1. Sterilization
Why Sterilization ?
Contaminated of surgical instruments will leads to
infections.
2. Contamination in Microbiology lab, leads to
difficulty in diagnosis and misdiagnosis.
3. Contamination in drug and food industries.

2. Sterilization : It is the process by which an article, surface or media are freed of all the micro-organisms either in vegetative or spore state. Disinfections: It is the process of removing the micro-organisms which are liable to produce infections.

3. Classification: 1. Physical methods A. Heat B . Filtration
C. Radiation
2. Chemical methods.
Various chemicals

4. HEAT Factors affecting by heat: 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
Mechanism: Kills by oxidation, protein denaturation & toxic effect of elevated levels of electrolyte
Types:
1. Flaming
2. Incineration
3. Hot air oven

6. 1. Flaming
Temp of flame 2500C – 3000C

7. 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

8. 2. INCINERATION
“ Complete burning of hospital waste until it becomes ash” Eg: Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable soiled bedding

10. 3. HOT AIR OVEN

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

12. Sterilization controls:
1. Chemical controls: Browne’s tubes
Color change from red to green
2. Thermocouples
3. 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

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14. MOIST HEAT
Mechanism: Lethal effect due to coagulation &
denaturation of proteins
Methods:
Temp below 1000C
Temp at 1000C
Temp above 1000C

15. Eg Salmonella, Brucella and Mycobacteria
TEMP BELOW 1000C
1. Pasteurization
630C – 30 min (Holder method)
720C – sec (Flash method)
Eg Salmonella, Brucella and Mycobacteria
2. Vaccine baths - 600C – 60 min
For vaccines of non-sporing bacteria
3. Water bath - 560C – 60 min – 3 days consecutive days
For serum / body fluids containing coagulable proteins
4. Inspissation – C – 30 min – 3 days
For media containing egg or serum – LJ, LSS

17. Tyndallisation (intermittent sterilization) -
TEMP AT 1000C
1. Boiling (1000C):
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): Arnold /Koch steamer
90 minutes at a stretch.
Tyndallisation (intermittent sterilization) -
1000C, 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

18. TEMP ABOVE 1000C
Autoclave (steam under pressure):

20. Uses: Used for rubber articles, dressings, sharp instruments, infectious medical waste, culture media.
Sterilization control
Thermocouples
106 spore of B. stearothermophilus. Incubate at 550C for 5 days

21. 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, sugar soln., filtrates of toxins & bacteriophages, in water bacteriology, in examination of Schistosoma eggs

22. Types of filters:
1. Earthenware candles-
Unglazed ceramic & diatomaceous earthen filters
Eg. Chamberland filters, Doulton filters
2. Asbestos filter- Seitz, Carlson
3. Sintered glass filter
4. Membrane filters – cellulose nitrate, cellulose acetate,
polycarbonate, polyester filters
5. HEPA (high efficiency particle air) filters – for large
volumes of air.

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27. RADIATION
Types:
1. Non ionizing –
Infra-red radiation ( rapid mass sterilization of syringes, etc)
Ultra Violet radiation (enclosed areas)
2. Ionising – Gamma, X ray, cathode ray
(plastics)

28. Disinfection
Strong disinfectants – for inanimate object
Mild disinfectant (antiseptic) – superficial application on living tissue

29. Categories of disinfectants
Alcohol
Aldehyde
Phenolics
Halogens
Dyes
Surface active agents
Gases
Metallic salts

30. Alcohols:
Ethanol, isopropyl alcohol have optimal bactericidal activity in aqueous solution at 70% conc.
Have limited activity against Mycobacteria and nonsporicidal. Action against viruses is good.
Due to volatile activity widely recommended as disinfectant of skin and surfaces.
Their efficacy in organic matter is low.
Methyl alcohol – to treat cabinets / incubators affected by fungal spores

31. Aldehydes:
1. Formaldehyde:
In aq. Soln (10%) is virucidal, bactericidal, sporicidal
Used in preservation of Anatomical specimens.
Has pungent smell, irritant to skin, eyes, mucus memb & toxic when inhaled
2. Glutaraldehyde –
less toxic, less irritant.
Used rubber endotracheal tubes, face masks metal instruments, polythene tubing.

32. Phenolics:
Carbolic acid – 5%
Powerful microbicidal.
General purpose disinfectant in hospital
Eg- Cresol, lysol
Chloroxylenol, chlorophenol, hexachlorophane – less toxic, less irritant, less active, more readily inactivated by organic matter

33. Mechanism: Kills by oxidation
HALOGENS
Mechanism: 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

34. DYES
Mechanism: Combine with nucleic acids
1. Aniline dyes
Eg- Brilliant green, malachite green, crystal violet
2. Acridine dyes
Eg-Proflavine, acriflavine, euflavine, aminacrine
Skin & wound antiseptics (Bacteristatic)

35. 1. Anionic surfactants – strong detergent
SURFACE ACTIVE AGENTS
Disrupt cell membrane
4 main groups-
1. Anionic surfactants – strong detergent
action, weak antimicrobial action
2. Non-ionic surfactants
3. Cationic surfactants – quaternary
ammonium compounds – cetrimide
4. Amphoteric surfactants – both detergent &
antimicrobial properties – Tego comps

36. GASES
Ethylene oxide:
It is a colourless gas highly inflammable and explosive. Mixed with carbon dioxide to eliminate its explosiveness.
Effective against all organisms including spores.
Used to sterilize heart lung machines, respirators dental equipments etc.
Not suitable for fumigation.

37. 2. Formaldehyde gas: Generated by adding pottacium permanganate to fomaldehyde solution. Room to be closed for 48 hrs. 3. Betapropiolactone (BPL): More effective than formaldehyde gas, suitable for fumigation of OT.

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

39. THANK YOU