INTRODUCTION:

Sterilization (or sterilisation) refers to any process that eliminates, removes, kills, or deactivates all forms of life and other biological agents (such as fungi, bacteria, viruses, present in a specified region, such as a surface, a volume of fluid, medication, or in a compound such as biological culture media.¹

Pharmaceutical Importance of Sterilization

— Moist heat sterilization is the most efficient biocidal agent. In the pharmaceutical industry it is used for: Surgical dressings, Sheets, Surgical and diagnostic equipment, Containers.

— Dry heat sterilization can only be used for thermo stable, moisture sensitive or moisture impermeable pharmaceutical and medicinal.

— These include products like; Dry powdered drugs, Suspensions of drug in non aqueous solvents, Oils, fats waxes, soft hard paraffin silicone, Oily injections, implants, ophthalmic ointments and ointment bases etc.

— Gaseous sterilization is used for sterilizing thermolabile substances

Like: hormones, proteins, various heat sensitive drugs etc.²

Methods of Sterilization:

The various methods of sterilization are:

1. Physical Method

a. Thermal (Heat) methods

b. Radiation method

c. Filtration method (Mechanical method)

2. Chemical Method

a. Gaseous method

b. Liquid Sterilization

1. Heat Sterilization:

Heat sterilization is the most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell.

This method of sterilization can be applied only to the thermostable products, but it can be used for moisture-sensitive materials- for which dry heat (160-180°C) sterilization us used, for moisture-resistant materials for which moist heat (121-134°C) sterilization is used.³¹

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.

Dry Heat Sterilization:

Dry heat sterilization is:

· Incineration

· Red heat

· Flaming

· Hot air oven

Incineration:

It is the process that involves the combustion of organic substances contained in waste materials. It is used for soil dressing, pathological bedding.

Red heat:

It is used for straight wires, bacterial loops and spatulas.

Flaming:

It is the process of heating over fire till they become red hot. It is used for bacterial loops, wires and spatulas.³

Hot-air oven

Dry heat sterilization is usually carried out in a hot air oven, which consists the following:

— An insulated chamber surrounded by an outer case containing electric heaters.

— A fan

— Shelves

— Temperature sensor

— Door locking controls.

— Exhaust

— Diffusion wall

— Airflow Damper

— Glass wool

— Insulation

Principle:

Sterilizing by dry heat is accomplished by conduction. The heat is absorbed by the outside surface of the item, and then passes towards the center of the item, layer by layer. The entire item will eventually reach the temperature required for sterilization to take place. Dry heat does most of the damage by oxidizing molecules. The essential cell constituents are destroyed and the organism dies. The temperature is maintained for almost an hour to kill the most difficult of the resistant spores.⁴

Procedure of operation:

— Articles to be sterilized are first wrapped or enclosed in containers of cardboard, paper or aluminum.

— Then, the materials are arranged to ensure uninterrupted air flow.

— Oven may be preheated for materials with poor heat conductivity.

— The temperature is allowed to fall to 40°C, prior to removal of sterilized material.

Advantages of dry heat sterilization

— A dry heat cabinet is easy to install and has relatively low operating costs

— It penetrates materials

— It is nontoxic and does not harm the environment

— And it is noncorrosive for metal and sharp instruments.

Disadvantages for dry heat sterilization

— Time consuming method because of slow rate of heat penetration and microbial killing.

— High temperatures are not suitable for most materials.⁵

2. Moist Heat Sterilization:

Principle: Moist heat is more efficient for sterilization in contrast to dry heat; it destroys microorganisms by the irreversible denaturation of enzymes and structural proteins. It may be used in three forms to achieve microbial inactivation. Moist heat sterilization involves the use of steam in the range of 121-134 °C.³⁰

Autoclave:

High-pressure steam sterilization is used to kill all microorganisms, including spores. It is the best and most widely used methods of sterilization. Example: Pressure cooker. Vapour pressure within the autoclave increases that enables steam to reach higher temperatures.

The autoclave is a tough double walled chamber in which air is replaced by pure saturated steam under pressure.

