Experiment -3: Methods of sterilization

Sterilization:-Itis defined as the process where all the living microorganisms, including bacterial spores, are killed. Sterilization can be achieved by physical, chemical, and physiochemical means. Chemicals used as sterilizing agents are called chemisterilants.

It is a process by which an article, surface, or medium is made free of all microorganisms either in vegetative or spore form. Sterilization is the process of killing or removing bacteria and all other forms of living microorganisms and their spores from preparations and articles.

Disinfection: Destruction of all pathogens or organisms capable of producing infections but not necessarily spores. All organisms may not be killed but the number is reduced to a level that is no longer harmful to health.

Antiseptics: Chemical disinfectants which can safely be applied to living tissues and are used to prevent infection by inhibiting the growth of microorganisms

Asepsis: Technique by which the occurrence of infection into an uninfected tissue is prevented.

Why do we need Sterilization?

• Microorganisms capable of causing an infection are constantly present in the external environment and constantly present in the external environment and on the human body the human body

• Microorganisms are responsible for contamination and infection

• The aim of sterilization is to remove or destroy them from materials or from surfaces from materials or from surfaces

What to sterilize?

• It is mandatory to sterilize :

– all instruments that penetrate soft tissues and bone.

– Instruments that are not intended to penetrate the tissues, but that may come into contact with oral tissues.

• If the sterilization procedure may damage the instruments, then, sterilization can be replaced by a disinfection procedure

Uses of sterilization

• • • Sterilization of Media and reagents used in the microbiology and tissue culture laboratory.

    • Food and drug manufacturing to ensure safety from contaminating organisms.

    • Food: Sterilization has helped to reduce food poisoning.

    • Medicine and surgery: Surgical instruments and medications that enter in the body must be sterilized to a high sterility assurance level. Example: Artificial pacemaker.

    • In pharmaceuticals: This is also used in the manufacture of parenteral pharmaceuticals.

    • Research and laboratory: The success of a research facility is often dependent upon an efficient sterilization process.

    • Vaccine and biological productions: The success of vaccine and biological production is often dependent on an efficient sterilization process.

How can microorganisms be killed?

1. Denaturation of proteins (e.g., wet heat, ethylene oxide)

2. Oxidation (e.g., dry heat, hydrogen peroxide)

3. Filtration

4. Interruption of DNA synthesis/repair (e.g., radiation)

5. Interference with protein synthesis (e.g., bleach)

6. Disruption of cell membranes (e.g., phenols)

Ideal sterilization/disinfection process

• Highly efficacious

• Fast

• Good penetrability

• Compatible with all materials

• Non-toxic

• Effective despite the presence of organic material

• Difficult to make significant mistakes in the process

• Easily monitored

Methods of sterilizations

There are two types of sterilization:

1. Physical and

2. Chemical.

1. Physical sterilization includes:

1. 1. Heat

      1. Dry heat

• Red heat or Flaming

• Incineration

• Hot air oven

1. 1. 2. Moist Heat

Sterilization below100°C

• Pasteurization

• Inspissation

• Vaccine bath

Sterilization at 100°C

• Boiling

• Tyndallisation

• Steam sterilization

v Sterilization above 100°C

• Autoclave

1. 2. Radiation

   3. Filtration

2. Chemical sterilization includes:

1. 1. 1. 1. 1. 1. Alcohols

               2. Aldehydes

               3. Phenolics

               4. Oxidizing agents

               5. Quaternary ammonium compounds

               6. ethylene oxide gas

               7. Others

Sterilization by heat

• Heat is the most practical, reliable, and inexpensive method of sterilization.

• It is used for the sterilization of objects and materials that can withstand high temperatures.

There are two types of sterilization by heat

1. 1. 1. Dry heat

      2. Moist Heat

Red heat: Used to sterilize inoculating loops and needles. Heat metal until it become red hot.

Materials are held in the flame of a bunsen burner till they become red hot.

