products to be labelled “STERILE”. Such control must then ensure , the absence of viable microorganism from these product. There are basically two types of control: Controls on the process of sterilization i.e. sterilization indicators. Sterility testing of the products. Slide 87: Monitoring of the sterilization process can be achieved by the use of physical, chemical or biological indicators of sterilizer performance. PHYSICAL INDICATORS : PHYSICAL INDICATORS MOIST HEAT: A Master process record (MPR) is prepared as part of the validation procedure for a particular autoclave and for each specified product and load. Thermocouple is placed in each load. 2. DRY HEAT: Temperature record chart is made of each sterilization cycle and is compared against a master temperature record. Slide 89: 3. FILTRATION: Bubble point pressure test is a technique employed for determining the pore size of filters device immediately after use. 4. RADIATION: A Plastic dosimeter gives an accurate measure of the radiation dose absorbed and is considered to be the best technique currently available for the radiation sterilization. CHEMICAL INDICATORS: : CHEMICAL INDICATORS: Chemical monitoring of a sterilization process is based on the ability of heat, steam, sterilant gases and ionizing radiation to alter the chemical or physical characteristics of a variety of chemical substances. i) Browne's tubes: containing reaction mixture and an indicator. Types of Browne's tubes : Types of Browne's tubes BIOLOGICAL INDICATORS: : BIOLOGICAL INDICATORS: BIs Are the Most accepted means of monitoring the sterization process because they directely determine the most resistant microorganisms. STERILIZATION : STERILIZATION Sterilization Method Marker organisms Steam sterilization Bacillus stearothermophyilus Dry-heat sterilization Bacillus subtilis Ethylene oxide Bacillus subtilis sterilization Ionizing radiation Bacillus pumilus sterilization Sterility Testing as per IP : Sterility Testing as per IP The test for sterility are intended for detecting the presence of viable forms of micro-organisms in or on pharmacopoeial preparations. The test must be carried out under conditions designed to avoid accidental contamination of the product during the test. Precautions taken for this purpose should not adversely affect any micro-organisms which should be revealed in the test. The tests are based upon the principle that if micro-organisms are placed in a medium which provides nutritive material and water, and kept at a favourable temperature, the organisms will grow and their presence can be indicated by a turbidity in the originally clear medium. Slide 97: A laminar flow biological safety cabinet ☺ Method of Sterilization for the accessories to beused : ☺ Method of Sterilization for the accessories to beused ☺ Steam sterilization ☺ (121°C, 15 lbs Pressure for 15 min) ☺ Dry Heat sterilization ☺ (170°C/1 Hr or 140°C /3 hrs or Suitable temp. & times) ☺ Gas sterilization ☺ (Ethylene Oxide, Chlorine Dioxide) ☺ Sterilization by Radiation ☺ (Gamma sterilization, UV sterilization) ☺ By filtration ☺ (Membrane filtration & Cartridge filtration) Slide 99: A sterility test may be defined as — ‘a test that critically assesses whether a sterilized pharmaceutical product is free from contaminating microorganisms’. Selection of Test Method : Selection of Test Method ☺ Direct inoculation ☺ Membrane filtration method Open funnel method (Manifold method) Closed method Culture Media : Culture Media 1.Fluid thioglycollate media - For use with clear fluid products. Used for detection of aerobic bacteria and fungi 2. Alternative thioglycollate medium – For use with turbid and viscid products and for devices having tubes with small lumina Used for detection of anaerobic bacteria and fungi 3. Soyabean- casein digest medium : universal media The media used should comply with the following tests carried out before or in parallel with the test on the preparation being examined : The media used should comply with the following tests carried out before or in parallel with the test on the preparation being examined Sterility : Incubate portions of the (a) fluid thioglycollate medium / alternate Thioglycolate medium at 30º to 35º and (b) soyabean casein digest medium at 20º to 25º for not less than 7 days; no growth of micro-organisms occurs. Growth promotion test : Test each autoclaved load of each lot of the medium for its growth-promoting qualities by separately inoculating duplicate test containers of each medium with about 100 viable micro-organisms or each of the strains listed in Table-2 and incubating according to the conditions specified. The test media are satisfactory if clear evidence of growth appears in all inoculated media containers within 7 days. The tests may be conducted simultaneously with the use of the test media for sterility test purposes. The