In The Name of GOD

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2. Virology dept.school of public health tehran university of medical science 2

3. Historical Review (1): 3

4. Historical Review (2): 4

5. Definitions of terms (1): 5

6. Definitions of terms (2): 6

7. Categorizing of sterilization procedures : 7

8. Disinfectants: 8

9. chemical disinfectants-general consideration : Must not be toxic . Must be chemically inert to an object . Must be stable . Must have rapid disinfection effect . Must have broad spectrum of effectiveness .  9

10. Anti microbial power of chemical disinfectants depend on : Concentration of antimicrobial agent Time of exposure pH water hardness Organic matter 10

11. physical sterilization 11

12. Autoclave (Steam sterilization)1: 12

13. Autoclave (Steam sterilization)2: 13

14. Oven (dry heat sterilization) 1: 14

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17. Ultraviolet radiation : UV light consists of light of wavelengths between 210 and 328 nm, but wavelength of 260 nm is most effective in killing microorganisms . UV lamps are used to sterilize workspaces and tools used in biology laboratories and medical facilities UV light causes adjacent thymine molecules on DNA to dimerize . It is especially effective in inactivating viruses . It kills far fewer bacteria than one might expect because of DNA repair mechanisms. UV light is of limited use because it does not penetrate glass, cloth, paper or most other material , and it does not go around corners . 17

18. Filtration (1): 18

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25. Formaldehyde : Formaldehyde inactivates viruses and toxins without destroying their antigenic properties . The mechanisms of action of formaldehyde is assumed to be due to the reaction with cell proteins and DNA or RNA. Formaldehyde solution is too irritant to be used as a general disinfectant. Althought formaldehyde is a high-level disinfectant, the hospital uses of formaldehyde are limited by its irritating fumes and the pungent odor. 25

26. Glutaraldehyde (Cidex) : Usually used as a 2% alkaline-buffered solution at room temperature . Has a Wide range of bactericidal, virucidal and fungicidal activity. Slow activity against bacterial spores. Active against mycobacteria, but slower against mycobacteria avium-intracellular. Irritant to skin, eyes and respiratory mucosa . Little inactivation by organic matter, but penetrate slowly Most preparations are non-damaging to metals and other materials. 26

27. Disinfectants 27

28. Alcohol (1) : Overview : In the healthcare setting, “alcohol” refers to two water-soluble chemical compounds— ethyl alcohol and isopropyl alcohol. These alcohols are rapidly bactericidal rather than bacteriostatic against vegetative forms of bacteria. They also are tuberculocidal, fungicidal, and virucidal but do not destroy bacterial spores. The optimum bactericidal concentration is 60%–90% solutions in water. Mode of Action. The most feasible explanation for the antimicrobial action of alcohol is denaturation of proteins The bacteriostatic action was believed caused by inhibition of the production of metabolites essential for rapid cell division. 28

29. Alcohol (2) : 29

30. Alcohol (3) : 30

31. Alcohol (4) : 31

32. chlorine & chlorine compounds (1): Ca(OCl) 2+ H2O Ca ++ + H2O+ 2OCl- Ca(OCl) 2 + 2H2O Ca(OH)2 + 2HOCl How HOCl destroy microorganisms? 32

33. chlorine & chlorine compounds (2): 33

34. chlorine & chlorine compounds (3): 34

35. Iodine And Iodophor (1): Iodine , is able to penetrate the cell wall of microorganisms rapidly. It can be assumed that iodine reacts 1- with basic N-H functions that are parts of some amino acids and the bases of nucleotides . 2- By oxidizing the S-H group of the amino acid cycteine, through which the ability of connecting protein chains by disulfide bridge , as an important factor in the synthesis of proteins, is lost. The most important application of iodine in human medicine is the disinfection of skin. The previously used aqueous iodine have been replaced by the iodophors , which cause less unwanted side reactions, such as staining and irritation of tissue. 35

36. Iodine And Iodophor (2): Among the investigated iodophors, povidone-iodine has been described as the compound of choice. An iodophor is a complex of elemental iodine with a carrier that has at least three functions: 1- To increase the solubility of iodine 2- To provide a sustained release reservoir of the halogen 3- To reduce the equilibrium concentration of free molecular iodine. The carriers are neutral polymers, such as polyvinyl alcohols, polyacrylic acid, polyamids and poly saccharides. 36