» Inoculating wires or loops

» Tips of forceps

» Needles

Flaming: Materials are passed through the flame of a bunsen burner without allowing them to become red hot.

» Glass slides

» Scalpels

» Mouths of culture tubes

Incineration: Effective way to sterilize disposable items (paper cups, dressings) and biological waste. Materials are reduced to ashes by burning.

• • • • • • • • Soiled dressings

              • Animal carcasses

              • Bedding

              • Pathological material

Hot Air Sterilization:

Place objects in an oven. Which is Electrically heated and fitted with a fan to equal distribution of air in the chamber. Also Fitted with a thermostat that maintains the chamber air at a chosen temperature

Dry heat transfers heat less effectively to a cool body than moist heat.

Involves heating at atmospheric pressure and often uses a fan to obtain uniform temperature by circulation.

• Heat at 180^oC for a half-hour,

• 170^oC for 1 hr., or

• 160^oCfor 2 hrs.

• Times are the periods during which an object is maintained at the respective temp.

Uses of Hot Air Oven

– Sterilisation of

• Glassware like glass syringes, Petri dishes, pipettes, and test tubes.

• Surgical instruments like scalpels, scissors, forceps, etc.

• Chemicals like liquid paraffin, fats, etc.

Precautions :

1. Should not be overloaded

2. Arranged in a manner that allows free circulation of air

3. Material to be sterilized should be perfectly dry.

4. Test tubes, flasks, etc. should be fitted with cotton plugs.

5. Petri dishes and pipettes should be wrapped in paper.

6. Rubber materials and inflammable materials should not be kept inside.

7. The oven must be allowed to cool for two hours before opening, since glassware may crack by sudden cooling.

Sterilization controls

1. Spores of Bacillus subtilis subsp. niger

2. Thermocouples

3. Browne’s tube

Dry-Heat Sterilization: Disadvantages

• Less reliable than autoclaving

• Large temp differences may arise within the device.

• sharp instruments get dulled

• Many materials do not tolerate dry heat

Moist heat methods of sterilization

Sterilization below100°C: Pasteurization, Inspissation, Vaccine bath, etc.

Sterilization at 100°C: Boiling, Tyndallisation, Steam sterilization, etc.

Sterilization above 100°C: Autoclave

Sterilization below100°C

Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc.

Not sterilization

• Heat-tolerant microbes survive

• Classic Method of Pasteurization (Low-Temperature long Time Pasteurization): exposed to 65^oC for 30 minutes.

• High-Temperature Short-Time Pasteurization (HTST): Milk is exposed to 72^oC for 15 seconds.

Inspissation:

• Heating at 80-85°C for half an hour daily on three consecutive days

• Serum or egg media are sterilized

Vaccine bath:

• Heating at 60°C for an hour daily in a vaccine bath for several successive days.

• Serum or body fluids can be sterilized by heating at 56°C for an hour daily for several successive days.

Sterilization at 100°C

Boiling for 10 – 30 minutes may kill most of vegetative forms but spores with stand boiling.

Tyndallisation: Steam at 100C for 20 minutes on three successive days. Used for egg , serum and sugar containing media.

Steam sterilization: Steam at 100°C for 90 minutes. Used for media which are decomposed at high temperature.

Sterilization above 100°C

Autoclave :

ü Steam above 100°C has a better killing power than dry heat.

ü Bacteria are more susceptible to moist heat.

ü Invented by Charles Chamberland in 1879

Components of autoclave:

v Consists of vertical or horizontal cylinder of gunmetal or stainless steel.

v Lid is fastened by screw clamps and rendered air tight by an asbestos washer.

v A discharge taps for air and steam, a pressure gauge and a safety valve.

Sterilization conditions

• Temperature – 121 °C

• Chamber pressure -15 lb per square inch.

• Holding time – 15 minutes

• Others :

1. 126°C for 10 minutes

2. 133°C for 3 minutes

Sterilization controls:

1. Thermocouples

2. Bacterial spores- Bacillus stearothermophilus

3. Browne’s tube

4. Autoclave tapes

Difference between Dry Heat sterilization and Moist Heat sterilization

Filtration:

• Useful for substances which get damaged by heat.

