Introductory Microbiology (BST 251-3) Lecture 03 PDF

Introductory Microbiology (BST 251-3) Dr. Samanthi Udayangani Kumari  Principles and methods of sterilization 1. Physical Methods and their mode of action 1. Heat 1. Dry heat – Hot Air Oven 2. Incineration 3...

Introductory Microbiology (BST 251-3) Dr. Samanthi Udayangani Kumari  Principles and methods of sterilization 1. Physical Methods and their mode of action 1. Heat 1. Dry heat – Hot Air Oven 2. Incineration 3. Moist heat- Autoclave, Pressure Cooker 4. Tyndallization (fractional sterilization 2. Filtration - Types of filters , Laminar air flow 3. Radiation methods: UV radiation, X- rays, etc. 2. Chemical methods: Definition of terms – disinfectants, antiseptics, Sanitizers microbicides – bactericide, virucide, fungicide and sporicide, microbistatic - bacteriostatic and fungistatic agents. Use and mode of action of – alcohols, aldehydes, halogens, phenols, heavy metals, Detergents: quaternary ammonium compounds.  Sterilization Physical Methods Chemical Methods Heat Filtration Radiation Disinfectants Antiseptics Chemotherapeutic agents  Introduction Microorganisms are omnipresent Most of them are well adapted to various extreme environments Therefore they cause harm to human health and property Since they cause contaminations, infections and decay, it becomes necessary to remove or destroy them from materials and from areas This is the objective of Sterilization  Sterilization Is the process of killing, inactivating or removing all microorganisms from a given product or surface either in the vegetative or spore state Destruction of all forms of life, including the bacterial spores, viruses and prions Variety of chemical and physical methods are using  Disinfection Is the process of destroying harmful pathogens in their vegetative state and reducing the number of microorganisms to levels that no longer pose a threat to human health. The purpose of doing this is to prevent transmission of certain microorganisms with objects, hands or skin and prevent spreading the infection. Eg: – Drinking water treatment – Pasteurization of milk or foods – Disinfection of swimming pools – Removal of mold contaminated building materials  An ideal sterilant….. Is the one that could function effectively and efficiently in all circumstances Should possess the following characters  Should be capable of destroying all types of microorganisms including viruses, bacteria and fungi  Should not adversely affect medical instruments.  Should act rapidly, allowing use of instruments meant for various sterilization and disinfection methods.  Methods of sterilization The basis for the difference between microorganisms with regard to their amenability to sterilization methods is largely attributed to their morphology (structure) and biochemical composition. Eg. 1. Thick walled spores are substantially more resistant than thinner walled vegetative cells 2. Microorganisms lack walls are more susceptible than those with walls Appropriate choice of disinfection or sterilization strategy must consider a number of variables including, – Human toxicity – Cost – Duration of activity – Suitability of the treated item to withstand the given disinfection method  1. Physical Methods 1. Heat – Sun drying – Air drying – Dry heat – Moist heat  Sun drying – Sunlight plays an important role in the spontaneous sterilization since it shows appreciable anti-bacterial activity under natural conditions. The action is primarily due to its content of ultraviolet rays. – Direct sunlight has an active germicidal effect due to the combined effect of UV and heat rays.  Air drying – Drying is gradual withdrawal of water from cells. – Moisture is essential for the growth of microorganisms. Water makes up to 80% of the weight of bacteria. Hence drying in air has a deleterious effect on bacteria – Drying concentrate the cellular solutes leading to metabolic inhibition. – But spores are not affected by drying. Hence this method is not practical for spore forming bacteria.  Use of Heat – Heat acts by disrupting membranes and denaturing proteins and nucleic acids. – Heat is the most effective method of sterilization. – Materials that get damaged by heat can be sterilized at low temperatures. – The bacteriological loops is still sterilized in this way.  Heat The factors influencing sterilization by heat are Nature of the heat – Dry heat or moist heat Temperature and time Number of microorganisms present Type of the material to be sterilized Characteristics of the microorganisms  Dry Heat – The killing effect of dry heat is due to protein denaturation, oxidative damage and toxic effect of elevated level of electrolytes. – This causes oxidation of cells, but this method is slow and requires a higher temperature for sterilization than moist heat – Different methods of sterilization by dry heat are; Flaming Incineration Hot air oven  Flaming – The flame of bunsen burner reaches very high temperature at its hottest point – Direct exposure to such intense heat ignites and reduces the microbes to ashes and gas – Incineration in a flame of electric heating coil is the most rigorous of all heat treatments – During flaming, materials like inoculating loop, wire, the tip of forceps, and spatulars are placed for a few seconds in a very hot flame of a bunsen flame till they become red hot Incineration( burn (something) completely) – This is a good method for the complete destruction of infectious materials such as syringes, needles, contaminated clothes and pathological materials  Hot air oven – The hot air oven provides another means of dry heat sterilization – This is the most widely used method of sterilization by dry heat – The holding period of 160 oC for 2 hour is used to sterilize glassware, forceps, scissors, scalpels, glass syringes etc. – The oven is usually heated by electricity, with heating coils in the wall of chamber – The fan fitted in the instrument ensures even distribution of air. – The oven should not be overloaded. – The materials should be arranged so as to allow free circulation of air in between the objects. – Glassware should be perfectly dry before placing