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Control of Microbial Growth Definition Methods Physical methods of sterilization Basis for selecting the method of sterilization Sterilization indicators Control of Microbial Growth: Introduction Early civilizations practiced salting, smoking, pickling, drying, and exposure of food and clothin...

Control of Microbial Growth Definition Methods Physical methods of sterilization Basis for selecting the method of sterilization Sterilization indicators Control of Microbial Growth: Introduction Early civilizations practiced salting, smoking, pickling, drying, and exposure of food and clothing to sunlight to control microbial growth. Use of spices in cooking was to mask taste of spoiled food. Some spices prevented spoilage. In mid 1800s Semmelweiss and Lister helped developed aseptic techniques to prevent contamination of surgical wounds. Before then: Nosocomial infections caused death in 10% of surgeries. Up to 25% mothers delivering in hospitals died due to infection Control of Microbial Growth: D efinitions Sterilization: Killing or removing all forms of microbial life (including endospores) in a material or an object. Heating is the most commonly used method of sterilization. Commercial Sterilization: Heat treatment that kills endospores of Clostridium botulinum the causative agent of botulism, in canned food. Does not kill endospores of thermophiles, which are not pathogens and may grow at temperatures above 45oC. Control of Microbial Growth: D efinitions Disinfection: Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases. Usually involves the removal of vegetative or non-endospore forming pathogens. May use physical or chemical methods.  Disinfectant: Applied to inanimate objects.  Antiseptic: Applied to living tissue (antisepsis).  Degerming: Mechanical removal of most microbes in a limited area. Example: Alcohol swab on skin.  Sanitization: Use of chemical agent on food-handling equipment to meet public health standards and minimize chances of disease transmission. E.g: Hot soap & water. Control of Microbial Growth: D efinitions Sepsis: Comes from Greek for decay or putrid. Indicates bacterial contamination. Asepsis: Absence of significant contamination. Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery. Aseptic techniques are also used to prevent bacterial contamination in food industry. Control of Microbial Growth: Definitions Bacteriostatic Agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them. Suffix stasis: To stop or steady. Germicide: An agent that kills certain microorganisms.  Bactericide: An agent that kills bacteria. Most do not kill endospores.  Viricide: An agent that inactivates viruses.  Fungicide: An agent that kills fungi.  Sporicide: An agent that kills bacterial endospores of fungal spores.  Sterilization: The freeing of an article from all living microorganisms including bacteria, spores, viruses and fungi. Disinfection: Removal of some types of pathogenic organisms usually not including spores (for inanimate surfaces) Antisepsis: Removal of some types of pathogenic organisms from the body surfaces usually skin and mucous membrane.  Overview of Various Microbial Control Methods 9 Factors influencing ability to kill microbes Strength of the killing agent Time that the agent has to act Temperature of environment – rate of microbe death doubles with every 10˚C rise in temp. Type of microbe Environment around the area to be decontaminated Number of microbes to be killed  Choice of method depends on practical issues such as ease of use or material compatibility  Physical Methods: Heat Advantages – Non-toxic – Quick – Cheap Disadvantages – Can only be used on heat-resistant materials – No use for many plastics, electronics, tarnishes some metals Some terms connected with Heat Heat: Kills microorganisms by denaturing their enzymes and other proteins. Heat resistance varies widely among microbes.  Thermal death point (TDP) is the lowest temperature at which all bacteria in a liquid culture will be killed in 10 minutes  Thermal death time (TDT) is the length of time required to kill all bacteria in a liquid culture at a given temperature  Decimal reduction time (DRT) is the length of time in which 90% of a bacterial population will be killed at a given temperature (especially useful in canning industry) Kinetics of thermal destruction Heat is frequently used to kill microorganisms. The thermal death of bacterial cells and their spores is logarithmic, i.e. the death is due to inactivation of a single molecule which is responsible for the reproduction and activity of the cell. K = _1_ log _No_ t Nt