• To sterilize sera, sugars and antibiotic solutions.

• To obtain bacteria free filtrates of clinical samples.

• Purification of water.

• This method is commonly used for sensitive pharmaceuticals and protein solutions in biological research.

• A filter with pore size 0.2 µm will effectively remove bacteria. For viruses, a much smaller pore size around 20 nm is needed.

• Prions are not removed by filtration.

• The filtration equipment must be pre-sterilized.

• To ensure the best results, filtration is performed in a room with highly filtered air (HEPA filtration) or in a laminar flow cabinet or "flowbox", a device which produces a laminar stream of HEPA filtered air.

• The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 μm (filtration membranes are most popular for this purpose).

Types of filters:

1. Candle filters

2. Asbestos disc filters

3. Sintered glass filters

4. Membrane filters

5. Air filters

6. Syringe filters

Radiations

1. Ionizing radiations

2. Non -Ionizing radiations

Ionizing radiations:

1. X rays

2. Gamma rays

3. Cosmic rays

Gamma radiation is commercially used for the sterilization of disposable items. (cold sterilization)

Nonionizing radiation

• Wavelengths greater than 1 nm Excites electrons, causing them to make new covalent bonds

1. Affects 3-D structure of proteins and nucleic acids

• UV light causes pyrimidine dimers in DNA

• UV light does not penetrate well

• Suitable for disinfecting air, transparent fluids, and surfaces of objects

Chemical sterilization includes:

1. 1. Alcohols

   2. Aldehydes

   3. Phenolics

   4. Halogens

   5. Iodophores

   6. Oxidizing agents

   7. Quaternary ammonium compounds

   8. ethylene oxide gas

   9. Others

Alcohols:

v Kill bacteria (bactericidal), fungi and viruses.

v Act by denaturing proteins and disrupting cell membranes.

v Used as skin antiseptic to wipe microbes off skin before injections.

v Also used for disinfection of thermometer, probes and external surfaces of stethoscope.

Aldehydes:

v Inactivate proteins by forming covalent cross links with several functional groups.

v Commonly used aldehyde is FORMALIN (37% aqueous solution).

v used extensively to inactivate viruses & bacteria in vaccines.

v Also used to sterilize metal instruments.

Glutaraldehyde

v Action similar to formaldehyde

v More active and less toxic than formaldehyde

v It is used as 2% buffered solution

v It is available commercially as ‘cidex’

v For sterilization of cystoscopies, endoscopes and bronchoscopes

v To sterilize plastic endotracheal tubes, face masks, corrugated rubber anaesthetic tubes and metal instruments

Phenols and Phenolics:

v Phenol acts as protoplasmic poison, penetrates & disrupts cell wall and cause bacterial death by inactivation of essential enzyme systems.

v Used for decontamination of hospital environment including laboratory surfaces, & for noncritical medical & surgical items.

v Lethal effect is due to cell membrane damage

v Phenol (1%) has bactericidal action

v Phenol derivatives like cresol, chlorhexidine and hexachlorophane are commonly used as antiseptics

Cresols

v Lysol is a solution of cresols in soap

v Most commonly used for sterilization of infected glasswares, cleaning floors, disinfection of excreta

Halogens

v Chlorine and iodine are two commonly used disinfectants

v Chlorine is used in the form of bleaching powder, sodium hypochlorite and chloramine in water supplies, swimming pools, food and dairy industries

v Chlorine is used as Hypochlorites have a bactericidal, fungicidal, virucidal and sporicidal action

v Bleaching powder or hypochlorite solution are the most widely used for HIV infected material

v Chloramines are used as antiseptics for dressing wounds

Iodine

v Iodine in aqueous and alcoholic solution used as skin disinfectant

v Iodine often has been applied as tincture of iodine (2% iodine in a water-ethanol solution of potassium iodide)

v Actively bactericidal, moderate action against spores

v Also active against the tubercle bacteria and viruses

v Compounds of iodine with surface active agents known as iodophores

v Used in hospitals for preoperative skin degerming

v Povidine-iodine (Betadine) for wounds and Wescodyne for skin and laboratory disinfection are some examples of iodophores

