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

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

Z valueincrease in temperature required to reduce D by 1/10

Z = t1-t2 / logDt1-logDt2

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

Control of Microorganisms by Physical and Chemical Agents

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

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

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

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

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

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

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.

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)

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

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%)

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