= Physical and Chemical Methods of Sterilization LEARNING OBJECTIVES tthe end of this chapter, the student should beable to: 1 define important terms related to sterilization and disinfection, 2 compare the different physical and chemical methods of sterilization as to clasication, mechanism of action, and indication for use; and 5. explain the different fctors affecting the eflcacy ofa chemical agent. Definition of Terms |. Sterilization ~ the proces of killing or removing all microbial forms, including spores Disinfection ~ the process by which most microbial form on inanimate object are killed without necesatly destroying saprophytes and bacterial endospores which leads to ‘reduction inthe numberof organisms toa level that they cannot produce infection. psis— use of chemical agents on living tissue (eg. skin) to prevent the spread ‘of microorganisms either by inhibiting their growths or destroying them, Bactericidal or germicidal agent— agent, physical or chemical, that kills bacteria Bacteristatic agent ~ agent, physical or chemical, capable of inhibiting the growth of bacteria without necessarily killing them, Sporicidal, fungicidal, viricidal ~ agents capable of destroying spores, fungi, and viruses, respectivelyPhysical Methods of Sterilization Heating Heating is the most common physical method of sterilization. The rate of killing is expressed in thermal death time, ie. the minimum time required to kill a suspension of en organism at 2 predetermined temperatute and environment. The mechanisms of action of heating include: (1) formation of single-strand breaks inthe bacterial DNA; (2) coagulation and denaturation of proteins; (3) accumulation of toxic levels of electrolytes; and (4) alteration of cell membrane structure. Several factors can affect the process of sterilization through heating These include: 1, Nature ofthe heat~ moist heat has greater king ation than dey heat. 2, ‘Temperature and time—as temperature increases the time taken to sterilize decreases In other words, there is an inverse relationship berween time and temperature, 3. Number of microorganisms ~ the more microorganisms there are, the higher the ‘temperatute and the longer the duation ofthe process required to destroy al f them, 4, Nature of microorganisms ~spore-forming microorganisms are more dificult to destroy than non-spore-forming ones. 5. Type of material - the temperature eequited to sterilize materials depend on the sensitivity of the material to heat. Heatsensitive materials will require lower temperature than heat-resistant material 6. Presence of organic material ~the presence of organic material such as fats, proteins, and sugars may necessitate higher temperatures. ‘Types of Heat 1. Moist heat ~ preferred over dey heat because ofits more apd killing ction Its main mechanism of action is to cause coagulation and denaturation of proteins. The various methods of moist heat may be classified according to the temperature use. ‘These include: Temperature below 100°C + Pasteurization This isthe method of destroying disease-producing ongunitms in milk and milk products as well as other beverages. There are several variations of this :method based onthe temperature utilized, One method is called the conventional ‘method where the milk is heated at 60°C-65°C followed by rapid cooling.Physic end Chemical Methods of Striaton ‘The flash method invelves heating at 72°C for 15 seconds followed by quick cooling to 13°C. A newer pasteurization method developed is what they call ultea-high temperature (UHT) method where heating is done at 140°C for a period of 15 seconds and 149°C for 05 seconds, + Vaccine bath ‘This is used to destroy contaminating bacteria in vaccine preparations, ‘The vaccine preparation is heated in a water bath at 60°C for one hour. This procedure is not sporicidal, Only the vegetative forms of the bacteria se destroyed. + Serum bath ‘This is used to inactivate bacteria contaminating serum preparations and is done by heating at 56°C for several sucessve days. Similar to vaccine bath, conly the vegetative forms are destroyed since higher temperatures will cause coagulation of proteins present in the serum, + Inspissation ‘This technique is used to solidify and disinfect egg-containing and serum- containing media. The culture medium is placed in the slopes ofa device called tan inspssacor and is heated at 80°C-85°C for 30 minuces for theee successive ays. The bass for the method is that onthe fist day, vegetative forms will die and the spores that wil germinate