Asepsis Notes

8/12/2019 Asepsis Notes

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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

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