STERILIZATION TECHNIQUES

‘NEW STERILIZATION METHODS’

Presented by:

NAIR RAHUL RAGHAVAN

1st yr M.Pharm (Pharmaceutics), SRIPMS

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Contents

What is sterilization?

Methods of sterilizations(traditional methods)

New sterilization methods

Conclusion

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What is sterilization?

Sterilization can be defined as any process that

effectively kills or eliminates transmissible agents

(such as fungi, bacteria, viruses and prions) from

a surface, equipment, foods, medications, or

biological culture medium.

DISINFECTION:

Disinfection describes a process that eliminates

many or all pathogenic microorganisms, except

bacterial endospores

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PHYSICAL METHODS

HEAT STERILIZATION:

-Heat sterilization is the most widely used and reliable method of

sterilization, involving destruction of enzymes and other essential cell

constituents

i) Dry Heat (160-180˚C): Oxidative changes occur.

Sterilization for thermostable products, moisture sensitive materials

ii) Moist heat (121-134˚C): Hydrolysis & denaturation occurs

Sterilization is used for moisture-resistant materials5

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The efficiency with which heat is able to inactivate

microorganisms is dependent upon

>the degree of heat, the exposure time and

the presence of water.

The action of heat will be due to induction of lethal

chemical events mediated through the action of water

and oxygen.

In the presence of water much lower temperature

time exposures are required to kill microbe than in the

absence of water.

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Thermal methods includes:

i) Dry Heat Sterilization

Ex:1. Incineration

2. Red heat

3. Flaming

4. Hot air oven

ii) Moist Heat Sterilization

Ex:1.Dry saturated steam – Autoclaving

2. Boiling water/ steam at atmospheric pressure

3. Hot water below boiling point7

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Dry heat sterilization Temp range of 160-180˚C & exposure time up to 2 hrs

Good penetrability & non-corrosive nature: useful for sterilizing glass wares & metal surgical instruments. Also used for sterilizing non-aqueous thermostable liquids and thermostable powders.

Dry heat destroys bacterial endotoxins (or pyrogens): applicable for sterilizing glass bottles which are to be filled

aseptically

Application: Sterilization of dry powdered

drugs, Suspensions of drug in non aq. solvents,

Oils, fats, waxes, soft & hard paraffin,

Oily injections, implants, ophthalmic ointments

& ointment bases etc 8

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HOT AIR OVEN

Moist heat sterilization

Moist heat sterilization involves the

use of steam in the range of 121-

134˚C. Steam under pressure(upto

15lbs) is used to generate high

temperature needed for sterilization.

Saturated steam acts as an effective

sterilizing agent

Application: Sterilization of surgical

dressings & instruments, containers,

closures, medical devices, culture

media, etc

AUTOCLAVE

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Radiation sterilization

EMR e.g. Gamma rays and UV light &

Particulate radiation (e.g. Accelerated electrons).

Major target for these radiation is Microbial DNA.

Use: Sterilization of heat & moisture sensitive products.

Gamma-rays (from Cobalt 60) are used to sterilize antibiotic,

hormones, sutures, plastics and catheters etc.

UV light: Has Lower energy & poor penetrability

Surface sterilization of aseptic work areas; for treatment of

manufacturing grade water; but is not suitable for sterilization of

pharmaceutical dosage forms.10

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Filtration sterilization

Does not destroy but removes the microorganisms

Used for clarification and sterilization of liquids and gases

The major mechanisms of filtration are Sieving, Adsorption

and Trapping within the matrix of the filter material

Ex: HEPA FILTERS

Used in the treatment of heat sensitive injections &

ophthalmic solutions, biological pdts & air & other gases for

supply to aseptic areas.

Used in industry as part of the venting systems on

fermentors, centrifuges, autoclaves and freeze driers.

Membrane filters are used for sterility testing11

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2 types of filters used:

(a)Depth filters: fibrous or granular materials so packed as

to form twisted channels of minute dimensions. Made of

diatomite, porcelain, sintered glass or asbestos.

(b)Membrane filters: Porous membrane 0.1 mm thick,

made of cellulose acetate, cellulose nitrate,

polycarbonate, etc.

Membranes are supported on a frame and held in special

holders. Fluids are made to transverse membranes by

positive or negative pressure or by centrifugation.

