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Asepsis and Antisepsis
Dr. G. Ruhinda
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Introduction
Early civilizations practiced salting,smoking, pickling, drying, and exposureof food and clothing to sunlight to
control microbial growth.
Use of spices in cooking was to masktaste of spoiled food. Some spices
prevented spoilage.
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Introduction
In mid 1800s Semmelweiss and Listerdeveloped aseptic techniques to
prevent contamination of surgicalwounds.
Before then: Nosocomial infections caused death in 10%
of surgeries. Up to 25% mothers delivering in hospitals
died due to infection
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Definitions
Sterilization: Killing or removing allforms of microbial life (includingendospores) in a material or an object.
Heat is the most commonly used methodof sterilization.
Commercial Sterilization: Used on a large
scale e.g. Pharmaceuticals and foodindustry
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Definitions
Disinfection: Reducing the number ofpathogenic microorganisms to the pointwhere they no longer cause diseases.
Usually involves the removal of vegetativeor non-endospore forming pathogens.
May use physical or chemical methods.
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Definitions
Disinfectant: Applied to inanimate objects.
Antiseptic: Applied to living tissue.
Degerming: Mechanical removal of most
microbes in a limited area. Example: Alcoholswab on skin.
Sanitization: Use of chemical agent on food-
handling equipment to meet public healthstandards and minimize chances of diseasetransmission. E.g: Hot soap & water.
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Definitions
Sepsis: Gk. for decay or putrid. Indicatesbacterial contamination.
Asepsis: Absence of significant contamination.
Aseptic techniques are used to preventcontamination of surgical instruments,medical personnel, and the patient duringsurgery.
Aseptic techniques are also used to preventbacterial contamination in food industry.
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DefinitionsBacteriostatic Agent: Agent that inhibits thegrowth of bacteria, but does not necessarilykill them.
Germicide: Agent that kills certainmicroorganisms. 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 endosporesof fungal spores.
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Effectiveness of agent depends on
Number of Microbes: The more microbespresent, the more time it takes to eliminatethe population.
Type of Microbes: Endospores are verydifficult to destroy. Vegetative forms varywidely in susceptibility
Environmental influences: Organic material(blood, feces, etc) inhibit antimicrobials
Time of Exposure: Chemicals and radiationneed longer times.
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Physical Methods of ControlHeat Hot air oven Flaming Boiling Autoclave Tindilization
FiltrationRadiation
DesiccationLow temperatureOsmotic pressure
Dry heat
Moist heat
Sterilization
except boiling
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Physical Methods of Control
Heat: Kills microorganisms by denaturing theirenzymes 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 bekilled 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 bekilled. Used in canning industry.
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Moist Heat
In general, moist heat is much moreeffective than dry heat.
Boiling: Kills vegetative forms of bacterialpathogens, almost all viruses, and fungi
and their spores within 10 minutes orless.
Endospores and some viruses are not
destroyed this quickly. HBV: Survive up to 30 minutes of boiling.
Endospores: Survive up to >20 hours ofboiling.
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Moist HeatReliable sterilization with moist heat requirestemperatures above that of boiling water.
Autoclave: Hot steam under pressure is the
preferred method of sterilization Temperature of steam reaches 121oC at twice
atmospheric pressure.
Most effective when organisms contact steamdirectly or are contained in a small volume of liquid.
All organisms and endospores are killed within 15minutes.
Require mores time to reach center of solid or largevolumes of liquid.
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Autoclave
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Moist Heat: Pasteurization
Reduces microbes responsible forspoilage of beer, milk, wine, juices, etc.
Classic Method: 65o
C for 30 minutes. High Temperature Short Time (HTST):
72oC for 15 seconds (Used today).
Ultra High Temperature (UHT): 140oC for 3
seconds and then cooled very quickly in avacuum.
Advantage: Milk can be stored at roomtemperature for several months.
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Dry HeatKills by oxidation effects.
Direct Flaming: For inoculating loopsand needles. Heat metal until it has a
red glow.Hot Air Oven: Requires 2 hours at170oC for sterilization. Dry heat is
transferred less effectively to a coolbody, than moist heat.
Incineration: For hospital waste disposal
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Filtration
Liquid or gas is passed through ascreen like material with small pores.
For heat sensitive materials likevaccines, enzymes, antibiotics, andsome culture media.
High Efficiency Particulate Air Filters(HEPA): Used in operating rooms andburn units to remove bacteria from air.
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Filtration
Membrane Filters: Uniform pore size.Used in industry and research.
Different sizes: 0.22 and 0.45m Pores: Used to filter most
bacteria. Dont retain spirochetes,mycoplasmas and viruses.
0.01 m Pores: Retain all viruses and somelarge proteins.
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Radiation: IonizingExamples: Gamma rays, X rays, electronbeams, or higher energy rays.
Dislodge electrons from atoms & form ions.
Cause mutations in DNA and produceperoxides.
Used to sterilize pharmaceuticals and
disposable medical supplies.Disadv: Penetrates human tissues and maycause genetic mutations in humans.
