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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