Sterilization and Disinfection

Views:
 
Category: Education
     
 

Presentation Description

Basics and principles of microbial control method. Description of the various methods of sterilization and disinfection and the mechanism of actions of each method. Give examples about suitable sterilization and disinfection method for each item in the field of microbiology. Emphasis given to sterilization and disinfection of medical items and microbiological media.

Comments

Presentation Transcript

PowerPoint Presentation:

Sterilization and Disinfection Dr. Mahmoud M. Berekaa Associate Prof. Molecular Microbiology & Biotechnology Biosafety-DU-April-2013

PowerPoint Presentation:

Biosafety-17-4-2013 Sterilization

PowerPoint Presentation:

Sterilization Killing or removing all forms of microbial life (including endospores ) in a material or an object . Mainly due to: oxidation of cell component, denature proteins, nucleic acids, RNA and loss of membrane permeability. Biosafety-17-4-2013

PowerPoint Presentation:

Methods of Sterilization Biosafety-17-4-2013

PowerPoint Presentation:

Biosafety-17-4-2013

PowerPoint Presentation:

Procedure carried out in the absence of living things Sterile Techniques Procedures performed in a way to prevent contamination with infectious microorganisms Used to prevent contamination of surgical instruments , medical personnel , and the patient during surgery Aseptic Techniques Biosafety-17-4-2013

Other Terms:

Other Terms Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms) Degerming : Mechanical removal of microbes from limited area. e.g., Alcohol swab on skin, washing of hands with soap Sepsis: Bacterial contamination Antisepsis: Reduction or Inhibition of microbes found on LIVING TISSUE Biosafety-17-4-2013

Other Terms:

Other Terms Bacteriostatic Agent: Agent that inhibits the growth of bacteria, but does not necessarily kill them. Suffix stasis : To stop or steady . Germicide: Agent that kills certain microorganisms. Bactericide: Agent that kills bacteria . Most do not kill endospores. Viricide : Agent that inactivates viruses . Fungicide: Agent that kills fungi . Sporicide : Agent that kills bacterial endospores or fungal spores. Biosafety-17-4-2013

Microbial Control Methods:

Microbial Control Methods Biosafety-17-4-2013

PowerPoint Presentation:

Biosafety-17-4-2013

PowerPoint Presentation:

Sterilization By HEAT Biosafety-17-4-2013

PowerPoint Presentation:

Factors Affecting Sterilization Process Heat Sterilization Biosafety-17-4-2013

PowerPoint Presentation:

Biosafety-17-4-2013 Heat in Bunsen flame till red hot 250 0 C – 300 0 C Destroy vegetative cells by oxidation Points of forceps & Inoculation loops, scalpel blade, glass slides, mouths of test tubes or flasks Dry Heat

Dry Heat: Incineration:

Dry Heat: Incineration Incineration Oxidation of microbes to ashes and gas incineration of loops and needles using a Bunsen burner, Common practice in microbiology lab- Tabletop infrared incinerators c an also be used Biosafety-17-4-2013

PowerPoint Presentation:

870 0 C - 980 0 C Complete Burning to ASHES Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable soiled bedding Dry Heat INCINERATION Oven Biosafety-17-4-2013

Dry heat - HOT AIR OVEN:

Dry heat - HOT AIR OVEN Used for glassware, forceps, swabs, water impermeable oils, waxes & powders Holding temp & time: 160 0 C for 1 hr Biosafety-17-4-2013

USE of HOT AIR OVEN Important Notes:

Before placing in hot air oven Dry glassware completely Plug test tubes with cotton wool Wrap glassware in Kraft papers Don’t over load the oven To allow free circulation of air between the material Biosafety-17-4-2013 USE of HOT AIR OVEN Important Notes

PowerPoint Presentation:

Moist Heat Sterilization

PowerPoint Presentation:

One of the Key Cell Constituents Biosafety-17-4-2013

PowerPoint Presentation:

Lethal effect due to DENATURATION & coagulation of proteins Temp below 100 0 C Temp at 100 0 C Temp above 100 0 C Biosafety-17-4-2013 Moist Heat Sterilization

PowerPoint Presentation:

cell membrane Enzymes

PowerPoint Presentation:

Structure of Proteins Biosafety-17-4-2013

Moist heat: Temp below 1000C:

Moist heat: Temp below 100 0 C Pasteurization 63 0 C for 30 min (Holder method) 72 0 C for 15-20 sec (Flash method) 132 0 C for 1 sec (Ultra high temp) Vaccine baths: 60 0 C for 60 min For vaccines of non- sporing bacteria Water bath: 56 0 C for 60 min (for 3 days) For serum/body fluids containing coagulable proteins Inspissation: 80 to 85 0 C for 30 min (for 3 days) For media containing egg or serum