37. Iodine And Iodophor (3): Practical applications of iodine as a disinfectant : 37

38. Phenol is commonly found in mouthwashes, scrub soaps and surface disinfectants, and is the active ingredient found in household disinfectants. Phenol disrupt cell membranes, denature proteins and inactivates enzymes. When used on surfaces, it retains its antimicrobial action for several days. 38

39. The addition of halogens to phenolic molecules usually increases their effectiveness . Hexachlorophene and dichlorophene , which are halogenated phenols, inhibit staphylococci and fungi, respectively , on the skin and elsewhere. Hexachlorophene is an excellent skin disinfectant. In a 3% solution, it kills staphylococci and most other gram positive organisms. 39

40. QAC or quats , have four organic groups, attached to a nitrogen atom. A variety of quats are available as disinfecting agents, their chemical structures vary according to their organic groups . One problem with quats is that their effectiveness is decreased in the presence of calcium or magnesium ions . An even more serious problem with these agents is that they support the growth of some bacteria of the genus Pseudomonas rather killing them. 40

41. The key ingredient which defines its unique antiseptic property is an aromatic chemical compound known as chloroxylenol This makes up 4.8% of Dettol's total mixture, with the rest composed of pine oil, isopropanol, castor oil ,soap, caramel and water. Because several of the ingredients are insoluble in water, Dettol produces a white emulsion of oil droplets when diluted during use. 41

42. Apart from its low toxicity and low metal corrosivity, it is also relatively cheap compared to other disinfectants. It is effective against gram positive/negative bacteria, viruses, fungi and yeast. It is able to kill 98% of microbes in just 15 seconds. 42

43. Soaps and detergents remove microbes, oily substances and dirt. In fact vigorous hand-washing is one of the easiest and cheapest means of preventing the spread of disease among patients in hospitals, in medical and dental offices. Soaps contain alkali and sodium and will kill many species of Streptococcus , Micrococcus, Niesseria and will destroy influenza viruses. Many pathogens that survive washing with soap can be killed by a disinfectant applied after washing . 43

44. A common practice after hand-washing and rinsing hands and inanimate objects is to apply 70% alcohol solution. Cationic detergent although not effective in killing endospores, they do inactivate some viruses. Anionic detergents are used for as household cleaning agents. They are less effective sanitizing agents than cationic detergents, probably because the negative charges on bacteria cell walls repel them. 44

45. In the event of a spill of infectious or potentially infectious materials, the following clean up procedures should be used. 1- Wear gloves and protective clothing , including face and eye protection . 2- Cover the spill with cloth or paper towels to contain it. 3- Pour an appropriate disinfectant over the paper towels and the immediately surrounding area ( generally, 5% bleach solutions are appropriate ). 45

46. 4- Apply disinfectant concentrically beginning at the outer margin of the spill area, working toward the center. 5- After the appropriate amount of time , clear away the materials. 6- Dispose of contaminated materials into a leakproof , puncture-resistant waste disposal container. 46

47. Ethidium bromide is a large, flat basic molecule that resembles a DNA base pair . Because of its chemical structure, it can intercalate (or insert) into a DNA strand. The compound forms fluorescent complexes with nucleic acids and these can be viewed under UV light. Ethidium bromide (EtBr) is widely employed for the rapid visualization of nucleic acids in electrophoretic gels. It may be harmful by inhalation, ingestion, and skin absorption and should be handled only when wearing gloves . 47

48. Disposal of Ethidium Bromide : Correct procedure for the disposal of EtBr , depend on the nature of the waste materials and the concentration of EtBr that they contain. Treatment of EtBr with hypochlorite (bleach) is not recommended as the degradation products retain approximately 20% mutagenic activity . Solid Waste : Small amounts of solid waste, such as tissues, gloves or stained electrophoresis gel , should be placed in appropriate packaging and sent for incineration . 48

