1. Sterilization & Disinfection
Tukaram Prabhu K
Asst Professor
Microbiology
PCMS & RC

2. Lecture conducted for II yr MBBS (2008-2009 session)
The following lecture is complied from different textbooks and is for teaching - learning use only
This lecture is not a substitute for classroom lecture but an adjunct for the same
This lecture may be useful for BSc Nursing, BMLT & DMLT students too

3. DEFINITION
The process of freeing an article or a surface from all living microorganisms including viruses & bacterial spores
Various methods
Heat
Filtration
Radiation
Sterilant gases

4. HEAT Factors affecting Two methods Nature of heat – dry or moist
Temperature & time
No of organisms present
Characteristics of the organism
Type of material
Two methods
Dry heat
Moist heat

5. DRY HEAT
Kills by oxidation, protein denaturation & toxic effect of elevated levels of electrolyte
Types of processes
Flaming
Incineration
Hot air oven

6. Dry heat - FLAMING
2500C – 3000C
Points of forceps & Inoculation loops – heat in bunsen flame till red hot
Slow passage through flame to destroy vegetative bacteria on surface of scalpel blade, glass slides, mouths of test tubes

7. Flaming

8. Dry heat - INCINERATION
8700C C
Complete burning to ashes
Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable soiled bedding

9. Incineration

10. Dry heat - HOT AIR OVEN Holding temp & time: 1600C for 1 hr
Used for glassware, forceps, swabs, water impermeable oils, waxes & powders
Before placing in hot air oven
Dry glassware completely
Plug test tubes with cotton wool
Wrap glassware in Kraft papers
Don’t over load the oven
Allow free circulation of air between the material

11. Dry heat - HOT AIR OVEN
Sterilization controls: to check whether the equipment is working properly
Chemical controls: Browne’s tubes
Color change from red to green
Thermocouples
Biological controls: paper strips containing106 spores of Clostridium tetani
Place strips in oven along with other material for the sterilization
Later culture the strips in thioglycollate broth or RCM at 370C for 5 days
Growth in medium indicates failure of sterilization

12. Dry heat – Hot air oven

13. MOIST HEAT Lethal effect due to denaturation & coagulation of proteins
Temp below 1000C
Temp at 1000C
Temp above 1000C

14. Moist heat - Temp below 1000C
Pasteurization
630C – 30 min (Holder method)
720C – sec (Flash method)
1320C – 1 sec (Ultra high temp)
Vaccine baths - 600C – 60 min
For vaccines of non-sporing bacteria
Water bath - 560C – 60 min – 3 days
For serum / body fluids containing coagulable proteins
Inspissation – C – 30 min – 3 days
For media containing egg or serum – LJ, LSS

15. Inspissator Water bath

16. Moist Heat - Temp at 1000C Boiling - 1000C for 10 min
Kills all vegetative bacteria
Water should be soft, deionized or distilled
2% sodium bicarbonate promotes the process
Kills vegetative bacteria, hepatitis virus & some spores
Steaming (free steam) – min in Arnold /Koch steamer
For heat labile media – DCA, TCBS
Tyndallisation (intermittent sterilization) C, 30 min, 3 days
Nutrient media & media containing sugars or gelatin
I day all vegetative bacteria are killed. On II & III day spores that germinate are killed

17. Moist Heat - Temp above 1000C
Autoclave (steam under pressure) C, 15 min, 15 lbs
Used for rubber articles, dressings, sharp instruments, infectious medical waste, culture media
Principle – (refer Ananthanarayan & Paniker 7th edn Page 27)
Sterilization control
Thermocouples
Browne’s tube (red-green), Bowie & Dick tape (white-brown)
106 spore of B stearothermophilus. Incubate at 550C for 5 days

18. Moist Heat - Autoclave

20. Moist Heat - Autoclave

21. Steam Jacketed Horizontal Autoclave

22. FILTRATION
Aqueous liquids may be sterilized by forced passage through a filter of porosity small enough to retain any microorganisms present in them
Used to sterilize serum, carbohydrates soln, filtrates of toxins & bacteriophages, in water bacteriology, in examination of Schistosoma eggs

23. FILTRATION Types of filters Earthenware candles
Unglazed ceramic & diatomaceous earth filters
Eg. Chamberland filters, Doulton filters
Asbestos filter – Seitz, Carlson, Sterimat
Sintered glass filter
Membrane filters – cellulose nitrate, cellulose acetate, polycarbonate, polyester filters
Pore size: – 12 μm
HEPA filters – for large volumes of air
Sterilization control – bubble pressure test

24. Asbestos Filter holder Earthenware filter

25. Sintered glass filter

26. Membrane filters

27. RADIATION 2 types Non ionising – Ionising – Gamma, X ray, cathode ray
Infra Red radiation ( rapid mass sterilization of syringes, etc)
Ultra Violet radiation (enclosed areas)
Ionising – Gamma, X ray, cathode ray
(plastics, syringes, oil, metal foils)
Sterilization controls
Dosimeter – measures radiation dose
Colored discs
M radiodurans, B pumilus

28. STERILANT GASES Ethylene oxide Formaldehyde Betapropiolactone
More efficient than HCHO

29. DISINFECTION
The process of freeing an article or a surface from all or some of the living microorganisms but not necessarily bacterial spores
Strong disinfectants – for inanimate object
Mild disinfectant (antiseptic) – superficial application on living tissue

30. Factors affecting DISINFECTION
Conc of disinfectant
Time of action
pH of the medium
Temperature
Nature & number of organisms
Presence of extraneous material
Others – hardness of water, relative humidity

31. Categories of disinfectants
Alcohol
Aldehyde
Ethylene oxide
Dyes
Halogens
Phenolics
Surface active agents
Metallic salts
Diguanides
Amides

