Sterilization and Desinfection

8/10/2019 Sterilization and Desinfection

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STERILIZATIONAND

DISINFECTION


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Why do we disinfect?

To protect :Lab workers

General publicThe environmentTo stop the spread of diseaseTo stop the release of recombinant or exoticorganisms into the environment.Our research


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STERILIZATION

Sterilization is the total destruction of all

microbes, including the more resilient forms suchas:- bacterial spores,- mycobacteria,

- nonenveloped viruses,- and fungi.


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DISINFECTION

Disinfection is the process of elimination ofmost pathogenic microorganisms (excludingbacterial spores) on inanimate objects.


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DECONTAMINATION

Decontamination is the process of removal ofcontaminating pathogenic microorganisms fromthe articles by a process of sterilization ordisinfection.


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DEFINITIONS

Sanitization is the process of chemical or mechanicalcleansing, applicable in public health systems. Usuallyused by the food industry. It reduces microbes on eatingutensils to safe, acceptable levels for public health.

Asepsis is the employment of techniques (such asusage of gloves, air filters, uv rays, etc) to achievemicrobe-free environment.

Antisepsis is the use of chemicals (antiseptics) to makeskin or mucus membranes devoid of pathogenic

microorganisms.


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DEFINITIONS

Bacteriostasis is a condition where the multiplication ofthe bacteria is inhibited without killing them.

Bactericidal is that chemical that can kill or inactivatebacteria. Such chemicals may be called variouslydepending on the spectrum of activity, such asbactericidal, virucidal, fungicidal, microbicidal, sporicidal,tuberculocidal or germicidal.

Antibiotics are substances produced by one microbethat inhibits or kills another microbe. Often the term isused more generally to include synthetic and semi-synthetic antimicrobial agents.


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Microbes in decreasing order of resistanceto germicidal chemicals


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DISINFECTION

Disinfectants are those chemicals that destroypathogenic bacteria excluding bacterial spores,

from inanimate surfaces.Those chemicals that can sterilize are calledchemisterilants .Those chemicals that can be safely applied overskin and mucus membranes are calledantiseptics .


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DISINFECTION

Disinfection can be accomplished with:1. Heat disinfection2. Chemical disinfection

Objects that can be disinfected are: bedpans,patient skin before operation and surgeonhands before putting gloves.


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HEAT DISINFECTION

It is accomplished by boiling water at atmosphericpressure for at least 5 mins .


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CHEMICAL DISINFECTION

Classification of disinfectants1. Based on consistency

a. Liquid (E.g., Alcohols, Phenols)

b. Gaseous (Formaldehyde vapor, Ethylene oxide)2. Based on spectrum of activity

a. High level

b. Intermediate level

c. Low level3. Based on mechanism of action

a. Action on membrane (E.g., Alcohol, Detergent)

b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)


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Chlorine-Containing Compounds

Bleach

Active ingredient = Sodium Hypochlorite (NaOCl) (5%)

1 g/l available chlorine = general lab decon (1:50)

5 g/l AC = stronger lab cleanup (1:10)

Laboratory decontamination recommendation is a 10% dilution

10% = 0.5% NaOCl = 5 g/l available chlorine

Unstable: degrades over time after opening and/or dilution

Make dilutions fresh daily

Reactive: Corrosive to metals (stainless)


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Alcohol (ethyl or isopropyl)

Broad spectrum antimicrobialVegetative bacteria (incl. mycobacteria)

Viruses

Fungi

Not effective against sporesNon- toxic (when used in moderation)

Most effective at a dilution of 70% in water

Causes membrane damage and rapid protein denaturation


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Phenolic Compounds

Some of the earliest used germicides

Active against vegetative bacteria and viruses

Can be used against mycobacteriaNot sporicidal

Membrane active induce leakage of intracellular components


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Quaternary Ammonia Compounds

Effective against vegetative bacteria and some lipid-containing

virusesMycobacteriostatic and sporostatic

Commonly used as low-level disinfectants or sanitizing agents

Active against microbial membranes


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Formaldehyde and Gluteraldehyde

Formaldehyde (Paraformaldehyde/Formalin)Effective against vegetative bacteria, mycobacteria, and spores

Used to decontaminate large equipment/spaces

Suspected carcinogen

GluteraldehydeBroad spectrum of activity: vegetative bacteria and spores; fungi,viruses

Considered safer than formaldehyde

Active against microbial membranes and cell walls


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Chemical disinfection

Number of different agents are used according totolerance of objects and infectious agents.

