STERILIZATION AND DISINFECTION

LEARNING OBJECTIVES At the end of the topic, students will be able to: Define terms related to sterilization and disinfection Describe the effects of microbial control agents on cellular structures Understand and utilize correct sterilization and disinfection techniques Distinguish between sterilization and disinfection Compare and contrast moist and dry heat in terms of the time of treatment and suitable applications Describe how microorganisms are mechanically removed by filtration Explain modes of action of microbial control agents Explain how radiation kills cells

BASIC TERMS STERILIZATION DISINFECTION ANTISEPSIS BACTERIOSTASIS ASEPSIS

STERILIZATION The process of destroying all microbial forms. A sterile object is one free of all microbial forms, including bacterial spores.

DISINFECTION Disinfection is the elimination of pathogens, except spores, from inanimate objects.

ANTISEPSIS Use of chemical agents on skin or other living tissue to inhibit or eliminate microbes; no sporicidal action is implied. Antiseptics are formulated for application to living tissue

BACTERIOSTASIS Inhibits the growth of bacteria.

ASEPSIS No living microorganisms exists.

Actions of Microbial Control Agents Alteration of membrane permeability Damage to proteins Damage to nucleic acids

Alteration of Membrane permeability

Damage to proteins

Damage to nucleic acids

PHYSICAL CONTROL OF MICROORGANISMS PHYSICAL AGENTS HEAT Dry Heat Moist Heat 2) FILTRATION 3) ) RADIATION (COLD STERILIZATION) 4) COLD TEMPERATURE 5) DESICCATION 6) LYOPHILIZATION

DRY HEAT STERILIZATION It brings about the oxidation of cellular protein. It takes long time and high temperature is required. It can be used in different ways, some of them are; Direct heating or Flaming. Incineration. Hot Air Oven.

DIRECT HEATING OR FLAMING Use of flame to destroy microorganisms. Rapid. Forceps, wire loops and needles are sterilized by this method.

INCINERATION This is a method of destroying contaminated material by burning them in incinerator. For decontamination of waste items prior to disposal in land fill. Articles such as solid dressings, pathological material and bedding etc should be subjected to incineration. Continue…

INCINERATION This technique results in the loss of the article, hence is suitable only for those articles that have to be disposed. Temp. may exceed 1000oC. Reduces volume of waster by up to 95%(oxidation to ashes). Improper use may lead to emission of pathogens in smoke.

HOT AIR OVEN It is a double walled container which control temperature. Hot air is produced and circulate within the instrument. Even distribution of heat through out the chamber is achieved by a fan. Perforated trays are present on which material is kept for sterilization. Use of perforated trays is to circulate uniform hot air. Continue…

HOT AIR OVEN Temperature of 150-160oC should be maintained. Exposure time is at least 1 hour. A period of 2 hrs is required for the destruction of bacterial spores. The materials that can be sterilized in hot air oven include laboratory glass wares, instruments, closed containers, anhydrous material such as oil, greases and powders.

MOIST HEAT STERILIZATION Moist heat acts by coagulation and denaturation of microbial proteins. Coagulation of microbial protein is irreversible. More rapid and more effective than dry heat. Steam is used that destroys microorganisms more quickly and at low temp. Continue…

MOIST HEAT STERILIZATION It can be used in different ways, some of them are; Pasteurization. Tyndallization. Autoclaving.

PASTEURIZATION This process was originally employed by Louis Pasteur. Currently this procedure is employed in food and dairy industry. It can be used on heat sensitive liquids and medical devices. Continue…

PASTEURIZATION There are two methods of pasteurization; The Holder method (heated at 62.9oC for 30 minutes). Flash method (heated at 72oC for 15 seconds) quickly followed by cooling to 13oC so that protein becomes coagulated.