PowerPoint Presentation:

Biosafety-17-4-2013 HEAT Moist Heat Fluids are heated at temperatures below boiling point to kill pathogenic microorganisms without altering the fluid’s palatability. Conditions: 62℃, 30min or 71.7℃, 15sec Significance: kills vegetative pathogens Applications: milk, Juice Pasteurization

Moist Heat - Temp at 1000C:

Moist Heat - Temp at 100 0 C Boiling: 100 0 C for 10 min Kills vegetative bacteria, hepatic virus & some spores Use deionized or distilled water 2 % sodium bicarbonate promotes the process Steaming (free steam ): 30-60 min in Arnold /Koch steamer For heat labile media Tyndallisation (intermittent sterilization ): 100 0 C, 30 min, 3 days Nutrient media & media containing sugars or gelatin I day all vegetative bacteria are killed. On II & III day spores that germinate are killed

Moist Heat: Temp above 1000C:

Moist Heat: Temp above 100 0 C Autoclave (steam under pressure ): 121 0 C , 15 min, 15 lbs Uses: rubber articles, dressings, sharp instruments, infectious medical waste, culture media Sterilization control ( to check whether the equipment is working properly) Thermocouples Chemical Control: Browne’s tube (Color change from red to green) Bowie & Dick tape (white-brown )

Steam Jacketed Horizontal Autoclave:

Steam Jacketed Horizontal Autoclave Biosafety-17-4-2013

Moist vs. Dry Heat:

Moist vs. Dry Heat Biosafety-17-4-2013

PowerPoint Presentation:

Sterilization controls: to check whether the equipment is working properly Biological controls: paper strips containing 10 6 spore of B. stearothermophilus and Incubate at 55 0 C for 5 days paper strips containing 10 6 spores of Clostridium tetani Place strips in oven along with other material for the sterilization Later culture the strips in thioglycollate broth at 37 0 C for 5 days Growth in medium indicates failure of sterilization

Browne Sterilizer Control Tubes Multi-variable indicators for Dry Heat and Fluid sterilization:

Browne Sterilizer Control Tubes Multi-variable indicators for Dry Heat and Fluid sterilization Test critical parameters of Time and Temperature For Dry Heat Sterilizers & Steam autoclaves Colour change from red (unprocessed) to green (pass). Black Spot (Type 1) for 121°C - 15 min Steam Fluid Yellow Spot (Type 2) for 134°C - 3 min Steam Fluid Green Spot (Type 3) for 160°C - 60 min Dry Heat Blue Spot (Type 4) for 180°C - 12 min Dry Heat White Spot (Type 5) for 180°C - 35 min Dry Heat

PowerPoint Presentation:

For steam sterilization The easy to interpret; black stripes prove successful sterilization . Tape securely sticks to non woven, paper, board, metal, plastic and glass Manufactured from treated crepe paper and coated with high performance cross linked heat sensitive adhesive . Autoclave Tape Biosafety-17-4-2013

PowerPoint Presentation:

Sterilization By FILTRATION Biosafety-17-4-2013

PowerPoint Presentation:

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, serum , carbohydrates solution, filtrates of toxins & bacteriophages , in water bacteriology, in examination of Schistosoma eggs Filtration Earthenware candles Unglazed ceramic & diatomaceous earth filters e.g. Chamberland filters, Doulton filters Asbestos filter: Seitz, Carlson, Sterimat Sintered glass filter

PowerPoint Presentation:

Membrane Filters: cellulose nitrate, cellulose acetate, polycarbonate, polyester filters with 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.

PowerPoint Presentation:

Sintered glass filter Membrane filters Earthenware filters Asbestos filters Biosafety-17-4-2013

HEPA Filtrers:

HEPA Filtrers HEPA: high-efficiency particulate air filters Advantages: No thermal damage Used in operating rooms and burn units to remove bacteria from air. Disadvantages: viruses not eliminated and must be either liquid or gas Filter Sterilized fluid (a) Vacuum Pump suction Filter Liquid Pore (b) b: © Fred Hossler/Visuals Unlimited

PowerPoint Presentation:

Sterilization By RADIATION Biosafety-17-4-2013

PowerPoint Presentation:

RADIATION

PowerPoint Presentation:

Biosafety-17-4-2013

Ultraviolet Radiation:

Ultraviolet Radiation Wavelength: 200-300 nm Poor penetrating power Pyrimidine dimers Mutations induced Disinfection rather than sterilization Hospital rooms, operating rooms, schools, food prep areas, dental offices Treat drinking water purify liquids