High load K = Thermal death rate No= Initial number of organisms Low load Nt = number of organisms at time t. Time (min) This is not the case for all microorganisms specially for bacterial spores. Deviation from linearity is due to: 1) Heat activation 2) Presence of more than one-thermo resistant variant in the population hump Straight line tailing Time (min)  A. Dry Heat kills by oxidation, requires greater temperature and exposure time than moist heat Examples: Red heat (heating till red hot) points of forceps Inoculating loops Flaming (passing the article through Bunsen burner) Glass slides Scalpels Cotton wool Mouth of culture cover slips stoppers tubes  Incineration o Incineration oxidizes the cell components to ashes or gas. o Incineration is used to destroy medical wastes and contaminated animals by heating in Furnace at 800˚C to 6500˚C. Medical waste Incinerator  Hot air oven o This is the main method of sterilization by dry heat. The oven is heated by electricity and the temperature of the air is maintained constant using thermostat. o Useful for sterilizing dry glassware, instruments, dry and oily based materials in sealed containers such as light and heavy kaolin, zinc oxide, talc and sulphonamides, fats and oils. o Hot air oven is effective at a holding period of 1 hr at 160˚C (R.P.) 1 hr at 170˚C (E.P.) 2 hrs at 170˚C (I.P.) Where the holding time is calculated when the temperature reaches 160 or 170˚C, respectively. Precautions: o Glassware should be dry o Powders sterilized by hot air oven must be in shallow layers (small volume) o After sterilization, the oven must be allowed to cool gradually for about 2 hrs before the door is opened.  Infra-red radiation o The infrared rays are directed on the object to be sterilized and the treatment is usually 20 min at 180˚C. o This method is used for sterilization of surgical instruments.  B. Moist heat kills by protein coagulation (denaturation) Moist heat can be employed: (1) At temperature below 100˚C Pasteurization Tyndallization  Pasteurization o Pasteurization Developed by Louis Pasteur that uses a brief heat treatment to reduce the number of spoilage organisms and kill pathogens o Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc. o Classic Method of Pasteurization: Milk was exposed to 65°C for 30 minutes.  Pasteurization o High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72°C for 15 seconds. o Ultra High Temperature Pasteurization (UHT): Milk is treated at 140°C for 3 seconds and then cooled very quickly in a vacuum chamber.  Advantage: Milk that has been treated in this way can be kept at room temperature for 2 months with only minimal changes in flavor. These processes will destroy the non-spore forming pathogens such as Salmonella, Mycobacterium, Brucella abortus that may be found in milk.  Tyndallization o For sterilizing serum or body fluids containing coagulable proteins that can be affected by heat. o Tyndallization can be attained by heating for 1 h at 56˚C on several successive days (up to 8 times). o Care must be taken not allow temperature to rise above 59˚C or else inspissations may occur. o It is suitable only to solutions in which bacterial spores will have a chance to develop during the period between the first and second heating. (2) Moist heat at a temperature of 100˚C Boiling at 100˚C o Heating for 5-10 min is sufficient to kill all non- sporing o Does not ensure sterility o Used for o Addition of 2% sodium carbonate promotes sterilization  Steaming at 100˚C o Used in the sterilization of sugar media and gelatin media which may be decomposed by higher temperatures. A Koch steam sterilizer is employed. o Articles can be sterilized in two ways: o By single exposure at 100˚C for 90 min o By intermittent exposure at 100˚C for 30 min on 3 successive days  Sterilization by heating with bactericidal o Dissolve or suspend the medicament in either 0.2% w/v chlorocresol or 0.002% w/v phenyl mercuric nitrate in water for injection and heat at 98-100˚C for 30 min o Used for sterilization of injection which is deteriorated when heated at 115˚C. (3) Moist heat at temperature above 100˚C (sterilization by autoclaving) o Water boils when its vapor pressure equals the pressure of the surrounding atmosphere. o This occurs at 100˚C for normal atmospheric pressure. o Thus, when water is boiled within a closed vessel at increased pressure, the atmosphere at which it boils and that of the steam it forms will rise above 100˚C.  