Before using the autoclave, check the drain screen at the bottom of the chamber and clean if blocked. If the sieve is blocked with debris, a layer of air may form at the bottom of the autoclave.⁶

Construction:

Autoclaves, or steam sterilizers essentially consist of following:

i) A cylindrical or rectangular chamber, with capacities ranging from 400 to 800 liters.

ii) Water heating system or steam generating system

iii) Steam outlet and inlet valves

iv) Single or double doors with locking mechanism.

v) Thermometer or temperature gauge

vi) Pressure gauges

Procedure for Operation

For porous loads (dressings) sterilizer’s minimum temperature of 134°C, and employing a minimum temperature of 121°C are used. Ensure that there should be (includes heating up, holding/exposure, and cooling stages). They are easily operated for bottled fluid, sterilizer’s sufficient water in the autoclave to produce the steam. The stages of operation of autoclaves include air removal, steam admission and sterilization cycle.⁷

Procedure for Operation:

— The air in the chamber is flushed out and filled with saturated steam.

— Water is boiled to produce steam, which is released through the jacket and into the autoclave's chamber.

— Hot, saturated steam enters the chamber and the desired temperature and pressure, usually 121°C.

— At this temperature saturated steam destroys all vegetative cells and endospores.

— Moist heat is thought to kill so effectively by degrading nucleic acids and by denaturing enzymes and other essential proteins.

— It also may disrupt cell membranes. The chamber is closed tightly the steam keeps on filling into it and the pressure gradually increases.

— The items to be sterilized get completely surrounded by saturated steam (moist heat) which on contact with the surface of material to be sterilized condenses to release its latent heat of condensation which adds to already raised temperature of steam so that eventually all the microorganisms in what ever form are killed.

— The usual temperature achieved is 121 °C at a pressure of 15 pps. ie. at exposure time of only 15-20 mins. By increasing the temperature, the time for sterilizing is further reduced.⁸

Advantages of Autoclave:

— It is rapid and effective. It destroys microorganisms more efficiently than dry heat.

— Exposure at a lower temperature is possible.

— It can be used for a large proportion of the official injections.

— It is supplied with dry saturated steam porous materials and can be sterilized with no damage.

— Equipment or components of rubber and certain plastics such as nylon and PV.C withstand the conditions.

Disadvantages of Autoclave:

— Items sensitive to heat cannot be sterilized.

— It cannot be used for injections and articles such as plastics that deteriorate at 115°C.

— It is unsuitable for anhydrous materials such as powders and oils.

Applications of Moist Heat Sterilization:

This method is most essential biocidal agent. It is used for surgical dressings, sheets, surgical and diagnostic equipments.⁹

FILTRATION:

It is a method of sterilization in which microbial population is removed with the help of filters. The sample is passed through a filter of appropriate pore size. The microbial load remains on the filter and we get a sterile sample. In order to sterilize solutions which is heat sensitive, filtration is an excellent way to reduce the microbial population. The filters simply remove the microbes instead of killing them.²⁹

I. Depth Filters:

Consists of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter. The solution is passed through the filter which is sucked through this layer under vacuum and microbial cells are removed. The material used mostly is porcelain, asbestos or other similar materials.²⁸

II. Membrane filters:

Are also used and have replaced depth filters in recent times. These filters are made up of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride, and other synthetic materials. These filters vary in size with pore sizes mostly of 0.2 to 0.5 µm in diameter and used to remove most vegetative cells. These filters are mostly used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics and other heat sensitive solutions.[27] The other way this method is used is in the laminar flow biological safety cabinets where the air is sterilized by filtration. These cabinets contain high-efficiency particulate air (HEPA) filters, which remove 99.97% of 0.3µm particles. The safety cabinets are most useful as the culturing of any organisms requires contamination free air to reduce the growth of other undesired organisms or for the preparation of media, examining tissue cultures etc.[10]

Air filter: (HEPA filter):

It is high efficiency particulate air or originally called High-Efficiency Particulate Absorber (HEPA). It is used to describe filters that are able to trap 99.97 per cent of particles that are 0.3 microns. Air particles are circulated through HEPA filter by directions viz.

(a) Direct Impaction:

Large contaminants, such as certain types of dust, mold, and pollen, travel in a straight path, collide with a fibre, and stick to it.

(b)Sieving: The air stream carries a particle between two fibres, but the particle is larger than the gap, so it becomes ensured.²⁶

Advantages:

The method is suitable for sterilization of thermolabile medicaments, such as, blood, products, insulin and enzymes.

— All types of bacteria i.e., living as well as dead are removed from the preparation, both clarification and sterilization are done side by side.

— It is an excellent method for the rapid supply of a small volume of a parenteral solution in an emergency

Disadvantages:

The method is not a reliable one and therefore a sterility test is necessary.

