Sterilization

Sterilization- definition and types of sterilization. Physical methods of sterilization: Equipment used for sterilization, operation of autoclave and hot air oven, Sterilization by radiation and filtration (membrane). sterilization control and sterilization indicators.

Sterilization: Definition and Types of Sterilization

“Process to eliminate or destruct all forms of living microorganism both vegetative and spores is called sterilization”.

Types of Sterilisation/ Classification of Sterilisation: There are two types of sterilization. These are

• 1. Physical Methods of Sterilizations

  • a. Heat Sterilization: There are two types of heat sterilization.

    • i. Moist Heat Sterilization.

    • ii. Dry Heat Sterilization.

  • b. Radiation Sterilization.

• 2. Chemical methods of sterilization: There are two types of sterilization.

  • a. Gaseous sterilization,

  • b. Liquid Sterilization.

• 3. Mechanical sterilization:  Filtration sterilization

Methods of Sterilization

• A. Dry heat sterilization: Destruction of all vegetative forms and spores of microorganisms by using heat without moisture is dry heat sterilization. Heating at 160 degrees C for two hours kill all vegetative form and spores of bacteria.

• Equipment: A Hot Air Oven is used as equipment in dry heat sterilization.

• Construction of Hot Air Oven: It is a double-walled chamber made up of steel. Its door is also double-walled. Insulation materials are present in between two walls. This stops the heat loss from the hot air oven chamber. The asbestos gasket is present on the inner wall of the door. This makes the hot air oven air-tight and also stops heat loss from the chamber.

• The chamber has two or more perforated shelves. Perforated shelves allow the air circulation inside the chamber.

• It is electrically heated equipment. Heating elements are present at the bottom of the double-walled chamber. Some hot air ovens have also heating elements under the side wall of the chamber.

• Some hot air chamber has a blower to circulate air inside the chamber. The hot air oven with a blower is called  Forced Convection Hot Air Oven. A hot air oven without a blower is called a Natural Convection Hot Air Oven.

• The outside wall of the oven has a thermostat to increase and decrease the temperature of the chamber as per requirement. It has a thermometer fitted to note down the temperature of the hot air oven chamber. Its side wall has ventilators. Ventilators can be opened or closed as per requirement. 

  Principle: Heating is carried out by convection and radiation. Air moves inside a chamber naturally. Hot air goes up and cold air moves down. This causes air circulation in a natural convection oven. In a forced convection oven, hot air moves by a blower.

• Heats evaporate water from microorganism cells. This causes oxidative damage to cells, denaturation of cellular proteins, and increased electrolyte levels to cause toxicity. It leads to microorganism death.

• Operation of hot air oven: Wrap the glass apparatus/articles with clean paper. Plug the opening of glass containers with non-absorbent cotton wool. Place them over the perforated shelves of the hot air oven chamber. Do not place them over the floor of the hot air oven. Do not overload the hot air oven. Maintain space between to materials inside the hot air oven. Open the ventilators of the oven and switch on to heat the oven. Note down the temperature in the thermometer of the oven. At 60 degrees C close the ventilator. Allow the temperature to rise up to 160 degrees C. Maintain this temperature for 2 hours. Allow oven to cool down after two hours up to 40 degrees C. Remove the sterilized materials from the oven.

Advantages

• 1. Suitable for moisture-sensitive materials

• 2. Suitable for glass and metal apparatus and equipment

• 3. Suitable for assembled equipment

• 4. Economical, easy to install and operate.

Disadvantages

• 1. Not suitable to sterilize items like surgical dressings, rubber materials, plastic materials, etc..

• 2. Do not destroy the heat-resistant endospores

• 3. Time-consuming process than steam, chemical, or radiation sterilization.

Applications: Sterilisation of glass apparatuses, scissors, glass syringes, and assembled equipment.

B. Moist heat sterilization (Steam Sterilization): Destruction of all vegetative forms and spores of microorganisms by using moist heat is moist heat sterilization. Moist heat sterilization utilizes the latent heat of steam to kill vegetative forms and spores of microorganisms. The following temperature and time are required for moist heat sterilization.

• i. Maintaining temperature 115 degrees C/ pressure 10lb per square inch for 30 minutes or

• ii. Maintaining temperature 121 degrees C/ pressure 15lb per square inch for 15 minutes or

• iii. Maintaining temperature 126 degrees C/ pressure 20lb per square inch for 10 minutes or

• iv. Maintaining temperature 134 degrees C/ pressure 25lb per square inch for 05 minutes.