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CHEMICAL METHODS

GASEOUS STERILIZATION

-Gases like formaldehyde & ethylene oxide have biocidal activity

–MOA: Alkylations of sulphydryl, amino, hydroxyl and carboxyl groups on

proteins & amino gps of nucleic acids.

-Conc. Range 800- 1200 mg/L for ethylene oxide & 15-100 mg/L for

formaldehyde with operating temperatures of 45-63°C & 70-75°C

respectively.

-Sterilizing hormones, proteins, various heat sensitive drugs etc.

-Demerits: These gases are potentially mutagenic & carcinogenic. They

also produce acute toxicity including irritation of the skin, conjunctiva and

nasal mucosa13

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LIQUID STERILIZATION

Peracetic acid is sporicidal at low concentrations.

Merits: Water soluble & leaves no residue after rinsing. Has no

harmful health or environmental effects.

MOA: Disrupts bonds in proteins and enzymes and may also

interfere with cell membrane transportation through the

rupture of cell walls and may oxidize essential enzymes and

impair vital biochemical pathways.

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RECENT ADVANCES IN STERILIZATION

Sterilization, as a specific discipline, has been with us for

approximately 120 years, since the invention of the steam autoclave

by Charles Chamberland in 1879

We have seen progressive refinement in steam sterilizers: from the

early, manually operated equipment to modern microprocessor-

controlled, automatic machines.

Although the efficiency, reliability, and performance monitoring of

modern equipment is continually improving, the fundamental

process remains essentially the same.

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NEW STERILISATION METHODSChemical methods Physical methods Physicochemical

methods

Synergistic methods

-Surfacine -Pulsed light

sterilization

-Gas plasma

sterilization

-Psoralen and UVA

-Superoxidized

water

-Ultra high pressure

sterilization

-Ultrasound and

bactericide

-Chlorine dioxide -Hydroclave

-Glutaraldehye

-Ortho

phthalaldehyde

-Endoclens

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SURFACINE

Surfacine is a surface coating that kills microorganism on

contact, by selectively delivering Silver.

Used on animate or inanimate surfaces.

Despite the fact that silver ions possess antimicrobial efficacy

equal to or greater than other heavy metals ions, but is almost

non toxic to mammals.

Silver has a broad spectrum of activity (bacteria, yeast and fungi)

CHEMICAL METHODS OF STERILIZATION

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Mechanism of SURFACINE

It incorporates silver iodide in a surface-immobilized coating

(a modified polyhexamethylenebiguanide) that is capable

of chemical recognition and interaction with the lipid

bilayer of the bacterial cell membrane by electrostatic

attraction.

The intimate microbial contact with the surface results in

transfer of silver directly from the coating to the organism.

Microorganisms contacting the coating accumulate silver

until the toxicity threshold is exceeded; dead

microorganisms eventually lyse and detach from the surface.

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A 3-D polymeric network is immobilized onto the

substrate surface

The immobilized surface network is impregnated

with sub-micron particles of silver iodide that

result in the formation of AgCl-polymer complex

Solubility characteristics of this complex prevent

ionic silver from leaching into solutions contacting

the surface coating

Silver is available, however, to react with

bacterial cells contacting the coating

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Silver is preferentially transferred directly to

the microorganism causing a toxic accumulation

that results in cell death.

The silver halide reservoir within the polymeric

network replenishes the coating surface with

silver, allowing the coating to maintain high

surface anti microbial activity to microorganisms

that contact it for further challenges

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Advantages

Very low leaching of silver into solutions

Duration of activity: Long term

Reservoir capacity: High

Non toxic to humans

Disadvantage

Not active against viruses and very less activity against spores.

Applications:

Can be used in:

Medical devices: prostheses, catheters, endotracheal tubes etc

Dental care products

Food preparation & packaging

Water storage, treatment and delivery systems 21

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SUPEROXIDIZED WATER (STERILOX/MEDILOX)

Broad spectrum disinfectant, introduced recently.

Prepared by electrolyzing saline solution with titanium-

coated electrodes at 9 amp

The main products are Hypochlorous acid[HClO] as

well as free chlorine radicals & superoxide radicals

The product generated has a pH of 5.0-6.5 and an redox

potential of >950 mV.

MOA is not clear but probably relates to a mixture of

oxidizing species.