— The suspension and oily preparations cannot be sterilized by this method.

— There are chances of absorption of medicaments from a solution by the filter.

— Defects in the media are not immediately detectable.

— Aseptic technique is necessary.

— Highly trained staff is required.

— The process is only suitable for medicaments which are in solution form.¹¹

3. RADIATION:

Many types of radiation are used for sterilization like-

A. Electromagnetic radiation:

(Gamma rays and UV rays) Particulate radiation (Accelerated electrons). The major target for this radiation is microbial DNA. Gamma rays and electrons cause ionization and free radical production while UV light causes excitation. U.V. light has limited sterilizing power because of poor penetration into most materials. Generally used in irradiation of air in certain areas eg, Operating rooms and T.B. laboratories. Gamma radiation: Source Cobalt60 has greater energy than U.V. light, therefore more effective. Used mainly in industrial facilities e.g. sterilization of disposable plastic syringes, gloves, specimens containers and Petri Dishes.¹²

B. Ionizing radiation:

High penetration power, rapid action, not raise temperature, and break DNA of micro organism. Articles being sterilized are passed through the irradiation chamber on a conveyor belt and move around the raised source. Penetrates deep into objects and is an excellent sterilizing agent. It destroys bacterial endospores and vegetative cells of both prokaryotic and eukaryotic origin but not against viruses.¹³

C. Non- Ionizing radiation: Lethal radiation, low penetrate power. Eh: UV, IR.

4. Chemical method sterilization:

Chemical sterilization is the process of elimination of all viable microorganisms and their spores. It is of two type’s viz. using liquids and gaseous compounds. Liquid sterilization involves submerging equipment in a chemical fluid for enough time to kill all viable microorganisms and their spores. Gas sterilization involves exposing equipment to chemical gases in an enclosed heated or pressurized chamber. Liquid sterilizing agents are impractical or ineffective for sterilizing items. Gaseous agents are more effective sterilants because they are able to permeate small openings easily, Gas chemicals also sterilize faster than liquids because they usually are combined with high heat. Gas residue is also easier to remove from sterilized articles but requires more expensive equipment.¹⁴

Fig: 1 Chemical Methods

A. Alcohol

· Principle: It is based on denaturing coagulating proteins and dissolving membrane lipid of microorganisms.

Advantages:

— It is inexpensive and non-toxic.

— Widely available.

— Rapidly effective.

— Active against bacteria.

Disadvantages:

— Not effective against bacterial spores.

— Not effective with organic materials.

B. Aldehyde:

— Principle:

Aldehyde combines with important proteins and nucleic acids of the bacterial cells. These interactions of aldehyde with these cellular substances produce antimicrobial action Formaldehyde inactivates microorganisms by alkylating the amino acids and sulfhydryl groups of proteins and ring nitrogen atoms of purine bases.¹⁵

Advantage:

— They have good activity against spores, viruses and fungi.

Disadvantages:

— They are toxic.

— They need long exposure time for action, minimum 3 hours.

— Freshness and pH are critical.

— They are irritant and carcinogenic.

C. Phenolics Agents:

— Principle:

They act by destroying plasma membranes denature proteins. They also affect plasma membrane, inactivate enzymes, and denature proteins.²⁵

Advantages:

— They are stable, persist for long times after application, and remain active in the presence of organic compounds.

— They are active against wide range of organisms.

— They have good antimicrobial activity and are rapid bactericidal.

— They are more active in acid pH.

— They remain active on surfaces long after application.

Disadvantages:

They are having systemic toxicity.

— They are not effective against spores.

— They are not effective at low temperature

— They have a disagreeable odor

D. Halogen:

Mainly Iodine and Chlorine are used.

— Principle: iodine is an oxidizing agent. These agents can irreversibly oxidize and inactivate essential metabolic compounds like proteins with sulfhydryl groups. Chlorinated compounds show antimicrobial activity due to formation of hypochlorous acid in water. This hypochlorous acid is further decomposed in to nascent oxygen which shows strong oxidation reaction.

Advantages of Iodine compounds:

— Iodine compounds are effective against gram positive bacteria.

— They produce residual activity

— They retain microbial action in the presence of organic debris.

— They are available in solutions, sprays and in gel preparations.

Disadvantages of iodine compounds:

Iodine compounds when used alone are major irritants.

— They are weak against myco bacteria, fungi, viruses.