Equipment: Autoclave is used as equipment in moist heat sterilization.

• Construction of Autoclave: The autoclave is made of a stainless steel chamber with a lid. The outer side of the lid has a steam vent, pressure gauze, and safety valve. The inner side of the lid has a rubber gasket to make the autoclave air-tight. The lid closes the autoclave by using wing nuts and volts. An electrically heated element is present at the bottom of the autoclave chamber. The Autoclave has a perforated inner chamber/basket. Materials to be sterilized are placed inside/on the perforated inner chamber/basket.

• Principle: Hot saturated steam kills microorganisms. Moist heat has more penetration power than dry heat due to its latent heat. Moist heat penetrates all forms of microorganisms like vegetative microbes, capsulated bacteria, spores, etc. Saturated steam condensation at bacterial cell components releases the latent heat. Latent heat coagulates the cytoplasm and protein cellular contents. The temperature of saturated steam increases with the increase in pressure.

  • temperature 115 degrees C/ at pressure 10lb per square inch

  • Temperature 121 degrees C/ at pressure 15lb per square inch

  • Temperature 126 degrees C/ at pressure 20lb per square inch

  • Temperature 134 degrees C/ at pressure 25lb per square inch.

• Operation of Autoclave: Place a sufficient quantity of water inside the autoclave. Electrically heated elements must be under water. However, water should not touch the perforated inner chamber/basket inside the autoclave. Place the lid on the opening of the autoclave. Close the lid tightly using wing nuts and volts.

• Switch on to heat water inside the autoclave. Open the vent and allow steam to come out from the vent for 5 minutes. Close the vent and allow the pressure inside the autoclave to rise. At 10lb pressure per square inch temperature of steam inside the chamber shall be 115 degrees C. Maintain this temperature for 30 minutes. After 30 minutes, switch off the autoclave. Allow to decrease pressure inside the autoclave. Allow the autoclave to cool down to 40 degrees C. Open the lid and collect sterilized materials.

• Advantages:

  • 1. More efficient than dry heat sterilization

  • 2. Sterilisation at lower temperatures in a shorter duration

  • 3. Larger quantities can be sterilized

  • 4. Larger types of materials can be sterilized,

• Disadvantages: Not suitable for heat-sensitive materials.

• Applications: Sterilisation of culture media, Sterilisation of glass apparatuses, Sterilisation of reagents, Sterilisation of closures and containers.

Dr Pramila Singh

A. Radiation sterilization

There are two types of radiation sterilization. These are sterilization by ultraviolet light and sterilization by ionizing radiation.

• Sterilization by ultraviolet light: Ultraviolet rays destroy microorganisms and spores. Ultraviolet rays for sterilization are produced by mercury vapor lamps. Low current at high voltage through mercury vapor under an evacuated glass tube produces ultraviolet light. The antimicrobial activity of UV rays depends upon wavelength of UV rays. Its antimicrobial activity is highest at 265 nm.

• Disadvantages

  • 1. Low penetration power

  • 2. Effective only against microbes in air or surface’

  • 3. Dust or grease on UV lamp decreases radiation

  • 4. UV light is harmful to workers.

• Applications:

  • 1. To prevent infection in hospital

  • 2. To maintain an aseptic condition

  • 3. To sterilize heat-sensitive materials,

  • 4. To sterilize the working top

Sterilization by ionizing radiation:

X-rays and Gamma rays are ionizing radiations. They can kill microorganism. Radio isotopes produce gamma rays. The material to be sterilized is placed inside a container. Then they are exposed to ionizing radiation. Ionizing radiation is more effective than ultraviolet rays for sterilization.

• Applications: To sterilize plastic syringes, hypodermic needles, surgical blades, adhesive dressings, bones, heat-sensitive medicines, etc.

• Advantages:

  • 1. Gamma radiation has high penetration power

  • 2. Negligible rise in temperature

  • 3. Suitable to sterilize all types of materials like dry or moist

  • 4. Suitable to sterilize large quantities of materials

  • 5. Does not require aseptic precautions.

  • 6. Reliable and accurate method

• Disadvantages:

  • 1. Very expensive

  • 2. Process once started cannot be stopped

  • 3. Harmful to worker

  • 4. Cause changes in medication.