49. Disposal of Ethidium Bromide : – Concentrated Liquid Waste ( Containing > 0.5mg/ml) : Dilute solution with water to reduce the EtBr concentration to < 0.5 mg/ml To the diluted solution , add 0.2 volume of fresh 5% hypophosphorous acid and 0.12 volume of fresh 0.5M sodium nitrite . Incubate 24 hours at room temperature. ( A check for loss of fluorescence can be used to monitor completion of the inactivation process.) Add a large excess of 1M sodium bicarbonate before discarding. 49

50. Disposal of Ethidium Bromide : – Dilute Liquid Waste ( Containing < 0.5mg/ml, e.g. electrophoresis buffer) Add 100mg powdered active charcoal to each 100ml solution. Keep at room temperature for 1 hour, shaking intermittently. Filter through a whatman No.1 filter Wrap the filter and charcoal in a plastic bag. Place inside a bag and send for incineration. 50

51. Cleaning of Equipment and Laboratory Surfaces Contaminated With EtBr: Unplug all electrical equipment before decontamination and wear appropriate protective equipment , including rubber gloves, lab coat and goggles. Make up decontamination solution just prior to use. This consist of 4.2 g of sodium nitrite and 20 ml hypophosphorous acid (50%) in 300 ml H2O. Wash the contaminated surface once with a paper towel soaked in the decontamination solution , taking care to avoid wetting electrical components. Then wash five times with water-soaked paper towels using a fresh towel each time. 51

52. Cleaning of Equipment and Laboratory Surfaces Contaminated With EtBr: 4) Soak all the towels in decontamination solution for 1 hour before disposal by incineration. 5) Use a portable UV lamp to check that decontamination is complete. EtBr absorbs a broad range of UV light, so short (245nm), medium(300-315nm) or long(365-6nm) wavelength lamps can be used. Appropriate eye protection must be worn to guard the user against UV light while the lamp is switched on. 6) Neutralize the used decontamination solution with sodium bicarbonate and discard as aqueous waste. 52

53. Working with RNA? What fun! Those little, nearly indestructible RNases are everywhere - on your skin and mucous membranes, in the water and on lab surfaces. Here are 10 ways to keep the RNases at bay, and keep your precious samples safe: 1. Clean everything; bench surfaces, pipettes, electrophoresis equipment and anything else you can think of with an RNase cleaning product, such as RNaseZap from Ambion (or 0.5% SDS followed by 3%H2O2). Establish a regular cleaning routine; a quick daily clean and a deeper weekly or monthly clean… and stick to it. 53

54. 2. Treat your solutions. Good old DEPC is a fine way to keep your solutions RNase free. Use 0.5 mL DEPC/L, incubate for 2 hr, autoclave for 45 minutes minimum. 3. Designate a workspace, and a set of pipettes, if possible, that are dedicated to RNase-free work. 4. Use barrier tips. Barrier tips stop cross-contamination of your reagents and samples by preventing aerosols reaching the barrel of your pipette. They are a must-have for RNA work. 54

55. 5. Wear gloves and a lab coat. The obvious ones are the best. Gloves and a lab coat will stop you from contaminating your samples with your own RNases. 6. Bake your glasswear. No enzyme can withstand baking for 300°C for 2 hours, but your glasswear can. 7. Use RNase-free enzymes. Enzymes isolated from bacteria (e.g. DNase) can be full of RNase. Make sure you use certified RNase-free enzymes on your RNA samples where possible. 55

56. 8. Use an RNase inhibitor when it’s not possible to keep things completely RNase-free. 9. Store RNA in ethanol at -80°C. Make aliquots if the sample is to be used a number of times to avoid freeze/thaw cycles. Before use, centrifuge to pellet the RNA, air dry then resuspend in an RNase-free buffer. 10. Be completely paranoid, work as far away from your colleagues as possible, and shower in RNaseZAP five times per day. Just kidding ! 56

57. Microbiology , principles and explorations, Jacquelyn G. Black. Disinfection in Healthcare, Petter hoffman. Antiseptic and Disinfectants: Activity, Action, and resistance. Gerald Mcdonnel and A.denever russel. Mechanisms of action of disinfectants, S.P.Denyer, B. Stewart. Antiviral Activity of alcohol for surface disinfection, WR Moorer. Disinfection Sterilization and Preservation , Lea & Febiger Philadelphia 57

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