sterility test is considered invalid if the test medium shows inadequate growth response. If freshly prepared media are not used within 2 days, store them in dark, preferably at 2º to 25º Finished media, if stored in unsealed containers, may be used for not more than one month provided that they are tested within one week of the time use. Method A : Membrane Filtration : Method A : Membrane Filtration The method needs exceptional skill and special knowledge; it also calls for the routine use of positive and negative controls. A suitable positive control is the occasional use of a known contaminated solution containing a few micro-organisms of different types (approximately 10 microbial cells in the total volumes employed). (a) Apparatus A suitable unit consists of a closed reservoir and a receptacle between which a properly supported membrane of appropriate porosity is placed. A membrane generally suitable for sterility testing has a nominal pore size not greater than 0.45 µm and diameter of approximately 47 mm, the effectiveness of which in retaining micro-organisms has been established. Preferably assemble and sterilise the entire unit with the membrane in place prior to use. Where the sample to be tested is an oil, sterilise the membrane separately and, after thorough drying, assemble the unit, using aseptic precautions. Diluting fluids Fluid A : Dissolve 1 g of peptic digest of animal tissue (such as bacteriological peptone) or its equivalent in water to make 1 litre, filter or centrifuge to clarify, adjust to pH 7.1 ± 0.2, dispense into flasks in 100 ml quantities and sterilise at 121° for 20 minutes. Method B : Direct Inoculation : Method B : Direct Inoculation Quantities of sample to be used The quantity of the substance or preparation being examined which is to be used for inoculation in the culture media varies according to the quantity in each container and is given in Table 3 along with the volume of medium to be used (b) Method of test : (b) Method of test For aqueous solutions and suspensions : The tests for microbial contamination are carried out on the same sample of the preparations being examined using the above stated media. When the quantity in a single container is insufficient to carry out the tests, the combined contents of the two or more containers are used to inoculate the above stated media. Remove the liquid from the test containers with a sterile pipette or with a sterile syringe or a needle. Aseptically transfer the specified volume of the material from each container to a vessel of the culture medium. Mix the liquid with the medium but not aerate excessively. Incubate the inoculated media for not less than 14 days, unless otherwise specified in the monograph, at 30° to 35° in the case of fluid thioglycollate medium and at 20° to 25° in the case of soyabean-casein digest medium. Observation and Interpretation of Results : Observation and Interpretation of Results Slide 109: At intervals during the incubation period, and at its conclusion, examine the media for macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation being examined passes the test for sterility. If evidence of microbial growth is found, reserve the containers showing this and, unless it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined and hence that the tests for sterility are invalid and may therefore be recommenced, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the tests for sterility. If evidence of microbial growth is found, isolate and identify the organisms. If they are not readily distinguishable from those growing in the containers reserved in the first test, the preperation being examined fails the tests for sterility. If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second retest using twice the number of samples. If no evidence of microbial growth is found in the second retest, the preparation being examined passes the tests for sterility. If evidence of growth of any micro-organisms is found in the second retest, the preparation being examined fails the tests for sterility. Objectives, by the end of these lectures you should be able to: : Objectives, by the end of these lectures you should be able to: Define Sterilisation and Sterility State the reasons why the above is necessary Give examples of materials and objects requiring sterilisation Describe the methods used to achieve sterility and the principles upon which they are based State the factors that have to be considered when setting up a sterilisation protocol Expected questions???? : Expected questions???? 1. Define the following term i. D-value(2007,2009) ii. Z-value (2007,2009,2010) iii. Antiseptic iv. Sterilization v. Preservatives 2. Differentiate the following : i. Sterilization and Disinfection (2006) ii. Dry heat sterilization and steam sterilization. 