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Applications:

Freshly generated superoxidized water is rapidly effective (<2 minutes) against microorganisms (Mycobacterium tuberculosis, M. chelonae, poliovirus, HIV, MRSA, E.coli, Candida albicans, Enterococcus faecalis, Pseudomonas aeruginosa)

Advantages:

Basic materials i.e saline and electricity, are cheap & the end product (water) is not damaging to the environment.

Nontoxic to Humans

Noncorrosive and nondamaging to endoscopes

Disadvantage:

Equipment used to produce the product may be expensive because parameters such as pH, current, and redox potential must be closely monitored. 23

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CHLORINE DIOXIDE(ClO2)

Prepared by reacting Hypochlorus acid and sodium or potassium

chlorate.

MOA

It is a molecular free radical and disinfects by oxidation

Organic materials in bacterial cells react with chlorine dioxide,

causing several cellular processes to be interrupted

Chlorine dioxide reacts directly with amino acids and the RNA in the

cell ->proteins synthesis is blocked-> cell death

Application:

-Drinking water disinfection

-Can be used against anthrax (ClO2 is effective against spore forming

bacteria)

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Advantages:

Alternative to chlorine- Better disinfectant activity than chlorine. Deactivates chlorine resistant Giardia and Cryptosporidium

No odour nuisance, unlike chlorine.

Unlike chlorine, prevents formation of harmful halogenated disinfection by-products.

Low contact time required.

Disadvantages:

Explosive

Safety equipment must be used while handling as it causes irritation, watery eyes

Highly unstable when in contact with sunlight.

More expensive than chlorine. 25

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GLUTARALDEHYDE

Trade names: Cidex, Sonacide, Sporicidin, Hospex.

Cold sterilant- used to sterilize a variety of heat sensitive instruments

viz. endoscopes, bronchoscopes, dialysis equipment, anesthesia &

respiratory equipment, transducers & spirometry apparatus

Concentration: 2-3% -is active against a wide range of microorganisms:

Gram positive & negative, mycobacteria & spores

MOA:

Causes Alkylation of sulfhydryl, hydroxyl, carboxyl, and amino groups of

microorganisms ->alters RNA, DNA, and protein synthesis.

Aqueous solution of glutaraldehyde is not sporicidal. Only when it is

activated(made alkaline) using alkylating agents (pH 7.5-8.5) does the

solution become sporicidal.26

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Spectrum of activity:

Active against MRSA(methicillin resistant Staphylococcus aureus),

VRE(vancomycin resistant enterococci), Influenza A virus, E.coli,

Salmonella typhi, P.aeruginosa, Klebsiella, Avian rotavirus.

Inactive against Mycobacterium chelonae, M.avium-intracelulare,

M.xenopi, fungal ascospores, Cryptosporidium

Engineering controls:

The goal of engineering controls is to keep the vapours from

entering the work room & the employee’s breathing zone by

containing & removing at the source of release.

General room ventilation

Local exhaust hoods

Transfer procedures 27

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Use and handling:

While transferring, pour the liquids carefully and minimize

splashing and agitation

Rinse soaked instruments under running water.

Use adequate ventilation

Use protective equipments: Gloves, Glasses, gas masks, etc

Transportation and storage:

Should be done in closed containers wih tight fitting lids to

minimize potential for spills

Store in a cool, secured and properly labelled area

Dispose off outdated solutions properly

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Advantages:

Biodegradable

Non carcinogenic (unlike formaldehyde)

Non corrosive to metals, rubbers, plastics

Relatively inexpensive

Excellent material compatibility

Disadvantages:

Side effects due to glutaraldehyde vapours: Respiratory and dermal irritant, occupational asthma, itching of eyes, rhinitis.

Pungent & irritating odour

Slower mycobacterial activity as compared to OPA

Exposure to vapours should be monitored

Protective equipment to be used during handling and transfer of gas.29

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ORTHO-PHTHALDEHYDE(OPA)OPA received clearance by FDA in Oct. 1999.

MOA – similar to glutaraldehyde

Advantages:

Potential advantages compared with glutaraldehyde:

Active against glutaraldehyde resistant Mycobacterium

Non-irritant to the eyes and nasal passages

Has excellent stability over a wide range of pH (pH 3-9)

Does not require exposure monitoring, and has a barely perceptible

odour.

