Sterilization

sterilization basics

1. STERILIZATION AND DISINFECTION

2. CONTENTS DEFINITIONS STERILIZING AGENTS PHYSICAL AGENTS CHEMICAL AGENTS CONCLUSION REFERENCES

3. DEFINITIONS STERILIZATION - The process by which an article, surface or medium is freed of all micro-organisms either in the vegetative or spore state. DISINFECTION - Destruction of all the pathogenic organisms or organisms capable of giving rise to infection but not necessarily bacterial spores. ANTISEPTICS - Chemical disinfectants which can safely applied to living tissues and are used to prevent infection by inhibiting the growth of microorganisms .

4. ASEPSIS - Technique by which the occurrence of infection into an uninfected tissue is prevented. DECONTAMINATION – Process of rendering an area or article free of danger from contaminants, including microbial, chemical, radioactive hazard. BACTERICIDAL - Agents which are able to kill the micro-organisms. BACTERIOSTATIC - Agents which only prevent the multiplication of bacteria . CLEANING – The process which removes visible contamination, but does not necessarily destroy microorganisms.

5. STERILIZING METHODS Physical agents Chemical agents

6. Sunlight Heat a. Dry heat b. Moist heat 4. Filtration 5. Radiation 6. Sonic & Ultrasonic vibration PHYSICAL AGENTS CHEMICAL AGENTS Alcohols Aldehydes Dyes Halogens Phenols Surface active agents Metallic salts Gases

7. SUNLIGHT Appreciable BACTERICIDAL activity. Action by combined effect of UV rays and HEAT rays. Recognised pathways : Direct - UVB rays damage nucleic acid prevents microbial replication. Linden, K. and Murphy J.R. 2017. Physical Agents. In: J.B. Rose and B. Jiménez-Cisneros, (eds) Global Water Pathogen Project, Part 4 Management of Risk from Excreta and Waste Michigan State University, E. Lansing, MI, UNESCO. Natural method of disinfec tion of water in tanks, rivers and lakes.

8. HEAT Most RELIABLE method of sterilization of articles. Factors influencing sterilization by heat: Nature of heat : Moist heat/Dry heat. Temperature and time : Inversely proportional. Number of microorganisms . Nature of microorganism : Spores are highly resistant to heat. Type of material . Presence of organic material .

9. 1. DRY HEAT G ood penetrability and non-corrosive nature of dry heat makes it applicable for sterilizing glass wares and metal surgical instruments. Destroys   bacterial endotoxins (or pyrogens ) which makes it applicable for sterilizing glass bottles.

10. a. Red Hot Held in a Bunsen flame until red hot along their whole length, almost up to the tip of their metal holder. Items sterilized by red heat – Inoculating wire loops. Points of forceps. Needles.  

11. Only exposed to the flame for a few seconds & not heating to red hot. Scalpel blades glass slides mouth of culture tubes, flasks. b. Flaming

12. Excellent & safe method. Material destroyed completely – reduced to ashes. Used for – Pathological wastes, surgical dressing, animal carcasses, plastics like PVC/ polythene. Avoid – Materials made of polystyrene as they emit black dense smoke. c. Incineration

13. Most widely used method by dry heat Glass-wares - glass syringes, petri dishes, flasks, pipettes and test tubes . Surgical instruments like Scalpels , scissors, forceps. Chemicals - liquid paraffin, fats, sulphonamides powders , glycerol and glove powder, grease d. Hot air oven

14. Heating elements fitted in chamber – fans for even distribution of heat. Thermostat – maintains the chamber air at a chosen temperature. TEMPERATURE: 160˚C – 2 hours. 170˚C – 60 minutes. 180˚C – 30 minutes. Temperature to fall up to 40°C (approx. 2 hours).