in the oven. – The oven must be allowed to cool slowly for about two hours before the door is opened, since the glassware may crack due to sudden or uneven cooling Hot air oven  Moist heat – Moist heat acts by denaturation and coagulation of proteins – To kill vegetative bacteria, yeast and molds, a temperature of 80 oC for 5 – 10 minutes is required, fungal spores 80 oC for 30 minutes and bacterial spores 121 oC for 15 minutes in pressure of 15lbs/sq – Sterilization by moist heat is done by four methods Steam under pressure Stream at atmospheric pressure (non pressurized) Boiling Pasteurization Autoclave  Autoclave Autoclaving is the most effective and most efficient method of sterilization. Sterilization is achieved using moist heat (or steam) under pressure. As the pressure increases, the temperature at which water boils and the temperature of the steam produced both rise. It is not the pressure by itself that is killing microbes, but the increased temperature it produces. The most efficient pressure - temperature combination for achieving sterilization is 15 psi (15 pounds per square inch), which yields at 121°C. The duration of the process is adjusted according to the bulkiness of the items in the load and how full the chamber is. All autoclaves operate on a time / temperature relationship. Exposure to 121°C for 15 minutes is generally required for sterilization.  Health and commercial industries use an autoclave for this purpose and a comparable home appliance is the pressure cooker. The autoclave is an excellent device to sterilize heat-resistant materials such as glass ware, clothes, rubber, metalic instruments, liquids, paper, etc. But it is ineffective for sterilizing substances that repel moisture (oils, waxes, powders, etc)  Pasteurization – Fresh beverages such as milk, fruit juices, beer, vine are easily contaminated during collection and processing. – For pasteurization of milk, the temperature employed is either 63 oC for 30 minutes or 72 oC for 15-20 seconds followed by cooling quickly to 13 oC – The important aim of pasteurization is to prevent the transmission of milk borne disease agents – Pasteurization is also has the advantage of extending milk storage time. Tyndallization / Fractional Sterilization This involves heating the material at 100°C for 20 minutes on 3 consecutive days intermittent with incubation at 37°C. On subsequent exposure to heat, vegetative cells will be destroyed and any spores that are present will germinate during the incubation period and are destroyed during heating. Repeating the procedure for 3 days ensures germination of all the spores and the destruction of all vegetative cells. Used to sterilize culture media and solutions of chemicals which can't be heated above 100°C. 2.Filtration During filtration, the fluid is filtered through a layer of material with openings large enough for the fluid to pass through but too small for microorganisms to pass through Most filters are perforated by very precise, uniform pores. The pore diameters vary The filters with smallest pores help in removing viruses A sterile liquid filtrate is produced by suctioning the liquid through a sterile filter into a sterilized container Filtration helps to remove bacteria from heat labile liquids such as sera and solutions of sugars or antibiotics used for the preparation of culture media It has been employed as an alternative to sterilize milk and beer without altering their flavour and is also an important step in water purification.  Different filters There are different types of filters, namely; – Seitz filter - made of asbestos which is compressed into a filter pad which is used to sterilize fluids. – Berkefeld filter - a water filter made of diatomite (a siliceous sedimentary rock). – Chamberland - Pasteur filter - a water filter made of unglazed porcelain. – Membrane filter / Millipore filter - made of biologically inert cellulose esters, Teflon or polyvinyl chloride and the pore sizes are ranging from 0.01- 10μm. Membrane filtration Laminar Air Flow Laminar Flow Cabinets create particle-free working environments by projecting air through a filtration system and exhausting it across a work surface in a laminar or uni-directional air stream. Air flow - steady, uniform velocity. Room air is taken into the unit and passed through a pre-filter to remove gross contaminants (lint, dust etc). The air is then compressed and channeled up behind and through the HEPA filter (High Efficiency Particulate Air filter) in a laminar flow fashion, that is the purified air flows out over the entire work surface in parallel lines at a uniform velocity. The HEPA filter removes nearly all of the bacteria from the air. To qualify as a true HEPA filter, the air filter must be able to capture at least 90% of all particles 0.3 microns or larger in diameter that enter it. Laminar Flow Cabinets -both horizontal and vertical cabinets. There are many different types of cabinets with a variety of airflow patterns for different purposes Laminar air flow 3. Radiation Radiation is defined as energy emitted from atomic activities and dispensed as high velocity through matter or space. It can behave as waves or particles, depending upon the conditions Two types of radiation are used – Non-ionizing (low frequency); eg. Infrared and ultraviolet rays – Ionizing (higher frequency); eg. Gamma and x-ray etc  Ionizing radiation – Sterilization by ionizing radiation is a highly effective alternative for sterilizing materials that are sensitive to heat or chemicals – Because it sterilize in the absence of heat – X-rays, gamma rays and cosmic rays are highly lethal to DNA and other vital constituents – They have high penetrating power Non ionizing radiation – Here electromagnetic rays with wavelength longer than those of visible light are used – These are to a large extent absorbed as heat, hence infrared radiation can be consider as a form of hot air sterilization – Ultra-violet radiation is an effective means of sterilizing work surfaces glass and air, but does not penetrate – UV has a damaging effect on human tissue, including sunburn, retinal damage, cancer and skin wrinkles