3. Define sterilization. List the different methods used for sterilization with suitable examples. (2004,2007,2008) 4. Explain the different sterilization monitors or indicators. (2003,2007) 5. Whether sterility testing for product containing antimicrobial agent is necessary. 6. Name the biological indicator used for monitoring Moist heat sterilization process. 7. How is milk sterilized? 8. Write note on Pasturization. (2003) 9. Explain in details sterility test as per I.P. (2002,2004,2007,2009) 10. Write a Classification of sterilization method. What do you mean by Sterilization cycle? How will you monitor Sterilization process? (2006,2010) 11. Enlist the advantage and disadvantages of radiation sterilization.(2010) 12. Suggest suitable methods of sterilization for the following(2007) i. Culture media ii. Powders iii. Oils iv. Official aq. Injection. 13. What do you mean by survivor curve? (2004) 14. Name the biological indicator used for monitoring Moist heat sterilization process.(2006) Definitions : Definitions Page 184 Control of Microbial Growth: Chemical Antimicrobials• Previously described physical factors (e.g., temperature) affecting microbialgrowth. Here focus on chemical antimicrobials affecting microbial growth. : Control of Microbial Growth: Chemical Antimicrobials• Previously described physical factors (e.g., temperature) affecting microbialgrowth. Here focus on chemical antimicrobials affecting microbial growth. Two main categories of antimicrobial agents: • non-chemotherapeutic agents: (e.g., antiseptics, disinfectants); used to inhibit or kill microbes on living sufaces (topically) or inanimate objects, but are too toxic for internal use inside the human body. • chemotherapeutic agents: (growth factor analogs, antibiotics) can be used internally to control microbes that cause infectious diseases. • Based on degree of selective toxicity to the human or domestic animal host. • Some Important (suffix) Terminology: • “-static”: antimicrobials that inhibit microbial growth without killing them, e.g., bacteriostatic, fungistatic. • “-cidal”: antimicrobials that kill microbes, e.g., bacteriocidal, fungicidal. • “-lytic”: antimicrobials that kill microbes by lysing them, e.g., bacteriolytic. Distinguish these 3 different categories by examining the effect of theantimicrobial agent on a growing bacterial culture: : Distinguish these 3 different categories by examining the effect of theantimicrobial agent on a growing bacterial culture: 3 types of action of antimicrobial agents. At the time indicated by the arrow, a growth-inhibitory concentration of the agent was added to the exponentially growing culture. Note that the bacteriostatic agent inhibits growth without killing cells, bacteriocidal and bacteriolytic agents kill. Cell # decline when lysed. Rate of Microbial Death : Rate of Microbial Death Bacterial death occurs at a constant rate I.e. 90% killed every minute of contact Rate of Microbial Death : Rate of Microbial Death Plotting microbial death Death curve Logarithmically Linear Arithmetically Exponential curve Factors Affecting Antimicrobials : Factors Affecting Antimicrobials 1- Number of microbes present More microbes = longer time to kill Also called load Factors Affecting Antimicrobials : Factors Affecting Antimicrobials 2 – Environmental factors Organic matter Often inhibits antimicrobials Feces Vomit Blood Temperature Temperature dependent reactions Warm temperatures are preferred Factors Affecting Antimicrobials : Factors Affecting Antimicrobials 3-Time of exposure “contact time” Extended times for endospores Longer times can offset lower temperatures Milk pasteurization 4-Microbial characteristics Virus vs gram+ vs gram - Actions of Microbial Agents : Actions of Microbial Agents Actions of microbial agents Alter membrane permeability Damage phospholipids or proteins in plasma membrane Cellular contents leak out Interferes with growth Actions of Microbial Agents : Actions of Microbial Agents Damage to proteins and nucleic acids Denatures proteins Enzyme Proteins necessary for bacteria metabolism Shape necessary for function Hydrogen bonds broken shape changes Covalent bonds are broken Sulfhydryl bonds – SH All can be broken Nucleic Acids DNA and RNA Can no longer replicate or synthesize proteins DISINFECTION : DISINFECTION It is the process of destruction or removal of microorganisms and reducing them to a level not harmful to health. Generally kill the sensitive vegetative cells but not the heat resistance endospores. Applied on inanimate (nonliving) object such as working areas,floor,dishes,benchs etc. Main difference with sterilization = the lack of sporocidal activity • Categorized into 3 levels: – High, – Intermediate – Low: Difference between Sterilization and Disinfection : Difference between Sterilization and Disinfection Killing or removing all forms of microbial life (including