Sterilization and Disinfection

Sterilization and Disinfection

• Sterilization involves killing or removing all microbial forms, including spores.
• Disinfection involves killing most microbial forms on inanimate objects, without necessarily destroying saprophytes and bacterial endospores.
• Disinfection leads to a reduction in the number of organisms to a level that cannot produce infections.

Important Terms

• Antisepsis uses chemical agents on living tissue, like skin, to prevent the spread of microorganisms.
• Antisepsis involves inhibiting their growth or destroying them.
• A bactericidal or germicidal agent is a physical or chemical agent that kills bacteria.
• A bacteriostatic agent is a physical or chemical agent that inhibits the growth of bacteria without necessarily killing them.
• Sporicidal, fungicidal, and viricidal are agents that destroy spores, fungi, and viruses, respectively.

Physical Methods of Sterilization

• Heating is the most common physical method of sterilization.
• The rate of killing is expressed in thermal death time, which is the minimum time required to kill a suspension of an organism at a predetermined temperature and environment.
• The mechanisms of action of heating include formation of single strand breaks in bacterial DNA
• Other mechanisms of action of heating include coagulation and denaturation of proteins, accumulation of toxic levels of electrolytes, and alteration of cell membrane structure.

Factors Affecting Sterilization Through Heating

• Moist heat has a greater killing action compared to dry heat.
• As temperature increases, the time needed to sterilize decreases.
• The more microorganisms present, the higher the temperature and longer duration of the process required to destroy all of them.
• Spore forming microorganisms are harder to destroy than non-spore forming ones.
• The temperature needed to sterilize materials depends on their heat sensitivity, with heat sensitive materials requiring lower temperatures than heat resistant materials.
• The presence of organic materials like fats, proteins, and sugars may require higher temperatures.

Types of Heat

• Moist heat is preferred over dry heat because it kills more rapidly.
• Moist heat's main mechanism is causing coagulation and denaturation of proteins.
• Dry heat's effectiveness depends on the penetration of heat.
• Dry heat sterilizes materials in enclosed tubes, oils, jellies, powders, and glassware like test tubes and Petri dishes.

Methods of Moist Heat Sterilization

• Methods are classified according to temperature used
• Includes Pasteurization, Vaccine bath, Serum bath, and Inspissation (below 100°C)
• Includes Boiling and fractional sterilization (Tyndalization) at 100°C
• Includes the Autoclave, which uses steam under pressure (above 100°C).

Pasteurization

• Destroys disease-producing organisms in milk, milk products, and beverages.
• The conventional method heats milk at 60-65 °C, followed by rapid cooling.
• The flash method heats at 72 °C for 15 seconds.
• A flash method cools quickly to 13 °C.
• The Ultra High Temperature (UHT) method involves heating at 140 °C for 15 seconds to 149 °C for 0.5 seconds.

Vaccine Bath

• Destroys contaminating bacteria in vaccine preparations.
• The vaccine preparation is heated in a water bath at 60 °C for one hour.
• The procedure is not sporicidal.
• Only vegetative forms of bacteria are destroyed.

Serum Bath

• Inactivates bacteria contaminating serum preparations.
• Occurs by heating serum to 56 °C for several successive days.
• Is similar to a vaccine bath, only vegetative forms are destroyed.
• Higher temperatures will cause coagulation of proteins in the serum.

Inspissation

• Solidifies and disinfects egg and serum containing media.
• Media is placed in the slopes of an inspissator, a device that heats media at 80-85 °C for 30 minutes for three successive days.

Boiling

• Uses water at a boiling temperature of 100 °C.
• Is not sporicidal and will destroy only vegetative forms.
• Adding 2% sodium bicarbonate can enhance the killing action.

Fractional Sterilization (Tyndallization)

• Intermittent sterilization
• Material is exposed to live stem at 100 °C for 30-90 minutes for thrree consecutive days
• Can sterilize culture media such as TCBS and selenite broth
• Only vegetative forms of the bacteria are destroyed.

Autoclave (Steam Under Pressure)

• The most efficient method of sterilization because it can destroy all microbial forms.
• When the pressure reaches 15 pounds per square inch (psi), the temperature inside the vessel is 121 °C.
• Takes only 15-20 minutes to sterilize material
• Sterilizes instruments, surgical bandages, culture media, and other contaminated materials that can withstand high temperature and high pressure.

