Disinfection and Disinfection and SterilizationSterilization

——For the course of Medical Microbiology for MBBS foreign students, Class 2006/2011, SYSU

September 18, 2007

Mengfeng Li （黎孟枫） , M.D. Department of Microbiology, Zhongshan School of

Medicine, SYSU, Guangzhou, China

limf@mail.sysu.edu.cn

Medical Microbiology Medical Microbiology

Early needs and success for disinfection Early needs and success for disinfection (Early 1800’s) —— a historic story(Early 1800’s) —— a historic story

• By mid-1800’s, almost half of post-operative patients died of sepsis (then By mid-1800’s, almost half of post-operative patients died of sepsis (then called “hospital disease”). called “hospital disease”). A common report by surgeons was: operation A common report by surgeons was: operation successful but patient diedsuccessful but patient died..

• A hypothesis: A hypothesis: ① ① exposing moist body tissue to oxygen exposing moist body tissue to oxygen sepsis;sepsis; ②② best best prevention: keeping air away from wounds by means of plasters, collodion or prevention: keeping air away from wounds by means of plasters, collodion or resins.resins.

• Having tried methods to encourage clean healing with no success, surgeon Having tried methods to encourage clean healing with no success, surgeon Joseph Lister discarded the concept of direct infection by bad air but Joseph Lister discarded the concept of direct infection by bad air but postulated that sepsis might be caused by a postulated that sepsis might be caused by a 'pollen-like dust'pollen-like dust‘, although he did ‘, although he did not know yet the “dust” was living microbes. not know yet the “dust” was living microbes.

• When Louis Pasteur suggested the presence of When Louis Pasteur suggested the presence of living organismsliving organisms in the air, in the air, Lister made the connection with wound sepsis: the Lister made the connection with wound sepsis: the microbesmicrobes in the air were in the air were likely causing the sepsis and should be destroyed before they entered the likely causing the sepsis and should be destroyed before they entered the wound.wound.

• Lister had previously heard that 'Lister had previously heard that 'carbolic acidcarbolic acid' was used to treat sewage, and ' was used to treat sewage, and that fields treated with the affluent were freed of a parasite causing disease in that fields treated with the affluent were freed of a parasite causing disease in cattle. He then cattle. He then began to clean wounds and dress them with carbolic acid..began to clean wounds and dress them with carbolic acid..

Joseph Lister operated with carbolic acid (石炭酸） spray

The milestone:

case of James Greenlees

-Aug. 12, 1865, James Greenlees, 11-year-old

-Struck by a horse-drown cart

-Compound fractures

-Taken to Royal Infirmary

-Under the care of Joseph Lister

Why disinfection and Why disinfection and sterilization?sterilization?

• Contagious diseasesContagious diseases• Hospital infection (e.g., OR, ID ward) or other Hospital infection (e.g., OR, ID ward) or other

opportunistic infection opportunistic infection • Lab contaminationLab contamination• Etc. Etc.

• Microbes:Microbes:- usually easy to grow in environment; usually easy to grow in environment; - but also can be inhibited or killed by certain but also can be inhibited or killed by certain

environmental (physical or chemical) environmental (physical or chemical) factors/conditions.factors/conditions.

Terminology Terminology

•DisinfectionDisinfection 消毒消毒•SterilizationSterilization 灭菌灭菌•BacteriostasisBacteriostasis 抑菌 抑菌 •AntisepsisAntisepsis 防腐防腐•Asepsis Asepsis 无菌 无菌

DisinfectionDisinfection （消毒）（消毒）

• Process of reducing or Process of reducing or eliminating eliminating living pathogenic living pathogenic microorganismsmicroorganisms in or on in or on materials, so they are no longer materials, so they are no longer a health hazard.a health hazard.

For example: use of alcohol before For example: use of alcohol before drug injection.drug injection.

SterilizationSterilization （灭菌）（灭菌）• Process of destroying Process of destroying all all

microbialmicrobial forms. A sterile object forms. A sterile object is one free of all microbial forms, is one free of all microbial forms, including bacterial including bacterial sporesspores..

• More thorough than disinfectionMore thorough than disinfection

BacteriostasisBacteriostasis （抑菌）（抑菌）

• Process of inhibiting the growth Process of inhibiting the growth of microorganisms, of microorganisms, in vivoin vivo (mostly)(mostly) or or in vitroin vitro..