A. MEMBRANE FILTER

 It is a thin flat membrane made of cellulose derivatives like cellulose acetate, cellulose nitrate, etc. It has 400 to 500 million pores in one square centimeter. Pore size varies from 0.01 µm to 14 µm. These pores are uniform in size and occupy 80% of the membrane surface area. A membrane filter is also known as a bacteria-proof filter membrane. It is used in filtration sterilization. A bacteria-proof filter membrane retains bacteria and allows liquid to pass during filtration. A bacteria-proof filter membrane is also called a Millipore filter. A metallic holder is used to hold a bacteria-proof filter membrane.

Principal: All filters in medical laboratories work on the same mechanism. Filters physically trap particles larger than their pore size. It also retains some smaller particles through the electrostatic attraction of the particles to the filters. Factors like

• 1. Electrostatic charges on the filter,

• 2. Electrostatic charges on organisms and

• 3. Nature of the fluid filtered.

The size of bacteria is 1 µm to 5 µm. The size of the pore in the membrane filter is less than 1µm thus it filters all bacteria and makes the filtrate bacteria free.

Care:

• 1. The membrane filter is very delicate thus prefilter should be used with the membrane filter. The prefilter provides support to the membrane filter and decreases membrane filter clogging during filtration.

• 2. Avoid excessive positive pressure and negative pressure during filtration. It may break the membrane filter.

• 3. Avoid prolonged filtration. It promotes microbial growth and entry of microbes into sterile solution.

Safe operating procedure: A membrane filter holder is used to hold the membrane filter. The membrane filter holder has an upper disc and a lower disc made of stainless steel. The tripod stand is attached to the lower disc. There is one outlet in the lower disc and one inlet in the upper disc. A membrane filter is attached at the upper surface of the lower disc. A prefilter is placed on the membrane filter. The upper disc is placed over pre-filter. The assembly is closed tightly to make it airtight. Enter slurry through the inlet nozzle present in the upper disc. Allow the liquid to pass through an outlet in the lower disc. Filtrate is collected in a closed container.

Either air pressure is applied upon the filter membrane or a vacuum is created below the membrane filter to increase the filtration rate. The filtrate obtained shall be free from bacteria.

Application: It is used to sterilize liquid products. Bacteria collected on the filter membrane can be used on culture media for optimum growth of bacteria for their further study.

• 1. It is used in diagnostic cytology

• 2. It is used to clarify and sterilize non-viscous microbiological liquid and culture media.

• 3. It is used for microfiltration, ultrafiltration, nanofiltration, and in reverse osmosis.

Advantages

• 1. Sterilisation and clarification of product

• 2. Suitable for heat-sensitive materials.

• 3. Removes both live and killed bacteria

• 4. Most suitable for small-volume parenterals.

Disadvantages

• 1. Not reliable, leakage in the filter membrane may occur during sterilization.

• 2. Suitable for only liquid solution

• 3. Not suitable for suspension and oily preparation

• 4. Require aseptic technique

B. SINTERED GLASS FILTER:

It is made of borosilicate glass. Borosilicate glass is finely powdered, and sieved to obtain the desired size of borosilicate glass powder particles. Powdered particles are heated to melt and packed to form a disc shape. The disc is fused to a funnel of suitable shape and size. The funnel containing sintered glass is numbered. This numbering depends upon pore size.

Principal: During filtration high pressure difference is used. It filters out all insoluble constituents and bacteria present in the liquid.

Care: Sintered glass filter is made of glass filter that needs proper cleaning before and after use to remove contaminants.

• 1. Clean the disc with diluted hot hydrochloric acid followed by several rinsing with distilled water.

• 2. Wash it as quickly as possible after its use. Use a soft brush to clean all parts,

• 3. Scrub the soft brush gently to avoid scratching on glass parts. Rinse it thoroughly

• 4. Dry it at room temperature or in the oven. Do not exceed oven temperature above 100 degrees C. Above 100 degrees C, there will be damage to a sintered glass filter.

• 5. Do not use hot concentrated phosphoric acid or hot alkaline solution for cleaning. It decreases the life of the sintered filter and increases the size of the pores.

Safe working procedure: Slurry is placed in a sintered glass funnel. Pressure is applied over the funnel or a vacuum is created under the funnel to create pressure difference. Collect filtrate through the neck of the sintered glass funnel.