32. ALCOHOL Ethanol, isopropyl alcohol Skin antiseptics at 70%
Less sporicidal & virucidal activity
Denature bacterial proteins
Isopropyl alcohol better fat solvent, more bactericidal and less volatile
Methyl alcohol – to treat cabinets / incubators affected by fungal spores
Others – benzyl alcohol, chlorbutol, phenylethanol

33. ALDEHYDE Formaldehyde - 10% used
In aq. soln is virucidal, bactericidal, sporicidal
Used to fumigate wards, sick rooms, labs
Expose to ammonia to remove residual formaldehyde
Has pungent smell, irritant to skin, eyes, mucus memb & toxic when inhaled
Glutaraldehyde – less toxic, less irritant
Endotracheal tubes, metal instruments, polythene tubing
Β propiolactone (BPL) – condensation product of ketane & formaldehyde
More efficient for fumigation but is carcinogenic
0.2% generally used

34. ETHYLENE OXIDE Highly inflammable, mixed with inert gases – CO2, N
Especially for heart lung machines, respirators, sutures, syringes, dental equipments

35. DYES Combine with nucleic acids Aniline dyes Acridine dyes
Brilliant green, malachite green, crystal violet
Acridine dyes
Proflavine, acriflavine, euflavine, aminacrine
Skin & wound antiseptics
Bacteriostatic, more active against GP bacteria

36. HALOGENS Kills by oxidation
Iodine – 2.5% in 70% alcohol, Skin antiseptic
Iodophores (iodine + non-ionic surface active agent) – betadine – non staining, less irritant, less toxic
Chlorine – disinfect water supplies, swimming pools
Sodium hypochlorite – 1% for HIV
Organic chloramines – antiseptic for wound dressings

37. PHENOLICS Carbolic acid – 2-5% Cresol, lysol
Powerful microbicidal, very corrosive
General purpose disinfectant in hospital
Cresol, lysol
Chloroxylenol, chlorophenol, hexachlorophane – less toxic, less irritant, less active, more readily inactivated by organic matter

38. SURFACE ACTIVE AGENTS Disrupt cell memb, 4 main groups
Anionic surfactants – strong detergent action, weak antimicrobial action
Non-ionic surfactants
Cationic surfactants – quaternary ammonium compounds – cetrimide, benzalkonium chloride - bacteriostatic
Amphoteric surfactants – both detergent & antimicrobial properties – Tego comps

39. METTALIC SALTS Mercuric salts – ointments
Silver salts – AgNO3 – to prevent infection of burns, ophthalmia neonatorum
Copper salts – antifungal, antialgae – water reservoirs, swimming pools

40. DIGUANIDES Chlorhexidine – burns, skin disinfection
Picloxydine –hospital equipment, floors

41. AMIDES
Propamide
Dibromopropamide
Antiseptic cream, eye ointments

42. TESTS FOR DISINFECTANTS
To determine efficacy of disinfectants
Phenol Co-efficient method
Rideal Walker test
Chick Martin test
Compares disinfectant with phenol
Kelsey & Sykes Capacity test
Determines dilution of disinfectant to be used
Kelsey & Mauer In-use test (stability test)
Checks end result of disinfection

43. PRIONS
BACTERIAL SPORES Sterilisation
High Level Disinfection
Bacillus subtilis
Clostridium sporogenes Intermediate Level disinfection
MYCOBACTERIA
M. tuberculosis var bovis
NON LIPID OR SMALL VIRUSES Low Level Disinfection
Polio virus, Rhino virus
FUNGI
Trichophyton, Candida, Cryptococcus
VEGETATIVE BACTERIA
Pseudomonas, Staphylococcus, VRE
LIPID OR MEDIUM SIZED VIRUSES
HBV, HIV, HSV, HCV, EBOLA, CMV

44. Categories Sterilisation
High level disinfection (HLD) kills all microorganisms except high number of bacterial spores - aldehydes, hydrogen peroxide, ortho-phthaldehyde
Intermediate level disinfection (ILD) kills all vegetative bacteria including M. tuberculosis var bovis, all fungi and most viruses - phenolics, iodophores, chlorine compounds, alcohols
Low level disinfection (LLD) kills most vegetative bacteria but not M. tuberculosis var bovis, some fungi and some viruses - Hospital type germicides- quaternary ammonium compounds

45. Plasma technology Glow discharge or low temperature plasmas
Plasma has sufficient energy to disrupt molecular bonds
Direct current, radiofrequency, microwave power is used to produce the plasma
Used in disinfection of OTs

46. A known HIV positive patient is admitted in an isolation ward after an abdominal surgery following an accident. The resident doctor who changed his dressing the next day found it to be soaked in blood. Which of the following would be the right method of choice of discarding the dressings :
a) Pour 1% hypochlorite on the dressing material and send it for incineration in a appropriate bag
b) Pour 5% hypochlorite on the dressing material and send it for incineration in a appropriate bag
c) Put the dressing material directly in an appropriate bag and send for incineration
d) Pour 2% Lysol on the dressing material and send it for incineration in a appropriate bag

47. Heat labile instruments for use in surgical procedures can be best sterilized by
Absolute alcohol
Ultra violet rays
Cholorine releasing compound
Ethylene oxide gas

48. Sterilizing agents include
Cyclohexidene
Ethylene oxide
Diethyl ether
Gallamine

49. Sharp instruments are not sterilized by
Dry heat
Boiling
2% glutaraldehyde
Autoclaving

50. Which is a form of cold sterilization
Gamma rays
Beta rays
IR rays
Autoclave

51. Sterilization of serum containing media
Autoclaving
Filtration
Gamma radiation
Incineration

52. Sterilizing agents include
Ether
Alcohol
Chlorhexidine
Dry heat
Ethylene oxide