Disinfection & Sterilization

Disinfection & Sterilization Professor Samuel Aguazim

Learning Objectives • At the end of this lecture, the student should be able to • Define disinfection and sterilisation • Describe the common substances and processes used to achieve these outcomes • Evaluate issues influencing choice of method

Definitions • Cleaning • process which physically removes contamination but does not necessarily destroy micro-organisms • prerequisite before decontamination by disinfection or sterilisation of instruments • organic material prevents contact with microbes, inactivates disinfectants • Disinfection • using an agent that destroys germs or other harmful microbes or inactivates them, usually referred to chemicals that kill the growing forms (vegetative forms) but not the resistant spores of bacteria

Definitions • Antisepsis • destruction of pathogenic microorganisms existing in their vegetative state on living tissue • Sterilization • any process, physical or chemical, that will destroy all forms of life, including bacterial, fungi, spores, and viruses

Germicide: • any agent that kills vegetative cells of microorganism but not the resistant stage(spores) is a germicide. • Types: • Bactericides • fungicides

Sanitization: • A sanitizer does not kill all the microbes but brings down their number to a safe level when they cannot cause any harm.

Pasteurization • This is a form of disinfection( originated by louis pastuer) used for killing microbes which bring about unwanted fermentation. This is now widely practised in the processing of milk so that it is free of disease causing bacteria. In this process milk is heated to a high temperature and suddenly cooled to eliminate bacteria

Microbiostasis • Microbiostatic agents create a condition wherein microbes are prevented from multiplying but not immediately killed. Microbiostasis means keeping the biological activity of microbes in a static condition( not active).

Preservative: • these are certain chemicals added to the food substance that prevent microbial growth. • Preservative are not harmful to living tissues

Physical Heat Filtration Irradiation Quarantine Chemical Choice of method depends on practical issues such as ease of use or material compatibility Proctoscope need not be as free of contamination as an artificial heart valve Cleaning of objects needed before attempt at sterilization Methods

Factors influencing ability to kill microbes • Strength of the killing agent • Time that the agent has to act • Temperature of environment • rate of microbe death doubles with every 10˚C rise in temp. • Type of microbe • Environment around the area to be decontaminated • Number of microbes to be killed

Effect of antimicrobial agents on microbial cells • 1. coagulation and denature of proteins: the action of many physical(heat) and chemical disinfectants coagulate the protoplasm into a solid mass as in a hard boiled egg or curdled milk. • Strong coagulant agents • *formalin, phenol and alcohols • Other chemicals like chlorine, iodine, cresol etc will combine with the proteins and denature them without necessarily coagulating them.

Injury to cell wall: • lowering surface tension injures the cells walls. Substances which reduce surface tension are called surfactants.

Injury to cell membranes • Lipid dissolving surfactants disrupt the cytoplasmic membranes and expose the cell contents as a result of which metabolic processes get disturbed leading to the death of microbial cells

Enzyme inhibition by removal of SH groups • Oxidizing agents such as chlorine, iodine and hydrogen suppress the growth of microbes largely through their ability to react with and render the sulfhydryl groups sueless.

Inhibition of metabolism: • many antimicrobial agenst inhibit the essenti; metabolic pathways of microbes thereby killing them. • Examples: • Flouride inhibits glycolysis • Arsenic compounds block TCA(tricarboxylic acid cycle or kreb cycle) • Cyanide inbits cytochrome oxidase

Chemical antagonism • A chemical substance which can inhibit the action of the enzyme(on the substrate) by combining with the active site and rendering it useless is known to bring about chemical antagonism or competitive inhibition. • Example: sulfonamide drugs competes with para amino benzoin acid(PABA)( PLAYS ROLE IN FORMATION OF FOLIC ACID)

Methods of antimicrobial activity(sterilization) • Sterilization is achieved by the following methods. • 1. PHYSICAL METHODS • *dry heat • *moist heat • *filteration • 2. radiation methods • 3. ultrasonic methods. • 4. chemical methods.

Physical Methods: Heat • Advantages • Non-toxic • Quick • Cheap • Disadvantages • Can only be used on heat-resistant materials • No use for many plastics, electronics, tarnishes some metals

Pasteurisation First used with milk: 72°C for 20 seconds Heating to 80°C for 1 minute will kill most vegetative organisms Examples: bed-pan washer, proctoscope Dry heat (hot air oven) used on waxes, oils (wet heat usually preferred) USED FOR TEST TUBES, CONICAL FLASKS, PETRIDISHES AND PIPETLETS. Incineration the ultimate sterilization used for disposal of hospital waste Wet heat Boiling limited use as spores may be resistant, boilers may be misused Low temperature steam disinfection (75°C for 30 mins) Used for e.g. ventilator tubing Autoclaving High-tempoerature steam plus pressure (same principle as pressure cooker) USED FOR SOLID AND LIQUID MEDIA FOR MICROBIAL CULTURES,HEAT STABLE LIQUIDS,HEAT RESISTANT EQUIPMENTS AND INSTRUMENTS, GLASSWARE,RUBBER PRODUCTS AND SURGICAL INSTRUMENTS Physical Methods: Heat

Autoclaving • Requires steam penetration • Cannot work on sealed containers (which can explode!) • Risks of a pressure vessel • Steam condenses on contact, releasing latent heat of evaporation • Condensation leads to vaccum, draws in more steam