Use: It has good mechanical strength to filter liquids for bacteriological and cytological studies.

F. SEITZ FILTER:

It consists of a compressed asbestos pad present on the perforated plate. Seitz filter has a 2mm thickness.

Principal: It uses an asbestos pad filter and sieve mechanism to filter. Material is absorbed in the filter media.

Care:

• 1. Avoid excessive positive pressure and negative pressure during filtration. It damages the Seitz filter.

• 2. Avoid prolonged filtration. It promotes microbial growth and entry of microbes into sterile solution.

• 3. Test it for cracks and leaks before the start of filtration.

• 4. Validate it regularly by monitoring microbe load in filtrate.

• 5. It contains an asbestos pad. Asbestos pad shade fibers. Use the proper device below the Seitz filter to block asbestos fibers.

Safe working procedure: Fill the upper part of the filter container with slurry. Asbestos deposited with slurry and filtrate passes out. Filtrate is collected in a container. The rate of filtration can be increased by creating a pressure difference above and below the seitz filter.

Dr Pramila Singh

Chemical Methods of Sterilisation

There are two types of chemical sterilization. These are

• 1. Sterilisation by heating with bactericides

• 2. Gaseous sterilisation

Sterilization by heating with bacteriocides: It is used to sterilize aqueous preparations not stable at moist heat sterilization temperature. Aqueous preparation is heated at 98 to 100 degrees C with bacteriocides like Chlorocresol, Benzalkonium chloride, phenylmercuric nitrate, chlorhexidine acetate, etc.

Gaseous sterilization: Gaseous chemicals such as formaldehyde, ethylene oxide, Beta propiolactone, etc are used in gaseous sterilization. Formaldehyde is an alkylating agent. Ethylene oxide is a better choice than formaldehyde. Beta propiolactone is liquid at room temperature, noninflammable, and has a lower penetration power than ethylene oxide.

Antiseptics are antimicrobial agents applied on living surfaces to kill or prevent the growth of microorganisms to reduce or prevent microbial infection or sepsis.

Disinfectants are antimicrobial agents applied on nonliving surfaces (inanimate surfaces) to kill or prevent microorganism growth to reduce their number to prevent infection. They do not kill spores.

Classification/Types of antiseptic and disinfectants

• · Acids: Ethanol, Isopropyl Alcohol

• · Aldehydes: Formaldehyde, Glutaraldehyde,

• · Phenol Derivatives: Phenol, Cresol, Paraben, Thymol, Resorscinol, Chlorhexidine, Chlorhexidine Gluconate, Chloxylinol, Hexachlorophene

• · Halogens: Chlorine, Iodine, Iodophores, Chloramine

• · Oxidising Agents: Hydrogen peroxide, Potassium permanganate, Benzoyl peroxide

• · Dyes: Gentian Violet, Methylene Blue, Brilliant Green

• · Heavy Metals: Mercury Compounds, Silver Compounds, Zinc Compounds

Formaldehyde: Formaldehyde is an alkylation agent. It was used in the old days to sterilize syringes, catheters, and other heat-sensitive materials. Nowadays it is only used for the fumigation of empty rooms.

• Properties:

• 1. Physical Properties: Formaldehyde is a colorless gas at room temperature. highly soluble in water. Normally it is stored as an aqueous solution called formalin. Formalin contains 37 to 50% formaldehyde in water. Formaldehyde and formalin have strong pungent distinctive odors and are highly flammable in nature.

• 2. Chemically it acts as a reducing agent.

• Uses:

• 1. Sterilisation: It was used in the old days to sterilize syringes, catheters, and other heat-sensitive materials. Nowadays it is only used for fumigation of empty rooms.

• 2. Disinfectant: It is used as a disinfectant in various healthcare and cosmetic industries

• 3. Preservative: It is used to preserve personal care products and biological samples like tissues, organs, etc

• 4. Fumigation: It is used to control pests in agricultural and storage facilities

• 5. Preservative: It is used in personal care products as preservatives

Ethylene oxide

• Properties: It is a colorless, flammable gas at room temperature. It has a sweet and ether-like odor. It can be liquefied easily. The boiling temperature of ethylene oxide is 10.8 degrees C. It is highly soluble in water and organic solvents. It is not used alone due to its highly inflammable properties. It is mixed with some inert gases such as carbon dioxide, dichlorodifluoromethane, etc.

