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    BT UNIT 1 Sterilizatn

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    BT UNIT 1 Sterilizatn

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    isi ion Sterilization and Disinfectio Sterilization is defined as the ps a anicle, a surface or aimed of all micro organisms including-viruses, bacteria, their spores, ‘and fungi, both pathogenic and non-pathogenic. Disinfectionis a process of lestruction ocremo- val of organisms.capable of giving rise to infection, Disiniectants are capable of killing vegetative bacteria, fungi, viruses and rarely bact ‘Therefore, disinfection must sterilization is, possi -Antisepsis is the destruction ot inhibition of promanisms in living tissues thereby TanTing or preventing the h armful disinfectant thai ‘0 38 an antiseptic; ~~ Various’ agents disinfection may be di A. Physical agents 1, Sunlight 2. Drying 3. Heat 4. Filtration 5. Radiations B. Chemical agents 1. Phenols and cresols 2. Halogens 3. Metallic salts 4. Aldehydes 5. Alcohols 1 effects of infection. A used in sterilization and vided into: lied to living tissue is referred 50 6. Dyes ; 7. Vapour-phase disinfectants 8. Surface active disinfectants A. PHYSICAL AGENTS". L 1. SUNLIGHT > UViegs Ey, At Sunlight possesses, ultraviolet rays which along with heat i ‘al rays are Tesponsible for aj ieee ppreciable germicidal activity. These however Sanner ETERS through glass, ic Hori ecg Hable, certain chemical disinfection S88, lemical Taection,ileave, “ontro) Sid ORTESS Thee Nes Sensitive, this met 0 TrateaT Fert POe Fequired for-ste Proportional to th hod shou rilization ro ; Ne temperaty, higher the temperature shorter 4 Im addition, factors influens me Dp include the number of 1 Groowsans eae and spores n-and ing ab crpggeanismns and the n of the material in which mf€foorganisms are heated. ‘There are two types. of heat: dey heat, and we heat Dry heat is believed to wi sing destructive oxidatio of essential cell suDry heat at 1OO*EFor 60 minutes and “TSC for 60 minutes can kill ll vegetative bacteria and fungal spores respectively. Bacterial spores can. be killed by dry heat at 160°C for one hour or 180°C. for 20 minutes. On the whole dry heat is less efficient sterilization process than moist heat. \The moist heat causes denaturation and coagu- lation of proteins. When steam condenses on c ooler siiface, it releases ifS Tatent heat and raises the temperature ofits surface.sIf spores are present, steam condense ON MENTE Increases th their water content bacterial spores varies with different strains of the same species. For example, spores of most strains of Clostridium tetani are killed by boiling at 100°C for 10 minutessHowever, some strains resist boiling for 1-3 hours, The spores of some strains of C. botulinum resist boiling at 100°C up to 8 hours. However, all spores are killed by autoclaving at 121°C for 10-30 minutes. Sterilization and Disinfer |. Sterilization by dry heat (A) Red heat Ingculating wires and loops, points of forceps and spatulas ate zed by holding them almost vertical in a bunsen burner flame until red hot (Table 5.1). (8) Flaming Scalpel blades, needles, mouths of culture tubes and bottles, glass slides and cover slips are sterilized by passing the article through the bunsen flame without ving them to become red hot. ‘(incineration This is an efficient method for rapidly destroying contaminated materials such as soiled dressings, pathological materials, etc. (D) Het-air even dishes, pipettes and flasks, metal instruments such as forceps, scissors and scalpels, sealed materials such as oils, greases and dry powders which are impervious to steam and swab sticks packed in test tubes. It is not suitable for materials like fabrics which may be damaged by heat. “Hot-air oven is electrical fot-air oven is electrical Reread is sited with a thermostat that mait the chamber air at a cA Table 5.1. Sterilization by dry Heat Mode of Instrument Temperature Sterilization of Advantages! Sterilization and time disadvantages Red heat Bunsen bumer.. Till red hot inoculating wires and loops, Sterilization is rapid i ae poirits of forceps and spatulas and thorough Flaming Bunsen burner Waving through te Scalpel blades, needles, glass 1. Surtace sterization flame yshth slides, cover slips and, mouths _is possible hetonreetg de! he’ of culture tubes and bottles. 