15. Should not be overloaded. Arranged in a manner which allows free circulation of air. Material to be sterilized should be perfectly dry. Test tubes, flasks etc. should be fitted with cotton plugs. Rubber materials and inflammable materials should not be kept inside . Precautions:

16. A D V A N T A G E S D I S A D V A N T A G E S

17. STERILIZATION CONTROL Biological controls:   Spores of Bacillus atrophaeus or Clostridium  tetani  on paper strips inoculated in thioglycolate or cooked meat medium & incubated for 3 to 5 days at 37°C.  B) Chemical control:  Browne’s tube No. 3  which shows a green color after sterilization at 160°C for 60 minutes (color changes from red to green). C) Physical control:  Thermocouples and temperature chart recorder used.

18. 2. MOIST HEAT Temp below 100˚C Temp at 100˚C Temp above 100˚C  Pasteurization Inspissation Water bath Boiling Koch & Arnold steamer/ Tyndallization   Autoclave

19. a. Pasteurization Used in dairy industry for milk. All non sporing pathogens like Mycobacteria, Brucella & Salmonella are destroyed . Coxiella burentii causative agent of Q fever survives holder method but killed by flash method METHOD TEMPERATURE TIME COOLING Holder method (LTLT) 63⁰C 30 mins 4˚C Flash process (HTST) 72⁰C 15-20 sec 13˚C

20. P rocess of heating an article on 3 successive days at 80-85⁰C for 30 minutes by a special instrument called Inspissator . Egg based media – e.g. Lowenstein-Jenson medium and Dorset’s egg medium. Serum based media- e.g. Loeffler’s serum slope. b. Inspissation

21. It is used to incubate samples in water. Disinfection of serum, body fluids and vaccine. Bacterial vaccine are disinfected at 60⁰C for 1 hour. Serum or heat labile body fluids can be disinfected at 56 ⁰C for 1 hour. c. Water bath

22. 100˚C for 10-30 min kills all vegetative bacteria but not the spores. Addition of 2% sodium bicarbonate may promote sterilization. Uses - For the disinfection of medical and surgical equipment – when sterility is not essential in emergency or under field conditions. d. Boiling

23. An exposure of steam100 ˚C for 20 min on three consecutive days is known as Tyndallization . Koch or Arnold steam sterilizer . Similar to that of inspissation. Uses Used for sterilization of gelation, egg , serum or sugar containing media. e. Tyndallization

24. Principle – water boils when its vapor pressure equals that of surrounding atmosphere. When pressure inside a closed vessel increases, the temperature at which water boils also increases. Steam condenses to water & gives up its latent heat to the cooler surface it comes in contact with. AUTOCLAVE

25. USES: Sterilizing culture media containing water, aqueous solutions, rubber materials, dressing materials, linen, gloves, instruments & pharmaceutical products . PRESSURE (Psi) Temperature (˚C) Time (mins) 15 121 15 20 126 10 30 134 5

26. A D V A N T A G E S D I S A D V A N T A G E S

27. Sterilisation CONTROLS Thermocouples Bacterial spores- Bacillus stear ot h erm ophilu s Browne’s tube Autoclave tapes

28.   132 -134˚C at 28-30 lb/inch for 4 min Air is mechanically removed from the chamber and load through a series of vacuum and pressure pulses. This allows the steam to penetrate porous areas of the load that couldn’t otherwise be reached with simple gravity displacement. RECENT DEVELOPMENT: PREVACUUM AUTOCLAVE

29. FILTRATION Removal of microbes by passage of a liquid or gas through a screen like material with small pores. Heat sensitive materials. Separation of bacteriophages & bacterial toxins from bacteria. Isolating viruses or organisms which are scanty in fluids .

30. Types of Filters Earthenware filters (Candle filters) Asbestos disc (Seitz) filters Used widely for purification of water for industrial and drinking purposes Made up of a disc of asbestos (magnesium trisilicate) Membrane filters Water purification & analysis Sterilization & sterility testing Bacterial count of water

31. AIR FILTERS Large volumes of air may be rapidly freed from infection by passage through high efficiency particulate air (HEPA) filters. Remove fine particles of size 0.3mcm or larger. Widely used in laminar air flow (LAF ) cabinets/chambers.