• Uses:

• 1. Sterilization: It is used to sterilize delicate diagnostic equipment. It is highly effective at 40 degrees C and in the presence of moisture.

• 2. It has high penetration power. It is suitable for the sterilization of materials packed in plastic or cellophane envelopes.

• 3. It is suitable to sterilize heat-sensitive materials such as rubber and plastic items.

Phenol compounds: Phenol compounds are also called carbonic acid or hydroxybenzene compounds.

• Properties: It is a white crystalline solid at room temperature. It is also available as a colorless liquid. It has a characteristic sweet odor slightly soluble in water and is readily soluble in organic solvents like ether, acetone, and alcohol.

• Uses: Antiseptic and disinfectant. It has bactericidal properties.

Alcohol: Alcohol, especially ethyl alcohol and isopropyl alcohol is commonly used in medical laboratories.

• Properties: Alcohol evaporates at room temperature and does not leave residue on the surface. It is a colorless liquid with good solvent properties.

• Uses

• 1. Disinfectant: It is the most commonly used disinfectant for working surfaces, equipment, and glassware.

• 2. Hand Sanitizer: cleaning hands and killing living microbes on hands and palm surfaces is called hand sanitization. Alcohol is a good hand sanitizer. Laboratory personnel sanitize their hands before working to reduce the risk of contamination.

• 3. Cleaning and Decontamination: Alcohol is used to clean and decontaminate glass slides coverslips and other medical equipment. It removes dirt, stains, and residues from the surface without leaving any residues.

• 4. Tissue processing: Alcohol is used in tissue processing, tissue fixation, or tissue embedding. Alcohol helps to dehydrate and preserve tissue specimens.

• 5. Preservation of microorganisms: It is used to preserve and store microbial culture in laboratories.

• 6. SpecimenTransport: Alcohol is used to preserve and transport clinical specimens such as urine samples.

• 7. Staining Procedure: Alcohol is used as a solvent to prepare a stain solution.

• 8. Fixation of blood smear: Alcohol is used to fix the blood smear in hematology.

Hypochlorite Compounds: The most commonly used hypochlorite is Sodium Hypochlorite also called bleach.

• Properties: It is a strong oxidizing agent and bleaching agent. It releases oxygen on its breakdown. Its pH is above 11. Its alkaline property makes it a strong disinfectant. It can damage metals and act as corrosive.

• Uses

• 1. Surface disinfectant

• 2. Sterilization

• 3. Instrument and apparatus decontamination

• 4. Sample decontamination

Definition of Phenol Coefficient: The Phenol coefficient is a numerical value to express the ability of disinfectant and antiseptic to kill or inhibit the growth of the microorganism. It is measured by comparing the concentration of test disinfectant or antiseptic to the concentration of phenol required to achieve the same level of antimicrobial activity.

Phenol coefficient = Minimum bactericidal concentration of phenol/ Minimum bactericidal concentration of test disinfectant.

A phenol coefficient greater than one indicates that the test disinfectant is stronger than phenol. A phenol coefficient lower than one indicates that the test disinfectant is weaker than phenol.

Determination Phenol coefficient by Rideal Walker method

Materials and Equipment

• 1. Phenol

• 2. Test disinfectant or antiseptic

• 3. Sterile culture Medium (Nutrient Agar)

• 4. Test microorganism

• 5. Glass apparatus: Petri dish, Test tubes and Pipettes

• 6. Incubator

• 7. Microbiological loop or spreader

• 8. Gloves and other protective kits.

Procedure

• 1. Prepare solutions of different strengths like 0.1%, 0.05%, 0.025%, etc of both phenol and test disinfectant or antiseptic separately

• 2. Inoculate sterile culture medium with test microorganisms such as Staphylococcus aureus using a sterile microbiological loop.

• 3. Allow the culture media to solidify

• 4. Careat a well in inoculated culture media.

• 5. In each well, add a specified volume of a particular dilution of phenol and test disinfectant.

• 6. Incubate the culture plates at 37 degrees C for 24 hours

• 7. After incubation, observe the bacterial growth around the well.

• 8. The concentration at which no bacterial growth is observed is called minimum bactericidal concentration (MBC).

• 9. Calculate the phenol coefficient by the following formula

• 10. Phenol coefficient = MBC of Phenol / MBC of the Test Disinfectant.

It is older method and not in use.