For example: bacteriostatic For example: bacteriostatic antibioticsantibiotics

Antisepsis Antisepsis （防腐）（防腐）• Process of inhibiting or preventing Process of inhibiting or preventing

growth of microbes, mostly growth of microbes, mostly in in vitrovitro and and not not bactericidal or bactericidal or sporicidal (sporicidal ( 杀芽胞的 杀芽胞的 ))

For example: use of chemical agents For example: use of chemical agents on skin, other living tissues or on skin, other living tissues or food/beverage.food/beverage.

AsepsisAsepsis （无菌）（无菌）

• A state where no living A state where no living microorganism exists. microorganism exists.

For example: OR (Operating Room)For example: OR (Operating Room)

Controlling Microorganisms Controlling Microorganisms with Physical Conditions with Physical Conditions

• High High TemperatureTemperature (heat) (heat)

• RadiationRadiation • Ultrasound Ultrasound

• FiltrationFiltration

• Low Temperature Low Temperature

• DesiccationDesiccation （干燥） （干燥）

High High Temperature Temperature ————Dry heat and Moist heat protein

denaturation and clotting; DNA strand breakdown

static action cidal action .

Dry heatDry heat ：： protein oxidationprotein oxidation

• IncinerationIncineration （焚烧）（焚烧）– most thorough (>500℃)most thorough (>500℃)– disposals and corpesdisposals and corpes

• Flaming (burner)Flaming (burner) （烧灼）（烧灼） – test tube opening, transferring looptest tube opening, transferring loop

• Hot air sterilization/BakingHot air sterilization/Baking （干烤）（干烤）– 160-170℃, 2h160-170℃, 2h– Glassware, china, syringes, needles, etcGlassware, china, syringes, needles, etc

• Infrared heat: similar to bakingInfrared heat: similar to baking

Moist heatMoist heat ：： denaturing proteins and melt lipidsdenaturing proteins and melt lipids

• AutoclavingAutoclaving （高压蒸汽灭菌）（高压蒸汽灭菌）– Most commonly used and effectiveMost commonly used and effective– 121℃ (103121℃ (103.4kPa).4kPa),, 15- 15-20min20min– killing both vegetative organisms and killing both vegetative organisms and

endospores endospores

• BoilingBoiling– 100 ℃ (105℃ with 2% Na100 ℃ (105℃ with 2% Na22COCO22) , 15-20min) , 15-20min

– cidal for vegetative cells but not necessarily cidal for vegetative cells but not necessarily sporesspores

• Regular Steam (Arnold Sterilizer)Regular Steam (Arnold Sterilizer)– 100 ℃, 15-20min100 ℃, 15-20min– cidal for vegetative cells but not necessarily cidal for vegetative cells but not necessarily

sporesspores

• PasteurizationPasteurization （巴氏消毒法）（巴氏消毒法）– to kill pathogens in readily perishable to kill pathogens in readily perishable

objects (objects (milkmilk, , winewine))– flash method (flash method ( 瞬间法瞬间法 ): 71.6℃, 15s ): 71.6℃, 15s – holding method (holding method ( 持续法持续法 ): 62.9℃, 30 min): 62.9℃, 30 min

• Fractional sterilizationFractional sterilization （间歇蒸气灭菌法）（间歇蒸气灭菌法）– alternating exposure and cooling time for a alternating exposure and cooling time for a

consecutive period:consecutive period:Steam heating (100 , 30 min)℃Steam heating (100 , 30 min)℃ 30 for ℃30 for ℃

endospores to endospores to germinategerminate 100 , 30 min to kill ℃100 , 30 min to kill ℃germinated endospores germinated endospores 30-37 overnight for ℃30-37 overnight for ℃remaining endospores to remaining endospores to germinategerminate 100 , 60 min to ℃100 , 60 min to ℃kill last remaining kill last remaining germinated endosporesgerminated endospores

– for sugar- or milk-containing culture media for sugar- or milk-containing culture media

Moist Heat Moist Heat vsvs Dry Dry HeatHeat

Moist heat Dry heatMoist heat Dry heat

Penetrating potencyPenetrating potency higher higher lowerlower

Temp for protein clottingTemp for protein clotting lower lower higherhigher

Extra heat releasedExtra heat released yes yes nono

from condensationfrom condensation

Sterilizing potency: Moist heat >> Dry heatSterilizing potency: Moist heat >> Dry heat

