Physical and Physical and Chemical Agents Chemical Agents

for Microbial for Microbial ControlControl

Chapter 11

Control of microbesControl of microbes

Physical and chemical Physical and chemical methods to destroy or reduce methods to destroy or reduce

microbes in a given areamicrobes in a given area

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OVERVIEW

Why do we want to control Why do we want to control microbes?microbes?

How do we do it?How do we do it?

Relative resistance of microbesRelative resistance of microbes

Highest resistanceHighest resistance– Bacterial endosporesBacterial endospores– PrionsPrions

Moderate resistanceModerate resistance– Pseudomonas sp.Pseudomonas sp.– Mycobacterium Mycobacterium

tuberculosistuberculosis– Staphylococcus aureusStaphylococcus aureus– Protozoan cystsProtozoan cysts

Least resistanceLeast resistance– most vegetative cellsmost vegetative cells– Fungal sporesFungal spores– enveloped virusesenveloped viruses– YeastYeast– Protozoan trophozoitesProtozoan trophozoites

Methods that kill microbesMethods that kill microbes______________________________ – a process that – a process that destroys destroys allall viable microbes, including viruses & endosporesviable microbes, including viruses & endospores– Heat, sterilantsHeat, sterilants

______________________________– a process to – a process to destroy destroy vegetativevegetative pathogens, not endospores pathogens, not endospores– disinfectantsdisinfectants or or germigermicidecides – s – chemicalchemical - kills - kills

pathogenic microorganismspathogenic microorganisms– __________________________________________ – destroy/inhibit vegetative – destroy/inhibit vegetative

pathogens on exposed body surfacespathogens on exposed body surfaces– Sepsis – infection/growth in body Sepsis – infection/growth in body – Asepsis – preventing entry of pathogens and infectionAsepsis – preventing entry of pathogens and infection

Methods that reduce numbersMethods that reduce numbers

______________________________– any cleansing technique that – any cleansing technique that mechanically removes microbesmechanically removes microbes and reduces and reduces numbers to safe levelsnumbers to safe levels– ______________________________– reduces the number of microbes – reduces the number of microbes

on skinon skin

______________________________________________________ – controls – controls numbers by preventing growth (multipication)numbers by preventing growth (multipication)

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Microbial deathMicrobial death

Permanent termination of an organism’s Permanent termination of an organism’s vital processesvital processes

microbiological definition:microbiological definition:

Involves permanent loss of reproductive Involves permanent loss of reproductive capability, even under optimum growth capability, even under optimum growth conditionsconditions

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Factors that influence microbial killing

How antimicrobial agents workHow antimicrobial agents work: : Cellular targets of control Cellular targets of control

(mode of action)(mode of action)

1.1. Cell wallCell wall

2.2. Cell membraneCell membrane

3.3. Cellular synthetic processes (DNA, RNA)Cellular synthetic processes (DNA, RNA)

4.4. ProteinsProteins

Cell wallCell wall

Action:Action:– Block synthesisBlock synthesis– DigestionDigestion– Disrupt surfaceDisrupt surface

w/o cell wall, bacteria will lyse (especially w/o cell wall, bacteria will lyse (especially gram positive… remember?)gram positive… remember?)

Examples: penicillin, detergents, alcoholExamples: penicillin, detergents, alcohol

mode of actionmode of action

Surfactants disrupt mode of actionmode of action

Affect on synthesis (DNA, RNA)Affect on synthesis (DNA, RNA)

Proteins – have many functions in the cell!Proteins – have many functions in the cell!

Antimicrobials can block: DNA synthesis Antimicrobials can block: DNA synthesis (master code), transcription, translation(master code), transcription, translation

Mutagens (radiation = permanent Mutagens (radiation = permanent inactivation of DNA)inactivation of DNA)

Antimicrobial therapy (drugs)Antimicrobial therapy (drugs) Chemicals – some destroy nucleic acidsChemicals – some destroy nucleic acids

mode of actionmode of action

Heat, pH, heavy metals can alter proteinsmode of actionmode of action

Practical concernsPractical concerns

Does the application require sterilization?Does the application require sterilization?

Is the item to be reused? (time, $$)Is the item to be reused? (time, $$)

Can the item withstand heat, pressure, radiation, Can the item withstand heat, pressure, radiation, or chemicals?or chemicals?

Is the method suitable?Is the method suitable?

Will the agent penetrate to the necessary Will the agent penetrate to the necessary extent?extent?

Is the method cost- and labor-efficient & is it Is the method cost- and labor-efficient & is it safe?safe?

