STERILIZATION STERILIZATION AND AND

DISINFECTIONDISINFECTIONDr. Daniel ShelverDr. Daniel Shelver2-213 Med School2-213 Med Schooldshelv@lsuhsc.edudshelv@lsuhsc.edu

5-48845-4884

Ignaz Semmelweis

1846: Physician at Vienna General 1846: Physician at Vienna General HospitalHospital

Interested in ‘childbed fever’ (often fatal Interested in ‘childbed fever’ (often fatal uterine infection that followed childbirth) uterine infection that followed childbirth) in obstetrical clinic there. in obstetrical clinic there. Two clinics, one staffed by physicians & Two clinics, one staffed by physicians &

medical students: mortality rate: medical students: mortality rate: 13% 13% other other staffed by midwives: mortality rate: staffed by midwives: mortality rate: 2%2%

Physicians/students were performing Physicians/students were performing autopsies, then after either not washing hands autopsies, then after either not washing hands or washing with soap, proceeding to deliver or washing with soap, proceeding to deliver babiesbabies

Ignaz Semmelweis

For the physician/medical student ward, For the physician/medical student ward, Semmelweis enforced a policy of Semmelweis enforced a policy of handwashing with chlorinated lime; handwashing with chlorinated lime; dropped mortality rate to 2%.dropped mortality rate to 2%.

Despite success, his policies were Despite success, his policies were rejected because they went against rejected because they went against current scientific beliefs (germ theory not current scientific beliefs (germ theory not developed yet); ultimately he was fired by developed yet); ultimately he was fired by Vienna General.Vienna General.

Sterilization and Sterilization and disinfectiondisinfection

Goal: reduce or completely Goal: reduce or completely eliminate the presence of potentially eliminate the presence of potentially harmful life formsharmful life forms BacteriaBacteria VirusVirus FungiFungi AlgaeAlgae Bacterial spores (in general the hardest Bacterial spores (in general the hardest

to kill; can survive even boiling water!)to kill; can survive even boiling water!)

IMPORTANT TERMSIMPORTANT TERMS

Sterile/sterilization Sterile/sterilization Absence of any life form/process Absence of any life form/process

of rendering something sterile of rendering something sterile ((must kill sporesmust kill spores))

Disinfection Disinfection Agent that frees from infection – Agent that frees from infection –

kills bacteria/inactivates virus, but does kills bacteria/inactivates virus, but does not necessarily kill bacterial sporesnot necessarily kill bacterial spores

IMPORTANT TERMSIMPORTANT TERMS Antiseptic Antiseptic

Substance that opposes growth or action Substance that opposes growth or action of microorganisms, either by killing or inhibiting of microorganisms, either by killing or inhibiting growthgrowth

Term is usually used in reference to body Term is usually used in reference to body surface or woundsurface or wound

SanitizeSanitize Utilization of agent that reduces microbial Utilization of agent that reduces microbial

contaminants to safe levels as determined by contaminants to safe levels as determined by public health requirementspublic health requirements

““-cide“-cide“ Kills microbes/inactivates virusesKills microbes/inactivates viruses

““-static“-static“ Arrests growth of microbes/inhibits replication Arrests growth of microbes/inhibits replication

of virusof virus

SterilizationSterilization Physical AgentsPhysical Agents - -

HeatHeat Moist heat – uses an autoclave that introduces Moist heat – uses an autoclave that introduces

steam under pressure to a closed chambersteam under pressure to a closed chamber Most effective, practical and practical methodMost effective, practical and practical method Most hospitals/laboratories use it as a first Most hospitals/laboratories use it as a first

choice method of sterilizationchoice method of sterilization Not applicable to heat-labile substances (ie., Not applicable to heat-labile substances (ie.,

antibody solution, antibioticsantibody solution, antibiotics Inactivates life forms by Inactivates life forms by

denaturation/coagulation of proteins; denaturation/coagulation of proteins; destruction of DNAdestruction of DNA

Process sterilizes more quickly than dry heatProcess sterilizes more quickly than dry heat

SterilizationSterilization

Dry heatDry heat Inactivates life forms by oxidation of Inactivates life forms by oxidation of

proteinsproteins Does not dull sharp edges, unlike Does not dull sharp edges, unlike

autoclavingautoclaving Can sterilize closed containers and Can sterilize closed containers and

oils/petroleum jellies, which are not oils/petroleum jellies, which are not penetrated with steampenetrated with steam

Takes longer to sterilize than autoclavingTakes longer to sterilize than autoclaving

Radiation Radiation Ultraviolet radiation Ultraviolet radiation

Destroys DNA & inhibits its replicationDestroys DNA & inhibits its replication Induces formation of unfavorable compounds Induces formation of unfavorable compounds

inin

bacterial habitats (ozone, hydrogen peroxide)bacterial habitats (ozone, hydrogen peroxide) Most effective against airborne Most effective against airborne

microorganismsmicroorganisms Does not kill all viruses or bacteriaDoes not kill all viruses or bacteria Often used to prevent airborne spread of Often used to prevent airborne spread of

bacteria in hospitals, microbiology labs, etc.bacteria in hospitals, microbiology labs, etc.

