Sterilization PDF

Sterilization Sub: BP3005 Pharmaceutical Microbiology Unit II Department of Pharmaceutical Sciences & Technology  Terminologies Sterilization: Latin sterilis, unable to produce offspring or barren It is the process by whi...

Sterilization Sub: BP3005 Pharmaceutical Microbiology Unit II Department of Pharmaceutical Sciences & Technology  Terminologies Sterilization: Latin sterilis, unable to produce offspring or barren It is the process by which all living cells, viable spores, viruses, and viroids are either destroyed or removed from an object or habitat Sterilizing agent Sterilant Disinfectant: killing, inhibition, or removal of microorganisms that may cause disease Destruction of potential pathogens (non-endospore forming), but disinfection also substantially re- duces the total microbial population usually Disinfecting agents are chemical, used to carry out disinfection and are normally used only on inanimate objects Disinfecting agents: chemicals, radiation, boiling water, steam etc.  Antiseptics: When chemical or other treatment directed at living tissue They also reduce the total microbial population Same chemical agent may be disinfectant for one use and an antiseptic for another (e.g. Dettol/ Savlon) Because they must not destroy too much host tissue, antiseptics are generally not as toxic as disinfectants Sanitization Lowers microbial count to safeguard public health and minimize disease transmission Example: spraying if disinfectant like 1% sodium hypochlorite solutions against COVID Degerming: Mechanical removal of microbes rather than killing e.g. swabbing of skin with alcohol  Rate of Microbial Death A microbial population is not killed instantly when exposed to a lethal agent Microbial population die at a constant rate Population death, like population growth, is generally exponential or logarithmic Time Deaths/ Number of Survivors (min) min 0 0 1,000,000 1 900,000 100,000 2 90,000 10,000 3 9000 1000 4 900 100 5 90 10 6 9 1 Fig source: Mirobiology by Tortora 13th Edition  Factors influencing effectiveness of antimicrobial treatments ▪ Number of microbes: ↑microbial load, ↑ time to eliminate entire population ▪ Population composition: ▪ The effectiveness of an agent varies greatly with the nature of the organisms being treated ▪ Bacterial endospores are more resistant compare to vegetative form ▪ Younger cells are usually more readily destroyed than mature organisms ▪ Influence of environment: ▪ Presence of organic matters inhibits action of chemical antimicrobials ▪ Biofilms resists biocides (use of warm disinfectants) ▪ Nature of suspending medium → affects heat treatments (fats/ proteins) ▪ Time of exposures: Chemical antimicrobials often require extended exposure to affect more-resistant microbes or endospores ▪ Microbial characteristics  Actions of Microbial Control Agents 1. Alteration of Membrane Permeability: ▪ plasma membrane of microbes is the target of many microbial control agents ▪ Damage to the lipids or proteins of the plasma membrane by antimicrobial agents causes cellular contents to leak into the sur-rounding medium and interferes with the growth of the cell 2. Damage to Proteins and Nucleic Acids: ▪ Enzymes, which are primarily protein, are vital to all cellular activities ▪ Bonds which helps to form the shape of proteins (e.g. -SH-, -H- are susceptible to breakage by heat or certain chemical ▪ Breakage of these bonds results in denaturation of the protein ▪ Damage to these nucleic acids by heat, radiation, or chemicals lethal to the cell: As the cells can no longer replicate, nor can it carry out normal metabolic functions such as the synthesis of enzymes  Different types of Sterilization Methods 1. Physical Method A. Heat i. Moist Heat ii. Dry Heat B. Filtration C. Irradiation 2. Chemical Method A. Gaseous B. Liquid  1. Physical Method of Sterilization ❑ Heat ▪ Kill microorganisms by denaturing their enzymes ▪ Two types: 1. Moist Heat 2. Dry Heat ▪ It is essential to have a precise measure of the heat-killing efficiency ▪ Thermal death point (TDP): the lowest temperature at which a microbial suspension is killed in 10 minutes ▪ Thermal death time (TDT): It is the shortest time needed to kill all organisms in a microbial sus-pension at a specific temperature and under defined conditions ▪ Decimal reduction time (D) or D value: is the time required to kill 90% of the microorganisms or spores in a sample at a specified temperature  A Theoretical Microbial Heat-Killing Experiment ❑ A microbial population is not killed instantly when exposed to a lethal agent. Population death, like population growth, is generally exponential or logarithmic—that is, the population will be reduced by the same fraction at constant intervals ❑ If the logarithm of the population number remaining is plotted against the time of exposure of the microorganism to the agent, a straight line plot will result ❑ When the population has been greatly reduced, the rate of killing may slow due to the survival of a more resistant strain of the microorganism  In a semilogarithmic plot of the population remaining versus the time of heating the D value is the time required for the line to drop by one log cycle or tenfold D values are used to estimate the relative resistance of a microorganism to different temperatures through calculation of the z value The z value is the increase in temperature required to reduce