Types of Control: Types of Control: I. Methods of I. Methods of PhysicalPhysical Control Control

1.1. HeatHeat

2.2. Cold temperaturesCold temperatures

3.3. DesiccationDesiccation

4.4. RadiationRadiation

5.5. FiltrationFiltration

1. Heat1. HeatMoist vs. dryMoist vs. dry

Moist heatMoist heat = lower temp and shorter time = lower temp and shorter time

Causes coagulation/denaturation of Causes coagulation/denaturation of proteinprotein

Dry heatDry heat = higher temp/longer time = higher temp/longer time

Dehydrates cell, removes water, Dehydrates cell, removes water, denatures proteins, oxidation (burning)denatures proteins, oxidation (burning)

Physical ControlPhysical Control

Physical ControlPhysical Control

1. Heat – moist heat1. Heat – moist heatMoist heatMoist heat uses hot water or steam uses hot water or steam

sterilization sterilization ______________________________ 15 psi/121 15 psi/121ooC/10-40min C/10-40min (steam (steam

under pressure)under pressure)

intermittent sterilizationintermittent sterilization – 100 – 100ooC 30-60 min for 3 C 30-60 min for 3 days days (unpressurized steam)(unpressurized steam)

disinfectiondisinfection BoilingBoiling at 100 at 100ooC for 30 minutes to destroy non-C for 30 minutes to destroy non-

spore-forming pathogensspore-forming pathogens ______________________________ kills kills Salmonella, ListeriaSalmonella, Listeria & &

overall microbe countoverall microbe count

Physical ControlPhysical Control

Physical ControlPhysical Control

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PasteurizationPasteurization

PasteurizationPasteurization – heat – heat kill kill potential agents of infection and potential agents of infection and spoilage spoilage without destroying the without destroying the food flavor or valuefood flavor or value6363°C°C–66–66°C°C for 30 minutes (batch for 30 minutes (batch method)method)71.671.6°C for 15 seconds (flash °C for 15 seconds (flash method)method)Not sterilizationNot sterilization – kills non-spore- – kills non-spore-forming pathogens and lowers forming pathogens and lowers overall microbe count; does not kill overall microbe count; does not kill endospores or many endospores or many nonpathogenic microbesnonpathogenic microbes

Dry heatDry heat uses higher uses higher temperatures than moist temperatures than moist heat, can also sterilizeheat, can also sterilize

incinerationincineration – 600-1200 – 600-1200ooC C combusts & dehydrates combusts & dehydrates cellscells

dry ovensdry ovens – 150-180 – 150-180ooC- C- coagulate proteins coagulate proteins

1. Heat – dry heat1. Heat – dry heatPhysical ControlPhysical Control

Thermal deathThermal deathPhysical ControlPhysical Control

Thermal death time (TDT) – shortest length of time required to kill all test microbes at a specified temperature

Thermal death point (TDP) – lowest temperature required to kill all microbes in a sample in 10 minutes

2. Cold temperatures2. Cold temperatures

______________________________– – slowsslows the growth of the growth of microbesmicrobes

refrigeration 0-15refrigeration 0-15ooC & freezing <0C & freezing <0ooCC

used to used to ________________________food, media and food, media and culturescultures

Physical ControlPhysical Control

3. Desiccation3. Desiccation

gradual gradual removal of waterremoval of water from cells, leads from cells, leads to metabolic inhibitionto metabolic inhibition

not effective microbial control – many cells not effective microbial control – many cells retain ability to grow when water is retain ability to grow when water is reintroducedreintroduced

______________________________ = freeze-drying= freeze-drying

Note: cold and dessication ARE NOT good methods of disinfection or sterilization.

Physical ControlPhysical Control

4. Radiation4. Radiation

1.1. ______________________________ radiationradiation – deep – deep penetrating power, breaks DNApenetrating power, breaks DNA

– gamma rays, X-rays, cathode raysgamma rays, X-rays, cathode rays– used to sterilize medical supplies & food used to sterilize medical supplies & food

productsproducts

2.2. ______________________________ radiationradiation – little – little penetrating power to sterilize air, water & penetrating power to sterilize air, water & solid surfacessolid surfaces

– uv lightuv light creates thymine dimers, which creates thymine dimers, which interfere with replicationinterfere with replication

Physical ControlPhysical Control

Physical ControlPhysical ControlIonizing radiationIonizing radiation

Physical ControlPhysical ControlNonionizing radiation - UVNonionizing radiation - UV

UV treatment of wastewater Physical ControlPhysical Control

Other “waves”Other “waves”

Sound (high frequency)Sound (high frequency)

Can also be used to disrupt cells Can also be used to disrupt cells (vibrations) or generate heat(vibrations) or generate heat

Ultrasonic devices are used clean dental, Ultrasonic devices are used clean dental, medical instruments before sterilizationmedical instruments before sterilization