the following day will also die. Temperature of 100°C + Boiling This method involves wslizing water at boiling termperature of 100°C. It is ‘not sporicidal and will destroy only the vegetative forms. The killing action can be enhanced by the addition of 2% sodium bicarbonate. Certain metal articles and glasswares can be disinfected using this method for 10-20 minutes without ‘opening the ld of the boiler. + Fractional sterilization (Iyndallization) ‘This method is also known as intermittent sterilization and involves exposing ‘the material to be sterilized to live steam at 100°C for 30-90 minutes for three consecutive days, depending on the material ro be sterilized. This sterilization ‘method can be used to sterilize culture media such as TCBS and selenite broth. ‘The vegetative forms ae killed on the frst day and the spores chat will germinate will be destroyed on the next successive days, Only vegetative forms of the ‘bacteria are destroyed with this method.Temperature above 100°C + Autoclave (Steam under pressure) This isthe most eflcieat method of sterilization because it can destroy all microbial forms. The temperature for sterilizing is dependent on the pressure of the steam. When the prestuce reaches 15 pounds per square inch (psi), the temperature inside the vessel reaches 121'C, Because ofthe high temperauce and pressure it would take ony 15-20 minutes co stelize the material. Ths method is used to sterilize instruments, surgical bandages, culture media, and other contaminated materials that can withstand hightemperature and high pressure. Figure 72 Inssator and weter bath2. Dry eat ~ the effectiveness of dry heat depends on the penetration of heat through the material to be sterilized. Its used to sterilize materials in enclosed tubes il, jellies, powders, and glasswares such a est tues and Per dishes. a. Red ame ‘This method is used to sterilize articles Ike bacteriological wire loops, straight wires, tips of forceps, and searing spatula. The materials are held over the flame of 1 Bunsen burner unt they become red ot. Itis limited only to articles tha can be Dated to rednes in lame, 1 Open flame (Flaming) Tis method also makes use ofthe Bunsen burer or alcohol lamp. The material tobe sterilized is passed over the flame several times but is not heated to redness, I's aimed at burning the organism into ashes and is used to sterilize such etcles ae mouths of test tubes, sealpls, pass slides, and cover slips. Only vegetative forms are destroyed. In addition, cracking of the glassware may occur. Figure 73 @ Stetlzation of inoculation lop using red la and being striized using open lame test tube ‘This method is aimed at burning the organism into ashes. The contaminated | material is burned using an incinerator. Articles that must be incinerated | include soiled dressings and beddings, animal carcasses, and pathological matecial, ‘This wil result in loss ofthe article and hence must be used only for articles that have to be disposed. Some material such as polystyrene emit dense smoke and must not be incinerated|. Hotairoven ‘The use of the hot air oven was first introduced by Louis Pasteur. Articles to be sterilized are placed in the oven with a temperature of 160°C for a period of one hour. This can be used to sterilize metallic instruments such as forceps, scalpels, and scissors. It can ako be used to sterilize certain glaswares (eg, peti Gishes, pipette, flasks) and it isthe only method used to sterilize powders and ointments, The disadvantage of using this method is that because ai is a poor conductor of hei, then hot air wil have poor penetration of the materials to be sterilized. In addition, cotton wool and paper may get slightly chanred and glasses can become smoky. Infrared In this method, the articles to be sterilized are placed in a conveyor belt and passed through a tunnel that is heated by infrared radiators. The temperature to which the materiale are subjected to is 180°C fora period of 7.5 minutes. It can be used to sterilize metallic equipment and glosware Dessication ‘This method is based on the principle of depriving the microorganism of moisture. Tis used mainly for food preservation, such asin the preparation of dried fish and fruits. It may destroy vegetative forms. Endospores ae resistant to drying. Freezing Freezing is not a reliable method of sterilization because most pathogenic organisms are resistant to low temperatures. Its main use in the laboratory is for the preservation ‘of microorganisms in process called pbilzation