Like glutaraldehyde, OPA has excellent material compatibility. 30

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Disadvantages:

OPA stains proteins gray (including unprotected skin) and

thus must be handled with caution (i.e., use of gloves, eye

protection, fluid-resistant gowns when handling contaminated

instruments, contaminated equipment, and chemicals).

Limited clinical studies of OPA are available.

Disposal:

If OPA disposal in the sanitary sewer is restricted, glycine

(25 g/gallon) can be used to neutralize the OPA and make

it safe for disposal.

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ENDOCLENS Used for sterilization of flexible endoscopes

Consists of a computer-controlled endoscope-reprocessing machine that uses performic acid as a sterilant. The sterilant is produced by automatic mixing of the two component solutions of hydrogen peroxide and formic acid

The system's major features are:

an automatic cleaning process

capability to process two flexible scopes asynchronously

filter water rinsing and scope drying after sterilization

hard-copy documentation of key process parameters

user-friendly machine interface

total cycle time (scope testing, washing, sterilization, and drying) is less than 30 minutes.

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PHYSICAL METHODS OF STERILIZATION

PULSED LIGHT STERILIZATION

It is a nonthermal method for sterilization that involves the use of

intense, short duration pulses of a broad spectrum to ensure

microbial decontamination.

MOA:

It appears that both the visible and infrared regions, combined

with the high peak power of pulsed light, contribute to killing

microorganisms.

Various mechanisms have been proposed to explain the lethal

effect of pulsed light, all of them related to the UV part of the

spectrum and its photochemical and(or) photo-thermal effect.33

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Photo chemical mechanism

Primary target of pulsed light is nucleic acids because DNA is a target

molecule for these UV wavelengths.

The germicidal effect of UV light has been attributed primarily to a

photo-chemical transformation of pyrimidine bases in the DNA of

bacteria ,viruses ,and other pathogens to form dimers.

Formation of such bonds prevents DNA unzipping for replication & the

organism becomes incapable of reproduction. Without sufficient repair

mechanisms , such damage results in mutations, impaired replication

and genetranscription, and ultimately the death of the organism.

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Photo thermal mechanism:

The lethal action of pulsed light also can be due to a photothermal effect.

Disinfection is achieved through bacterial disruption during their temporary

over heating resulting from the absorption of all UV light from a flashlamp.

This over heating can be attributed to a difference in UV light absorption

by bacteria and that of a surrounding medium.

The water content of bacteria is vaporized, that induces membrane

disruption.

Types of damage induced by pulsed UV light are:

Photolysis

Loss of colony-forming ability

Inability to support phage growth (enzyme inactivation)

Destruction of nucleic acid. 35

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CLARANOR STERILIZATION EQUIPMENT:

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Operation:

The pulse is produced in 2 steps:

1. A 20 kV pulse lasting a few nanoseconds makes the xenon-filled

lamp conductive while creating an electric arc in the lamp

2. The capacitor charged at 3000 V discharges in this arc during a

300 microsecond pulse, which ionizes the gas in the lamp and

generates a plasma emitting a very high intensity white light

(20,000 times greater than sunlight on the earth's surface)

The energy of the lamp, which is delivered over a very short period

(0.3 ms), produces a power of 1 MW, half of which is dissipated in

heat and the other half in optical energy.37

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Advantages:

Total DNA destruction

Quick process

No chemicals used.

Worker-friendly (safe and easy to use)

Minimum space requirements.

Water circulated for cooling of lamps can be recycled.

Disadvantages:

Pulsed light is a surface treatment. The decontaminated areas are those

which receive the light pulse

Applications:

Sterilization of caps, cups, lids and other packaging materials

Sterilization of food packaging

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HYDROCLAVE The Hydroclave is essentially a double-walled

(jacketed) cylindrical, pressurized vessel, horizontally mounted, with one or more side or top loading doors, and a smaller unloading door at the bottom.

CHARACTERISTICS:

Sterilizes the waste utilizing steam, similar to an autoclave, but with much faster & much more even heat penetration

Hydrolyses the organic components of the waste such as pathological material.

Removes the water content (dehydrates) the waste.

Breaks up the waste into small pieces of fragmented material.