2. Rapid method Hot air Hot airoven 160°C for 1 hour or Glassware, metal instruments, Can be used for loads 180°C for 20 minutes sealed materials such as oils, that cannot be pene- greases and dry powder, swab trated by steam. slicks packed in test tubes, etc. 52 Textbook of Microbiology chasen temperature and GRR that distributes hot air )- Temtast not be ov rerloaded ulation of air through Table 5.2. Biological controls of different sterilization methods Biological control ‘Method of i 160°C or 180°C respectiv Sterilization controls : Two types of controls are available. A. Biological control : An envelope contai a filter paper strip impregnated with 10° spores of Bacillus subtilis subsp. niger (NCTC 10075 or ATCC 9372) is placed within the load (Table 5.2). After sterilization is over, the strip is removed and inoculated into tryptone soy broth and incubated at 37°C for five days. No growth of B. ‘subtilis subsp. niger indicates proper sterilization, B. Chemical control : A Browne's tube contai- ning red solution is placed within the load. A change of colour of the solution to green indicates proper sterilization, Il. Sterilization by moist heat Sterilization by moist heat means killing of the micro- organisms with hot water or steam. Moist heat is divided into 3 forms (Table 5.3): Thermometer Fig. 5.1. Hot air oven. hot-airoven | sterilization be = Hot-air oven Bacillus subtilis subsp. niger ‘Autoclave Bacillus stearothermophilus Low temperature Bacillus stearothermophilus steam-formaldehyde Ethylene oxide Bacillus globigh (a red-pigmented variant of Bacillus subtilis) Bacillus pumilis Serratia marcescens, Pseudomonas diminuta lonizing radiations Filtration ‘A. At temperatures below 100°C. B. Ata temperature of 100°C. (a) Boiling water (b) Free steam C. At temperature above 100°C. A. Moist heat at temperatures below 100°C Heat-labile fluids may be disinfected (not sterilized) by Heating at temperatures below 100°C. Such treatment is sufficient to kill mesophilic vegetative bacteria. This includes: ~ w-Pasteurization of milk and butter. The temper- ature employed is either 63°C for 30 minutes (holder method) or 72°C for 20 seconds (flash metiiod) followed by rapid cooling to 13°C or lower. By this method non-sporing organisms such as mycobaciéria, Brucella and salmonellae are destroyed. However, Coxiella burnetil, causative agent of Q fever may survive pasteurization by holder method. + Heat-labile fluids such as serum may be disinf- ected by heating at 56°C for one hour. If tem- perature rises above 59°C it will coagulate. + Vaceines prepared from non-sporing bacteria may be inactivated in a water bath at 60°C for one hour. + Household utensils and patient’s clothing may be disinfected by washing in water at 70- oo Instrument Tomporaturo and timo. C for 1 hour or 65-75°C Jor 10 min forslenser 3 th’ on Mode of sterilization Bolow 100°C Wator bath ‘at 100°C Boiling 100°C tor 10-20 min, water bath toms steaming at Amold 100°C for 20 min. on steamer 3 succossivo days 100°C Nyro xebon st er bation 121°C for 15-20 min. Is Psy Above 100°C Autoclavo $0°C for several minutes, Items which cannot withstand heat at 100°C maybe disinfe by steam_at_sub-atmospheric pressuri temperature of 75 Searothermophilus (NCTC 10003 or ATCC foi 7959 asbeen used to test the efiesey of TST VOUS serilizers (Table 5.2). ary formal: at ww erp OLAS HUB Aly 4 Uap B, Moist heat at a teniperature of 100 (a) Boiling at 100°C : Boiling at 100°C for 10- Oiminutes kills all vegetative bacteria and some bacterial spores. fore, it recommended for sterilization of instruments Steam at_normal (b) Free steam at aimospheric pressure is at 100°C. But, in as latent i casing on the article to be sterilized releases its Jatent heat, A Koch or Arnold steam sterilizer. consists SF'a vertical metal-cylinder- with 0,2, removable conial id having a small opening Yc for the escaping steam. Water is added on the bottom and there is a perforated-shelf above Tablo 5.3. Storilization by molst hoat Storilization of Sonum, body tlulds, vaccines and spatulas Glass, motal and rubbor Culturo modia containing sugar and golatin Cultura modia and othor laboratory matorials Fone single exposu decompose for 20 n employed. Th mittent sterilization. First exposure tc all vegetative bactet a May bo usod for disinfoction, ~Mést vogotativo mosophilic bacteria aro killod. Kills all vogotative bactoria ‘and somo sporas. Provonts decomposition of modin, Sporos of thormo- philic bactoria may escape killing. Most rollablo method of storilization. complete steri but for media ng sugar and gelatin, which may get on long heating?iin exposure of 100°C ‘ cutive days is is known as tyndallization or inte and any spores pres ing Perforated shelf SSS Tinie Water level (Fig, 5.2).