32. Standard Filtration Rate of MPPS ISO / European (ISO29463 / EN 1822) ≥ 99.95% US Standard (MIL-STD-282) ≥ 99.97% HEPA standard must satisfy certain levels of efficiency

33. RADIATION rapid mass sterilization of syringes and catheters bacteriological laboratory, inoculation hoods, laminar flow and operation theatres sterilization of disposable items such as plastic syringes, swabs, culture plates, cannulas, catheters

34. NON IONISIZING RADIATION: Types Produced by Mode of action Uses Comments Infrared rays : Sterilization of prepacked Low energy items such as syringes Use of UV light (Infrared and UV lamps DN A damage and catheters. is limited by ultraviolet rays) Ultraviolet rays: penetration and Used for disinfecting hazardous. operation threatres and laboratories. IONISIZING RADIATION: High energy For the sterilization of They are ionizing type antibiotics, hormones expensive. (Gamma rays and Cobalt-60 DN A damage and other prepacked They are very X-rays and based disposable items such effective due to cosmic rays) instruments as catheters, gloves, high penetrative syringes, infusion sets, power. oils, animal feeds, etc.

35. CHEMICAL METHODS OF STERILIZATION

36. IDEAL REQUIREMENTS Broad & wide spectrum of activity Be active in the presence of organic matter Be effective in acidic or alkaline media Have high speedy action High penetration power Be stable Be compatible with other agents Not corrode metals Not interfere with healing Not cause local irritation or sensitivity

37. SPAULDING CLASSIFICATION

38. ALCOHOLS Used as skin antiseptics and act by denaturing bacterial proteins. ETHYL (70%) & ISOPROPYL alcohol (70%)- widely used ISOPROPYL ALCOHOL - better fat solvent more bactericidal action less volatile Methyl alcohol is effective against fungal spores but weak bactericidal action. DISADVANTAGES Relatively inefficient in the presence of blood and saliva. Causes corrosion of metals.

39. ALDEHYDES Two aldehydes are currently of considerable importance: Formaldehyde Both in liquid and gaseous states. Aqueous solution is a bactericide, tuberculocide, fungicide, virucide and sporicide. A 10% aqueous solution is routinely used. However, penetration is poor and the surface must be exposed to the gas for upto 12hrs . for effective sterilization. GASEOUS STATE

40. USES:  Anatomic specimen preservation % Formalin + 0.5% sodium tetraborate – sterilised clean instruments . Fumigating sick rooms, laboratories. Disinfecting - clothing, bedding, furniture (under controlled conditions) in hospitals. Formaldehyde leaves a residue and instruments must be rinsed before use. Can cause hypersensitivity reactions

41. Glutaraldehyde High-level disinfectant and chemical sterilant . It is used as 2% buffered solution. Destroys vegetative cells in 10 – 30 mins & spores in 10hrs. It is recommended for use on surgical instruments where residual blood may be present. Kills by denaturation of proteins.

42. ADVANTAGES: High biocidal activity. Tuberculocidal , sporicidal. Used only as immersion sterilization / disinfectant . Active in organic debris. Doesn’t degrade rubber & plastic items. Use : corrugated rubber anesthetic tubes and face masks. plastic endotracheal tubes. Dental instruments. Optic fiber endoscopes. DISADVANTAGES: It can damage many metal items e.g . nickel coated impression trays & carbon steel burs It can cause hypersensitivity on repeated exposure S hould be thoroughly rinsed in sterile water prior to use.

43. HALOGENS Iodine in aqueous and alcoholic solution used as skin disinfectant. Active bactericidal agent and moderate action on spores. T incture of iodine: 2% iodine solution in 50% ethyl alcohol and sterile water. Used as an antiseptic for application over skin and mucous membrane . IODINE DISADVANTAGES: Allergy Irritation Stains skin & clothes.

