Microbiology presentation instrument ppt

Micro

1. STERILIZATION AND DISINFECTION PhD, Associate Professor Sarsembayev Khussein Samir

2. Microorganisms are omnipresent causing contamination, infection, decay Disinfection & sterilization are essential for ensuring that medical/ surgical instruments do not transmit infectious pathogens to patients Hospital infection prevention & control policy must identify whether cleaning, disinfection or sterilization is indicated for various hospital areas/activities

3. History . Semmelweis  introduced the concept of hand washing with lime Florence  Later laid down the principles of proper hygiene and nursing procedures Lister  Introduced the concept of antiseptic surgeries using carbolic acid Louis Pasteur  Put forth the concept of sterilization techniques using hot air oven, autoclave and sterilizers

4. Classification of sterilization methods CHEMICAL AGENTS

5. Level of sterilant/ disinfectants according to their microbicidal action

6. Performs sterilization of medical devices, equipment & consumables Processing area of CSSD- four unidirectional zones starting from an unsterile area to a sterile area separated by physical barrier Decontamination area- washing by manual/ automated washers Packaging area- items are enclosed safely to allow penetration of sterilant Sterilization area- steam sterilizer/ ETO/ plasma sterilizer Sterile storage area Central Sterile Supply Department (CSSD)

7. Spaulding’s Classification of Medical Devices Risk category Definition Recommended method Medical equipment or surfaces Critical device (high risk) Items that enter a normally sterile site Sterilization Surgical instruments, implants/prosthesis, rigid endoscopes, syringes, needles Semi-critical device (intermediate risk) Items in contact with mucous membranes or body fluids Disinfection ( HLD ) Respiratory equipment, non-invasive flexible endoscopes, bedpans, urine bottles Non-critical (low-risk) Items in contact with intact skin Disinfection (ILD or LLD) Non- critical patient items, n on- critical environmental surfaces

8. Definitions Sterilization - A process by which an article, surface or medium is freed of all microorganisms (pathogenic/ nonpathogenic) either in their vegetative or spore state Can be carried out by physical or chemical methods Physical methods- moist heat, dry heat Chemical methods- ETO gas, Hydrogen peroxide Results in reduction of ≥10 6 log CFU of microorganisms & their spores

9. Disinfection- destruction/ removal of all disease causing organisms except spores. Results in reduction of atleast ≥10 3 log CFU of most microorganisms Antiseptics - disinfectants which can safely be used on living tissues (used for asepsis ) High level disinfectant (HLD) Intermediate level disinfectant (ILD) Low-level disinfectants (LLD) Capable of killing all microorganisms & bacterial spores when used in sufficient concentration under suitable conditions Destroy all microorganisms, but not bacterial spores Destroy vegetative bacteria & enveloped viruses; variable action on nonenveloped viruses & fungi, but no action on tubercle bacilli & spores

10. Sanitization - disinfection used in connection with food & catering equipment Decontamination/cleaning- process by which an area or article is rendered free of contamination (microbial, chemical or radioactive) to a level at which items are considered as safe without protective attire Results in reduction of at least ≥1 log CFU of most of microorganisms but not spores

11. Level of disinfectant Spores Tubercle bacilli Non enveloped viruses Fungi Vegetative bacteria Enveloped viruses 1. Sterilant YES YES YES YES YES YES 2. Disinfectant a) HLD +/- YES YES YES YES YES b) ILD NO YES YES YES YES YES c) LLD NO NO +/- +/- YES YES

12. PHYSICAL AGENTS i . Sunlight ii. Drying iii. Dry heat a. Flaming b. Incineration c. Hot air oven iv. Moist heat 1. Temperature below 100 C a. Pasteurization b. Inspissation c. Vaccine bath/water bath 2. Temperature at 100 C a. Boiling b. Koch’s/Arnold’s steam sterilizer c. Tyndallization

13. 3. Temperature above 100 C a. Autoclave v. Filtration depth filters membrane filters vi. Radiation Ionizing radiation: y rays, X ray, cosmic ray Non ionizing radiation: UV, Infra red vii. Ultrasonic & sonic vibration