D to 1/10 its value or to reduce it by one log cycle when log Dis plotted against temperature Another way to describe heating effectiveness is with the F value The F value is the time in minutes at a specific temperature (usually 250°F or 121.1°C) needed to kill a population of cells or spores  In a semilogarithmic plot of the population remaining versus the time of heating the D value is the time required for the line to drop by one log cycle or tenfold D values are used to estimate the relative resistance of a microorganism to different temperatures through calculation of the z value The z value is the increase in temperature required to reduce D to 1/10 its value or to reduce it by one log cycle when log Dis plotted against temperature Another way to describe heating effectiveness is with the F value The F value is the time in minutes at a specific temperature (usually 250°F or 121.1°C) needed to kill a population of cells or spores  Dry Heat Sterilization ❑ Sterilizing by dry heat is accomplished by conduction ❑ The heat is absorbed by the outside surface of the item, then passes towards the centre of the item, layer by layer ❑ Dry heat does most of the damage by oxidation of cell constituents and denaturation of proteins ❑ Examples of dry heat sterilization: 1. Incineration 2. Flaming 3. Hot air oven ❑ Dry air heat is less effective than moist heat ❑ Dry heat does not corrode glassware and metal instruments as moist heat does, and it can be used to sterilize powders, oils, and similar items ❑ Most laboratories sterilize glass petri dishes and pipettes with dry heat ❑ Not suitable for heat-sensitive materials like many plastic and rubber items ❑ Hot Air Oven ▪ It employs 160-180 °C for 1-2 hrs ▪ Articles to be sterilized are first wrapped or enclosed in containers of cardboard, paper or aluminum ▪ Then, the materials are arranged to ensure uninterrupted air flow ▪ Oven may be pre-heated for materials with poor heat conductivity ▪ The temperature is allowed to fall to 40 °C, prior to removal of sterilized material Hot Air Oven  Moist Heat Sterilization ❑ Moist heat kills microorganisms primarily by coagulating proteins (denaturation) ❑ Breakdown of H-bonds which hold the proteins in their 3D structure ❑ One type of moist heat “sterilization” is boiling ❑ Boiling kills vegetative forms of bacterial pathogens, many viruses, fungi and their spores within about 10 minutes ❑ But some bacterial endospores can resist boiling for more than 20 hours: Hence boiling is not always reliable ❑ Reliable sterilization with moist heat requires temperatures above that of boiling water ❑ These high temperatures are most commonly achieved by steam under pressure in an autoclave  Types of Autoclave A Horizontal Autoclave A Vertical Autoclave and its components  Pasteurization ❑ The intent of pasteurization of milk was to eliminate pathogenic microbes ❑ It involved brief heating at 55 to 60°C to destroy these microorganisms ❑ Pasteurization does not sterilize a beverage, but it does kill any pathogens present and drastically slows spoilage by reducing the level of nonpathogenic spoilage microorganism ❑ Milk can be pasteurized in two ways: ❑ Older Method: Milk was hold at 63°C for 30 minute ❑ Flash pasteurization or high-temperature short-term (HTST) pasteurization: ❑ Quick heating to about 72°C for 15 seconds, then rapid cooling ❑ Milk flows continuously past a heat exchanger ❑ Lowers total bacterial counts, so the milk keeps well under refrigeration ❑ Ultra high-temperature (UHT) sterilization: ❑ Milk and milk products are heated at 140 to 150°C for 1 to 3 seconds ❑ UHT-processed milk does not require refrigeration and can be stored at room temperature for about 2 months without flavor change  Filtration ❑ Do not directly destroying contaminating microorganisms ❑ Filtration removes microorganisms ❑ 2 types of filters: 1. Depth Filter: ▪ Consist of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter ▪ The solution containing microorganisms is sucked through this layer under vacuum ▪ Microbial cells are removed by physical screening or entrapment and also by adsorption to the surface of the filter material ▪ Examples: diatomaceous earth (Berkefield filters), unglazed porcelain (Chamberlain filters), asbestos etc 2. Membrane filters: ▪ These circular filters are porous membranes, a little over 0.1 μm thick ▪ Made of cellulose acetate, cellulose nitrate, poly-carbonate, polyvinylidene fluoride, or other synthetic materials ▪ Wide variety of pore sizes are available ▪ Membranes with pores about 0.2 μm in diameter are used to remove most vegetative cells  Membrane Filter Cross section of the membrane filtering unit A complete filtering setup Syringe Filters Bacillus megaterium on an Ultipor nylon membrane Enterococcus faecalis resting on a polycarbonate with a bacterial removal rating of 0.2 μm (×2,000) membrane filter with 0.4 μm pores (×5,900)  HEPA Filters ❑ Used for air borne microorganisms ❑High-efficiency particulate air (HEPA) filters remove 99.97% microorganisms larger than 0.3 μm ❑ Laminar flow biological safety cabinets force air through HEPA filters, then project a vertical curtain of sterile air across the cabinet opening ❑ This protects a worker from microorganisms being handled within the cabinet and prevents contamination of the room Types of HEPA filters/ Laminar flow hoods Vertical Laminar Flow unit/ Biological Safety Cabinets