44. POVIDONE-IODINE Maintains extended duration of effect, while reducing risk of irritation and cytotoxicity. O ral care applications is the 1% PVP-I gargle, which contains 0.1% available iodine . Given to patients with chronic periodontitis, irrigating extraction sockets

45. CHLORINE: In the form of: Bleaching powder Sodium hypochlorite Chloramine Disinfection is due to release of free chlorine with protein to kill micro-organism . Used in water supplies, swimming pools, food and dairy industries . CLOROX & PUREX (5.25%) - household sanitation. Sodium hypochlorite – root canal irrigant (2.5%) & D akin’s sol (0.5%)

46. ADVANTAGES: It causes tissue dissolution It has antibacterial and bleaching action It causes lubrication of canals Economical Easily available DISADVANTAGES: Irritant to tissue periapically If comes in contact, it may cause gingival inflammation Bleach the clothes Bad odour and taste Vapours can irritate the eyes

47. PHENOLS ACTION: penetrating and disrupting cell wall denaturation of intracellular proteins. Phenol is active against gram positive bacteria. Bactericidal at 1% and fungicidal at 1.3 %. Commonly used Phenol derivatives are: 1. Cresol 2. Chlorhexidine 3. C hloroxylenol 4. H exachlorophenes

48. Lysol is a solution of 50% cresols in soap. Active against a wide range of organisms. uses: 1. I nfected glass ware. 2. Cleaning of floors. 3. Disinfection of excreta . Formacresols used as R.C medicament. Cresols :

49. N on-corrosive and non-irritating to intact skin. It is used as antiseptic and for disinfecting surgical instruments. Skin cream and soap: 0.8%, Commercial 4.8 % solution used for surgical antisepsis. Chloroxylenol (Dettol )

50. Chlorhexidine Active against gram positive and gram negative bacteria. At low conc. - bacteriostatic effect . At high conc . - bactericidal effect . USES: RC irrigant post dental surgeries denture stomatitis adjunct to oral hygiene Mouth rinses - 0.2% & 0.12% Sprays – 0.1% & 0.2%

51. GASES Formaldehyde gas 150 grams of KMnO4 + 280 ml Formalin Employed for fumigation of operation theatres, wards and laboratories etc. Sterilization is achieved by condensation of gas on exposed surfaces. The doors should be sealed and left unopened for 48hours.

52. b. Ethylene oxide (ETO) Highly lethal to all kinds of microbes including spores. I ts alkylates the amino, carboxyl, hydroxyl and sulfhydryl groups in protein molecules. Uses: Sterilizing plastic and rubber articles, respirators, heart-lung machines, sutures, dental equipment and clothings. D isposable plastic syringes, Petri dishes . Its high penetrating power can sterilize prepackaged materials.

53. c. Betapropiolactone (BPL) Condensation product of ketone and formaldehyde Has low penetrating power but has a rapid action For sterilization of biological products 0.2% BPL is used Capable of killing all microorganisms and is very active against viruses Uses: In the liquid form it has been used to sterilize vaccines, tissue grafts, surgical instruments and sera as a vapor-phase  disinfectant  in enclosed spaces.

54. Recent vapour phase disinfectants Hydrogen peroxide Used to decontaminate biological safety cabinets Peracetic acid It is an oxidising agent One of the high level disinfectants Used in plasma sterilizers

55. CONCLUSION  “PREVENTION IS BETTER THAN CURE”- a proverb well suited to sterilization. Aim of sterilization is to break the chain of cross infection between patients by killing micro-organisms including spores. Effective instrument cleaning prior to sterilization should be carried out – reduces the bacterial load and renders sterilization very effective . Sterilization by steam is considered as the most efficient, cost effective and safe method of sterilizing dental instruments. The sterilization process performed should not damage the instruments and should be repeatable, standardizable, verifiable and a documented method .

56. REFERENCES Textbook of microbiology 5 th edition R.Ananthanarayan and C.K.Jayaram Paniker. Microbiology for dental students T.H.MELVILLE and C RUSSELL. Textbook of microbiology for dental students 3 rd edition. Prof. C P Baveja. Linden, K. and Murphy J.R. 2017. Physical Agents. In: J.B. Rose and B. Jiménez-Cisneros, (eds) Global Water Pathogen Project, Part 4 Management of Risk from Excreta and Waste.