RadiationRadiation • Ultraviolet (UV) radiationUltraviolet (UV) radiation

– mechanism: blockage of DNA replication by mechanism: blockage of DNA replication by forming forming thymidine dimmersthymidine dimmers

– microbicidal activity of UV depends on:microbicidal activity of UV depends on:• length of exposure length of exposure

• wavelength: wavelength: 2200-300 nm, with the best effect of 00-300 nm, with the best effect of 265-266nm265-266nm

• bulb life (4000hr)bulb life (4000hr)

– very poor penetrating powervery poor penetrating power– for air or surface disinfectionfor air or surface disinfection

(OR, ID ward, labs) (OR, ID ward, labs) – causing eye damage, burnscausing eye damage, burns

and mutation in skin cells and mutation in skin cells

• Ionizing RadiationIonizing Radiation （电离辐射）（电离辐射）– X-rays, gamma rays and high-speed X-rays, gamma rays and high-speed

electrons electrons – generating more energy and penetrating generating more energy and penetrating

power than UVpower than UV– to sterilize pharmaceuticals, disposable to sterilize pharmaceuticals, disposable

medical supplies (e.g., syringes, gloves, medical supplies (e.g., syringes, gloves, catheterscatheters, , sutures) and foodssutures) and foods

• Microwave Microwave – penetrating penetrating non-metalnon-metal materials (glass, materials (glass,

plastics, china)plastics, china)

• UltrasoundUltrasound– more effective for gram-negative bacteriamore effective for gram-negative bacteria– Lack of thoroughness Lack of thoroughness ———— survivors survivors

remainremain

• FiltrationFiltration – sterilize heat- or chemical-sensitive sterilize heat- or chemical-sensitive

solutionssolutions– not effective for virus, ricketia, not effective for virus, ricketia,

mycoplasmamycoplasma

Seitz filter

• DesiccationDesiccation– static effect bystatic effect by inhibiting microbial inhibiting microbial

enzymesenzymes– not effective against endosporesnot effective against endospores– mainly for food reservation mainly for food reservation

• Low Temperature (-20℃ Low Temperature (-20℃ ～ ～ -70℃)-70℃)– inhibits microbial growth by slowing inhibits microbial growth by slowing

down microbial metabolismdown microbial metabolism– a special form: lyophalization (freeze-a special form: lyophalization (freeze-

drying), used for long-term (years) drying), used for long-term (years) reservation of bacteria stocks reservation of bacteria stocks • fast freezing + dryingfast freezing + drying

• protecting agents (glycerol, serum)protecting agents (glycerol, serum)

Control Microorganisms with Control Microorganisms with Chemical Agents Chemical Agents

(Disinfectants and Antiseptics)(Disinfectants and Antiseptics)

Antimicrobial modes of action of Antimicrobial modes of action of disinfectants and antisepticsdisinfectants and antiseptics

• Denaturation of bacterial proteins by Denaturation of bacterial proteins by disrupting disrupting hydrogen and disulfide bondshydrogen and disulfide bonds—— —— phenol (high conc.), alcohol, heavy-metal (high phenol (high conc.), alcohol, heavy-metal (high conc.), conc.), acids, alkalies, aldehydes)acids, alkalies, aldehydes)

• Damage to bacterial membrane (lipids and/or Damage to bacterial membrane (lipids and/or proteins), causing leakage of intracellular proteins), causing leakage of intracellular moleculesmolecules—— —— phenol (low conc.), surfactants, dyesphenol (low conc.), surfactants, dyes

• Interference of bacterial enzyme and metabolismInterference of bacterial enzyme and metabolism—— —— oxidants, heavy-metals (low conc.), alkylating oxidants, heavy-metals (low conc.), alkylating agentsagents

• Phenol and phenol derivativesPhenol and phenol derivatives ———— altering membrane permeability and altering membrane permeability and

denaturing denaturing proteinsproteins

•0.01% - 0.05% Chlorhexidine0.01% - 0.05% Chlorhexidine （洗必泰）（洗必泰）—— —— vaginal wash, OR hand-washvaginal wash, OR hand-wash