Autoclaving • Best results if air excluded • Downward displacement autoclaves • Usually for lab use: steam displaces air through outlet • Steam heated jacked used to help drying • High pre-vacuum autoclaves • Air excluded before steam enters

Autoclaving • Need to establish correct cycle and hold times • Typical settings: 121˚C @ 15 psi for 15 min. or 121˚C @ 30 psi for 3 min • Need to maintain autoclaves and monitor effectiveness • Temperature & pressure charts • Chemical indicators (Browne’s tubes, Bowie-Dick test) • Spore tests

Physical Methods • Filtration • Used on labile fluids and on air supplies • Gamma-Irradiation(ionizing radiation) • Used on disposable plastics, e.g. in sealed packs • Only in specialised centres . CATHODE RAYS Used for surgical appliances

Non ionizing radiation • Less penetrating(have less energy) and can be controlled and made unidirectional. • Example • Ultraviolet rays • Wave length length of 250 is very toxic to bacterial spores as well as vegetative cell. • Commercial: u.v lamps • Used for biological fluids such as blood plasma and vaccine and make them free of viruses • Also used for food processing.

ULTRASONIC METHODS • High frequency sound waves( about 20 kilocycles per second) have microbicidal properperties. • Ultrasonic waves can be produced by instruments called SONICATORS. • Sonication is used to disrupt biological membranes of bacteria by exposing them to high frequency sound waves. • Not very useful method for sterilization. It is however useful in isolating the cell organlles and other intra cellular components as it disrupts the structural integrity of the cell

Use depends on spectrum of antimicrobial activity and compatibility with materials Also limited by dangers of chemicals themselves Examples Halogens Alcohols Alkylating agents Ethylene oxide Phenolics cetrimide (QAC) chlorhexidine (diguanide) Chemicals

Hypochlorites (household bleach) & chlorine Advantages active against viruses, spores, fungi Disadvantages inactivated by organic matter, freshness & pH critical (go off if diluted), corrosive to metals Practical Uses 0.1% hypochlorite used as general disinfectant Strong hypochlorite (0.25%) used in lab & on wounds Extra strong (1%) used on HBV blood spills Chlorine used to treat drinking water and control Legionella Halogens

Halogens • Iodophors & iodine • Advantages • Some activity against viruses, spores, fungi • Disadvantages • inactivated by organic matter, can stain skin, irritant, expensive • Practical Uses • Pre-op skin disinfection • Povidone iodine used as surgical scrub, as powder on ulcers

Alcohols • Isopropanol & ethanol • Advantages • kill vegetative bacteria on clean surfaces in 30 seconds • Disadvantages • inactive against spores, fungi • Inflammable • Need to be at correct %age with water (65-80%) • Practical uses • Skin antisepsis before venepuncture • Hand rubs • Disinfection of e.g. trolley tops

Alkylating agents • Glutaraldehyde and Formaldehyde • Advantages • Good activity against spores, virues, fungi • Disdvantages • Glutaraldehyde only moderately active against TB • Need long exposure time for full effect (3 hours) • freshness & pH critical • TOXIC! • Practical uses • Disinfection of endoscopes • Blood spills • Fumigation

Ethylene oxide • Highly toxic flammable gas, kills spores! • Used for bulky items such as heart lung machines • Can be used on glutaraldehyde-labile endoscopes • Use limited by safety issues

Phenolics & QACs • Clear soluble phenolics (e.g. Hycolin) used as disinfectant on soiled surfaces, relatively inactive against spores and viruses • Hexachlorophane used as surgical scrub • Contents of antiseptic soap and hand wash • Quaternary ammonium compounds, e.g. cetrimide usually only used in combination with other agents; good detergent properties. • Used for surgical instrument, skin ointments, lotions.

Chlorhexidine (a diguanide) • Used as general purpose antiseptic for skin and mucous membranes in many formulations, e.g. Hibiscrub, Hibisol, Savlon • Advantages: relatively non-toxic and good against S. aureus • Disadvantages: can support growth of e.g. P. aeruginosa

Factors determining usefulness of chemical disinfection • Spectrum of antimicrobial activity • is it the right agent for the job? • Used at correct concentration • concept of 'in use concentration’ • diluted down from high concentration • stored for <24 hours • no topping up of old solutions

Factors determining usefulness of chemical disinfection • Time of exposure • You cannot disinfect an endoscope in 5 minutes glutaraldehyde! • Correct pH? • Inactivating materials • Pus, blood vomit, cork, soaps etc • Is disinfectant sterile? • Many cases of Gram-negatives living in disinfectants! • Microbiological “in-use” testing

The Problem of CJD and TSEs • Creutzfeld-Jakob syndrome and other transmissible spongiform encephalopathies caused by highly resistant proteinaceous particles, prions • can survive 3 years of environmental exposure and are unusually resistant to conventional decontamination methods • Iatrogenic CJD documented in three circumstances • use of contaminated medical equipment (2 cases) • use of extracted pituitary hormones (> 130 cases) • implantation of contaminated grafts from humans (cornea, 3 cases; dura mater, > 110 cases)

The Problem of CJD and TSEs • Current advice on decontamination • Incinerate instruments used on known cases • Quarantine instruments used on suspected cases • Improved need for instrument tracking

Summary • Definitions • Physical methods • heat (wet heat with pressure=autoclaving), filtration, irradiation • Chemical methods • Halogens, alkylating agents, EtOxide, alcohols, etc. • Problem of CJD

Disinfection & Sterilization