Principle of autoclaving o In the autoclave, all parts of the load to be sterilized must be permeated by steam. o Ideally, the steam should be hot and saturated (i.e. at the point of condensing to liquid water) o Steam at certain temperature and pressure comes into contact with a cooler article o It rapidly condenses onto its surface & gives latent heat to the article o The article rapidly reaches the temperature of steam  Composition Autoclave consists of a vertical or a horizontal cylinder. One end has an opening which is meant for placing materials to be sterilized. The lid is provided with a pressure gauge, to measure the pressure A safety valve (discharge tap) is present to permit the escape of air from the chamber  Packaging for Autoclaving Be sure the material should be autoclaved – No volatile chemicals, sharps, red bag waste, radioactive materials Utilize containers and autoclave bags appropriate for autoclaving – Clear or orange bags Do not overfill containers Autoclave clean items and waste separately Do not allow material to be autoclaved to touch the sides or top of the chamber  Packaging for Autoclaving Load material to allow efficient steam penetration Check that all containers including bags are vented – Loosely close autoclave bags – Vent lids on bottles containing solutions o In autoclaving, four parameters to be considered: 1-Steam 2-Pressure 3-Temperature 4-Time o Air must be removed and steam must reach the pressure value for the required time at the required temperature. The directions for using the simple autoclave include several stages: o Air evacuation stage o Steam penetration stage o Holding stage o Chamber evacuation stage o Drying stage  Air evacuation o Air removed through discharge tap – The mixture of steam with air results in lower temperature being achieved at the chosen pressure – air pockets - no condensation or latent heat transfer - incomplete and patchy sterilization o Vacuum created (all the air has been eliminated from the autoclave)  Steam penetration o By closing the discharge tap, the steam takes place of the vacuum and penetrates all corners o once this state is achieved - penetration is complete  Holding time o The time necessary to maintain same state to complete sterilizing the object (destroys spores) (when the pressure reaches any of the mentioned) The minimum holding times are as follows: 2 min at not less than 134˚C (30 Ib per sq. in. gauge pressure) 12 min at not less than 121˚C (15 Ib per sq. in. gauge pressure) 30 min at not less than 115˚C (10 Ib per sq. in. gauge pressure)  Chamber evacuation o Turn off the heater and allow the autoclave to cool down until the pressure gauge indicates that the inside is at the atmospheric pressure (0 Ib per sq. in.) o If the tap is opened while the chamber pressure is still high and the pressure is reduced too rapidly, liquid media tend to boil violently and spill from their containers. o Leave the door open to dry the objects. o Autoclaving is the most reliable method and the most widely used for sterilization of culture media, pipette tips and surgical supplies, besides laboratory waste bags o Anhydrous materials (oil, greases, powders) cannot be sterilized by steam o Steam cannot penetrate hollow needles or instruments packed in moisture resistant materials  Deficiencies of the simple autoclave 1- The method of air discharge is insufficient. 2- Not furnished with a thermometer. 3- It lakes means of drying the load after sterilization. That is why we moved to Steam-Jacketed Autoclave Testing the efficiency of a sterilizer Two types of tests are possible: 1- Direct tests: (sterility tests on the products) 2- Indirect tests: a. Instrumental: Adequate instrumentation to ensure that the sterilizing temperature was maintained throughout the process for the proper time. b. Cultural: Preparation containing bacterial spores are placed into packs in the load and tested for sterility after exposure. The spores of Bacillus stearothermophilus are used. The selected spores are resistant than those of the most resistant pathogens Testing the efficiency of a sterilizer C- Chemical Chemical indicators: o The efficiency of sterilization is indicated by the change in the color or the melting point of the indicator substance (Browne tubes) also using autoclave tape  Radiation Acts By Destroying DNA or Damaging It Its Efficiency is Dependent on the Wavelength, Intensity, and Duration Ionizing radiation Non-Ionizing radiation X-rays Ultraviolet light Gamma rays Microwave Electron Beams Sterilization Disinfection  Ultraviolet Radiation Most lethal at wavelength of 260 nm that can be produced by mercury vapor lamps. This is the wavelength most actively absorbed by DNA. Once absorbed by DNA, it causes adjacent thymine molecules to dimerize (pyrimidine dimers) that could lead to inhibition of DNA replication.  