14. 2. Chemical agents i . Alcohols a. Ethyl alcohol b. Isopropyl alcohol ii. Aldehydes a. Formaldehyde b. Glutaraldehyde c. Orthophthaldehyde iii. Dyes- aniline, acridine dyes iv. Halogens- chlorine, iodine & iodophores v. Phenolics - cresol, lysol , chloroxylenol vi. Biguanides - chlorhexidine gluconate

15. vi. Surface active agents: Quarternary ammonium compounds vii. Metallic salts: Mercuric chloride, copper salts viii. Gases Ethylene oxide Formaldehyde Betapropiolactone ix. Gas plasma sterilants a) Low temperature steam formaldehyde b) Ethylene oxide(ETO) c) Betapropiolactone d) Plasma sterilization

16. Physical agents

17. 1. Sunlight Ultraviolet rays of sunlight has bactericidal activity leading to spontaneous natural sterilization Ultraviolet rays are filtered by ozone layer & impurities in atmosphere but in tropical countries where there is less impurity there is appreciable bactericidal effect 2. Drying Water constitutes 80% of body weight of bacteria therefore drying has deleterious effect on bacterial growth but bacterial spores are unaffected

18. 3. Heat Most reliable method of sterilization, method of choice unless contraindicated Dry heat: kills by protein denaturation, oxidative damage, toxic effect of elevated electrolytes Moist heat : kills by protein denaturation & coagulation

19. Dry heat methods Red heat: process of sterilization in which inoculating loops & wires, tips of forceps & scissors are held vertically in flame until red hot Flaming: Scalpel blades, glass slides, mouth of culture tubes, bottles are exposed to flame for few sec w/o heating them to become red hot Incineration : efficient method of sterilization for destroying contaminated materials such as soiled dressings, animal carcasses & pathological materials Disadvantage Polystyrene materials cannot be incinerated because they emit clouds of dense toxic smoke but plastics like poly vinyl chloride & polythene can be incinerated

20. 4. Hot air oven Most widely used method of dry heat sterilization for sterilizing loads that might be damaged by moist heat but can tolerate high temperature (160 C- 180 C) Acts by oxidation of cell constituents Used to sterilize glass wares ( petridishes , pipettes, tubes, measuring cylinders, flasks, glass syringes) forceps & scissors scalpels swab sticks & some pharmaceutical products like liquid paraffin, dusting powder, fats Operated electrically with heating elements in walls of chamber

21. A fan is fitted inside for even distribution of air A temperature indicator, a control thermostat & timer, open mesh shelving & adequate wall insulation Sterilization temp is set to ensure that sterilization hold time starts only when required temp is reached as detected by the thermocouples

22. Method of operating hot air oven Materials to be sterilized are cleaned, dried & packed loosely to allow free circulation of air & optimum heat transfer Glass test tubes with slip on aluminium caps are placed vertically in metal racks Ends of pipettes stoppered to a depth of 2 cm with non-absorbent cotton wool & kept in metal canisters Powders, fats, oils, greases are sterilized in sealed metal containers

23. Temperature- time cycle Temperature (C) Holding time ( mins ) 160 120 170 60 150 150 Oven is cooled for 2 hrs before door is opened Sterilization controls :- Spores of Bacillus atrophaeus are used as sterilization control Spores of nontoxigenic strains of Clostridium tetani are used to test efficacy of dry heat sterilization Browne’s tube: Containing red colour solution which turns green on proper sterilization

24. Moist heat Temp below 100 C Pasteurization Method used for control of microorganisms in beverages like fruits/ vegetables juices, beer & milk Holder method- Heated at 63 C for 30 min Flash method- heated at 72 C for 20 sec followed by rapid cooling to 13°C UHT Sterilisation - milk & milk products are heated at 140-150°C for 1-3 sec

25. Water bath- vaccines or serum containing coagulable protein are kept in a water bath at 60 C for 1 hour. eg . Typhoid vaccine Inspissation (fractional sterilization)- media like Lowenstein Jensen’s medium can be heated at 80-85 C for 30 min on 3 successive days by a special instrument called Inspissator