or feeze-drying where the organism is rapidly frozen then dehydrated in high vacuum and stored ina vacuum-sealed contanes. Filtration ‘This is a form of mechanical sieving that does not kill microorganisms but merely separates them from the fuid, A cellulose ester filter with a pore size of 0.22 um-0.45 ym is used which can filter all microorganisms except viruses and the three smallest bacreria— “Myeplasma, Rickettsia, and Chlamydia, It is used for liquid solutions that will be destroyed by heat of freezing such as serum, antibiotic solutions, sugar solutions, or urea solution. This ‘method can be used to remove bacteria from culture media or to prepare suspensions of viruses and phages.Radiation 1. Ultraviolet Light (UVL)/Non-ionizing radiation ~ the effective UVL wavelength isin ‘the range of 200 n-280 ne, with 260 nm asthe most effective. This corresponds with the maximum absorption of bacterial DNA. UVL act by inducing formation of thymine- thymine dimers resulting in lethal frameshift mutations. Microorganisms such as bacteria, viruses, and yeasts can be inactivated within seconds. However, UVL is not sporicidal and is more frequently used for surface disinfection. It is used to disinfect hospital ward, ‘operating rooms, laboratories, nd other rooms inthe hospital that need tobe sterilized. ‘The disadvantage of UV say is that thas low penetrance. It is also limited by the lifespan ‘of the UV bulb In addition, there are some bacteria that have DNA repai systems that ‘an counteract the damage done by UV says. Cate should also be observed by the handler ‘because UV rays can be harmfl tothe skin and eyes. 2. Ionizing radiation ~ ionising ays have greater penetrance than UV rays, It exerts its cffect by causing formation of fee cadicals that chemically interact with proteins and nuclei acids, resulting in cell death, Iti not routinely used because of its potential to harm human tissues. There are two types of ionizing radiation used for sterilization purposes: elactrom beams and eltctromagnetic ray. 4. Electron beams Electron beams are particulate in nature. A linear accelerator from a heated cathode is used to generate high speed electrons. It canbe used to sterilize syringes, loves, dressing packs, food, and some pharmaceuticals, Tthas lower penetrance and requires sophistieated instruments ', Electromagnetic ays (Gamma rays) Electromagnetic rays are produced from nuclear disintegration of selected radioactive isotopes. They have greater penetrance than electron beams but reguize longer exposure time. The high energy radiation produced cause damage to the microorganisms nucleic acid. Tes bactericidal, fungicidal, vrcidal, and sporicidal. It is used commercially to sterilize disposable Petri dishes, plastic syringes, vitamins, antbiotieshoemones, Fabris and glaswae Sonic and Ultrasonic Vibrations Some bacteria can be killed after exposure to certain frequency of sound waves. Exposure to sound waves at a frequency of approximately 20,000 eycles/second for one hour can kill some bacteria and viruses. High fequency sound waves at by distupting cells. They are used to lsinfect and clean instruments and to reduce microbial loa.Osmotic Pressure "This method is based on the principle of osmosis, so that when the concentration ofthe fluid surrounding the organism is altered, this will cause the bacterial cll to collapse. This is ‘sed for preservation of fruits in syrup and meats in brine, Chemical Methods of Sterilization Chemicals can inhibit the growth of pathogenic organisms, ether temporarily or permanently Several factors can affect the eflacy of a chemical agent. These include: 41, Concentration and potency ofthe chemical agent. In general, a higher concentration is bactericidal whereas a lower concentration may only be bacteriostatic. This isnot true for alcohol For alcohol the effective bactericidal concentration is at 50% to 80%. Duration of exposure, The longer the time of exposure to the chemical agent, the better the killing ction, "Temperature. higher temperature speeds up the rate of@ chemical reaction and thus accelerates kiling ation, However, there are also certain chemical agents that exert ‘optimal effet at lower emperaures. Nature of the surrounding medium. The pH of the medium and the presence of extraneous materials lke pus or blood decreases the efficiency of the chemical agent. ‘These materials may inactivate or lower the concentration of the chemical agent of may bind the chemical agent to its surface. [Nature ofthe organism. This refers