— They are absorbed into skin and can be toxic. They can cause burns.

Advantages of Chlorine Compounds: They are cheap and readily available.

— Chlorine is highly soluble in water.

— They are toxic to most microorganisms. They remove iron and manganese and ammonia nitrogen during oxidation. They destroy taste and other odour compounds.

Disadvantages of Chlorine Compounds:

— Chlorine is a poisonous and toxic gas.

— They are corrosive, requires special non-metal conduits.¹⁶

E. Heavy Metals

— Principle:

Heavy metals and their compounds have antimicrobial activity due to combination with cellular proteins and inactivating their function. Example: mercuric chloride inhibits the action of enzyme by acting on sulfhydryl group of enzyme and form inactive enzyme. High concentration of heavy metal salt coagulates cytoplasmic proteins of microorganisms that cause death of the cells.¹⁷

Advantages:

— They are powerful biocides.

— They form complex with proteins of microorganisms and converts inactive form microorganisms.

Disadvantage: They are highly toxic to the animals.

F. Surface Active Agents

— Principle: They denature proteins, interference with glycolysis and damage membrane of microorganisms. They also damage the cell cytoplasmic membrane and alter structure.¹⁸

Advantages:

They are effective against vegetative bacteria.

— They are widely available.

— They are less expensive.

— They are non-irritant.

Disadvantages: They are ineffective against spores.

— They are not effective against non-enveloped viruses.

— They may become contaminated.

G. Dyes:

— Principle: They have their inhibitory effect by interfering with cellular oxidation processes.

Advantages:

— They are bacteriostatic in high dilution.

— They are more active against Gram positive bacteria.

Disadvantages:

— They are less active against Gram negative.

— They have low bactericidal activity.¹⁹

Gaseous Agents for Sterilization:

The main gaseous agents are used for sterilization are ethylene oxide, Formaldehyde, beta propiolactone etc. They act on principle of denaturing proteins. Gas can be hazardous to people.20

· They often highly explosive.

— Some time gases are extremely poisonous.

— They are potentially carcinogenic.

Factors:

— Efficiency of sterilization method is influenced by the concentration of ethylene oxide.

— The humidity of the sterilizing atmosphere.

— The temperature of sterilization.

— Time of exposure

— Physical nature and permeability of the load.

Formaldehydes:

It is also a group of alkylating agent. It inactivates microorganisms by alkylating amino acid and sulfhydryl groups of proteins and ring nitrogen atoms of purine bases.²¹

Ethylene Oxides:

It is a type of alkylating agent that is used for gaseous sterilization. It is high penetrating and can sterilize items within plastic bags. Ethylene oxide have high antimicrobial activity, it kills even endospores. It is used for sterilization of heat sensitive materials such as spices, oils, plastics etc.²²

B-Propiolactone:

It binds to DNA, thereby inactivating it. It is a clear liquid with a strong odor and has the ability to kill endospores. It has been used in either liquid form or as a vapor for the sterilization of medical instruments and tissue grafts, and it is a common component of vaccines, used to maintain their sterility. It is also used for the sterilization of nutrient broth as well as blood plasma, milk and water.²³ Penetration power of B-propiolactone is less than ethylene oxide but it is more active in killing microorganisms. Due to its carcinogenic effects, it is not commonly used.

H. Chemical Sterilisation

Hydrogen Peroxide Sterilization:

This method disperses a hydrogen peroxide solution in a vacuum chamber, creating a plasma cloud. This agent sterilizes by oxidizing key cellular components, which inactivates the microorganisms. The plasma cloud exists only while the energy source is turned on. When the energy source is turned off, water vapor and oxygen are formed, resulting in no toxic residues and harmful emissions. The temperature of this sterilization method is maintained in the 40-50°C range, well-suited for use with heat-sensitive and moisture-sensitive medical devices.²⁴

The instruments are wrapped prior to sterilization, and can either be stored or used immediately.

AUTHORS’ CONTRIBUTIONS:

Both authors participated in the literature search, interpretation of the articles reviewed analysis of the data, and review of the manuscript. All authors have read and approved the paper.

CONFLICT OF INTERESTS:

The authors declare that they have no conflict of interest.

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Received on 08.07.2021 Modified on 29.08.2021

Res. J. Pharmacology and Pharmacodynamics.2021;13(4):155-161.

DOI: 10.52711/2321-5836.2021.00029