Physical ControlPhysical Control

5. Filtration5. Filtrationphysical removal of physical removal of microbes by passing a microbes by passing a gas or liquid through gas or liquid through filterfilter

Pores of filter large Pores of filter large enough for liquid but too enough for liquid but too small for microbe (<1 small for microbe (<1 μμm)m)

used toused to

Physical ControlPhysical Control

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OVERVIEW

Types of Control: Types of Control: II. Methods of II. Methods of ChemicalChemical Control Control

Categories:Categories:• HalogensHalogens• PhenolicsPhenolics• ChlorhexidineChlorhexidine• AlcoholsAlcohols• Hydrogen peroxideHydrogen peroxide• Detergents & soapsDetergents & soaps• Heavy metalsHeavy metals• AldehydesAldehydes

Uses:•Disinfectants •antiseptics•sterilants•degermers •preservatives

Chemical antimicrobialsChemical antimicrobials

10,000 manufactured today10,000 manufactured today

About 1,000 routinely usedAbout 1,000 routinely used

Society is obsessed with “killing germs” – Society is obsessed with “killing germs” – to the point of being excessiveto the point of being excessive

Result: widespread overuse Result: widespread overuse resistance resistance of pathogens, death of natural floraof pathogens, death of natural flora

Qualities of chemical antimicrobialsQualities of chemical antimicrobials

Rapid action, even in low concentrationRapid action, even in low concentrationWater/alcohol soluble, stabileWater/alcohol soluble, stabileBroad spectrum w/o being toxicBroad spectrum w/o being toxicPenetration, sustained actionPenetration, sustained actionResitance to inactivationResitance to inactivationNoncorrosive, nonstainingNoncorrosive, nonstainingSanitizing and deodorizingSanitizing and deodorizingInexpensive and availableInexpensive and available

Levels of activityLevels of activity

Factors that affect activityFactors that affect activity

Type of microorganism being treatedType of microorganism being treatedMaterial being treatedMaterial being treatedAmt of contaminationAmt of contaminationExposure timeExposure timeStrength/action of germicideStrength/action of germicide

Appendix C shows procedures for testing Appendix C shows procedures for testing effectivenesseffectiveness

Ways to express Ways to express strength/concentrationstrength/concentration

Dilution (1:200 is one part chemical in 200 Dilution (1:200 is one part chemical in 200 parts dilutant such as water)parts dilutant such as water)ppm – parts per millionppm – parts per millionPercent – 70% alcohol, or mg/mlPercent – 70% alcohol, or mg/ml

______________________________ solutions – have water solutions – have water as the solventas the solvent______________________________– are dissolved in – are dissolved in alcoholalcohol

Chemical control - categoriesChemical control - categories

1.1. HalogensHalogens

2.2. PhenolicsPhenolics

3.3. ChlorhexidineChlorhexidine

4.4. AlcoholsAlcohols

5.5. Hydrogen peroxideHydrogen peroxide

6.6. Detergents & soapsDetergents & soaps

7.7. Heavy metalsHeavy metals

8.8. AldehydesAldehydes

1. Halogens1. Halogens

Ionic (halide) or nonionicIonic (halide) or nonionic

Mostly Mostly ______________________________

Germicidal and sporicidialGermicidal and sporicidial with long exposure with long exposure

Affect protein structure (bonds)Affect protein structure (bonds)

1. Halogens1. Halogens

Chlorine – ClChlorine – Cl22, hypochlorites (chlorine , hypochlorites (chlorine

bleach - OCl), chloraminesbleach - OCl), chloramines– In water – release hypochlorous acid (HOCl)In water – release hypochlorous acid (HOCl)– Denaturation of proteins by disrupting disulfide Denaturation of proteins by disrupting disulfide

bondsbonds

– Can be sporicidalCan be sporicidal

– 0.6-1 ppm Cl0.6-1 ppm Cl22 to clean water to clean water

– Bleach – sanitization/disinfectionBleach – sanitization/disinfection

Iodine - IIodine - I22, iodophors (betadine), iodophors (betadine)

– Denature proteins – similar to Chlorine but not Denature proteins – similar to Chlorine but not affected by organic matter or pHaffected by organic matter or pH

– Broad spectrum microbicide, can be sporicidalBroad spectrum microbicide, can be sporicidal– Milder medical & dental degerming agents, Milder medical & dental degerming agents,

disinfectants, ointments, topical antisepticdisinfectants, ointments, topical antiseptic– Betadine (iodophor) = iodine + neutral Betadine (iodophor) = iodine + neutral

polymer; allows for slow release and polymer; allows for slow release and increased penetration – used as antisepticincreased penetration – used as antiseptic

1. Halogens1. Halogens

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2. Phenolics2. Phenolics

Phenol ring (aromatic carbon ring) + Phenol ring (aromatic carbon ring) + groupsgroups

Disrupt cell membranes & precipitate Disrupt cell membranes & precipitate (denature) proteins; bactericidal, (denature) proteins; bactericidal, fungicidal, virucidal, fungicidal, virucidal, not sporicidalnot sporicidal– ______________________________– ________________________antibacterial additive to soapsantibacterial additive to soaps– Mouthwash! (thymol)Mouthwash! (thymol)– Can be VERY toxicCan be VERY toxic! Not typically used as ! Not typically used as

antiseptics.antiseptics.