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Forms of Radiation
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Radiation: UV light (Non-ionizing)
Damages DNA by producing thyminedimers, which cause mutations.
Used to disinfect operating rooms,nurseries, cafeterias.
Disadvantages: Damages skin, eyes.Doesnt penetrate paper, glass, and
cloth.
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Radiation: MicrowaveHeat is absorbed by water molecules.
May kill vegetative cells in moist foods.
Bacterial endospores, which do not
contain water, are not damaged bymicrowaves.
Solid foods are unevenly penetrated by
microwaves.Trichinosis outbreaks have beenassociated with pork cooked inmicrowaves.
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Dessication
In the absence of water, microbescannot grow or reproduce, but somemay remain viable for years.
Neisseria gonnorrhea: Only survives
about one hour.Mycobacterium tuberculosis: Maysurvive several months.
Viruses are fairly resistant todessication.
Clostridium spp. and Bacillus spp.: Maysurvive decades.
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Low Temperature
Effect depends on microbe and treatmentRefrigeration (0oC to 7oC): Bacteriostaticeffect. Reduces metabolic rate, so theycannot reproduce or produce toxins.
Freezing(1/3 of vegetative forms may survive 1 yr. Most parasites are killed by in few days
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Osmotic Pressure
High concentrations of salts and sugars in foodsis used to increase the osmotic pressure andcreate a hypertonic environment.
Plasmolysis: As water leaves the cell, plasmamembrane shrinks away from cell wall. Cellmay not die, but usually stops growing.
Yeasts and molds: More resistant to highosmotic pressures.
Staphylococci spp. that live on skin are fairlyresistant to high osmotic pressure.
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Chemical methods of control
Phenol and Phenolics
Halogens
AlcoholsHeavy Metals
Quaternary Ammonium Cpds (Quats)
AldehydesGaseous Sterilizers (ethylene oxide)
Oxidizing agents: Peroxygens
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DisinfectantsPhenol (carbolic acid) first used by Lister 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 innurseries.
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Halogens: Iodine
Tincture of iodine (alcohol solution) Combines with amino acid tyrosine in
proteins and denatures proteins.
Stains skin and clothes, somewhatirritating.
Iodophors: Slow releasing, take severalminutes to act. Used as skin antiseptic in surgery. Not
effective against bacterial endospores.
Examples: Betadine and Isodine
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Halogens:ChlorineWhen 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 organicmaterials.
Sodium hypochlorite (NaOCl): Is active
ingredient of bleach.Chloramines: Consist of chlorine and ammonia.Less effective as germicides.
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Heavy Metals
Copper, Selenium, Mercury, Silver & Zinc.
Very tiny amounts are effective.
1% silver nitrate used to protect infants
against gonorrheal eye infections (nolonger used)
Organic mercury compounds like
merthiolate and mercurochrome are usedto disinfect skin wounds.
Copper sulfate is used to kill algae inpools and fish tanks.
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Heavy Metals
Selenium: Used for fungalinfections and also used in dandruff
shampoos.Zinc chloride is used inmouthwashes.
Zinc oxide is used as antifungalagent in paints.
Q t A i C d
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Quaternary Ammonium Cpds(Quats)Widely used surface active agents.Effective vs G+ve, less effective vs gram-ve
Destroy fungi, amoebas, and enveloped viruses.
Zephiran, Cepacol, used in lab spray bottles.Pseudomonas strains that are resistant
Adv: Strong antimicrobial action, colorless,odorless, tasteless, stable, and nontoxic.
Diasadv: Form foam. Organic matter interfereswith effectiveness. Neutralized by soaps andanionic detergents.
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Aldehydes: Formaldehyde gas
Inactivate proteins by forming covalentcrosslinks with several functional groups.
Used as formalin, a 37% aqueous
solution.Formalin was used extensively to preservebiological specimens and inactivate
viruses and bacteria in vaccines.Irritates mucous membranes, strong odor.
Used in embalming.
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Aldehydes: Glutaraldehyde
Less irritating and more effective thanformaldehyde.
It is a sterilizing agent.
A 2% solution of glutaraldehyde (Cidex) is: Bactericidal, tuberculocidal, and viricidal in 10
minutes.
Sporicidal in 3 to 10 hours.Used to disinfect hospital instruments.
Used in embalming.
Eth l O id G
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Ethylene Oxide: GaseousSterilizers
Chemicals that sterilize in a chamber similarto an autoclave.
Denature proteins, by replacing functional
groups with alkyl groups.Kills all microbes and endospores, butrequires exposure of 4 to 18 hours.
Toxic and explosive in pure form.Used to sterilize mattresses and largeequipment.
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PeroxygensOzone: Oxidize cellular components and disruptsmembranes and proteins.
Used along with chlorine to disinfect water.
Hydrogen Peroxide: Effective in disinfection of inanimate objects.
Sporicidal at higher temperatures.
Benzoyl Peroxide:Used in acne medications.
Peracetic Acid: Sterilant
Kills bacteria and fungi in less than 5 minutes.
Kills endospores and viruses within 30 minutes.
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Thank You