•3% - 5% carbonic acid 3% - 5% carbonic acid oror 2% Lysol 2% Lysol —— —— floor floor or surface disinfectionor surface disinfection

• AlcoholsAlcohols —— —— denaturing bacterial proteins and denaturing bacterial proteins and

membranesmembranes

•70% 70% - 75% ethyl or isopropyl alcohol- 75% ethyl or isopropyl alcohol （乙醇或异（乙醇或异丙醇）丙醇）—— —— skin and thermometer disinfectionskin and thermometer disinfection

– ineffective against endospores and non-ineffective against endospores and non-enveloped viruses enveloped viruses

• Heavy metals (Heavy metals (HgHg2+2+ 、、 AgAg++))

—— —— denaturing proteins and denaturing proteins and inactivating enzymesinactivating enzymes

•2% mercurochrome2% mercurochrome （红汞）（红汞） or 0.1% merthiolateor 0.1% merthiolate（硫柳汞）（硫柳汞）—— —— skin, mucosa and woundskin, mucosa and wound

– bacteriostatic, ineffective against endosporesbacteriostatic, ineffective against endospores

•1% silver nitrate1% silver nitrate （硝酸银）（硝酸银）—— —— eye drops for eye drops for newborns to prevent gonococcal ophthalmianewborns to prevent gonococcal ophthalmia（淋菌性眼炎）（淋菌性眼炎）

• OxidantsOxidants

—— —— oxidation, protein precipitationoxidation, protein precipitation

•0.1% potassium permanganate0.1% potassium permanganate （高锰酸钾）（高锰酸钾）—— —— skin, fruits/vegetablesskin, fruits/vegetables

•3% peroxide3% peroxide （过氧化氢）（过氧化氢）—— —— small trauma small trauma wound, skin, mucosawound, skin, mucosa

•0.2% - 1% peroxyacetic acid0.2% - 1% peroxyacetic acid （过氧乙酸）（过氧乙酸）—— —— plastics, glasswareplastics, glassware

•0.2 – 0.5 ppm cholorines —— 0.2 – 0.5 ppm cholorines —— water and water and swimming poolswimming pool

• SurfactantsSurfactants

—— —— damaging bacterial membranes, damaging bacterial membranes, inactivating inactivating enzymesenzymes, protein , protein precipitationprecipitation

•0.05-0.1% bromogeramine0.05-0.1% bromogeramine （新洁尔灭）（新洁尔灭）—— —— OR hand-wash, skin, surgical instruments OR hand-wash, skin, surgical instruments

• Alkylating agentAlkylating agent （烷化剂）（烷化剂）

—— —— alkylating proteins and nucleic acidsalkylating proteins and nucleic acids

•formalin (formaldehyde) formalin (formaldehyde) ———— surface surface disinfection, air, surgical instrumentsdisinfection, air, surgical instruments

•glutaric dialdehydeglutaric dialdehyde （戊二醛）（戊二醛）———— high-high-precision instruments, endoscopesprecision instruments, endoscopes

•50mg/L epoxy ethane50mg/L epoxy ethane （环氧乙烷）（环氧乙烷）———— surgical instruments and dressingsurgical instruments and dressing

• DyesDyes

—— —— inhibiting bacterial growth by inhibiting bacterial growth by interfering with oxidationinterfering with oxidation

•2% - 4% methyl violet2% - 4% methyl violet （龙胆紫）（龙胆紫）—— —— wound wound disinfection disinfection

• Acids and alkalies Acids and alkalies

—— —— destroying cell membrane and cell wall, destroying cell membrane and cell wall, denaturing proteinsdenaturing proteins

•5-10ml/m5-10ml/m3 3 acetic acid evaporation acetic acid evaporation ———— air air disinfectiondisinfection

•quicklime [Ca(OH)quicklime [Ca(OH)22] ] ———— floor and excretion floor and excretion (feces, urine, sputum, pus) disinfection(feces, urine, sputum, pus) disinfection

Effectiveness of antimicrobial agents Effectiveness of antimicrobial agents are affected by ——are affected by ——

• The concentration/intensity and nature of The concentration/intensity and nature of the disinfectant; the disinfectant;