26. Temp at 100 C Boiling: vegetative bacteria die almost immediately but spores require a longer time, about 20- 30 mins are required to kill; not ideal for surgical instruments Steam at atmospheric pressure: (Koch’s/ Arnold steam sterilization)- A single exposure of 90 mins usually ensures sterilization Tyndallisation : exposure to steam at 100°C, used for sugar & gelatin media, 20 min for 3 successive days to kill spores

27. Temp above 100 C Autoclave: Principle- water boils when it’s vapour pressure equals the surrounding atmosphere Pressure inside a closed vessel is made to rise, so water boils at higher temp, releasing steam Steam has good penetrating power & condenses to give up it’s latent heat to the article, creating a vacuum, drawing in more steam to the area till temp of the surface is raised to that of steam

28. May be vertical or horizontal cylinder with a steam jacket (water compartment) Made up of gunmetal/ stainless steel The lid is made airtight with screw clamps & asbestos washer Upper side consists of: Discharge tap for air & steam Pressure gauge Safety valve Heating is done electrically

29. Used to sterilize All critical & semi-critical items that are heat & moisture resistant Surgical instrument, anesthetic equipment, dental instruments, implanted medical devices, surgical drapes, linens For sterilization of various culture media & aqueous solutions Surgical dressings Pharmaceutical products Discarding cultures ( biohazardous waste) Autoclavable plastic containers

30. Method of operating autoclave Water level is adjusted, articles are placed on perforated tray, lid is closed & autoclave is switched on Conditioning phase Safety valve is adjusted to required pressure Discharge tap is closed after complete removal of air After desired pressure is reached (15 lbs psi), the safety valve opens & excess steam escapes Exposure phase - From this point holding time is calculated (15 mins) Exhaust phase - It is switched off & allowed to cool till pressure inside is equal to that of the atmospheric pressure & discharge tap is opened & air is let into autoclave

31. Precautions Discharge tap should not be opened when inside pressure is still high because it may lead to violent boiling of the liquid media & spillage or explosion If it is opened after the inside pressure has fallen below atmospheric pressure, an excessive amount of water would have evaporated & lost from the media

32. Sterilization conditions- Temp/ time cycle 121 C for 15 min 126 C for 10 min 134 C for 3 min

33. Sterilization controls Biological indicator Geobacillus stearothermophilus ( thermophile ) is used to test efficacy of sterilization by moist heat Its spores grow at 55- 60 C & require an exposure of 12 mins at 121 C to be killed Paper strips impregnated with 10 6 spores are placed in paper envelops kept in lowermost & innermost parts of the load After sterilization, the strips are inoculated into suitable medium & incubated at 55 C for 5 days If spores germinate & color of media changes from purple to yellow then it is proved that sterilization is improper

34. Unsterilized tube Sterilized tubes

35. Chemical indicator Autoclave tape: a heat sensitive material which undergoes color change if sterilization parameter is achieved Put on the external surface of each pack to indicate that the pack has been directly exposed to sterilization method

36. Filtration Performed for heat sensitive liquids- serum, sugars & antibiotic solutions Only bacteria is removed There are two types of filters: Depth filters Membrane filters

37. Depth filters Porous filters that retain particles throughout the depth of filter Made up of mats of metallic, polymeric or inorganic material Rely on density & thickness of filter to trap particles Egs . Candle filters Unglazed porcelain Asbestos filters Sintered glass filters

38. Uses: Industrial applications Food, beverages & chemicals Advantages: Can retain a large mass of particles before becoming clogged Flow rate of fluid is high Low cost Disadvantages: Some of the particles still come out of filtrate Not suitable for filtration of solution containing bacteria

39. Membrane filters Made up of cellulose nitrate, polyvinylidene chloride & have pores of uniform & predetermined size Most common method for use in hospitals, sterility testing & for preparation of solutions for parenteral use Average pore size of 0.22 µm removes most bacteria allowing viruses to pass through

40. Air filters: surgical masks & HEPA filters Surgical masks- (3 ply mask)- polypropylene filter layer in between 2 layers of non woven fabric b. Large volumes of air can be rapidly freed from infective agents by passage through high efficiency particulate air (HEPA) filters which remove 99.97% particles of 0.3µ or larger. Used in Biosafety cabinets, OTs. ULPA filter: Ultra low particulate air filter can remove at least 99.999% of dust, pollen, mold, bacteria & any air borne particles with a size of 0.12µm or larger

