Basic Microbiology Techniques Lecture 16 PDF

Basic Microbiology Techniques Lecture 16 Sterilisation BMT_LT16_AS/CE_ExamSeries_Sep24 1  Objectives 1. Explain some common terms used in the control of microbial growth. 2. List various methods of sterilization such as heat, chemical, radiation and filtr...

Basic Microbiology Techniques Lecture 16 Sterilisation BMT_LT16_AS/CE_ExamSeries_Sep24 1  Objectives 1. Explain some common terms used in the control of microbial growth. 2. List various methods of sterilization such as heat, chemical, radiation and filtration 3. Outline the procedures of different sterilization methods 4. Outline the principles of different sterilization methods 2  Some common terms used Sterilization Disinfection Anti-microbial agents Microbiostatic agents Microbicidal agents Physical, chemical and mechanical methods 3  Modes of Microbial Control Microbicidal kills microbes reduces cell number Modes of action Microbiostatic Stops / inhibits growth The number of the viable cells remains constant. 4 Classification of Sterilization Methods Physical Chemical Mechanical Heat Liquid Filtration Sterilants Gaseus Radiation Sterilants 5  4 main types of sterilization covered in this lecture Heat – Physical Radiation - Physical Chemical Sterile/membrane filtration - Mechanical Sterilization is a form of microbial control but not all microbial control is sterilization 6  Heat Sterilization – Physical Method Most effective and widely used It is microbicidal through the destruction of enzymes and other essential cell constituents. The effects occur more rapidly in a fully hydrated state, (requires lower temperature and less time) 7 Different modes of Heat Sterilization Moist heat Steam under pressure Dry heat Red Heat Hot air with low moisture content (Oven) Ethanol flaming 8  Moist Heat Control Boiling - not sterilisation 100°C for at least 20 minutes, destroys vegetative cells of pathogens but not endospores Pasteurisation - not sterilisation Heat treatment process for food e.g. Heating at 135°C for 15 seconds or 63°C for 30 minutes to kill vegetative cells of most pathogens. Steam under Pressure (Autoclaving) - sterilisation 121°C at a pressure of 15 psi for 15 minutes, destroys all viable microorganisms including endospores. 9 Steam under Pressure / Steam Sterilization / Autoclaving – Moist Heat Sterilisation (above 100C) Water boils at 100°C under normal atmospheric pressure (760 mm Hg) Boiling point increases when the pressure is increased. Autoclave: Pressure at 15 PSI (775 mm Hg), water boils at 121°C Steam under pressure has higher penetrating power, kills all viable microbes including endospores and viruses Condensed liquid ensures moist killing of the microbes. Autoclave ensures complete sterility, used for – Sterilization of culture media, glassware, etc. – Decontamination of instruments and culture media contaminated by microorganisms. 10  Dry Heat Sterilization 1. Process of destroying microorganisms by applying moisture-free heat 2. Based on the principle of conduction: heat passed from outer surface to the next layer inside the item until the entire item reaches the temperature needed for sterilization. 3. Dry heat destroys microorganisms by denaturation and lysis of proteins. 4. Dry heat sterilization is suitable for moisture-sensitive substances and materials that cannot be or are difficult to be sterilised by moist-heat. – Powders – Oily materials – Petri dishes and pipettes 11  Hot Air Oven – Dry Heat Sterilisation Examples of temperatures and duration needed (the lower the temperature, the longer the duration needed):  170°C for 30 min  160°C for 60 min  150°C for 150 min Ovens Can be used for sterilization of glassware, Sterilization requires higher Petri dishes, and powder samples. temperature or longer duration 12 Red Heat / Ethanol Flaming – Dry Heat Sterilisation Sterilisation by red heat and ethanol flaming have been covered under streak plate and spread plate topics respectively Can you still remember what you learnt? 13  Comparison Moist Heat VS Dry Heat More effective, requires lower Requires higher temperatures temperatures and shorter and longer durations. durations. Does not corrode metals. Moisture may corrode some May cause thermal shock to materials like metal. glassware. Suitable for materials like Suitable for powders and oils. rubber, plastic, textile, heat stable liquids, and materials Not suitable for heat sensitive through which steam is materials. permeable. 14 Radiation - Physical Method 15  Radiation - Physical Method Non-ionising radiation (UV light and infrared ray) UV light (around λ = 260nm, most effective) Leads to abnormal bond formation in DNA which may result in mutation. Used for surfaces of biological safety cabinets (BSC), air and water Blocked by glass, water and dirt. Ionising radiation (X-rays, gamma rays, high-speed electrons) Effective against vegetative cells and endospores. But not always against viruses. Used on food, medicine and heat sensitive products (plastics) Causes damage and mutation to DNA, chemical changes in cell organelles and production of toxic substances. 