endospores) in a material or an object. Bacterial spore are destroyed Sterilization is done by any of the Physical, Chemical and Mechanical method. Done for animate and inanimate object Eg. Autoclave, Hot air oven etc.. It is the process of destruction or removal of microorganisms and reducing them to a level not harmful to health. bacterial spore are not destroyed Disinfection is done by using any of the chemical disinfectant such as phenol, formaldehyde Applied on inanimate object Phenol, bezalkonium chloride etc. Slide 126: Resistance of different pathogens to chemical and/or physical methods Ideal Properties required : Ideal Properties required Broad spectrum Non toxic Fast acting Odorless Surface compatibility Economical Easy to use Solubility and miscibility Not affected by physical factor Mechanism of killing : : Mechanism of killing : DENATURATION 1. STRONG MINERAL ACIDS AND ALKALIES 2. ORGANIC ACIDS (lactic acid, citric acid, propionic acid) INHIBITION OF ENZYME ACTIVITY OXIDAZING AGENTS (-SH group oxidized to S-S /disulfid/) 1. HALOGENS: chlorine, hypochlorites (free chlorine), iodine 2. HYDROGENE PEROXIDE 3. DYES: brilliant green, crystal violet (triphenylmethanes - dermatology), acridine dyes (bactericidal) ALKYLATING AGENTS: formaldehyde, glutaraldehyde (spores are sensitive!) SOLUBLE SALTS OF HEAVY METALS (mercury, silver, and others) Slide 131: DAMAGE OF THE CELL MEMBRANE 1. ORGANIC SOLVENTS: disorganization of the cell membrane 2. ALKOHOLS: disorganization of the lipid structures (50-70% ethanol as skin disinfectant, isopropyl alcohol: higher toxic effect) 3. SURFACE ACTIVE AGENTS (detergents):reduction of the interface tension (wetting agents, emulsifiers (cationic compounds/quaternary ammonium compounds: benzalkonium chloride for skin, but spores and viruses are not sensitive) 4. PHENOLIC COMPOUNDS: lysis (bactericidal), irreversible inactivation of oxidases and dehydrogenases, alkyl derivates: cresol (tricresol) chloro derivates: hexachlorophene, chlorhexidine NOT EFFECTIVE ON SPORES AND VIRUSES! Chemical Agents : Chemical Agents Phenolics Alcohols Halogens Heavy metals Quaternary Ammonium Compounds Aldehydes Sterilizing Gases Evaluating Effectiveness of Chemical Agents Chemical Agents: Phenolics : Chemical Agents: Phenolics Aromatic organic compounds with attached -OH Denature protein & disrupt membranes Phenol, orthocresol, orthophenylphenol, hexachlorophene Commonly used as disinfectants (e.g. “Lysol”); are tuberculocidal, effective in presence of organic matter, remain on surfaces long after application Disagreeable odor & skin irritation; hexachlorophene once used as an antiseptic but its use is limited as it causes brain damage Phenols : Phenols Phenols First used by Lister Rarely used now Irritates skin Throat sprays and lozenges 1 % solution Antibacterial Phenolics : Phenolics Phenolics Derivatives of phenol Increased antibacterial activity Decrease irritation to tissue Often with soap or detergent Injure plasma membrane Active in presence of organic material Phenolics : Phenolics Phenolics Good for disinfecting pus, saliva and feces Effective against Mycobacterium Cell wall high lipid content Very effective Cresols O – phenylphenol Lysol Bisphenols : Bisphenols Phenol derivatives Hexachlorophene pHisoHex Prescription antibacterial lotion Gram + in newborns Staph Strep Bisphenol : Bisphenol Triclosan Anti-bacterial soaps Kitchen cutting boards Some cases of resistance Inhibits synthesis of fatty acids Effective against g+ and g- Pseudomonas aeruginosa Biguanides : Biguanides Chlorhexidine Broad spectrum Used on skin and mucus membranes Scrubs Washes Low toxicity Damaging to eyes Damages plasma membrane Mycobacteria, endospores and protozoa are resistant Effective on some viruses Lipohilic viruses Chemical Agents: Alcohols : Chemical Agents: Alcohols Ethanol; isopropanol; used at concentrations between 70 – 95% Denature proteins; disrupt membranes Kills vegetative cells of bacteria & fungi but not spores Used in disinfecting surfaces; thermometers; “ethanol-flaming” technique used to sterilize glass plate spreaders or dissecting instruments at the lab bench Chemical Agents: Halogens : Chemical Agents: Halogens Act as oxidizing agents; oxidize proteins & other cellular components Chlorine compounds Used in disinfecting municiple water supplies (as sodium hypochlorite, calcium hypochlorite, or chlorine gas) Sodium Hypochlorite (Chlorine Bleach) used at 10 - 20% dilution as benchtop disinfectant Halazone tablets (parasulfone dichloroamidobenzoic acid) used by campers to disinfect water for drinking Chemical Agents: Halogens : Chemical Agents: Halogens Iodine Compounds Tincture of iodine (iodine solution in alcohol) Potassium iodide in aqueous solution Iodophors: Iodine complexed to an organic carrier; e.g. Wescodyne, Betadyne Used as antiseptics for cleansing skin