Reduces the waste substantially in weight and volume

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STEPS INVOLVED IN THE TREATMENT CYCLE

a) Loading

b) Heat-up and fragmentation

c) Sterilization period

d) De-pressurization and De-hydration

e) Unloading

a) Loading

In smaller units, materials to be sterilized are packed &

loaded manually

In large sized units, a combination of conveyors, hoppers

and tippers are available to load the waste into large top

loading doors.40

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b) Heat-up and fragmentation

After loading, the vessel doors are closed, and the outer jacket of

the vessel is filled with high temperature steam, which acts as an

indirect heating medium for heating the waste

During heat-up, the shaft and mixing arms rotate, causing the

waste to be fragmented & continuously tumbled against the hot

vessel walls.

The moisture content of the waste will turn to steam & the vessel

will start to pressurize.

At the end of this period, the correct sterilization temperature and

pressure are reached, and the sterilization period is initiated

automatically

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c) Sterilization period

By computer, the temperature & pressure are maintained

for the desired time to achieve sterilization.

The mixing/fragmenting arms continue to rotate during

the entire sterilization period, to ensure thorough heat

penetration into each waste particle.

Sterilization time of 15 minutes at 132 C, or 30 minutes at 121 C

d) De-pressurization and De-hydration

After sterilization period ends, the vessel is de-pressurized via a steam condenser, which causes initial waste dehydration due to depressurization.

The steam to the jacket will remain on, agitation continues, and the waste loses its remaining water content through a combination of heat input from the jacket and continued agitation.

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e) Unloading

Due to the unique construction of the mixing arms, the opposite rotation causes the fragmented waste to be pushed out of the vessel discharge door, into a waste container, or onto a conveyor.

The vessel is now ready for another treatment cycle, having retained most of its heat for the treatment of the next batch.

Uses:

The Hydroclave can sterilize:

Bagged waste, in ordinary bags

Sharps containers and needles

Liquid containers

Cardboard containers

Metal objects

Plastics43

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Advantages:

Performance

Guaranteed high level of sterilization, including wet waste, metals, liquids and sharps.

Automatic operation, and not dependent on operator skill for sterility.

No infectious or harmful emissions.

Economic

Low operating cost with low energy consumption.

Low maintenance costs.

No costly bags, filters or chemicals in the process.

Very large weight and volume reduction of the waste.

Dry waste, regardless of its original water content.

Low odour, due to the dryness. 44

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Hydroclave Autoclave

•Low operating cost by recycling

steam.

•No special bags required

•Treats wet or liquid loads easily

•Strong weight reduction

•Strong volume reduction

•Consistent high sterility

•Higher operating cost, no steam

recycling

•High temp. bags required

•Cannot treat wet or liquid loads

•Weight increase

•No volume reduction

•Spotty sterility

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Also called High hydrostatic pressure sterilization(HHP)/

Pascalization. Mainly used in food sterilization technology

It is a cold sterilization technique by which products, already sealed

in its final package, are introduced into a vessel and subjected to a

high level of isostatic pressure (200–600MPa/43,500-87,000psi)

transmitted by water, for few seconds to few minutes.

Pressures > 400 MPa at cold

(+ 4ºC to 10ºC)inactivate vegetative

flora(bacteria, virus, yeasts, moulds

& parasites) present in food,

extending its shelf life

ULRA HIGH PRESSURE (UHP) STERILIZATION

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MOA:

• Under UHP the volume of the pdt is compressed, results in the deeper

penetration of proteins & other macromolecules into the pdt,

resulting in the destruction of its 3-D structure

• Thus the food's proteins are denatured, hydrogen bonds are fortified,

& noncovalent bonds in the food are disrupted, while the product's

main structure remains intact -> causes starch and protein

denaturation and enzyme inactivation, all of which may have an

effect on the texture of the product. Pressure is instantly and evenly

applied to the product, regardless of its size, shape and volume.

• Because UHP sterilization is not heat-based, covalent bonds are not

affected, causing no change in the food's taste

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Advantages:

Being a cold sterilization the characteristics of the fresh product are

retained, nutritional properties, flavor and taste remain almost intact.

Destroys pathogens (Listeria, Salmonella, Vibrio, Norovirus, etc.)

Extends product shelf life: improved customer satisfaction.

Avoids or reduces the need for food preservatives: Clean label foods

(Natural/Additive Free).

Only needs water (which is recycled) & lower electricity consumption:

Environment friendly.