• Length of exposure;Length of exposure;• Species and number of the microbe(s); Species and number of the microbe(s); • Temperature and humidity;Temperature and humidity;• Acidity (pH);Acidity (pH);• Presence of organic substances;Presence of organic substances;• Presence of chemical antagonistsPresence of chemical antagonists• The nature of the material bearing the The nature of the material bearing the

microbesmicrobes

Summary 1. Application of Summary 1. Application of chemical disinfectants chemical disinfectants

Patient excretionPatient excretion Chlorines, 5% carbonic acid, 2% Chlorines, 5% carbonic acid, 2% LysolLysol

Skin (hands)Skin (hands) 2% Lysol, 2% Lysol, 0.2-0.4% peroxyacetic acid0.2-0.4% peroxyacetic acid（过氧乙酸）（过氧乙酸） for HBVfor HBV, 70% ethyl , 70% ethyl

alcohol, 2% alcohol, 2% mercurochromemercurochrome （红汞）（红汞）

MucosaMucosa oraloral- 3% peroxide; - 3% peroxide; uri-reproductiveuri-reproductive- - 0.01-0.05% Chlorhexidine0.01-0.05% Chlorhexidine （洗必泰）（洗必泰） , , 0.1% potassium permanganate0.1% potassium permanganate （高锰酸（高锰酸钾）钾） ; ; newborn eyesnewborn eyes- 1% silver nitrate- 1% silver nitrate

Drinking waterDrinking water ChlorinesChlorines

Toilets, sewageToilets, sewage quicklime [Ca(OH)quicklime [Ca(OH)22]]

Air (OR, ID ward)Air (OR, ID ward) formalin steam (12.5-25ml/mformalin steam (12.5-25ml/m33,12-,12-24h),24h), formalin 40ml + potassium formalin 40ml + potassium permanganate permanganate 30g/m30g/m33;;

HBV ward- peroxyacetic acid 3g/mHBV ward- peroxyacetic acid 3g/m33 90min90min

Glassware, china,Glassware, china, 0.5% 0.5% iodophores, iodophores, 0.2-0.4% 0.2-0.4% peroxyacetic peroxyacetic

Rubber, metal Rubber, metal acidaciddevicesdevices

Potency DefinitionPotency Definition ExamplesExamples

High Killing all microbes including High Killing all microbes including glutaric dialdehydeglutaric dialdehyde （戊二醛），（戊二醛）， endospores and TBendospores and TB formaldehyde peroxyacetic acidformaldehyde peroxyacetic acid（过氧乙（过氧乙 酸），酸）， epoxy ethaneepoxy ethane （环氧乙烷）（环氧乙烷）

Medium Killing all non-spore microbesMedium Killing all non-spore microbes alcohol, chlorines, iodophoresalcohol, chlorines, iodophores including TBincluding TB

Low Killing vegetative bacteria Low Killing vegetative bacteria chlorhexidinechlorhexidine （洗必泰），（洗必泰）， and lipophilic (enveloped) and lipophilic (enveloped) bromogeraminebromogeramine （新洁尔灭）（新洁尔灭） viruses, but resisted by viruses, but resisted by

endospores, TB and hydrophilic endospores, TB and hydrophilic (non-enveloped) viruses(non-enveloped) viruses

Summary 2. Potency levels of Summary 2. Potency levels of chemical disinfectantschemical disinfectants

Spore-killing disinfectinsSpore-killing disinfectins

– glutaric dialdehydeglutaric dialdehyde （（戊二醛）戊二醛） , , formaldehydeformaldehyde （（甲醛甲醛 ), ),

Iodines, HIodines, H22OO22, , epoxy ethaneepoxy ethane （（环氧乙烷）环氧乙烷）

Non spore-killing disinfectinsNon spore-killing disinfectins

– alcohols, phenols, alcohols, phenols, chlorhexidinechlorhexidine （洗必泰）（洗必泰） , ,

bromogeraminebromogeramine （新洁尔灭）（新洁尔灭）

Summary 3. Spore-killing effects Summary 3. Spore-killing effects of chemical disinfectantsof chemical disinfectants

Disinfection and Disinfection and SterilizationSterilization

——For the course of Medical Microbiology for MBBS foreign students, Class 2006/2011, SYSU

September 18, 2007

Mengfeng Li （黎孟枫） , M.D. Department of Microbiology, Zhongshan School of

Medicine, SYSU, Guangzhou, China

limf@mail.sysu.edu.cn

Medical Microbiology Medical Microbiology