16  Radiation - Physical Method Infrared (IR) Radiation Infrared Tunnel Radiation is absorbed and converted into heat https://www.ukessays.com/essays/ biology/development-infrared-ray- energy. sterilization-2915.php A tunnel containing an IR source is used. The instruments and glassware to be sterilized are kept in a tray and passed through the tunnel on a conveyer belt, moving at a controlled speed. During this movement, the instruments will be exposed to the radiation, which will result in a temperature of about 180°C for about 8 min. IR is applicable for mass sterilization of packaged Infrared items like syringes and catheters. Chamber https://heaterlamps.en.made-in- china.com/product/BvmQXewCrKrT/ China-Multifunctional-IR-Sterilization- Chamber.html 17 Example of an Ionising Radiation Facility https://solasindustries.com/gamma-sterilization/ 18  Summary Physical Antimicrobial Methods Moist Dry heat Radiation heat Boiling Red heat Ionising Pasteurisation Ovens Non-ionising Steam under pressure Ethanol Flaming (UV, Infrared) 19 Chemical Sterilization: Gaseous or Liquid 20  Chemical Sterilization The process of removal of microorganisms by the use of chemical microbicidal agents Used for heat-sensitive materials like plastics, fibre optics, and biological specimens Chemical (either in liquid or gaseous state) used for sterilization. The chemical method of sterilization can be categorized as liquid and gaseous sterilisation. 21  Gaseous Sterilization The process of exposing equipment or devices to different gases in a closed heated or pressurized chamber. More effective than liquid as gases can penetrate tiny spaces/openings and provide more effective results. Gases are commonly used along with heat treatment. Issue of release of some toxic gases during the process which needs to be removed regularly from the system. 22  Liquid Sterilization This process of sterilization which involves the submerging of equipment in the liquid sterilant to kill all https:// www.wikihow.com/ Sterilize-Medical- viable microorganisms and Instruments their spores. Not as effective as gaseous sterilization but appropriate in conditions where a low level of contamination is present. https://www.steris.com/healthcare/ knowledge-center/infection- prevention/cleaning-and-disinfecting- surgical-instruments 23  Hypochlorite Sodium hypochlorite, commonly known as bleach, frequently used as a disinfecting agent. Submerging devices for a short period in liquid bleach might kill some pathogenic organisms but to reach sterilization, submersion for 20-24 hours is required. It is an oxidizing agent and acts by oxidizing organic compounds. It is corrosive to metallic equipment. Appropriate concentrations of hypochlorite can be used for the disinfection of workstations and even surfaces to clean blood spills and other liquids. 24 Relative size of human cells, bacteria and viruses 25  Sterile/ Membrane Filtration – Mechanical Method Membrane filtration (microfiltration) - For heat sensitive culture media Vacuum filtration tower 26 Source: Membrane Filtration assessed on 05 Mar 2017 http://www.sswm.info/content/membrane-filtration  Membrane Filtration – Mechanical Method 1. Removes rather than destroys microorganisms. 2. The filter prevents both living microbes and non-living particles from passing through, can be used for clarification and sterilization of liquids and gases. 3. It involves filtering/sieving and trapping within the matrix of the filter material. 4. Filters membranes have tiny pores - allow liquid to pass through but prevent bigger particles such as bacteria from passing through. 5. Applications: sterilization of heat-sensitive liquids such as injections and ophthalmic solutions as well as air and other gases for supply to aseptic areas 27  Membrane Filtration - Sterilization of liquids Membrane filters Assembled pressure-operated filter holders for syringe mounting and in-line use Vacuum filtration tower devices for filtration of liquid Filtration under pressure is generally considered most suitable The filtrate is sterile after filtration 28  4 main types of sterilization covered in this lecture Heat – Physical Radiation - Physical Chemical Sterile / Membrane Filtration - Mechanical 29  References Foundations in Microbiology, Basic Principles. Talaro K. P. 5th edition. (McGraw-Hill) – Chap 11 Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. Willey, Sherwood & Woolverton: Prescott’s Principles of Microbiology, international edition, McGraw-HillHigher Education. Chapter 8 & https://microbenotes.com/physical-methods-of-sterilization/ 30