Disadvantages:

For the inactivation of bacterial endospores require synergistic action

of very high pressure(>600MPa) & temperature(>60*C)48

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PHYSICOCHEMICAL METHODS OF STERILIZATION

GAS PLASMA STERILIZATION

Discovered by Sir William Crookes in 1879. This technology was

patented in 1987 and marketed in U.S in 1993

Plasma is defined as an ionized gas with an equal no. of +ve & -ve

ions. 4th state of matter. Its properties are similar to those of both

gases and liquids.

Gas plasma is generated in an enclosed

chamber under deep vacuum (low pressure)

using radiowaves or microwaves to excite

gas molecules (hydrogen peroxide) to

produce ionized gas particles49

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MOA: It operates synergistically via three mechanisms:

1. Free radicals interactions

2. UV/VUV radiative effects

3. Volatilization

UV/VUV radiation causes

-Formation of thymine dimers in DNA, inhibiting bacterial replication.

-Base damage

-Strand breaks

Volatilization: It is able to vaporize microbiological matter, causing physical destruction of spores.

Application: STERRAD System enable sterilization of surgical instruments,

rigid and flexible endoscopes, cameras, catheters,etc.

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Steps involved in plasma steriliztion:

1. The Vacuum Phase: Internal pressure is reduced by evacuating the chamber

2. The Injection Phase:

Liquid peroxide is injected into the chamber, evaporating the aqueous hydrogen

peroxide solution & dispersing it into the chamber, where it kills bacteria on any surface

it can reach

3. Diffusion phase:

The H202 vapor permeates the chamber, exposing all load surfaces to the Sterilant and

rapidly sterilizes devices and materials without leaving any toxic residues

4. The Plasma Phase

An electromagnetic field is created in which the H2O2 vapour breaks apart, producing a

low-temperature plasma cloud that contains ultraviolet light and free radicals.

5. The Vent Phase

The chamber is vented to equalize the pressure enabling the chamber door to be opened.

There is no need for aeration or cool-down. Devices are ready for immediate use.51

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Advantages:

The process is usually at room temperature and hence poses no dangers

associated with high temperatures (unlike autoclaves)

Byproducts are generally water and oxygen-harmless to the environment

Time of treatment is fast(1 min or less)

Disadvantages:

Weak penetrating power of the plasma. Complications arise in:

-Presence of organic residue

-Packaging material

-Complex geometries

-Bulk sterilization of many devices

High power consumption

Can corrode certain materials52

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SYNERGISTIC STERILIZATION METHODS

PUVA (Psoralen and UVA)

Psoralen is a coumarin derivative which occurs naturally in the seeds

of Psoralea corylifolia, as well as in the common fig, celery, parsley

and in all citrus fruits

MOA: Psoralen intercalates into DNA and on exposure to ultraviolet

(UVA) radiation can form monoadducts and covalent interstrand

cross-links (ICL) with thymine, resulting in apoptosis.

Uses:

-Sterilization of Blood plasma and platelets

-It is also active against viruses ex.HIV, hepatits, etc

-Other uses of PUVA: Treatment of psoriasis, eczema, vitiligo53

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ULTRASOUND AND BACTERICIDE:

Low(20kHz) or High(250kHz) frequency ultrasound energy is

synergistic with low concentration Glutaraldehyde

This technique is effective in killing bacterial endospores.

Advantages:

-Heat sensitive materials can be sterilized(plastics and rubber

equipments)

-Inexpensive and quick sterilization is possible.

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CONCLUSION

We have seen the new methods used in sterilization, which have

been developed in the past few decades and currently in use in

the industries for sterilizing medical instruments, foods and

pharmaceuticals.

Most of these methods are automated and efficient, environment

friendly, have low running cost, can sterilize on a large scale,

highly effective against certain resistant microorganisms and

ensure complete sterility of products.

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REFERENCE The theory and practice of industrial pharmacy by Lachman and Lieberman

Gas Plasma Sterilization in Microbiology: Theory, Applications, Pitfalls and New Perspectives by

Hideharu Shintani, Akikazu Sakudo

Disinfection, sterilization and preservation by Seymour Stanton

Hospital sterilization by Anand Nagaraja Prem

www.Medscape.com

www.mddionline.com

Thank You…. 56

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