41. Liquid filtration Used for bacteriological examination of water in hospital settings - dialysis water To remove bacteria from pharmaceutical fluids that are heat labile & cannot be purified by any other means Sterilisation control: Brevundimonas diminuta Serratia marsecens

42. Radiation Non ionizing radiation interferes with DNA replication but do not penetrate glass, dirt films water etc. Infrared - rapid, mass sterilization of pre-packed items (syringes, catheters), serum, vaccines, toxins Ultraviolet- enclosed areas like OTs, labs, inoculation hoods, biosafety cabinets in which dangerous microorganisms are handled & also in sterilization of drinking water - The recommended dose is 250- 300nm wavelength of UV rays for 30 minutes

43. Radiation Ionizing radiation : X rays, gamma rays and cosmic rays are used. Lethal to DNA and other vital constituents of cell Penetrative power is high Uses: plastic items, syringes, swabs, catheters, animal feeds, cardboard, oils, greases, fabrics and metal foils. Non ionizing radiation : Infra red and ultraviolet rays are used. Uses: pre packed items such as syringes and catheters.

44. Ultrasonic and sonic vibration It is high frequency sound waves. They are credited with bactericidal powers Microorganisms vary in their sensitivity to them Causes their destruction As the bubble collapses , alternate high and low pressure areas impinge on microorganism High frequency sound waves cause formation of microscopic bubbles in fluid

45. Ionizing (cold sterilization) X-rays, Gamma rays (from cobalt 60) - very high penetrative power & causes breakage of DNA but temp is not increased They induce structural defects in microbial DNA, inhibit DNA synthesis, leading to cell death Used for- Disposable rubber or plastic syringes, tissue for transplant, phamaceuticals , catheters, metal foils, oils, gloves that are unable to withstand heat Sterilisation control: Bacillus pumilus

46. Incineration Used for treatment of BMW materials It burns waste at a very high temperature 870- 1,200°C converting into ash

47. Microwave For disinfection of soft contact lenses, dental instruments, dentures, urinary catheters MOA: Microwaves are radio-frequency waves, which are (2450 MHz); produce friction of water molecules which generates heat

48. Chemical agents

49. High- level disinfectants (HLD) Kill bacterial spores when used in sufficient concentration under suitable conditions 1. Aldehyde- Formaldehyde, glutaraldehyde, ortho-phthalaldehyde MOA- They combine with nucleic acids, proteins, inactivate them by cross-linking & alkylating molecules

50. ™: ™Advantages: ™Disadvantages: Glutaraldehyde Ortho- phthalaldehyde Formaldehyde Uses Semicritical items- 2% or 2.4% (e.g. Cidex ) Disinfection time- 20 min (for spores 10- 14 hrs) Fogging, cleaning of floor & surfaces of critical areas (e.g. Bacillocid Extra) Uses Semicritical items- 0.55 % Disinfection time- 5-10 min ™Uses preservation of anatomical specimen Preservation of stool specimen embalming agent Advantage- It remains active in the presence of organic matter, has excellent material compatibility Advantages- it does not require activation, better odor, less eye irritation, acts faster (5- 10 min) Disadvantage- It has a pungent odour , can produce eye irritation, occupational asthma, contact dermatitis Disadvantage- does not kill spores effectively, stains skin gray Disadvantage- Produces irritating fumes, pungent odor, potential carcinogen, corrosive to metals, skin irritation, asthma

51. 2. Peracetic Acid Used in automated machines or as manual immersion; 0.1- 0.2%, used for 5- 15 min Use- endoscopes, arthroscopes, surgical, dental instruments ™ Disadvantages- Expensive, has material compatibility issues, causes chemical irritation, eye damage 3. Hydrogen Peroxide (H 2 O 2 ) MOA- hydroxyl free radicals that attack various cell components

52. Uses: Sporicidal only at >4- 5% 3% is used for environmental surface disinfection, fogging, for wound cleaning 3- 6% to disinfect soft contact lens, tonometer biprisms, ventilators, fabrics, and endoscopes, etc. 6- 7.5% as chemical sterilant in plasma sterilization Vaporized H 2 O 2 is used for industrial sterilization of medical devices and for decontamination of areas Advantages: rapid action, nontoxic, has detergent properties with good cleaning ability, is active in presence of organic material Disadvantages: expensive, has material compatibility issue, chemical irritation, corneal damage