Modes of Action of Ionizing Versus Non-ionizing Radiation:

Modes of Action of Ionizing Versus Non-ionizing Radiation Ionizing: ejects electron, causing ions to form Biosafety-17-4-2013

PowerPoint Presentation:

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. Microwave Radiation Biosafety-17-4-2013

Plasma technology:

Plasma technology Glow discharge or low temperature plasmas Plasma has sufficient energy to disrupt molecular bonds Direct current, radiofrequency, microwave power is used to produce the plasma Used in disinfection of many bacteria Biosafety-17-4-2013

PowerPoint Presentation:

DISINFECTION

PowerPoint Presentation:

The process of freeing an article or a surface from all or some of the living microorganisms but not necessarily bacterial spores Strong disinfectants: for inanimate object Mild disinfectant (antiseptic ): superficial application on living tissue Disinfection Biosafety-17-4-2013

PowerPoint Presentation:

Ideal Disinfectant

Dependents on: Conc., length (time), temperature Chemical reaction 2x increase in rate with each 10°C:

Disinfectant Performance Dependents on: Conc. , length (time), temperature Chemical reaction 2x increase in rate with each 10°C Biosafety-17-4-2013

PowerPoint Presentation:

DISINFECTANTS AND MODE of ACTION Biosafety-17-4-2013 Control of Microorganisms By CHEMICALS

Categories of disinfectants:

Categories of disinfectants Alcohol Aldehyde Ethylene oxide Dyes Halogens Phenolics Surface active agents Metallic salts Diguanides Amides Biosafety-17-4-2013

PowerPoint Presentation:

Chemical control of microorganisms Biosafety-17-4-2013

Chemicals with Surface Active Action: Detergents:

Act as surfactants Cationic detergents are more effective because the positively charged end binds with negatively charged bacterial surface proteins Soaps are weak microbicides but gain germicidal value when mixed with agents such as chlorhexidine or iodine Chemicals with Surface Active Action: Detergents

SOAPS and SURFACE ACTIVE AGENTS:

SOAPS and SURFACE ACTIVE AGENTS Soaps & surfactants DISRUPT cell membrane Soaps are Na- or K- salts of fatty acids , a natural product There are 4 main groups of surface Active Agents Anionic surfactants: strong detergent action, weak antimicrobial action Non-ionic surfactants Cationic surfactants: Quat ernary a mmonium c ompounds : cetrimide , benzalkonium chloride: bacteriostatic Amphoteric surfactants: both detergent & antimicrobial properties

PowerPoint Presentation:

Quats are cationic detergents that act by disrupting lipid bilayers  Benzalkonium chloride  Cetylpyridinium chloride

PowerPoint Presentation:

Quats are bactericidal , fungicidal, viricidal (enveloped) , and amoebicidal Most effective against Gram-positive bacteria, less effective against gram-negative bacteria. Quats do not kill endospores , Mycobacteria spp., nor non-enveloped viruses Disadvantages: rapidly inactivated by organics including cotton, soap and anionic detergents Advantages: Strong antimicrobial action, colorless, odorless, tasteless, stable, and nontoxic. Biosafety-17-4-2013

ALCOHOL:

ALCOHOL Ethanol, isopropyl alcohol Skin antiseptics at 70% Kills bacteria and fungi Less effective on spores (Less sporicidal activity) & non-enveloped viruses (Less virucidal activity) Denature bacterial proteins and dissolves lipids Flammable; also may damage rubber, plastic, etc. Methyl alcohol: to treat cabinets / incubators affected by fungal spores Others: benzyl alcohol, chlorbutol , phenylethanol

PowerPoint Presentation:

Denature bacterial proteins and dissolves lipids Biosafety-17-4-2013

ALDEHYDE Inactivate proteins by forming covalent crosslinks with several functional groups :

ALDEHYDE Inactivate proteins by forming covalent crosslinks with several functional groups Formaldehyde: 10% used In aq. soln is virucidal , bactericidal, sporicidal Used to fumigate wards, sick rooms, labs Expose to ammonia to remove residual formaldehyde Has pungent strong smell , irritant to skin, eyes, mucus membrane & toxic when inhaled 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 .