to the innate resistance of the microorganism to isinfectants. Microorganisms vary in their resistance to disinfectants, Bacteria that produce endospores may be resistant to most chemical agens. Mycobacterial cell wall is lipd-rich that makes i difficult forthe chenscals to penetrate it. Gram-negative bacteria have an cuter membrane that confers resistance to disinfectants. [Number of organisms/Size of inoculum. The larger the number of microorganism resent, the more time needed fr a disinfectant to destroy ll of them, A chemical agent, to be effective as» disinfectant or antiseptic, must be chosen caefilly bused on the specific purpore, pathogen, and envionment. A good chemical agent must possess the following characteristics 1. Teshould be broad spectrum, able to destroy a wide variety of microorganisms 2, It shouldbe fast-acting able to destry microbes within a short period of time 3, should be active inthe presence of organic matter. 4. Ie should be ative in any pH. 5. Itshould be stable.6. Tshould be non-toic, non-allergenic, non-intative, and non-corrosive. 7. Itshould be soluble in wate ad easy to apply 8. It should lave a residual antimicrobial film onthe treated surface 9, Ttshould have high penetrating power, 10. Ttshould not be expensive and must be easily avaiable, 11, Ttshould be safe uncer storage and shipping for reasonable periods of ime, 12, Itshould no have «bad odor. Classification of Chemical Disinfectants ‘Chemical disinfectants may be classified based on the following: (1) consistency (Liquid or guseous)s (2) spectrum of activity (high lve, intermediate level, low lve) or (3) mechanism of ston Mechanism of Action Damage to the cell membrane Damage tothe cell membrane can cause smaller molecule to leak out of the bacterial cell, and interfere with the active transport and energy metabolism within the cell, Chemicals under this include the following: 1. Surface active agents compounds have long chain hydrocarbons that are fat-soluble and charged ions that are water-soluble. They concentrate on the surface of ‘membranes and disrupt membrane resulting in leakage of cell components. These agents are active against vegetative microbial forms including Mycobacteria as well as ‘enveloped viruses. They are widely used as disinfectants in homes and hospitals but their activity is reduced inthe presence of hard water and organic matte. 2. Cationic agents ‘These are detergents where the fat-soluble portion is positively charged due to combination with 2 quaternary nitrogen atom. These are called quaternary ammonium compounds and ae effecve at alkaline pH, Examples ae cetrimide and benzallonium chloride, b. Anionicagents These ate n Examples are s0 emulsification snd ae tively charged agents that contain long chain hydrocarbons aps and bile salts. They remove diet through the process of acidic pH.2, Phenolic compounds ~ these act by disrupting cll membranes as well a causing precipitation of proteins and inactivation of enzymes. These are coal-tardesivativs ‘that acts disinfectant at high concenteaton and as antiseptic alow concentrations. PPhenols are bactericidal and fungicidal with good activity against Mycobacteia but have poor att against spores and most vrs. 2 Phenols no longer used as a disinfectant because itis toxic to human cel It is sed asa gold stndaed in the chemical evaluation of new chemical agent sing the phenol coeficien test. '. Cresols ae phenol dtivatves more potent and safer thin phenol. An example is Lysol. Chlotheridine i used as skin disinfectant ifn isopropanol solution. The acs reparation is used for wound igation. Its main ue ia antiseptic hand wath 4. Chlorexyenols are used for topical purposes. They are elective against gram-ponive bacteria . Hexachlorophene isa chlorinated diphenyl which has greater activity agsinat gram-positive bacteria simile to chlorxpendl Teiclosan, an orgnic phenyl ther has good activity against gram-positive bacteria and a number of gram-negative bacteria including Paudemonar e as some sctivgyon fang and viruses 3. Alcohols ~ disorganize the lipid structure of the cell membrane, dbydrate cell and cause denaturation and coagulation of cellar proteins. The microbial killing property of alcohol i cen beter in 2 70% aqueous solution compared to absolute alcohol The disadvantage of using alcohols is chat they are skin iiants and are also Sammable. 