53. Intermediate-level disinfectants Alcohol MOA: kill all microorganisms except spores by denaturation of proteins Uses: Alcohol (60- 80%) is used for various purposes Alcohol based handrub (ABHR) Disinfecting smaller non-critical instruments ( thermometers), which are immersed in alcohol for 10–15 minutes Disinfection of small medical items/surfaces such as rubber stoppers of multiple-dose medication vials or vaccine bottles and hubs of the central line

54. Disinfection of external surfaces of equipment such as stethoscopes, ventilators, manual ventilation bags, ultrasound machines Disinfection of non-critical surfaces such as laboratory bench, medication preparation areas Spirit (70% alcohol): Used as skin antiseptic Disadvantages Flammable and must be stored in cool, well-ventilated area Evaporate rapidly, making exposure time difficult to achieve unless the items are immersed May damage tonometer tips, lenses Inactivated by organic matter

55. 2 . Phenolics (carbolic acid) Phenol was the first widely used antiseptic & disinfectant for surgery in 1867 by Joseph Lister ( the father of antiseptic surgery ) MOA: protoplasmic poison, disrupt cell wall, precipitate cell proteins Uses Cresol, lysol for disinfecting environmental surfaces (e.g. bedside tables, bedrails, laboratory surfaces) & noncritical medical devices 5% phenol is mycobactericidal used for disinfection of sputum specimen Used as antiseptics: chloroxylenol™ Advantages: Phenolics retain activity in presence of organic materials Disadvantages: They can cause hyperbilirubinemia in infants, toxic to skin

56. 3. Halogens 1. Iodine MOA- disruption of protein & nucleic acid Tincture of iodine: iodine (2%) in potassium iodide, can cause staining, skin allergy Iodophor (e.g. povidone iodine): It is prepared by complexing iodine with carrier ( povidone ) which helps in sustained-release of iodine. It is nonstaining , no skin toxicity - Used as antiseptics 5% topical solution and ointment is used for wound cleaning 7.5% is used for hand scrub 10% is used for surgical skin preparation 1% is used as an oral antiseptic, for mouth wash - Used as disinfectant for medical equipment, such as hydrotherapy tanks, thermometers

57. 2. Chlorine & Hypochlorite Preparations: Liquid form (sodium hypochlorite or household bleach) Powder form (calcium hypochlorite or bleaching powder) Other forms: include sodium dichloroisocyanurate ( NaDCC ) available as tablets & chlorine dioxide MOA: All preparations yield hypochlorous acid ( HClO ), which causes oxidation of cellular materials, destruction of vegetative bacteria/ fungi

58. ™ Uses (free chlorine): used for disinfection of municipal water supplies & swimming pool water ™ Uses (sodium hypochlorite): available at 5.25- 6.15% (50,000 ppm of available chlorine); contact time 10- 20 min Large blood spill: 0.5% (1:10 dilution or 5,000 ppm) Small blood spill: 0.05% (1: 100 dilution, or 500 ppm) Pre-treatment of liquid waste before disposal: 1% (1:5 dilution, 10,000 ppm) Laundry items : 0.1% (1 in 50 dilution 1,000 ppm „ Surface disinfectant: 0.5% (1:10 dilution or 5,000 ppm) C. difficile (diarrheal stool): Hypocholorite is sporicidal only >0.5% (5000 ppm)

59. Advantages: Hypochlorites are broad spectrum, rapid in its action, non-flammable, low cost ™ Disadvantages: Inactivated by organic matter Toxic to skin/ mucosa, carcinogenic Daily preparation Corrosive, damages fabrics, carpets Leaves residue, requires rinsing or neutralization Offensive odors Bleaches the fabrics & carpets