Ultraviolet Radiation UV radiation cause the formation of thymine dimers on DNA.  Ultraviolet Radiation Its value is limited by its very low penetration as non passes dirt, glass, water, or other substances. If a surface is dusty, then complete inactivation of all microorganisms may not occur This type of radiation is also harmful if someone is directly exposed to it, as it may damage the skin and eyes. Due to its poor penetration, UV radiation is only useful for disinfecting outer surfaces.  UV Irradiation UV treatment system used to disinfect water.  Ionizing Radiation Unlike X-ray and electron beams which have too little penetration, gamma radiation can penetrate deeper into objects, and is used to sterilize food, drugs, plastic disposables and medical supplies. The source of these gamma rays is usually cobalt- 60 which has a half-life of 5 years. Ionizing radiation are very lethal to cells, their DNA being destroyed either by direct ionization or by the chemical effect of ionizing water. The time for sterilization is long about 2 days but radioactivity is never induced.  Gamma Irradiation (Cobalt 60) Materials which are sterilized using this type of radiation do not become radioactive, and irradiation of food does not change its nutritional value. But, due to initiation of some chemical reactions, the flavor of the food is often spoiled. (a) 51 Gamma radiation machine used to sterilize fruits, vegetables, meats, fish, and spices (b) Radora symbol  Cellular Effects of Radiation 52  Applications of Radiation Ionizing radiation: Alternative sterilization method Materials sensitive to heat or chemicals (thermolabile materials) Some foods (fruits, vegetables, meats) Non-ionizing radiation: Alternative disinfectant Germicidal lamp in hospitals, schools, food preparation areas (inanimate objects, air, water) – Has been used for water treatment  Physical Agents Heat Radiation Dry Moist Ionizing Non Ionizing Incineration Steam Under X Ray, Cathode, Pressure UV Gamma Dry Oven Sterilization Boiling Water/Hot Water Sterilization Disinfection Pasteurization Disinfection  Filtration Filtration is the passage of a liquid or gas through a screen like material with pores small enough to retain microorganisms. It helps remove bacteria from heat labile liquids such as sera and solutions of sugar, Antibiotics The following filters are mainly used Candle filters Asbestos filters Sintered glass filter Membrane filters  Candle filters Widely used for purification of water Two types (a) Unglazed ceramic filter – Chamberland filter (b) Diatomaceous earth filters – Berkefeld filter They are sterilized by steaming or autoclaving  Asbestos filter This type consists of a disk of an asbestos composition through which the fluid is passed. The disk is inserted into a metal holder that ensures a tight joint being made. Used for sterilization of large amounts of serum to be used in the preparation of media. Disposable single use discs High adsorbing tendency They are sterilized by steaming or autoclaving Eg: Seitz filter Asbestos discs Seitz metal holder  Sintered glass filter Prepared by heat fusing powdered glass particles of graded size Cleaned easily, reusable, brittle, expensive. Attached to filtering apparatus and sterilized by autoclaving or hot air oven  Membrane filters Made of cellulose esters or other polymers They have several advantages over the widely used Seitz filters as they are less adsorptive and the rate of filtration is much greater. Also, the bacteria is retained on the surface of the membrane filters that can be subsequently grown and detected. Uses Water purification & analysis Sterilization & sterility testing Preparation of solutions for parenteral use Membrane Filter (a) Membrane filtration system. (b) Membrane filter close-up.  Membrane filters They have perforated membrane of varying pore sizes (0.22 to 0.45 um)  Filtering Air surgical masks used in hospitals and Labs cotton plugs on culture vessels High-efficiency particulate air (HEPA) filters used in laminar flow biological safety cabinets (remove 99.97% of particles) as the filter has 0.3µm pores to trap organisms HEPA filter  Testing the efficiency of filters A suspension of small sized bacterium such as Serratia marcescens is added to the fluid before filtration and cultures are made from the filtered fluid on suitable media and incubated The filter is out of order if the filtrate gave a red color colonies Physical Antimicrobials Agent Mechanisms of Comments Action Moist Heat, Denatures proteins Kills vegetative bacterial cells boiling and viruses Endospores survive Moist Heat, Denatures proteins 121°C at 15 