Using phenol with cleaning component destroy themembrane of microorganisms.Using 70% alcohol for skin which denatures proteinsof microorganisms.

Use soap containing hexachlorophene for hands.In case of hepatitis use 5% solution of chloraminesor heat disinfection


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TESTING OF DISINFECTANTS

Kochs method Spores of Bacillus anthracis were dried on silkthread and were subjected to action of disinfectants. Later it waswashed and transferred to solid medium.

Rideal Walker method This method relies on the estimation ofphenol coefficient . Phenol coefficient of a disinfectant is calculated bydividing the dilution of test disinfectant by the dilution of phenol thatdisinfects under predetermined conditions.

Chick Martin test This test also determines the phenol coefficient ofthe test disinfectant. Unlike in Rideal Walker method where the test iscarried out in water, the disinfectants are made to act in the presenceof yeast suspension (or 3% dried human feces).


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STERILIZATION

CLASSIFICATION OF METHODS

Physical Agents

1. Heat2. Radiation3. Filtration

Chemical Agents

In practice, certain methods are placed under sterilization which in fact donot fulfill the definition of sterilization such as boiling for 1/2 hr andpasteurization which will not kill spores.


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HEAT

Nature of heat Moist heat is more effective than dry heat.

Temperature and time are inversely proportional. As temperatureincreases the time taken decreases.

Number of microorganisms More the number of microorganisms, higherthe temperature or longer the duration required.

Nature of microorganism Depends on species and strain ofmicroorganism, sensitivity to heat may vary. Spores are highly resistant toheat.

Type of material Articles that are heavily contaminated require highertemperature or prolonged exposure. Certain heat sensitive articles must besterilized at lower temperature.

Presence of organic material Organic materials such as protein, sugars,oils and fats increase the time required.

Factors affecting sterilization by heat


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DRY HEAT

Red heat Articles such as bacteriological loops, straight wires, tips offorceps and searing spatulas are sterilized by holding them in Bunsenflame till they become red hot.


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DRY HEAT

Flaming This is a method of passing the article over a Bunsen flame.


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DRY HEAT

Incineration This is a method of destroying contaminated materialby burning them in incinerator. Articles such as soiled dressings;animal carcasses, pathological material and bedding etc should besubjected to incineration.


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Hot air oven

Sterilization cycle This takes into considerationthe time taken for the articles to reach thesterilizing temperature, maintenance of thesterilizing temperature for a defined period(holding time) and the time taken for the articles

to cool down. Different temperature-timerelations for holding time are:

60 min. at 160 C

40 min. at 170 C

20 min. at 180 C.

Increasing temperature by 10 degrees shortensthe sterilizing time by 50 %.

The hot air oven must not be opened until thetemperature inside has fallen below 60 C toprevent breakage of glasswares.


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Sterilization control

Physical Temperature chart recorder andthermocouple.

Chemical Brownes tube No.3 green spot color

changes from red to green. Dry Heat Indicator Labels (DHI Labels )

green color changes from green to dark-brown.

Biological : 10 6 spores of Bacillus subtilis var niger or Clostridium tetani on paperstrips are placed inside envelopes and thenplaced inside the hot air oven. Uponcompletion of sterilization cycle, the stripsare removed and inoculated intothioglycollate broth and incubated at 37 Cfor 3-5 days. Proper sterilization shouldkill the spores and there should not be

any growth.


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MOIST HEATAt temperature 100 C

Tyndallization

Steaming for aprox. 20 min. on 3 or 4 successive occasions, separatedby 24 hour intervals at room temperature.The incubation intervals permit any dormant, resistant endospores togerminate and become active.


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MOIST HEATAt temperature above 100 C

AutoclaveSterilization can be effectively achieved at a temperature

above 100 C using an autoclave.In an autoclave the water is boiled in a closed chamber. As thepressure rises, the boiling point of water also raises.