60. 4. Heat-based Methods Pasteurization: Developed by Louis Pasteur, used to disinfect respiratory/ anesthesia equipment, by immersing in hot water (70°C for 30 min) Boiling at 100°C Steaming at 100°C: When the autoclave is used without closing the pressure valve, the temperature does not rise beyond 100°C. It may be useful for disinfecting those items which cannot withstand high temperature of autoclave Inspissation: egg-based culture media such as Lowenstein- Jensen medium are sterilized by heating at 80- 85°C for 30 min on 3 successive days to kill spores

61. 5 . Ultraviolet (UV) Radiation MOA: Non ionizing radiation, destroys nucleic acid through induction of thymine dimers, kills most microorganisms; not spores Uses: Disinfection of drinking water, titanium implants & contact lenses Disinfection of air and/or surfaces as in operating rooms, isolation rooms, biologic safety cabinets Disadvantages: Effectiveness influenced by organic matter In isolation rooms, it may cause skin erythema & keratoconjunctivitis in patients. UV lamps should be placed at least above 2-meters height from the floor level

62. Low-level disinfectants Destroy vegetative bacteria, enveloped viruses, variable action on nonenveloped viruses, fungi, no action on tubercle bacilli/ spores 1. Quaternary Ammonium Compound (QAC) MOA: inactivation of energy producing enzymes, denaturation of essential cell proteins, disruption of cell membrane Use: environmental sanitation of noncritical surfaces, such as floors, furniture, walls, non-critical medical equipment that contacts intact skin (BP cuffs) Eg. Benzyl ammonium chloride, didecyl dimethyl ammonium bromide

63. 2. Chlorhexidine Gluconate (CHG)- biguanide disinfectant MOA: acts by disruption of cytoplasmic membrane Uses: Hand hygiene product: Hand rub (0.5%), hand wash (4%) Mouthwash (0.1- 0.2%) Body wash solutions Skin disinfectant before surgery (2%) Antiseptic for wound cleaning™ Advantages: residual activity, less irritant Disadvantages: slower action, activity is pH dependent, greatly reduced in presence of organic matter, dermatitis on prolonged use

64. Cleaning Products Enzymatic (proteolytic) cleaners- enzymes such as amylase, lipase, cellulase, protease which break down proteinaceous matter present on equipment Cleaning chemicals (detergents)- act by reducing surface tension, dissolving fat, organic matter; detergents used for surface, floor cleaning are different than that used for instrument cleaning Cleaning Methods Manual cleaning: by immersion of instruments into cleaning solution, or by wiping surfaces with cloth soaked with cleaning solution Automatic or mechanical cleaning : useful for cleaning hard-to-reach parts of surgical instruments Egs. ultrasonic washers

65. Methods to Test efficacy of sterilant/ disinfectant Tests for chemical disinfectants Rideal & Walker test or Phenol coefficient test: tests efficacy of a phenolic disinfectant to kill S. Typhi, when compared with that of phenol Chick Martin test: disinfectant acts in presence of organic matter to simulate natural conditions (modification of Rideal & Walker) Capacity (Kelsey- Sykes) test: tests capacity of a disinfectant to retain its activity when the microbiological load keeps increasing; used to test new disinfectants In-use (Kelsey- Maurer) test: simulates real-time situation; used to determine whether an in- use solution of disinfectant in hospital is microbiologically contaminated

66. 2. Tests for Sterilizers (Indicators) Physical Indicators- digital displays of sterilizer equipment (temperature, time, pressure) Chemical Indicators- use heat/ chemical sensitive materials which undergo a color change if the sterilization parameter is achieved Class I: indicator/ external pack control; used on external surface of each pack Class II: Bowie- Dick test or as equipment control, checks efficacy of air removal, air leaks, steam penetration™ Class IV and V: internal pack control indicator, verifies whether time, steam quality, temperature are attained

67. Chemical indicator: A. Type-I (autoclave tape; B. Type V (internal pack control indicator)

68. c. Biological Indicator (BI) Use bacterial spores to check effectiveness of sterilization The spores will be destroyed only when effective condition is achieved ™ Geobacillus stearothermophilus for steam sterilizer & gas plasma (hydrogen peroxide) ™ Bacillus atrophaeus for ethylene oxide sterilizer & dry heat sterilizer (hot air oven) Spore containing vials are incubated

69. Biological indicator: A. Vial; B. Incubator

70. thanks