Gaseous sterilization

v Chemical are used as gases or vapours. Many gases like sulphur dioxide, chlorine, ozone, formaldehyde, beta-propiolactone and ethylene oxide have bacterial effects. • The gases used for chemical sterilization are as follows,

v Formaldehyde : It has been used commonly for fumigating the rooms and blanket in the hospital.

v Beta-propiolactone : It is quite active at low concentration.

Ethylene oxide (ETO)

v Colorless liquid with a boiling point of 10.7 0 C

v Highly lethal to all kinds of microbes including spores

v Action is due to its alkylating the amino, carboxyl, hydroxyl and sulfhydryl groups in protein molecules

v In addition, it reacts with DNA and RNA

v Highly inflammable and in concentrations (>3%) highly explosive

v By mixing with inert gases such as CO 2 , its explosive tendency can be eliminated

Disinfectants can be divided into three groups

1. High level disinfectants: Glutaradehyde, hydrogen peroxide, per-acetic acid and chlorine compounds

2. Intermediate level disinfectants: Alcohol, iodophores and phenolic compounds

3. Low level disinfectants: Quarternary ammonium compounds

Factors that influence efficacy of disinfection/sterilization

1. Contact time

2. Physico-chemical environment (e.g. pH)

3. Presence of organic material

4. Temperature

5. Type of microorganism

6. Number of microorganisms

7. Material composition

STERILIZATION MONITORING

q There are 3 methods of monitoring sterilization:

1. Mechanical techniques

2. Chemical indicators

3. Biological indicators

q These parameters evaluate both the sterilization conditions and procedure’s effectiveness

MECHANICAL INDICATORS: Includes assessing cycle time, temperature and pressure by observing the displays on the sterilizers

CHEMICAL INDICATORS: They allow detection of equipment malfunctioning and help in identifying procedural errors

BIOLOGICAL INDICATORS: Most accepted for monitoring sterilization process

Terminologies

A suffix indicating that the antimicrobial agent will kill or destroy a certain group of microorganism

suffix “cide” – meaning to kill

ü Viricide – destroys virus

ü Fungicide – destroys fungi

ü Bactericide – destroys bacteria

ü Germicides : These are the substance which kill germs.

ü Mycocides : These are the agents which kill molds.

Suffix “static/stasis” – meaning to stand still

A suffix indicating that the agent will prevent the growth or multiplication of the type of organism but are not killed outright

ü Bacteriostatic - prevents the growth of bacteria

ü Fungistatic – prevents the growth of fungi

Differences between sterilization and Disinfection

Thermal death time: The thermal death time may be defined as the time required to kill a specific type of microorganisms at a given temperature under specific conditions. It not only depends on controllable factors like temperature, pH, etc. but also depends on some uncontrollable factors like contaminating microorganisms and their resistance to heat.

How sterilization destroys microbes?

1. Alteration of cell wall or cytoplasmic membrane;

v Cell wall maintains the integrity of cell.

v When disrupted cannot prevent cell from bursting and cellular contents leak out.

2. Protein denaturing

v Functions of protein depends on its specific 3-D shape.

v Various sterilization methods denature proteins and as a result 3-D shape and their functions are lost.

3. Interference with Nucleic Acid

v Nucleic Acid is considered as controlling centre of the cell.

v Various methods of sterilization methods can

i. Produce mutation,

ii. Interfere the process of transcription.

Ideal sterilization/disinfection process

v Highly efficacious

v Fast

v Good penetrability

v Compatible with all materials

v Non-toxic

v Effective despite presence of organic material

v Difficult to make significant mistakes in process

v Easily monitored