Sterilization Lab Procedures PDF

 Sterilization DR. Samah Hashem  Table of Contents 2.1 Physical Methods 01 Definition 2.2 Chemical Methods 02 Methods 2.3 Mechanical Methods 1 Definition  Disenfiction VS Sterilization Dis...

 Sterilization DR. Samah Hashem  Table of Contents 2.1 Physical Methods 01 Definition 2.2 Chemical Methods 02 Methods 2.3 Mechanical Methods 1 Definition  Disenfiction VS Sterilization Disenfiction Sterilization Any process aimed at destroying or removing the infectious capability of pathogenic microbes that generally occur Sterilisation refers to the anti-microbial on inanimate objects, is called process during which all microorganisms are disinfection. The chemicals used for killed or eliminated in or on a substance by disinfection can be classified according to applying different processes. their chemical structure and their mode of action.  Sterilization uses Food processing: to destroy all harmful microbes which cause food poilage Medical uses: sterilize surgical instruments to prevent infection causes. Isolation, purification and growing microorgnisms: 2 Methods  Sterilization methods Physical Radiation Heat Nonionizing Dry heat Moist heat Ionizing Hot Air Boiling (100 oC) Sterilizer Pasteurization (100oC) Mechanical Chemical Bacterial filters Liquids ( Alcohols and phenols) Gaseous (formaldehyde)  First: Physical Methods  1- Heat First: Dry Heat: The use of dry heat is based on the removal of the water content of microbes and subsequent oxidation. Hot Air Sterilizer Flame 160 oC/ 30 min. Used for ○ Cotton plugged glass ware ○ Inoculation needles ○ High with stand chemicals ○ Glass slides 180 oC/ 1h ○ Tube mouth ○ Glass equibments ○ Surgical instruments  Second: Moist heat The heat conductivity of water is several times higher than that of the air, therefore heat sterilize es more quickly and effectively in the presence of hot water or steam than dry heat. Boiling Boiling is the simplest and oldest way of using moist heat. The temperature of boiling water does not exceed 100°C at normal atmospheric pressure. Heat resistant, endospore-forming bacteria can survive the 10-30-minute heat treatment of boiling, so no sterilizing effect can be expected from boiling.  Pasteurization Pasteurization is a widespread method – named after Louis Pasteur – to reduce the number of micro organisms found in different heat sensitive liquids. Milk can be pasteurised by heating to 65°C for 30 minutes or to 85°C for 5 minutes. During ultra- pasteurisation milk is heat-treated at 135-150°C for 2 minutes in a heat exchanger. The temperature and time used for pasteurisation are suitable to control the presence of some pathogenic bacteria, however endospores and cells of heat resistant bacteria e.g. Mycobacterium species, can survive  1. Autoclaving To achieve sterilisation, generally 15 minutes of heat treatment at 121°C under 1.1 kg/cm2 pressure has to be applied. Most microbes are unable to tolerate this environment for more than 10 minutes. However, the time used for sterilisation depends on the size and content of the load. Autoclaves Operation of Autoclaves  How to use Autovalves 1- Open the lid of the autoclave and check that there is sufficient amount of distilled or deionised water in it. 2. Place the correctly packaged materials (e.g. laboratory equipment, culture medium in a flask) into the chamber of the autoclave. Stick a piece of autoclave indicator tape onto the surface of materials! 3. Close the lid of the autoclave. 4. Turn on the heating of the autoclave (the indicator lamp is lit). 5-. check the temperature and pressure increase inside the chamber of the autoclave. 6. The sterilisation time (15 minutes or more) begins only when the temperature equalization (to 121°C) in the chamber has occurred. 7. Turn off the power switch of the autoclave when the sterilisation cycle/period has ended and Allow the device to cool down to at least 60-70°C.  Autoclave uses Autoclaves are widely used in: 1- Micro- biological practise mainly for sterilisation of culture media, 2- Glassware and heat-resistant plastic products before their use, 3- For contaminated materials prior to disposal as municipal solid waste. 4- Tyndallisation (intermittent sterilisation) Sterilization at 100oC / 20 min./ 3 days Is an old and lengthy method of heat sterilisation named after John Tyndall. During this method, a medium or solution is heated to a temperature at 100 C for 30 minutes for three successive days, and the substances are placed in an incubator at 37°C or stored at room temperature in the intermittent periods. First Vegetative forms are destroyed during It is used for sterilizing: day the heat treatments. Protein media Skim milk agar Second Endospores which can germinate during the incubation period are destroyed polysaccharides day during the consecutive heat treatments Third A confirmatory step; This way, after the third day of heat treatment, no living cells day remain in the substance. 2- Radiations Other forms of energy [e.g. ultraviolet (UV) and ionizing radiation] Very strong "germicidal" effect can be achieved around 265 nm, because maximum UV absorption of DNA occurs at this wavelength. The main cause of cell death is the formation of pyrimidine dimers in nucleic acids UV (germicidal) lamps are widely used in hospitals and laboratories (e.g. in biological safety cabinets) for decontamination of air and any exposed surfaces. The disadvantage of the use of UV ra- diation is that it does not penetrate through glass, dirt films, water, and other substances.  Gamma radiations Among the high-energy ionizing The advantage of gamma radiation: radiation, γ-rays from radioactive 1- is its deep penetration through the nuclides 60Co are generally used for: packaging. 1- sterilisation of disposable needles, Its disadvantage is: syringes, bandages, medicines and the scattering in all directions, which certain food (e.g. spices). requires special circumstances for application  Second: Chemical Methods  It is used to inhibit or kill microbes. Some of the antimicrobial agents only inhibit the growth of microorganisms (e.g. bacteriostatic, fungistatic, and virostatic compounds) while others kill them (e.g. bacteriocidal, fungicidal, and virocidal agents).\ They should not be toxic to higher organisms, They should not enter detrimental reactions to the materials being treated with, they should not be bio- degradable, They should be environmentally friendly, easy to apply and economical.  Ethyl Alcohol ○ The action mechanism of alcohols depends on the applied concentration. ○ Alcohols pass through the cell membrane with altered permeability, denature the proteins inside the cell and have a dehydration effect as well. ○ Absolute alcohol (100% ethanol) provides the best de- hydration effect but does not coagulate the intracellular proteins. ○ 70% dilution of alcohols is the most effective way to kill the vegetative forms of bacteria and fungi, but less effective against spores. 2- Phenol called carbolic acid. Phenol denatures proteins, and irreversibly inactivates the membrane-bound oxidases and dehydrogenases. It is used in 2-5%. 3- The halogens (F, Cl, I, Br) and their derivatives are very effective disinfectants and antiseptic agents; mainly their non-ionic forms have antimicrobial activity. 4- Iodine is also a widely used disinfectant and antiseptic agent for washing hands before surgery. 5- Aldehydes, such as formaldehyde and glutaraldehyde, are broad-spectrum disinfectants. They are used for decontamination of equipment and devices.  Third: Mechanical Methods During filtration, liquids or gases are pressed through a filter, which (depending on its pore size) retains or adsorbs (e.g. asbestos filter pads) microbes, thereby the filtrate becomes sterile. The pore diameter of filters should be chosen carefully so that bacteria and other cellular components cannot penetrate.  Filtration uses: Air and Gases Heat labile liquids such as ○ Enzymes ○ Toxins ○ Urea ○ Vitamins Retaining bacteria for viral studies