4 BthyLalcoho ~ used as skin antiseptic, is bacterial and emove lipids fom skin surfers. bs lsopropyl alcohol ~ it has greater bactericidal activity than ey alcohol and isles volatile Ie can be used to dsingect surfaces. Inhalation of is fumes can «Benzyl aleohol~ it is used manly as preservative 4. Methyl alcohol ~ ic is fungicidal and sporicidal used in disinfecting inoculation hoods Denaturation of cellular proteins Substances that cause denaturation o- loss of the normal structure of proteins pave the ‘way for the eventual destruction of the bacterial cell. Denaturing agents include: (1) acids and alkalis, (2) aleohol and acetone, and (3) phenol and eresl‘Pryseal ang Chemical Metnc of Sterlzation Modification of the functional groups of proteins and nucleic acids 1. Heavy metals ~ cause damage to the enzyme activity of bacteria. They also cause precipitation of proteins and exidation of sulfhydryl groups. Heavy metals ae mostly ‘acreriostarc than bactericidal 4, Mereurals (eg, mercurochrome and merthiolate) are biocidal and are used as ancsepeics, These are active aginst viruses at dilution of 1-500 to 1:1000. b. Silver compounds (eg sive nitrate) are bactericidal. 19 silver nitrate solution is ‘sed clinically as weatment for ophthalmia neonatorum (Crede's prophylaxis) Siver sulfadiazine is used topically inthe treatment of burn wounds 2. Halogens—bactericial oxidizing agents that ease oxidation ofessental sulhydry groups of enaymes causing inactivation ofthe enzymes 1, Todine (tincture of iodine, iodophores is considered the best antiseptic because itis spoicdal, bactericidal, fungicidal, viscdal, and amoebicidal. It can be combined with neural cari polymers to produce iodophores (e.g, povidone iodine). A 109% solution of povidone-iodine is used for pre-operative and post-operative skin disinfection. ' Chlorine is mainly used in the treatment of water (chlorine gos). Hypochlortes are used for sanitizing dairy and food processing equipment. It is also a ‘common houschold disinfectant. At higher concentrations, iti used to disinfect swimming pools. «Hydrogen peroxide is a weak antiseptic and used only for cleaning wounds and inthe Asinfcton of surgical devices and soft plastic contact lenses. 3, Alkylating agents Aldehydes damage nucleic acids by allylation of amino-carbaxy-.or hydroxy groups. Teil ll microorganisms including spores. + Formaldehyde (formalin) is used for surface disinfection. It can be wsed to sterilize bedding and furniture. Iris also used to Kill Mycdacterium tubercles in sputum and fungi in athlete's fot + Ghutaraldchyde is sporicidl and used asa cold serlanein sterilizing medical equipment such as respiratory therapy machines and other equipment that can ‘be damaged by heat. Iris more potent than aldehyde. Itreqies alkaline pH for its action and exposure time ofa east 3 hours to he effective. bh Ethylene oxide is also sporicidal and is used in the gaseous sterilization of heat-sensiive materials or equipment like heat-hing machine, respiratory and dental equipment, and polyethylene tubes in anesthesia machines. It is more potent than glutaraldehyde bat slower-actng, Is highly flammable and is usually combined with 10% COp.It causes eye intation and is mutagenic and carcinogenic.Merobology an Parastoley: A Textbook and Labortory Manual forthe Health Scionces| * Sterilization i the process of removing all microbial forms, including spores + Disinfection isthe process of removing most ofthe microbial forms, It is meant to reduce the microbial load to prevent the development of infection + Factors that affect the effectivity of physical methods of sterlization include 1, Nature ofthe heat ‘Temperature and time Number of microorganisms 2. ‘ 4. Nature of microorganisms 5. Type of material 6. Presence of onzanic material ‘+ Heating isthe most common physical method of sterilization. It acts by the following mechanisms 1. formation of single-strand breaks in bacteri DNA 2. coagulation and denaturation of proteins 53. accumulation of taxi levels of electrolytes 4, alteration of cell membrane tractre + An effective chemical disinfectant must have a broad spectrum of activity; be fast-acting, inexpensive easy to apply, odorless, and non-toxic to human tissues. * Chemical agents used for disinfection and sterilization ae clasified based on their main mechanisms of action > Agents that cause damage tothe cell membrane include surfice active agents, phenolic ‘compounds, and alcohol. Acid and alkalis, alcohol and acetone, phenols and cresols all cause denaturation of proteins, * Modification of functional protein groups i the mechanism of action of heavy metals, halogens, and alkylating agents