-On.this shelf articles to be sterilized are placed. a Fig. 5.2 Steam sterilizer 54 Textbook of Microbiology in a favourable medium, will germinate and will be Killed on the subsequent occasions, Therefore, non= nutricnt media cannot be sterilized by thix method, C, Molst heat at temperature above 100°C aturated ste “nt than hot air be more efficient sterilizing, a CYAN provides )) el is quicker in heating. up the expoved articles. + I-can penetrate easily porous material such as cotton woo! stoppers, paper and cloth wrap- pers, bundles of surpical linen, and hollow app- aratus, eater lethal action of moixt When the steam meets the cooler surface of the article, it condenses into a small volume of water and liberates its considerable latent heat 10 that surface, for example, 1600 ml of steam at 100°C, ic, at atmospheric pressure condenses into Fmt of water at 100°C liberating 518 calories of heat. The Jarge contrat volume brings more steam to the same site and the process continues until the temperature of the article is raised to that of st “The water of condensation ensures moist conditions for'killing, of the exposed microorganisms, Pure steam must be used and the presence of air avoided ince air hinders penetration by the steam, “Waster boils when ity pressire equals the pressure of the” surrounding atmosphere. When. the “atmospheric pressure is raised then the boiling, temperature js also raised, At normal pressure water boils at 100°C but when it is boiled in a closed vessel a increased pressure the temperature a. which it boils and that of the steam it forms rises above 100°C. ‘This principle has been exploited in autoclave and | pressure cooker, ae a Shogo a by (Gon under _pressure end Uner_ py (autoclaving) is suitable for culture media and aqueaus-solitions (sin atmosphere of steam _ 4 bo ey prevents evaporation during heating), dressing material, linen, gloves, etc, Salisfactory sterilization i | ae can, ichics dat! 15 pounds per squa chi(psi) pressure equi —_—— vale 1OTANC In 15=20-mlgutes. In al and dependable method of ing different fact the only prac sterilization is steam under pressure fypex of autoclave: All the air must be removed from the autoclave chamber and the articles to be sterilized so that the fatter are exposed to pure steam, There are three «ons for this ( wis syringes, cl (“De tthe air being denser forms a separate-and ~~ cooler layer in the lower part of the autoclave, 80 it prevents adequate heating of arti there. ‘everal types of steam sterilizers are available, ‘The laboratory autoclave or pressure cooker type autoclave (Fig. 5.3) consists of a vertical or horizontal cylinder of gun metal or stainless steel in a supporting frame or case, The lid is fastened by screw clamp and rendered air tight by asbestos gasket, Lid bears a pressure gauge and a steam release valve or safety valve. The latter openg and closes when the steam pressure rises or falls the desired level respectively. On its upper part of the side, the autocave has a discharge tap forvair and steam and an air and steam knob. Heating is done by electricity, Water is added on the bottom of the autocl perforated shelf on which anticles to be sterilized are placed, The lid is closed, discharge tap is opened and safely valve is adjusted to the required pressure. AS heating continues, the steam and air mixture escape: ‘To know when all the air inside the autoclave has escaped the discharge tap is connected with oneent ber TBE wind the « of it is placed in water, When the air bubbles stop coming it indicates that all the air from inside the autoclave has been © noved. ‘Lhe _dischargelap-is-now: closed, Steam ure rises inside and when-itreaches the desired sel level (15,psi).the safety valve opens and excess pre: Safety valve Discharge tap for ar oC Air and steam and steam 1| release knob : H—Portorated t] she : : Fig. 5.3 Autoclave __ steam escapes. From this point the holding time (15 minutes) is counted. — When the holding time is over, the heating is stopped and autoclave allowed to cool till pressure gauge indicates that inside pressure has reached to the aimospheric pressure. The discharge tap is now operied and air allowed to enter the autoclave. The lid is now 6pened and the sterilized articles removed. ned when the pressure inside is ane nquranedia boll wolenily.andma On the other hand, if the articles are not removed for a long time after the normal atmospheric pressure has reached inside the autoclave, an excessive amount of water will be evaporated and lost from the media, Sterilization controls : Two types of controls are available: A. Biological control : An envelope containing filter paper strip impregnated with 10° spores of Earthenware filters & —— Sterilization and Disinfection 553 Bacillus stearothermophilus (NCTC 10003 or ATCC 7953) is placed with the load in the coolest and least accessible part of the autoclave chamber (Table 5.2). After sterilization is over the strip is removed and inoculated into tryptone soy broth and incubated aerobically at 56°C for 5 days. No growth of B. stea- rothermophilus indicates proper