p.s.i. for 30 min Autoclaving kills everything Moist Heat, Denatures proteins Kills pathogens in food Pasteurization products Dry Heat, Incineration of Used for inoculating loop Flaming contaminants Dry Heat, Hot Oxidation & Denatures 170°C for 2 hours; Used for air oven proteins glassware & instrument sterilization Filtration Separation of bacteria Used for heat sensitive liquids from liquid (HEPA: from air) Radiation, UV DNA damage (thymine Limited penetration dimers) Radiation, X- DNA damage Used for sterilizing medical rays supplies Physical Methods of Microbial Control: Dessication: In the absence of water, microbes cannot grow or reproduce, but some may remain viable for years. After water becomes available, they start growing again. Susceptibility to dessication varies widely:  Neisseria gonnorrhea: Only survives about one hour.  Mycobacterium tuberculosis: May survive several months.  Viruses are fairly resistant to dessication.  Clostridium spp. and Bacillus spp.: May survive decades. Physical Methods of Microbial Control: Low Temperature: Effect depends on microbe and treatment applied.  Refrigeration: Temperatures from 0 to 7oC. Bacteriostatic effect. Reduces metabolic rate of most microbes so they cannot reproduce or produce toxins. Freezing: Temperatures below 0oC. Flash Freezing: Does not kill most microbes. Slow Freezing: More harmful because ice crystals disrupt cell structure.  Over a third of vegetative bacteria may survive 1 year.  Most parasites are killed by a few days of freezing. Physical Methods of Microbial Control: Osmotic Pressure: The use of high concentrations of salts and sugars in foods is used to increase the osmotic pressure and create a hypertonic environment. Plasmolysis: As water leaves the cell, plasma membrane shrinks away from cell wall. Cell may not die, but usually stops growing.  Yeasts and molds: More resistant to high osmotic pressures.  Staphylococci spp. that live on skin are fairly resistant to high osmotic pressure.  Quiz What is the best method of sterilizing the following: Serum Plastic syringe Nutrient broth medium Paraffin oil Glass syringe Food Milk water  Aseptic technique The aim of aseptic technique is to prevent the access of organisms during the preparation and testing of sterile pharmaceutical products How to achieve??  Bacteriological clean air In the pharmaceutical industry, bacteriological clean air is required for two major purposes: For aeration of the fermentation processes in which aerobic organisms are required to grow in submerged culture. To produce satisfactory atmospheres for aseptic dispensing and filling under sterile conditions. Methods used for purifying air The methods used for purifying air include: 1- Mechanical filtration 2- Electrostatic precipitation 3- Heat 4- UV light 5- Chemicals  Mechanical filtration The majority of microorganisms are retained on the surfaces of the filters Electrostatic precipitation  Electrostatic precipitation The air passes through an ionizing unit containing alternately spaced, earthed tubes and thin wires. As a dust particle passes through the unit, some ions attached to its surface making it becomes positively charged. The air then passes into a collection cell containing a series of flat parallel plates of which, are earthed while the others are positively charged. The voltage gradient drives the charged particles on to the earthed plate where they adhere.  Heat All organisms would be destroyed, at about 300°C A short exposure is essential Coasts very high  UV light A lamp inside a screen is useful aid in aseptic processing  Chemicals Chemical substances which are used in asepsis include: Resorcinol: It is easily vaporized and also be sprayed in glycerol solution. It is effective in very low concentration (1 part 250 million) Hexyl resorcinol: It is non irritating but is more resistant and is active in greater dilution Propylene glycol: One part in 2 in 2 millions destroys vegetative bacteria Triethylene glycol: Extremely active, it is effective in a very low concentration. Sodium hypochlorite (5 ml if 1 gm solution per 1000 cu. ft )  Investigation of the sterility of air This is done by estimating the number of bacteria or bacterial carrying particles in a sample of air. The following procedures are used: 1- Settling plates: Open petri dishes of nutrient agar are exposed for a suitable time incubated and the colonies are counted. The method depends on the sedimentation of bacteria. 2- Impingement methods: The particles from a known volume of air to impinge on the surface of the solid culture medium This technique includes: a- Slit sampler b-Reuter sampler Air samplers