TYNDALLIZATION Also called Fractional Sterilization. John Tyndall gave the idea about tyndallization. The substance is heated at temp. of about 80-100oC for 30 mins and then the material is incubated. If spores are present, they will germinate and becomes vegetative cell. The procedure is repeated for 3 successive days. On heating between 80-100oC for 30 mins on successive days with incubation will result in resistant spores to germinate and the vegetative cells are killed on second and third days. Continue…

TYNDALLIZATION The success of process depend on the germination of spores. Time consuming. Heat sensitive materials such as microbiological media, solution of chemicals and biological materials can be sterilized by this process.

AUTOCLAVING Steam under pressure. rapid heating, penetration and moisture in abundance, which facilitates the coagulation of protein. Autoclave. Continue…….

AUTOCLAVING It is a double-jacketed steam chamber . Steam is generated by boiling water. An autoclave chamber is used in which steam at a pressure of 15psi, reaches a temp. of 121oC. Exposure time: 15-20mins. Continue…

AUTOCLAVING This procedure kills even the highly resistant spores of Clostridium botulinum. Minimal time required. Most dependable sterilant for lab use. Continue..

AUTOCLAVING USES: To sterile glass wares, bacteriological media To destroy pathological cultures. Decontamination of reusable supplies and equipments. Decontamination of infectious waste.

FILTRATION for sterilizing heat sensitive liquids. Microorganisms are not destroyed rather they are removed. Bacterial filters are used for this purpose. Most commonly used filters composed of Nitrocellulose and has a pore size of 0.22 um. This size will retain all bacteria and spores. Continue...

FILTRATION Liquid pharmaceutical preparations e.g. solution, IV fluids are often sterilized by filtration. For sterilization of large volume of heat sensitive liquids i.e. vitamin, hormone, enzyme, antibiotic etc.

FILTRATION ASSEMBLING A flask with side arm attached to rubber tube is connected with vacuum pump. Bacterial filter is present on the top. Vacuum is attached because under ordinary gravitational force, there is no pressure and the liquid can not pass through the pores of the filter. Continue…

FILTRATION ASSEMBLING

RADIATION For heat sensitive solids. Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are low energy rays with poor penetrative power e.g. UV-rays. Ionizing rays are high-energy rays with good penetrative power e.g. Gamma rays and X-rays. Since radiation does not generate heat, it is termed “Cold Sterilization".

Interfere with DNA replication. UV light creates thymine dimers UV-RAYS Interfere with DNA replication. UV light creates thymine dimers

UV-RAYS Greatest antimicrobial activity occurs at 250-260nm. Bacterial spores are quite resistant and require a dose up to 10 times greater than the vegetative bacteria. Rays are harmful to skin & eyes. It doesn't penetrate glass, paper or plastic. Used in hospitals to kill air-borne organisms esp. in operating rooms when they are not in use.

X-Rays (Roentgen Rays) Have higher energy and penetrating power than UV radiation. Damages DNA. For sterilization of heat sensitive solids such as sutures, surgical gloves and plastic items such as syringes.

Disrupts DNA and RNA in living organisms. GAMMA IRRADIATION Disrupts DNA and RNA in living organisms. Have shorter wavelength and higher energy. Great penetration. Co-60 is used which produces gamma rays. Materials that can be sterilized include; Media for growth of microorganisms. All disposable plastic materials. Syringes etc.

Mechanism of DNA disruption by X-Ray & Gamma Rays

LOW TEMPERATURE slows the growth of microbes refrigeration 0-15oC freezing <0oC used to preserve food, media and cultures

DESICCATION gradual removal of water from cells leads to metabolic inhibition not effective microbial control many cells retain ability to grow when water is reintroduced

LYOPHILIZATION Freeze drying Process of drying in which water is sublimed from the product after it is frozen. In this condition, lyophilized cultures of microorganisms remain viable for many years. PHARMACEUICAL USE: Used frequently for drugs of poor stability. Drugs are reconstituted into solution prior to injection.

REFRENCES ALCAMO, fundamentals of microbiology, 6th Edition, CH# 21 PELCZAR. Microbiology