sterilization. Spores of this organism withstand 121°C for-up to: 12 minutes and this has made the organism ideal for testing autoclaves. B. Chemical control : A Browne’s:tube contai- ning red solution is placed witfiin the load-A change of colour_of the solution to green indicates proper sterilization. whe gayi bia0w 4, FILTRATION Liquids such as sera and solutions of heat labile subsiances Such as sugars and urea, used for prep Aitiorof media; cam be sterilized by filtration. This method is also useful ion of bacteriopha- ges and bacterial toxins feom-bacteria-and.for, the isolation of organisms which are The filter disc retains the organism: be cultured. Mycoplasma and viruses, which are very small, cannot be kept back by the bacterial filters, therefore, serum sterilized by filtration cannot be employed for clinical use. Serratia marcescens and Pseudomonas diminuta have been used to test the efficacy of different filters (Table 5.2). Several types of filters are now available, They include: ati a3) Kaas? | Imports gramles are earthenware filters from kibséleuhe'(d foS8il diatomaceous earth), and chambefland filters, made of unglazed”porcelain. ‘They are made in the form of hollow candles with different grades of porosity. The fluid to be sterilized is forced by suction or pressure from inside to olisidé or-vice versa. After use they Cait be’ sterilized’ by scrubbing with stiff brush followed by boiling: and autoclaving. be 86 Toxlbook of Microblology int “il Asbostos (Soltz) filtors { y ‘They are made up ofa dive of asbestos. ‘Magneslum Us supported on a perforated metal dive etal funne! Inter with filler dine Titted ized by autoclaving, It is then fitted on to A ti within iS seri Sterile Mask through asiticone rubber bung, ‘The fluid (0 be sterilized is put into the funnel and flap connected to the exhaust pump through ity side tap, ed from the flask and filter Sintered glass filters These are made of finely ground ther. These are available in di \_e Membrane filters These are made up of cellulo used now-a-days for steri fluids. These are also known as milliporé filte They are available as discs of pore size varying from nies that develop can be counted and viable count calculated, V. Syringe filters Syringes fitted with membranes of 13 mm and 25 mm diameter are available. The fluid to be sterilized is forced through the dise by pressing the piston of the syringe, M-Air fitters Large volumes of air.may be rapidly-freed from infection by passage through HEPA (high efficiency particle arresters) fil 5 SAO / viola Ms 9 i tan0l@ ; ubeaste wa) rte) as 5. RADIATIONS ‘Two types of radiations (Table 5.4) are used; + Non-ionizing. + fonizing, Jd pNenlonizlng podlationas <<. 4, © "These include infrared and ultraviolet radiations, Infrared is used for rapid mash steriliz ion of, Pepacked items such as syringes and cattidien, Ultraviolet (UV) radiations le range of 25G- 260-nm-wavelength are highly mis with the wavelength of 253.7 nm, Most Vegelative bacteria are susceptible to UV radiations, Spores are highly resistant and Susceptibility of viru. tes is variable. Human immunodeficiency virus is Not inactivated by UY radiations. UV radiations nduce thymine dimers in DN, is in ih DNA replica 'onlzing radiations Theve include X-rays, : ese have. very nigh pees 9804 coomie rays highly lethal to all ean eve damage DNA by various mechan ee Comeetsisant than vegeta commercial plants use "ration of prepacked disperses ean syringes, transfusion sets, Cathetery, ne culture plates, ee. that are unabie ‘6 witha because there is no appreciaty increase temperature, Therefore, this methea Sesllzalon. High cos of installation fen spmnmercally Ballas puis has ban 8 the efficacy of ionizing radiations CTatne 5.2), est Plastic effective. Loy mercury vapour Jamps emit over 95% of | ‘Stortization and Disintectio Norionizing UV lamps, 250-260 (UV radiation) commercial nm wave- UV units and length for mercury 30 min, vapour lamps Tablo 5.4, Stotization by radiation pe of Instrument Dose reation seadioadiee aaa eaantagea! : wadionds disadvanta in Cobalt 60°" 2.5 = lonizing a rad rrartzaton Of antibiotics, hor- Excellent sterilizing agent, Sutures and yd disposabio i ooo syringes, transtu: catheters, cani Plates, etc, Disinfection of clean surfaces tke inoculation hoods, laboratories, Wards, operation theatres, etc. Penetrates deep objects, produc less can be used for heat Sensitive items. But it is expensive, Destroys microorganisms only on exposed surfaces, Hazardous for eyes and skin. - items such as plastic ision sets, Inulas, culture eat, CHEMICAL AGENTS Several chemical agents are used as antiseptics and disinfectants. An ideal antiseptic or disinfectant should: (@* Fave a wide spectrum of activity and must be effective against all microorganisms. Be active in the presence of organic matter. @-Be effective in both acid and alkaline media, Piet O-Tlave penetrating powerl + & ips GoBe stable, Crier vere! LeNot corrode metglel'8H'8 4) @ +ot cause {oct itritation or sensitization. Not be toxic if absorbed into circulation. er interfere with healing. e-Be cheap and easily available. - Following factors influence the disinfectants: wrP¢ ‘Concentration. — potency of ime of action. _ YPH of medium, _ Temperature, ‘Nature of organisms. ice of organic matter. 