SterilizationSterilization

RadiationRadiation Ionizing radiationIonizing radiation

Beta rays (electron) or gamma raysBeta rays (electron) or gamma rays ‘ ‘Chain reaction’ where bacterial/viral cell Chain reaction’ where bacterial/viral cell

components absorb electrons, then give off components absorb electrons, then give off high energy electrons, disrupting cell high energy electrons, disrupting cell componentscomponents

Sutures, plastic tubing, plasticwareSutures, plastic tubing, plasticware

SterilizationSterilization

FiltrationFiltration Used for solutions that are heat labile Used for solutions that are heat labile

or whose efficacy/safety is compromised or whose efficacy/safety is compromised by the introduction of chemicalsby the introduction of chemicals

Solution is filtered through a membrane Solution is filtered through a membrane that retains bacteria, fungi, algaethat retains bacteria, fungi, algae

Does not always remove viruses Does not always remove viruses (depends on pore size) – therefore not (depends on pore size) – therefore not ‘sterilization’ in the strictest sense‘sterilization’ in the strictest sense

SterilizationSterilization

Chemical AgentsChemical Agents Action (one or more of following)Action (one or more of following)

Oxidation of cellOxidation of cell Denaturation of proteinsDenaturation of proteins Inactivation of vital enzymesInactivation of vital enzymes Modification of permeability of cellModification of permeability of cell Disruption of cellDisruption of cell

Effectiveness against different life Effectiveness against different life forms varies (more so than physical forms varies (more so than physical methods)methods)

SterilizationSterilization Chemical AgentsChemical Agents - -

Hydrogen peroxide plasma – oxidizes*Hydrogen peroxide plasma – oxidizes* Hydrogen peroxide plasma that has been microwaved to Hydrogen peroxide plasma that has been microwaved to

increase available oxygen radicals, which are the killing increase available oxygen radicals, which are the killing agentagent

Chlorine dioxide –gas (used to decontaminate Chlorine dioxide –gas (used to decontaminate buildings where anthrax attacks occurred); oxidizes, buildings where anthrax attacks occurred); oxidizes, kills sporeskills spores

Ethylene oxide (gas, slow) alkylates*Ethylene oxide (gas, slow) alkylates* Useful for delicate instruments, optics Useful for delicate instruments, optics Somewhat impractical; toxicSomewhat impractical; toxic

Peracetic acid (Liquid solution) oxidizes*Peracetic acid (Liquid solution) oxidizes* Preferred method of sterilizing heat-sensitive instruments Preferred method of sterilizing heat-sensitive instruments

at LSUHSC – sterilizer equipment manufactured by Sterisat LSUHSC – sterilizer equipment manufactured by Steris Limited to instruments that will be used immediately Limited to instruments that will be used immediately

(objects come out wet)(objects come out wet)

* = used at LSUHSC* = used at LSUHSC

Evaluation of Evaluation of SterilizationSterilization

PhysicalPhysical ThermometersThermometers Pressure gaugesPressure gauges

BiologicalBiological SporesSpores

DisinfectionDisinfection High level (some overlap with sterilizers)!High level (some overlap with sterilizers)!

Glutaraldehyde – Alkylates cell componentsGlutaraldehyde – Alkylates cell components Peracetic acid – Oxidizes cell componentsPeracetic acid – Oxidizes cell components Chlorine dioxide – Oxidizes, liquid or gas (also a sterilizer)Chlorine dioxide – Oxidizes, liquid or gas (also a sterilizer) Hydrogen peroxide – Oxidizes cell componentsHydrogen peroxide – Oxidizes cell components

Intermediate levelIntermediate level Alcohols – denatures proteins – inhibited by dirt!Alcohols – denatures proteins – inhibited by dirt! Sodium Hypochlorite (bleach) – inhibited by alkaline pH Sodium Hypochlorite (bleach) – inhibited by alkaline pH

(i.e. alkaline detergents). Stronger (i.e. alkaline detergents). Stronger bacteriocidal/virucidal/sporacidal activity at higher bacteriocidal/virucidal/sporacidal activity at higher concentrations (can be high or low level disinfectant)concentrations (can be high or low level disinfectant)

Iodophor compounds – denature/oxidize proteins – not Iodophor compounds – denature/oxidize proteins – not influenced by pH – inhibited by dirt.influenced by pH – inhibited by dirt.