Dry Heat Sterilization

• Red flame sterilizes bacteriological wire loops, straight wires, tips of forceps, and searing spatulas by holding them over the flame of a Bunsen burner until red hot.
• The method can only be used to sterilize articles that can be heated to redness in flame.
• Open flame makes uses of a Bunsen burner or alcohol lamp
• Material to be sterilized is passed over the flame several times, but is not heated to redness.
• Used to sterilize article such as mouths of test tubes, scalpels, glass slides and cover slips by burning the organism into ashes.
• Incineration is aimed at burning the organism into ashes
• Contaminated material is burned using an incinerator
• Soiled dressings, bedding, animal carcasses and pathological material must be incinerated
• Materials such as polystyrene emit dense somke and must not be incinerated
• Hot air ovens were first introduced by Louis Pasteur
• Hot air ovens are a temperature of 160°C for one hour
• Hot air ovens can sterilize metal instruments such as forceps, scalpels, and scissors
• They also sterilize glassware, petri dishes, pipettes and flasks
• Used to sterilize powders and ointments.
• A disadvantage to hot air ovens is that they have poor penetration of heat in the material, since it doesn't conduct well
• Additionally, cotton wool and paper get charred slightly
• Infraread ray sterilization involves articles placed in a conveyor belt that passes through a tunnel heated by infrared radiators.
• Articles are exposed to 180°C for 7.5 minutes
• Infrared rays sterilize metallic equipment and glassware.

Desiccation and Freezing

• Dessication deprives the microorganism of moisture and is used mostly for food preservation
• Freezing is not a reliable method of sterilization because most organisms are resistant to low temperatures
• Freezing is used in the laboratory for the preservation of microorganisms in a process called lyophilization
• Lyophilization involves freeze-drying the organism in a high vacuum

Filtration

• Filtration is a form of mechanical sieving that does not kill microorganims
• Filtration separates organisms from the fluid.
• A cellulose ester filter with a pore size of 0.22 µm- 0.45µm is used, which can filter all microorganisms except viruses and the three smallest bacteria: Mycoplasma, Ricksettsia, and Chlamydia
• Filtration is used for liquid solutions that will be destroyed by heat or freezing (e.g. serum, antibiotic solutions, sugar solutions, or urea solution).

Radiation

• Electromagnetic radiation is used for sterilization purposes
• Ultraviolet light (UVL) is nonionizing
• An effective UVL wavelength is in the range of 200nm-280nm
• UVL acts by inducing the formation of thymine-thymine dimers resulting in lethal frameshift mutations.
• Bacteria, viruses and yeasts can be inactivated within seconds
• UVL is not sporicidal and is more frequently used for surface disinfection
• A disadvantage of UV rays is that they have low penetrance and its lifespan is limited by that of the UV bulb
• UV rays can be harmful to skin and eyes.
• Ionizing radiation has greater penetrance that UV rays
• It causes the formation of free radicals which cause cell death
• It isn't routinely used because it has the potential to harm human tissue
• Electron beams are particulate in nature
• Electron beams sterilize syringes, gloves, dressing packs food sand some pharmaceuticals
• Electron beams have lower penetrance and require sophisticated instruments
• Electromagnetic rays are produced from nuclear disintergration of selected radioactive isotopes
• Electromagnetic rays have greater penetrance than electron beams but require longer exposure time
• They cause damages to microorganismis' nucleic acid and are bactericidal, fungicidal, viricidal and sporicidal
• They sterilize disposable Petri dishes, plastic syringes, vitamins, antibiotics hormones fabrics and glassware commercially.

Sonic Vibration and Osmotic Pressure

• Exposure to sound waves at a frequency of approximately 20,000 cycles/second for an hour kills some bacteria and viruses by disrupting cells
• Sonic vibration disinfects and cleans instruments and to reduce microbial load.
• Osmotic pressure is based on the principle of osmosis
• An altered concentration of the fluid surrounding the organism will cause the bacterial cell to collapse
• Osmotic pressure preserves frutis in syrup and meats in brine

Chemical Methods of Sterilization

• There are chemicals that can be used for sterilization purposes

Factors Affecting Efficacy of a Chemical Agent

• A higher concentration is bactericidal, whereas a lower concentration may be bacteriostatic.
• Effective bactericidal concentration is 50-80% alcohol.
• The longer the exposure, the better the killing action.
• A higher temperature speeds up the rate and accelerates killing action can cause certain checmials to exert optimal effect at lower temperatures
• pH of the medium and the presence of extraneous materials like pus or blood decreases the efficacy of the chemical agent, which may inactivate or lower the concentration,
• Microorganisms vary in their resistance to disinfectants
• A larger # of microorganisms will mean more exposure time needed to destroy all microorganisms

Characterisitics of Good Chemical Agents

• It should be broad spectrum and able to destroy a wide variety of microorganisms.
• It should be fast acting and able to destroy microbes within a short period of time.
• It should be active in the presence of organic matter and work at any pH
• It should be stable, non-toxic, non-allergenic, non-irratative, and non-corrosive,
• It needs to be soluble in water and easy to apply.
• It needs ti leave be a residual antimicrobial film on the treated surface
• It should have a high penetrating power and most be available and not expensive
• It should also be safe for sotrage and while shipping and not have a bad odor

Classification of Chemical Disinfectants

• Chemical disinfectants may be classified based on consistency (liquid or gaseous), spectrum of activity (high, intermediate, low level), and mechanism of action
• Mechanism of action includes damage to the call membrane, denaturing cellular proteins, and the modification of the function groups of protein and nucleic acids

Damage to the Cell Membrane

• Chemicals can cause smaller molecule to leak out of the bacterial cell and can interfere with with the active transport and energy metabolism within the cell
• This includes surface active agents phenolic compounds and surface active agents

Sterilization and Disinfection: Surface Active Agents

• Surface-active agents have long-chain hydrocarbons that are fat-soluble and charged ions.
• These surface-active agents are water-soluble.
• They disrupt membranes leading to cell component leakage.
• The agents are active against vegetative microbial forms, which include mycobacteria and enveloped viruses
• Surface-active agents are disinfectants in homes and hospitals but their activity is reduced in the presence of hard water and organic matter.