1. PHENOLS AND CRESOLS These are obtained by distillation of coal tarand have @ powerful microbicidal action. They cause cell membrane damage thus releasing cell content and causing cell lysis. They are resistant to inactivation by organic matter and are active against gram- Positive and gram-negative bacteria, moderately active against. mycobacteria and.have little activity against spores and viruses. They are used mainly for discarded cultures, contaminated pipettes and other infected material. Phenol is ba icidal_at a concentration of 1%. At a concentration of 0.5% it is used for preservation of sera and vaccines. Tt is readily inactivated by organic matter. It is inactive against Pseudomonas. @& HALOGENS - ar Chlorine and iodine are the halogens which are used as disinfectants, The} ane Wetec and sporic @ They are fave in xery high dilutions and their actions veryrapid, In additionto chlorine el there are three types of chlorine compounds, the Ee chlorites, and the inorganic and organic: borat . ‘The disinfectant action of all the chlosi compounds is due to release of free chlorine, When clement chlorine or hypochlorites are added to water, book of Microbiology shlorous ach form hypoc! ctive dis- ‘5a Text acid. chlorine reacts wi 4 xi It is a strong OxI01s” f chlorine infectan. The oe of organic matter. influenced by the presen tof Chlorine has a ec Dn ns of hy] ° water supply and ae cae ad disinfection eget re Hypochirites havea wide in food and dairy industry. Hypo "itle Sod of activity against viruses and very cal 5 ill. They are available activity against tubercle bacilli. They are wal re in liquid or powder form as salts of calcium, lithiu and sodium. _ Todinejn aleoholic and aqueous solutionsis used, almost exclusively s kin disinfectant (antiseptic) “7 Like chlorine, itis also inactivated by organic matter. Itis also active against tubercle bacilli and a number of viruses. Mixtures of iodine with various surface active agents that act as carriers for iodine are known as iodophores, an example of this is e- It ccan be used as a bactericidal antiseptic for intact skin and for disinfection of superficial wounds. lis, also active against fungi and Trichomonas, of oh 0 3. METALLIC SALTS All metallic salts have some-degree-of foxicity for bacteria, The most toxic are those of nigreury’ and Silver and the Jeast toxic are those of sodium_and Potassium. Mexcury compounds act on with sulphydryl (SH) groups Proteins and other essen acterial intracellular compounds, Imercurithiosalicylate) is used in a dilution of 1 in 10,000 for preservation of Sera, In the past, 1% solution ‘silver Rirhte was instilled inthe ee in newborn babies for the prophy. taxis of gonococcal ophthalmia, 4. ALDEHYDES, .., Two aldehydes (formaldehyde and glutaraldehyde) importance, although are currently of considerable others also possess antimicrobial activity. k Formaldehyde is an irritant, water soluble gas. It is ‘highly Jethal to bacteria and their spores, Tifigi and viruses, It is cheap and can be used for sterilization 5 d a wide variety_of Atticley pniture..an\ F ; of.ro0ms, PE omaged by heat, such 8. clothing liable (0 nattresses, respirators, heq, r kets, m t voollen blan : ai ei tive instruments, tC. It may be uset na faa oran aqueous solution. It combines readily wiy, form i ive in the presence of i J is less effective in icra Initant residues of formaldehyde may aed by exposure of the disinfected articles e ia vapour. ° “Glatareide i e effective and less irritant than formaldehyde. It possesses high microbicidal activity-against bacteria and thei spores, mycelial and spore forms of fungi ayd various types of viruses, including human immuno- deficiency virus and enteroviruses. Two per cent alkaline buffered solution is used for sterilization of heat-sensitive instruments like cystoscopes, bronchoscopes, thermometers, etc. oy drafts 5. ALCOHOLS 7 6940? & Several alcohols possess antimicrobial activity. The antimicrobial activity of alcohols can be-attributed 'y to denature proteins, Alcohol solutions hiehnite 60-70% alcohol-are-most effective, and higher concentrations Proteins are are less potent because = Ala

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