Agricultural Microbiology Course PDF

Agricultural Microbiology Course 1 Sterilization  Table of Contents 2.1 Physical Methods 01 Definition 02 Methods 2.2 Chemical Methods 1 Definition  Disinfection VS Sterilization Disinfection...

Agricultural Microbiology Course 1 Sterilization  Table of Contents 2.1 Physical Methods 01 Definition 02 Methods 2.2 Chemical Methods 1 Definition  Disinfection VS Sterilization Disinfection Sterilization is defined as a process of complete is defined as a process of complete elimination of vegetative forms of elimination or destruction of all forms microorganisms except the bacterial of microbial life (i.e., both vegetative spores from inanimate objects. and spore forms), which is carried out by various physical and chemical methods.  Antisepsis VS Decontamination Antisepsis Decontamination Antisepsis is a process of removal of Decontamination is the process of germs from the skin. When it is related to removal of pathogenic microorganisms the patient’s skin, it means disinfection of from objects so that they are safe to living tissue or skin. When it is related to handle. the health care worker, it means reduction or removal of transient microbe from the skin.  Germicide VS cleaning Germicide cleaning Germicide: It is the agent that destroys Cleaning is defined as removal of visible germs. It includes both antiseptics and soil (e.g., organic and inorganic disinfectants. The type of microorganism materials) from the surfaces and is identified from the prefix (e.g., objects. Technically, it achieves virucide, fungicide, bactericide, sporicide, and tuberculocide). minimum reduction of ≥1 log CFU of microorganisms.  Sterilization uses Food processing: to destroy all harmful microbes which cause food spoilage Medical uses:sterilize surgical instruments to prevent infection causes. Isolation, purification and growing microorganisms. 2 Methods  METHODS OF STERILIZATION Physical methods Chemical methods  First: Physical Methods  Physical methods. Thermal Radiation Filtration (Heat) methods methods methods. (a) Thermal (Heat) methods  Flaming Dry Heat Physical Thermal (Heat) Sterilization: methods Hot air oven methods. Moist Heat Sterilization:  First: Dry Heat : Sterilization. The use of dry heat is based on the removal of the water content of microbes and subsequent oxidation. Hot Air Sterilizer Flame 160 °C/1h. Used for ○ Cotton plugged glass ware ○ Inoculation needles ○ High with stand chemicals ○ Glass slides ○ Tube mouth 180 °C/ 30 min ○ Surgical instruments ○ Glass equipment's Thermal (Heat) methods Dry Heat Sterilization: Boiling Moist Heat at a Temperature of 100°C: Moist Heat Tyndallisation Sterilization: Dry saturated steam – Autoclaving.  Second: Moist heat Sterilization. : The heat conductivity of water is several times higher than that of the air, therefore heat sterilizes 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.  2. Sterilization at 100°C / 20 min./ 3 days Tyndallisation (intermittent sterilisation) Arnold steam sterilizer 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 20 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. Vegetative forms are destroyed First It is used for sterilizing: during the heat treatments. day Protein media Skim milk agar Endospores which can germinate during Second polysaccharides the incubation period are destroyed day during the consecutive heat treatments A confirmatory step; This way, after the Third third day of heat treatment, no living day cells remain in the substance.  Autoclaving To achieve sterilization, generally 15 -20 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 practice 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.  Advantages of Autoclave Temp. > 100 C therefore spores killed. Condensation of steam generates extra heat. The condensation also allows the steam to penetrate rapidly into porous materials. Note: for all invasive procedures at operating room or clinics, autoclavable equipments should be used.  Monitoring of Autoclaves 1. Physical method: use of thermocouple to measure accurately the temperature. 2. Chemical method: it consists of heat sensitive chemical that changes color at the right temperature and exposure time. e.g. a)- Autoclave tape 3. Biological method: where a spore-bearing organism is added during the sterilization process and then cultured later to ensure that it has been killed. Spore test ( biological indicator) (b) Radiation method  Physical methods. RADIATION STERILIZATION: Gamma ray Ultraviolet Sterilizer Irradiation  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 radiation 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 (c)Filtration 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  Physical methods. Filtration (size) Sintered glass filters Membrane filters are Seitz filters are Candle filters are are an alternative thin filters that are usually made of made of clay-like type of filter that are made of cellulose asbestos mud. made of glass Advantages of Filtration: Relatively inexpensive, except for those with the smallest pore sizes Filters do not clog easily Suitable for heat sensitive liquids as filters do not use heat They can filter large volumes of fluid reasonably fast Disadvantages of Filtration Filters can only work on liquids and gasses Autoclaving is usually cheaper than filtration since filters are expensive to replace, especially nano-filters Glass filters are very brittle and can break easily Membrane filters rupture easily The solution in Sietz Filters might get absorbed by the filter pad itself Clogging may occur Long process  Second: Chemical Methods  chemical methods Alcoholic Halogens Hydrogen Phenol Peroxide Alcoholic: 50-70% aqueous solution has excellent antiseptic properties. Due to the solubility of lipids in 50-95% ethanol solutions, biological membranes are disintegrated. 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 dehydration effect but does not coagulate the intracellular proteins. Phenol: Phenol denatures proteins, and irreversibly inactivates the membrane-bound oxidases and dehydrogenases. Ex: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene, chlorhexidine, chloroxylenol (Dettol). They act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses. 3- Halogens They are oxidizing agents and cause damage by oxidation of essential sulfydryl groups of enzymes. 4- Hydrogen Peroxide Hydrogen peroxide produces hydroxyl-free radical that damages proteins and DNA. Application: It is used at 6% concentration to decontaminate the instruments, equipments such as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal. Thanks!