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SterilizationConcepts• Sterilization – removal of all
living cells from an object,
Factors that affect cell deathTime, number of cells, type f i i th d
g j ,including viruses, spores, viroids and prions
• Aseptic technique – techniques with sterile solutions
of microorganism, method of killing, concentration or intensity, temperature, pH, presence of organic
• -static – bacteriostatic, fugistatic (growth-inhibiting)
• -cide – bactericide, algicide, fungicide (killing)
presence of organic material, biofilm, development of resistance
u g c de ( g)• Level of risk – probability of
sterility
Killing kineticsKilling kinetics• TDP – thermal death point: the
lowest temperature at which all the bacteria in a population will be killed within 10 minutesbe killed within 10 minutes
• TDT – thermal death time: the length of time before all microorganisms in a population will be killed at a certainwill be killed at a certain temperature
• D-value and Z-value
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Decimal reduction time (D or D value)time required to kill 90% of microorganisms or spores intime required to kill 90% of microorganisms or spores in
a sample at a specific temperatureN0
Nt=N0xekt
k, slope
t
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Z valueincrease in temperature required to reduce D by 1/10
Z = t1-t2 / logDt1-logDt2
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The Bacterial EndosporeThe Bacterial Endospore
formed b some bacteria• formed by some bacteria (Bacillus and Clostridium)
• normally formed when• normally formed when growth ceases because of lack of nutrients
• Dormant (no metabolism)
• resistant to numerous• resistant to numerous environmental conditions– heat– radiation– chemicals– desiccation
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Control of Microorganisms by Physical and Chemical Agents
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PasteurizationPasteurization• LTH low temperature holding• LTH, low temperature holding,
63oC, 30 min
• HTST, high temperature short ti 72oC 15time, 72oC, 15 sec
• UHT, ultra high temperature, 140-150oC, 1-3 sec
• controlled heating at temperatures well below boiling
• used for milk, beer and other beverages
d t t ili b t• process does not sterilize but does kill pathogens present and slow spoilage by reducing the total load of organisms present
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The Autoclave or Steam SterilizerThe Autoclave or Steam Sterilizer
m st be carried o t• must be carried out above 100oC which requires saturated steam under pressurep
• carried out using an autoclave
• effective against all types of microorganisms including spores
• degrades nucleic acids, denatures proteins, and disrupts membranes
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RadiationRadiationUltraviolet (UV) RadiationUltraviolet (UV) Radiation• limited to surface
sterilization because it does not penetrate glass, dirt films water and otherfilms, water, and other substances
• has been used for water treatment
Ionizing Radiation• penetrates deep into
objects• destroys bacterial
endospores; not always effective against viruse
• used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food
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Filtration• reduces microbial population or sterilizes solutions of heat-
sensitive materials by removing microorganisms• also used to reduce microbial populations in air
depth filters– thick fibrous or granular filters that remove
microorganisms by physical screening, entrapment, and/ormicroorganisms by physical screening, entrapment, and/or adsorption
membrane filters– porous membranes with defined pore sizes that remove
microorganisms primarily by physical screeningmicroorganisms primarily by physical screening
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Phenol• Causes lysis of cells, destroys the cell membrane and
denatures proteinsff• The effect increases with increasing temperature and
decreasing pH• Bactericide: 1-2% solution• Skin irritant, corrosive,• Modification of phenol with chlorine and methyl groups has
resulted in disinfectants with reduced skin irritation and corrosive properties and increased antimicrobial activity.
• The effect is impeded by organic materialThe effect is impeded by organic material.• Hexachlorophene: commercial disinfectant, antiseptic, the
antimicrobial effect persists for an extended time, e.g. when used to wash the skin
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Alcohol• Bactericidal and fungicidal but NOT sporicidal
Denatures proteins and destroys membranes (lipids)• Denatures proteins and destroys membranes (lipids)• Ethanol: 70% solution most effective• Isopropanol: 70% solution most effective
P ti ti b tit ti f l h l• Preservative, aromatic substitution of alcohol• Phenyl alcohol, eye drops, contact lens solutions• Benzyl alcohol, injection solutions
O i id (E d )• Organic acids: (European code no.)Sorbic acid (E200)Benzoic acid (E210)
d i f dused in foods
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Halogen compoundsHalogen compounds• Strongly oxidizing f fi l t• Strongly oxidizing• denatures proteins• bactericide, fungicide • Cl, chlorine gas
• any of five elements: fluorine, chlorine, bromine, iodine, and astatine
• iodine and chlorine areg• I, iodophors (a combination
of iodine and surfactants)
• 1 µg/ml is used to disinfect
• iodine and chlorine are important antimicrobial agents
• 1 µg/ml is used to disinfect water
• Mechanism:Cl2 + H2O HCl + HOCl
chloramines R - SO2NCl2(chlorophors) give off Cl slowly,e.g. halazone tablets for water purificationOrganic material reduces the effect.
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FormaldehydeFormaldehyde• highly reactive molecules, sporicidal• Mechanism: causes binding of amino groups
between proteins, cross-binds DNA/RNA, alkylating• Boiling point –19C
N t l i t t t 80 C• Note: polymerises at temperatures < 80C• Very effective, 3-10 mg/l is bactericidal, compared
with ethylene oxide 500-1000 mg/lPoor penetration usually used for surface• Poor penetration, usually used for surface disinfection
• Formalin, 37% formaldehyde in water + 10% methanol (to prevent polymerisation)methanol (to prevent polymerisation)
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Quaternary ammonium compounds
• detergents that have antimicrobial activity and are• detergents that have antimicrobial activity and are effective disinfectants– organic molecules with hydrophilic and
hydrophobic endshydrophobic ends– act as wetting agents and emulsifiers
• cationic detergents are effective disinfectants• Effective against G+ bacteriaEffective against G bacteria• Alkali solution improves the effect• Soap (anionic detergents) reduces the effect
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Ethylene oxideEthylene oxide• used to sterilize heat-sensitive materials, microbicidal and
sporicidalsporicidal• Boiling point 10.7C, Very good penetration• Used with CO2, freon etc. to avoid explosions, > 3% ethylene
oxide in air is explosive!M h i th l id lk l t h d l b l d• Mechanism: ethylene oxide alkylates hydroxyl, carbonyl and sulfhydryl amino groups of enzymes etc.
• Effect depends on – gas pressure g p– exposure time– temperature– type of microorganism or spore
i t t t ( ti 28 33%)– moisture content (optimum 28-33%)
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Heavy metalsHeavy metals
• e g ions of mercury • Silver nitrate (AgNO3)• e.g., ions of mercury, silver, arsenic, zinc, and copper
• effective but usually
• Silver nitrate (AgNO3) (1% solution) is sometimes used to disinfect the eyes of effective but usually
toxic• combine with and
inactivate proteins; may l i it t t i
newborn babies.
• Copper sulphate (C SO4) f i id dalso precipitate proteins
• Mechanism: react with sulfhydryl groups in proteins
(CuSO4) fungicide and algicide, agricultural uses
proteins