PowerPoint Presentation:

Glutaraldehyde : less toxic, less irritant Endotracheal tubes, metal instruments, polythene tubing 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 preserving. Β eta- propiolactone (BPL ): condensation product of ketane & formaldehyde More efficient for fumigation but is carcinogenic 0.2% generally used

HALOGENS:

HALOGENS Kills by oxidation Iodine: 2.5% in 70% alcohol , Skin antiseptic Iodophores : (iodine + non-ionic surface active agent) – betadine – non staining, less irritant, less toxic Chlorine: disinfect water supplies, swimming pools Sodium hypochlorite: 1% for HIV Organic chloramines: antiseptic for wound dressings Biosafety-17-4-2013

Halogens:

Halogens Iodine: commonly used as an antiseptic against all microbes , fungi, and viruses Iodine: It inhibits protein synthesis and oxidizes –SH groups of amino acids Chlorine: Used as a disinfectant (10% bleach) Chlorine: Hypochlorous acid ( HOCl ) is a product, formed in water, that is the active form of the disinfectant Cl2 + H2O ------> H+ + Cl- + HOCl Hypochlorous acid

PHENOLICS:

PHENOLICS Carbolic acid: 2 - 5 % Powerful microbicidal , very corrosive General purpose disinfectant in hospital Stable, persistant , and especially effective when dealing with disinfecting materials contaminated with little organics Cresol , lysol Chloroxylenol , chlorophenol , hexachlorophane less toxic, less irritant, less active , more readily inactivated by organic matter

PowerPoint Presentation:

Affect plasma membrane , inactivates enzymes , and denature proteins Leave residual films, can irritate skin , don’t kill endospores, and are corrosive to rubber and plastics Some phenolics are mild enough for use as antiseptics while others are too harsh or otherwise dangerous to be employed on living tissue PHENOLICS Biosafety-17-4-2013

Heavy Metals or Metallic Salts:

Heavy Metals or Metallic Salts Most common: Ag, Cu, Hg (ointment), Ni, Zn, Ag(NO 3 ) 2 , CuSO 4 , ZnCl 2 , HgCl 2 Oligodynamic action: exert antimicrobial activity React with sulfhydral (–SH) groups of proteins, denaturing proteins Silver nitrate is used to treat Ophthalmia neonatorum in newborns as caused by Neisseria gonorrhoeae ( gonorrheal eye infections) Copper salts: antifungal, antialgae – water reservoirs, swimming pools

Oligodynamic action of Heavy Metals:

Oligodynamic action of Heavy Metals Biosafety-17-4-2013

Heavy Metals or Metallic Salts:

Heavy Metals or Metallic Salts Mercury: Organic mercury compounds like merthiolate and mercurochrome are used to disinfect skin wounds . Selenium Kills fungi and their spores. Used for fungal infections. Also used in dandruff shampoos. Zinc Zinc chloride is used in mouthwashes. Zinc oxide is used as antifungal agent in paints. Biosafety-17-4-2013

Peroxygens (Oxidizing Agents):

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. Peroxygens (Oxidizing Agents ) Oxidize cellular components of treated microbes. Oxidize DNA & Disrupt membranes and proteins. Biosafety-17-4-2013

Peroxygens :

B. Hydrogen Peroxide H 2 O 2 Used as an antiseptic. Bacteria and body tissues contain enzymes (catalase) that inactivate hydrogen peroxide Oxygen released upon inactivation can help oxygenate deep wounds and thus kill strict-anaerob e contaminants, e.g., Clostridium tetani Sporicidal at higher temperatures. Used by food industry and to disinfect contact lenses. C. Benzoyl Peroxide Used in spotted skin medications (an acne treatment) Peroxygens

Peroxygens :

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 . Peroxygens Biosafety-17-4-2013

DYES as Antimicrobial Agents:

DYES as Antimicrobial Agents Combine with nucleic acids Aniline dyes (crystal violet and malachite green, Brilliant green ) are very active against gram-positive bacteria and various fungi Yellow acridine dyes ( acriflavine , euflavine and proflavine ) sometimes used for antisepsis and wound treatment Limited applications because they stain and have a narrow spectrum of activity Biosafety-17-4-2013

PowerPoint Presentation:

Denature proteins, by replacing functional groups with alkyl groups. Kills all microbes and endospores , but requires exposure of 4 to 18 hours . Ethylene oxide chambers in hospitals used to sterilize mattresses and large equipment Especially for heart lung machines, respirators, sutures, syringes, dental equipments ETHYLENE OXIDE ( C 2 H 4 O )

ETHYLENE OXIDE:

ETHYLENE OXIDE Biosafety-17-4-2013 Ethylene oxide Used to sterilize heat- or moisture-sensitive items Not corrosive, not damaging to delicate instruments, Good penetration of materials, and Diffuses rapidly from material Highly inflammable, mixed with inert gases (CO 2 ) But is costly, toxic, carcinogenic, explosive , and relatively lengthy process

PowerPoint Presentation:

Dr. Mahmoud Berekaa