STERILIZATION AND DISINFECTION

Microorganisms are the agents of contamination, infection, and decay. Hence it becomes necessary to remove them from materials and areas. Early civilization practiced salting, smoking, pickling and exposure to sunlight .

Sterilisation; the killing of all forms of microbial life. Disinfection: A chemical or a process used on nonvital objects to kill vegetative pathogenic organisms but not necessarily spores or viruses  Antiseptic: A chemical or a process that is applied to living tissues such as skin or mucous membrane to reduce the number of microbes on living tissues mainly skin. Cleaning ( Decontamination ) is the removal of foreign material (e.g., soil, and organic material) from objects and is normally accomplished using water with detergents. Bactericidal (microbicidal, germicidal): kills bacteria (and/or other microorganisms). Bacteriostatic (microbiostatic): prevents growth of bacteria (and/or other microorganisms

Disinfection A process which destroys only the vegetative forms of bacteria ( but not spores and viruses ) . purpose - prevent transmission of certain microorganisms with objects, hands or skin and prevent spreading the infection A disinfectant: any chemical substance with either a bactericidal (kills) or a bacteriostatic ( inhibits) action. Antiseptic -- Agent applied to living tissue Disinfectant -- Agent applied to inanimate surface

Factors that influence the degree of killing  Types of organisms  Number of organisms  Concentration of disinfecting agent  Presence of organic material (e.g., serum, blood)  Nature (composition) of surface to be disinfected  Contact time  Temperature  pH  Biofilms  Compatibility of disinfectants and sterilants

Types of organisms Organisms vary in their ability to withstand chemical and phisical treatmant, e.g., - Spores – have coats rich in proteins, lipids and carbohydrates - Mycobacteria – cell walls are rich in lipids - Biofilms - microorganisms living together in communities - Prions – the most resistant known organisms to the action of heat, chemicals, and radiation .

Dry Heat Simplest method is exposing the item to be sterilized to the flame e.g. Bunsen burner- for sterilizing bacteriological loops, knives, blades. Hot air oven expose items to 160°C for 1 hour. It has a fan to circulate air for even distribution of heat in chamber. Oven without fan is dangerous. Used for Metals, Glassware, Ointment, Oils, Waxes, Powders i.e. items that are lacking water. Incineration: This is an excellent method of destroying materials such as contaminated cloth, animal carcasses and pathological materials. DRY HEAT KILL MICROORGANISMS BY OXIDATION and needs more time

Moist heat kills microbes by denaturing proteins

Moist Heat 1-Boiling – quite common especially in domestic circumstances. Kills vegetative forms of bacterial pathogens like Mycobacterium tuberculosis which causes tuerculosis, Salmonella typhi ( causes typhoid fever), and Brucella (causes malta fever ). Hepatitis virus: Can survive up to 30 minutes of boiling. Endospores: Can survive up to 20 hours or more of boiling

2- Tyndallization The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, This is repeated for 3 successive days. The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat- sensitive vegetative (growing) stage, which can be killed by the next boiling step.

3- pasteurization does not kill spores 1- 63-65 C for 30 min. 2- HTST (High Temperature Short Time) flash method- 72 C for 15 sec. 3- UHT (Ultra-High Temperature) 135 C for 1-2 sec. Application: in the food industry, eliminating food-born pathogens without affecting the taste (e.g. UHT milk)

Diseases prevented by pasteurization include: Tuberculosis caused by Mycobacterium tuberculosis,  Brucellosis ( B. abortus) ,  Diphtheria ( Corynebacterium diphtheriae) ,less frequent because of vaccination.  Typhoid fever ( Salmonella typhi), and   Escherichia coli O157:H7 which causes diarrhea . All are non-spore forming bacteria!!!!

The Autoclave works under the same principle as the pressure cooker The Autoclave works under the same principle as the pressure cooker. Because of increased pressure the boiling point of water is >100°C. The autoclave is a tough double walled chamber in which air is replaced by pure saturated steam under pressure. The air in the chamber is evacuated and filled with saturated steam. The chamber is closed tightly the steam keeps on filling into it and the pressure gradually increases. The usual temperature achieved is 121 °C at a pressure of 15 pps.i. at exposure time of only 15-20 mins . By increasing the temperature, the time for sterilizing is further reduced.

Radiation: Used in two forms: Ionizing radiation- e.g. Gamma radiation: has short wavelength and greater energy than U.V radiation, therefore more effective. Used mainly in medical industry e.g. sterilization of disposable plastic syringes, gloves, specimens containers and Petri Dishes. Non ionizing radiation – e.g U.V radiation - Reduces the number of bacteria in the air because it can not penetrate solid surfaces . Generally used in irradiation of air in certain areas e.g. Operating rooms and T.B. laboratories.

Filtration Best known example is the membrane filter made from cellulose acetate. Generally removes most bacteria but viruses and some small bacteria e.g. Chlamydias & Mycoplasmas may pass through. Thus filtration does not technically sterilize items but it is adequate for circumstances under which it is used. Main use: for heat labile substances e.g. sera, antibiotics. The recommended size filter that will exclude the smallest bacterial cells is 0.22 micron HEPA : High Efficiency particulate Air filters are used for air filtration in rooms of immunocompromised and laboratories.

Chemical sterilization and disinfection Chemical agents act by Protein coagulation Disruption of the cell membrane Damage DNA and RNA

Water is needed for protein denaturation !!! Alcohols Both ethanol and isopropanol (rubbing alcohol) are widely used, normally at a concentration of about 70%. The optimum bactericidal concentration is 60%–90% solutions in water (volume/volume) Concentrations of 60% to 95% are effective. They are bactericidal and fungicidal but are not effective against endospores or non-enveloped viruses. Uses: Disinfection of clinical thermometer. Disinfection of the skin – Venipuncture Water is needed for protein denaturation !!!

Aldehydes Formaldehyde & Glutaraldehyde are frequently used Formaldehyde is bactericidal, sporicidal & has a lethal effect on viruses. Glutaraldehyde is effective against Tubercle bacilli, fungi and viruses Halogens: Iodine in aqueous and alcoholic solution has been used widely as a skin disinfectant. Actively bactericidal with moderate against spores Chlorine and its compounds have been used as disinfectants in water supplies & swimming pools( Na-hypochlorite)

Chemical agents Gases  Ethylene oxide - the most commonly used for sterilization - alkylates of nucleic acids in the cell wall - active against ALL microorganisms and spores Application: widely in hospitals for materials that cannot withstand steam sterilization

Heavy metals - bind irrevesibly to the sulfhydryl groups of proteins - rather bacteriostatic Application: -rarly used in clinical applications - silver nitrate (1% eyedrop solution) used in the prevention of eyes infections caused by Neisseria gonorrhoeae and Chlamydia trachomatis in newborns