Surface Active Agents: Cationic Agents

• Cationic agents: detergents where the fat soluble portion is positively charged to a quarternary nitrogen atom
• Are called quarternary ammonium compounds and are effective at alkaline pH
• Anioninc agents: are negativily charged agents that contain long chain hydrocarbons, such as soaps and bile salts
• Remove dirt through the process of emulsification and are most effective at acidic pH.

Phenol and Alcohols

• Phenolic compounds disrupt the cell membrane and cause precipitation of proteins and inactivation of enzymes.
• Phenols are disinfectant at high concentration and as antiseptic at low concentrations, such as coal tar derivatives
• They are bactericidal and fungicidal, with great avtivity against mycobacteria but has low activity against spores and most viruses
• Alcohols disorganize the lipid structure of the cell membrane, dehydrates' and can cause denaturation and coagulation of cellular proteins.
• 70% aqueous alcohol has better microbial killing property that absolute alcohol but their disadvantage is that they are skin irritants and also highly flammable.
• Ethyl alcohol is used as a skin antiseptic, and removes lipids from skin surfaces it is bacteria
• Isopropyl alcohol has better bactericidal activity and is less volatile and can be use to disinfect surfaces as the fumes can cause narcosis.

Benzyl and Methyl Alcohol

• Benzyl alcohol is used as a perservative.
• Methyl alcohol is a fungicidal and sporicidial and is used for disinfecting inoculation hoods.

Cellular Protein Denaturation

• Substances that cause denaturation or loss of of the normal structure ot the protein allows for the destruction of bacterial cell.
• Acid, alkali, alcohol, acetone, phenol and cresol are denaturing agents

Modification of Functional Protein Groupings and Nucleic Acids

• Heavy metals cause damages to emzyme activity of bacteria
• They cause precipitation of protiens and oxidation of sulfhydryl gorup and are more bacteriostatic than bactericidal
• Mercruials are biocidal and used as antiseitics. These are active against viruses at dilution of 1:500 to 1:1000
• Silver nitrate is highly bactericidal and is used to treat opthalmia neonatorum. Silver sulfadiazine is used to treat burn wounds topically

Other Methods

• Halogens are bactericidal oxidizing agents oxidizing essential sulfhydryl groups, inactivating the emzymes.
• Iodine is considered the best antiseitics because it is sporicidal bactericidal, fungicidal, viricidal and amoebicidal, such as povidone iodine
• Clorine trears water and the hypochilorites sanitize dairy and food processing equipment and is also common in household detergents
• Hydrogen peroxide is a weak antiseitics and can be used only for cleaning wounds and soft plastic contact lenses

Alkylating Agents

• These damage nucleic acids by amino to carbonyl groups
• Kills all microoganism including spores
• Formaldehyde (Formlin) is used to disinfect surface but can be used to kill mycobacterium.
• Guteraldehyde is a cold steriliant that kills medical equipment and requires the use of alkaline Ph to be more potent
• Ethylene oxise is a sporicidal sterilizering gasses of sensitive material
• Highly Flammable and causes irritation and is mutagenic and can be combined with 10/02
• It is highly flamable and is combined with 10% C02-

Antimicrobial Susceptibility Testing and Antibiotics

• Antibiotics or antimicrobials are subatances produced from microoganisms or syntheticaly that are capabale of inhibiting or destroying microrganisms even in in low centrations. Natural sources include fungi and bacteria
• An ideal antimicrobial agent must have ability kill or inhibit growth, broad specturm of activity, avoid damages
• the microbial agent or inhibit its growth, broad spectrum of activity, avoide causes damage and adverse, remain stable and be effective on long term in tissue
• There are those which one limits action in topical agents
• and systemic ones that affect several body of systems

Agents Interfeering

• Agents can interfere with the synthesis of bacterial wall and peptdoglyca synthesis and destroing peptdogly
• Agent may atler functioning cell membranes such as polymxins and antifungal groups
• Agents can inhibit Protein Synthesis by ribosomes and deform proteins
• Other aganets interfere metabolic path ways

Nucleic Actions Agents

• Some act on Nuclei acids DNA topisomerase, a essential protiens
• Other inhibit RNA synthesis such that specifically kills B synthesis
• An organism develops resistence if not effect by action ofantibiotics
• Resistance, either innate or acquired, is genetically determined, but acquired resistance in from anti drugs to species
• Over use anti broad specurm incorrect diagnostic
• Resistance can come form transformation
• Transduction
• conjugation