134

C 3.0 atm 5 min


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Sterilization control

Physical method includes automatic processcontrol, thermocouple and temperature chartrecorder.

Chemical method Bowie Dick tape is applied toarticles being autoclaved. If the process hasbeen satisfactory, dark brown stripes willappear across the tape.

Biological method Ampoule biological testsincludes a paper strip containing 10 6 spores ofGeobacillus stearothermophilus.


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RADIATION

Non-ionizing wavelength UV Radiation has a wavelength of 200-280nm; it has a germicidal effecton microorganisms.

Common uses: Surface disinfection in hospitals, operating theatre andlaboratories.

Sunlight The microbicidal activity of sunlight is mainly due to the presence of ultraviolet rays in it. It is responsible for spontaneous sterilization in natural conditions.


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RADIATION

Ionizing wavelength

Particulate (Electron beam)Common uses: sterilisation of instruments such as syringes, gloves,dressing packs, foods and pharmaceuticals. Electromagnetic (Gamma rays)

Common uses: sterilisation of disposable petri dishes, plasticsyringes, antibiotics, vitamins, hormones and fabrics.


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FILTRATION

Filtration does not kill microbes, it separates them out. Membranefilters with pore sizes between 0.2-0.45 m are commonly used toremove particles from solutions that can't be autoclaved.It is used to remove microbes from heat labile liquids such asserum, antibiotic solutions, sugar solutions, urea solution.Filtration is aided by using either positive or negative pressureusing vacuum pumps.The older filters made of earthenware or asbestos are called depthfilters.


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Types of filters

Earthenware filters These filters are made up of diatomaceous earth orporcelain. They are usually baked into the shape of candle.

Asbestos filters These filters are made from chrysotile type of asbestos,chemically composed of magnesium silicate. They are pressed to form disc,which are to be used only once. The disc is held inside a metal mount,which is sterilized by autoclaving.Sintered glass filters These are made from finely ground glass that arefused sufficiently to make small particles adhere to each other. They areusually available in the form of disc fused into a glass funnel.Membrane filters These filters are made from a variety of polymericmaterials such as cellulose nitrate, cellulose diacetate, polycarbonate andpolyester. The newer ones are composed of cellulose diacetate. Thesemembranes have a pore diameter ranging from 0.015 m to 12 m. Thesefilters are sterilized by autoclaving.Air Filters Air can be filtered using HEPA (High Efficiency Particle Air)filters. They are usually used in biological safety cabinets. HEPA filters are atleast 99.97% efficient for removing particles >0.3 m in diameter.


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Asepsis

Sterilization Disinfection

Heat RadiationChemical Heat Chemical

Steam

Dry heat

Gas

Liquid

Gamma Boiling water Phenol

Heat orchloramines solution

Hexachlorophene

70% AlcoholEthylene oxide

Formalin

GlutaricAldehyde


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Microbiological Waste Management

Biohazardous and infectious (or potentially infectious) agents areroutinely handled and manipulated in biomedical and animallaboratories.

Biohazards include human samples and tissue cultures, bacteria,viruses, prions, parasites, toxins, recombinant DNA (rDNA) - whichis used in expression vectors such as plasmids, infected ortransfected/transduced cells, infected human or animal tissues,transgenic animals, and any material contaminated with a

biohazardous agent.To prevent the escape of biohazards into the environment,waste from biohazardous operations must be treated and/ordisposed of in a special way.


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Microbiological Waste Management

Microbiological waste includes:Discarded cultures and stocks of infectious agents and

associated biologicalsDiscarded cultures of specimens from medical, clinical,commercial, and industrial laboratoriesDiscarded, used disposable culture dishes

Discarded, used disposable devices used to transfer,inoculate, and mix cultures

A t bl M th d f T t t


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Acceptable Methods of Treatmentof Microbiological Waste

Steam Sterilization1. temperature of at least 121C2. pressure of at least 2 atm.3. time of at least 30 minutes


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Identifying Biohazardous Waste

All biohazardous waste containersmust be labeled with a biohazard label

(red or red/orange labelwith the universal biohazard symbol and

the word BIOHAZARD underneath).


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Biohazard containers

Sterilization and Desinfection

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