surfaces and wounds Halogens : Halogens Iodine (I2) Oldest Very effective Bacteria, endospores, various fungi, some viruses Exact mode is unknown Possibly combines with amino acids Tincture Iodine in an aqueous alcohol solution Iodophor Iodine attached to an organic molecule, slowly releases Iodine Do not stain like tinctures Chlorine : Chlorine Gas (Cl2) or in combination Hypochlorous acid (HOCl) forms in water Unknown mechanism of action NaOCl – sodium hypochlorite Clorox = bleach Used to disinfect drinking water, swimming pools, sewage Chlorine : Chlorine Chlorine dioxide (ClO2) Can kill endospores Anthrax Chloramines Chlorine and ammonia Release chlorine over long periods Slow acting Toxic to fish Chemical Agents: Heavy Metals : Chemical Agents: Heavy Metals Mercury, silver, zinc, arsenic, copper ions Form precipitates with cell proteins At one time were frequently used medically as antiseptics but much of their use has been replaced by less toxic alternatives Examples: 1% silver nitrate was used as opthalmic drops in newborn infants to prevent gonorrhea; has been replaced by erythromycin or other antibiotics; copper sulfate used as algicide in swimming pools Heavy Metals : Heavy Metals Inorganic mercury Mercuric chloride Mercurochrome Control mildew in paint Bacteriostatic Toxicity Heavy metals : Heavy metals Copper Copper sulfate Destroy algae Control mildew in paint Zinc Used to galvanize nails Zinc chloride Mouthwashes Zinc oxide Antifungal in paints, and adds pigment Chemical Agents: QuaternaryAmmonium Compounds : Chemical Agents: QuaternaryAmmonium Compounds Quaternary ammonium compounds are cationic detergents Amphipathic molecules that act as emulsifying agents Denature proteins and disrupt membranes Used as disinfectants and skin antiseptics Examples: cetylpyridinium chloride, benzalkonium chloride Chemical Agents: Aldehydes : Chemical Agents: Aldehydes Formaldehyde and gluteraldehyde React chemically with nucleic acid and protein, inactivating them Aqueous solutions can be used as disinfectants Quaternary Ammonium Compounds (Quats) : Quaternary Ammonium Compounds (Quats) Surfactants Surface-active agents Cationic detergents Strong bactericidal (alter plasma membrane) Gram + Gram – (less effective) Fungicidal Amoebicidal Virucidal (enveloped) Do not kill Endospores mycobacteria Quaternary Ammonium Compounds (Quats) : Quaternary Ammonium Compounds (Quats) Zephiran Benzalkonium chloride Cepacol Cetylphyridinium chloride Organic material interferes Rapidly broke down by soaps Pseudomonas can actually grow in quats Surfactants : Surfactants Decrease surface tension Soaps and detergents Soap breaks up oil film into tiny droplets Emulsification Acid anionic surfactants Used on dairy equipment EVALUATION OF ANTIMICROBIAL AGENT EFFECTIVENESS : EVALUATION OF ANTIMICROBIAL AGENT EFFECTIVENESS The best known disinfectant screening test is PHENOL COEFFICIENT TEST in which the potency of the disinfectant is compared with that of phenol. The higher the phenol coefficient value the more effective the disinfectant under these test conditions. A value greater than 1 indicate that the disinfectant is more effective than phenol. Table. Phenol coefficient (PC) of some disinfectants : Table. Phenol coefficient (PC) of some disinfectants Chemical Agents:Evaluating the Effectiveness : Chemical Agents:Evaluating the Effectiveness Phenol Coefficient Test A series of dilutions of phenol and the experimental disinfectant are inoculated with Salmonella typhi and Staphylococcus aureus and incubated at either 20°C or 37°C Samples are removed at 5 min intervals and inoculated into fresh broth The cultures are incubated at 37°C for 2 days The highest dilution that kills the bacteria after a 10 min exposure, but not after 5 min, is used to calculate the phenol coefficient Evaluation of Disinfectant : Evaluation of Disinfectant 1. Agar well diffusion method 2. Tube Dilution Methods 3. Phenol Coefficient test 4. Kelsey sykes method Slide 162: Tube dilution Method: . A series of increasing concentrations of the antimicrobial agent are prepared in the culture broth medium. Each tube is equally inoculated and incubated to allow microbial growth to proceed. Growth (turbidity) occurs in those tubes containing the antimicrobial at concentrations below the MIC. Tube cultures are non-turbid (clear, no growth) at MIC and higher concentrations of the antimicrobial agent. Disc agar diffusion method: : Disc agar diffusion method: The test organism is spread on the culture medium in an agar plate and then sterile antibiotic discs are applied. After incubation, the organism produces a confluent “lawn” of growth except in zones of inhibition around discs containing antibiotics to which the organism is susceptible. When a filter paper disc impregnated with a chemical is placed on agar the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a “zone of inhibition”. Chemical Agents:Evaluating the Effectiveness : Chemical Agents:Evaluating the Effectiveness Phenol Coefficient Test (cont.) The reciprocal of the maximum effective dilution for the test disinfectant is divided by the reciprocal of the maximum effective dilution for phenol to get the phenol coefficient For example:Suppose that, on the test with Salmonella typhiThe maximum effective dilution for phenol is 1/90The maximum effective dilution for “Disinfectant X” is 1/450The phenol coefficient for “Disinfectant X” with S. typhi = 450/90 = 5 Slide 168: Phenol coefficient test: standardizes the potency of bacteriocidal agents in comparison with phenol using specific test microorganisms. 1. A series of dilutions of phenol and the experimental antimicrobial agent are prepared in sterile tubes. 2. Viable test bacteria (Staphylococcus aureus and Salmonella typhi) are inoculated into each tube and incubated at 37 ｰC. 3. After 10 minutes, samples of inoculated tubes are transferred into fresh culture medium without disinfectant and incubated for several more days. 4. The broth culture tubes are evaluated for growth by turbidity. 5. The highest dilutions of the test antimicrobial agent and of phenol that effectively kill the test organisms within 10 minute exposure are used to compute the phenol coefficient as follows: 1 / [Effective dilution of test antimicrobial agent] 1/ [Effective dilution of phenol] Example: If the highest effective dilution of the test antimicrobial = 1/500 and that of phenol = 1/100, the phenol coefficient of the test antimicrobial = 500/100 = 5. Agents with a phenol coefficient > 1 are more effective than phenol. The higher the phenol coefficient value, the more effective the test antimicrobial is in killing bacteria under the test conditions. Note that phenol coefficient tests are only valid for bacteriocidal (not bacteriostatic) antimicrobials, since cells inhibited (but not killed) by the latter would resume growth when transferred to fresh medium. Investigation of disinfectant activity : Investigation of disinfectant activity Conditions Influencing Antimicrobial Activity : Conditions Influencing Antimicrobial Activity Several critical factors play key roles in determining the effectiveness of an antimicrobial agent, including: Population size Types of organisms Concentration of the antimicrobial agent Duration of exposure Temperature pH Organic matter Biofilm formation Kelsey-Sykes test : Kelsey-Sykes test Kelsey-Sykes test is a triple challenge test, designed to determine concentrations of disinfectant that will be effective in clean and dirty conditions. The disinfectant is challenged by three successive additions of a bacterial suspension during the course of the test. The duration of test takes over 30 minutes to perform. The concentration of the disinfectant is reduced by half by the addition of organic matter (autoclaved yeast cells), which builds up to a final concentration of 0.5%. Depending on the type of disinfectant, a single test organism is selected from S. aureus, P. aeruginosa, P. vulgaris and E. coli. The method can be carried out under 'clean' or 'dirty‘ conditions. The dilutions of the disinfectant are made in hard water for clean conditions and in yeast suspension for dirty conditions. Test organism alone or with yeast is added at 0, 10 and 20 minutes interval. The contact time of disinfectant and test organism is 8 min. The three sets of five replicate cultures corresponding to each challenge are incubated at 32oC for 48 hours and growth is assessed by turbidity. The disinfectant is evaluated on its ability to kill microorganisms or lack of it and the result is reported as a pass or a fail and not as a coefficient. Sets that contain two or more negative cultures are recorded as a negative result. The disinfectant passes at the dilution tested if negative results are obtained after the first and second challenges. The third challenge is not included in the pass/fail criterion but positive cultures serve as inbuilt controls. If there are no positive cultures after the third challenge, a lower concentration of the disinfectant may be tested. Slide 173: Interpretation of result: No growth occurs in 2 or more of the 5 tubes of the 18 min samples Factors Affecting Effectiveness ofDisinfection : Factors Affecting Effectiveness ofDisinfection • Cleaning – Residual particles harbor & shelter from disinfectant – Organic load restrict disinfectants effectiveness of alcohol, phenols, chlorine & iodines • Nature of object: crevices, hinges, lumens more difficult to disinfect. • Concentration of disinfectant: – Diluted during application – Lose potency with time • Time of contact • Physical and chemical environment: temperature, water hardness, pH. Formulation You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.