______________________________

3. 3. ______________________________ Hibiclens, HibitaneHibiclens, Hibitane

A surfactant & protein A surfactant & protein denaturant with broad denaturant with broad microbicidal propertiesmicrobicidal properties

Not sporicidalNot sporicidal

Used as skin Used as skin _________ _________ agents for preoperative scrubs, agents for preoperative scrubs, skin cleaning & burnsskin cleaning & burns

4. Alcohols4. Alcohols

Ethyl, isopropylEthyl, isopropyl in solutions of 50-90% in solutions of 50-90% (water needed for protein coagulation)(water needed for protein coagulation)

Act as surfactants dissolving membrane Act as surfactants dissolving membrane lipids and coagulating proteins of lipids and coagulating proteins of vegetative bacterial cells and fungivegetative bacterial cells and fungi

______________________________

Isopropanol = rubbing alcohol, but vapors Isopropanol = rubbing alcohol, but vapors can be toxiccan be toxic

5. Hydrogen peroxide5. Hydrogen peroxide

Weak (3%) to strong (25%)Weak (3%) to strong (25%)

______________________________ agent (steals electrons) agent (steals electrons)

Produce highly reactive hydroxyl free Produce highly reactive hydroxyl free radicals that damage protein & DNA while radicals that damage protein & DNA while also decomposing to Oalso decomposing to O22 gas (bubbles) gas (bubbles)

toxic to toxic to ______________________________ , overwhelms , overwhelms catalase in aerobescatalase in aerobes

Strong solutions are sporicidalStrong solutions are sporicidal

colonoscope

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6. Detergents & soaps6. Detergents & soaps

________ ________ compounds that work as compounds that work as ______________________________ Quaternary ammonia cpds (Quaternary ammonia cpds (____________________) ) act as surfactants that alter membrane act as surfactants that alter membrane permeability of some bacteria & fungipermeability of some bacteria & fungi– Not sporicidal, ineffective against TB, Not sporicidal, ineffective against TB,

hepatitis, pseudomonashepatitis, pseudomonas

Soaps- mechanically remove soil and Soaps- mechanically remove soil and grease containing microbesgrease containing microbes

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Figure 11.17

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7. Heavy metals7. Heavy metals

Solutions of silver & mercury kill vegetative Solutions of silver & mercury kill vegetative cells in low concentrations by cells in low concentrations by inactivating inactivating proteinsproteins

Metallic saltsMetallic salts

OligodynamicOligodynamic action action

Not sporicidalNot sporicidal

_________ _________ to humansto humans– Not used on broken skinNot used on broken skin

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8. Aldehydes8. AldehydesGlutaraldehyde & formaldehyde kill by Glutaraldehyde & formaldehyde kill by alkylating protein & DNA (-CHO is reducing alkylating protein & DNA (-CHO is reducing group)group)______________________________ = H on AA is replaced by = H on AA is replaced by the aldehyde (and crosslinked)the aldehyde (and crosslinked)______________________________ in 2% solution (Cidex) in 2% solution (Cidex) used as used as sterilantsterilant for heat sensitive for heat sensitive instruments instruments ______________________________ - disinfectant, - disinfectant, preservative, toxicity limits use (formalin is preservative, toxicity limits use (formalin is aqueous solution)aqueous solution)

Gases & aerosolsGases & aerosols

______________________________, propylene oxide, , propylene oxide, betapropiolactone & chlorine dioxidebetapropiolactone & chlorine dioxide

Strong Strong ______________________________ agents, agents, sporicidal (sterilization)sporicidal (sterilization)

________________________ ________________________ – uses ethylene oxide

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Miscellaneous antimicrobialsMiscellaneous antimicrobials

Dyes – crystal violet, acriflavineDyes – crystal violet, acriflavine

Acids, bases (alkalies) – destroy but some Acids, bases (alkalies) – destroy but some are corrosive and hazardousare corrosive and hazardous– Pickling – acetic acidPickling – acetic acid– Sauerkraut, olives – lactic acidSauerkraut, olives – lactic acid– Benzoic, sorbic acid – in lots of processed Benzoic, sorbic acid – in lots of processed

foods (preservatives = foods (preservatives = ______________________________ ) )