Low levelLow level Quaternary amine compounds – denature cell membranes; Quaternary amine compounds – denature cell membranes;

precipitate biological moleculesprecipitate biological molecules

Factors Affecting Killing Factors Affecting Killing DynamicsDynamics

1.1. Number microorganismsNumber microorganisms2.2. Nature of the microbial population Nature of the microbial population 3.3. Concentration of the agent usedConcentration of the agent used4.4. Time Time 5.5. Temperature (ex bleach more effective at Temperature (ex bleach more effective at

higher temperature)higher temperature)6.6. Extraneous material Extraneous material 7.7. pH (ex bleach effectiveness as pH pH (ex bleach effectiveness as pH

increased)increased)8.8. VolumeVolume

Antisepsis Antisepsis Used at LSUHSC:Used at LSUHSC:

Lotion soap (washing hands between uninfected Lotion soap (washing hands between uninfected patients, using bathroom) 15-30 secpatients, using bathroom) 15-30 sec

Weakly antiseptic; bacteria/viruses can still remain on Weakly antiseptic; bacteria/viruses can still remain on skinskin

Chlorohexadine 2% 15- 30 sec – contact with Chlorohexadine 2% 15- 30 sec – contact with infected patient or bodily fluids –disadvantage; infected patient or bodily fluids –disadvantage; some people allergic to this compound (use some people allergic to this compound (use iodine, antibacterial soap or alcohol instead) – iodine, antibacterial soap or alcohol instead) – advantage; advantage; its antiseptic activity persist after its antiseptic activity persist after dryingdrying

Chlorohexadine 4% 6 min – surgical scrubChlorohexadine 4% 6 min – surgical scrub Iodine – surgical scrub; often used after alcohol Iodine – surgical scrub; often used after alcohol

washwash Alcohols – very effective except do not kill spores; Alcohols – very effective except do not kill spores;

dries skin; activity does not persist after dryingdries skin; activity does not persist after drying Use of gloves (must be changed between patients) Use of gloves (must be changed between patients)

reduces chance of spreading agents but between reduces chance of spreading agents but between & before gloves!& before gloves!

Sterilization/Disinfection in Sterilization/Disinfection in the laboratorythe laboratory

SterilizationSterilization Autoclave – most commonly used: heat stable Autoclave – most commonly used: heat stable

media, dry goods that are heat stable media, dry goods that are heat stable (eppendorf tubes, glass culture tubes), (eppendorf tubes, glass culture tubes), biohazards (doesn’t matter if heat labile since biohazards (doesn’t matter if heat labile since destined for garbage)destined for garbage)

Filtration – heat labile solutions, antibiotic Filtration – heat labile solutions, antibiotic solutions, antibody solutions.solutions, antibody solutions.

Ionizing radiation: used on prepackaged Ionizing radiation: used on prepackaged disposable plastic plasticware bought from disposable plastic plasticware bought from manufacturer (i.e. sterile filtration apparatus)manufacturer (i.e. sterile filtration apparatus)

Disinfection/antisepsis in Disinfection/antisepsis in the laboratorythe laboratory

Disinfection of working areasDisinfection of working areas Cell culture areas, laboratory benches where bacteria Cell culture areas, laboratory benches where bacteria

will be culturedwill be cultured 70% ethanol (medium level disinfected) – kill all bacteria 70% ethanol (medium level disinfected) – kill all bacteria

except spores, many virusesexcept spores, many viruses 10% bleach for blood spills10% bleach for blood spills Formaldehyde vapor for severe contamination of cell Formaldehyde vapor for severe contamination of cell

culture hoodsculture hoods Flaming of inoculating loopFlaming of inoculating loop

Dry heatDry heat Baking of glasswareBaking of glassware

Dry heatDry heat Skin antisepsisSkin antisepsis

Used in combination with or without gloves gloves Used in combination with or without gloves gloves during cell culture: 70% ethanol or alcohol gel.during cell culture: 70% ethanol or alcohol gel.

Level

MHHMHMHL

MKN/AKills g-N/A