Sterilization &
Disinfection
Sardar hussain
Asst.prof.Biotechnology
Gsc, chitradurga
Sardar1109@gmail.com
Lecture conducted for I B.Sc,biotechnology
(2013-2014 session)

• The following lecture is complied from
different textboo...
DEFINITION
• The process of freeing an article or a
surface from all living microorganisms
including viruses & bacterial s...
HEAT
• Factors affecting
– Nature of heat – dry or moist
– Temperature & time
– No of organisms present
– Characteristics ...
DRY HEAT
• Kills by oxidation, protein denaturation &
toxic effect of elevated levels of electrolyte
• Types of processes
...
Dry heat - FLAMING
• 2500C – 3000C
• Points of forceps & Inoculation loops –
heat in bunsen flame till red hot
• Slow pass...
Flaming
Dry heat - INCINERATION
• 8700C - 9800C
• Complete burning to ashes
• Used for soiled dressings, animal
carcasses, patholo...
Incineration
Dry heat - HOT AIR OVEN
• Holding temp & time: 1600C for 1 hr
• Used for glassware, forceps, swabs, water
impermeable oils...
Dry heat - HOT AIR OVEN
• Sterilization controls: to check whether the
equipment is working properly
– Chemical controls: ...
Dry heat – Hot air oven
MOIST HEAT
• Lethal effect due to denaturation &
coagulation of proteins
– Temp below 1000C
– Temp at 1000C
– Temp above 1...
Moist heat - Temp below 100 C
0

• Pasteurization
• 630C – 30 min (Holder method)
• 720C – 15-20 sec (Flash method)
• 1320...
• Inspissator

Water bath
Moist Heat - Temp at 100 C
0

• Boiling - 1000C for 10 min
•
•
•
•

Kills all vegetative bacteria
Water should be soft, de...
Moist Heat - Temp above 100 C
0

• Autoclave (steam under pressure) - 1210C, 15
min, 15 lbs
– Used for rubber articles, dr...
Moist Heat - Autoclave
Moist Heat - Autoclave
Steam Jacketed Horizontal Autoclave
FILTRATION
• Aqueous liquids may be sterilized by forced
passage through a filter of porosity small
enough to retain any m...
FILTRATION
• Types of filters
– Earthenware candles
• Unglazed ceramic & diatomaceous earth filters
• Eg. Chamberland filt...
• Asbestos Filter holder
Earthenware filter
• Sintered glass filter
Membrane filters
RADIATION
• 2 types
– Non ionising –
• Infra Red radiation ( rapid mass sterilization of syringes, etc)
• Ultra Violet rad...
STERILANT GASES
• Ethylene oxide
• Formaldehyde
• Betapropiolactone
– More efficient than HCHO
DISINFECTION
• The process of freeing an article or a
surface from all or some of the living
microorganisms but not necess...
Factors affecting DISINFECTION
•
•
•
•
•
•
•

Conc of disinfectant
Time of action
pH of the medium
Temperature
Nature & nu...
Categories of disinfectants
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Alcohol
Aldehyde
Ethylene oxide
Dyes
Halogens
Phenolics
Surfac...
ALCOHOL
• Ethanol, isopropyl alcohol
–
–
–
–

Skin antiseptics at 70%
Less sporicidal & virucidal activity
Denature bacter...
ALDEHYDE
• Formaldehyde - 10% used
–
–
–
–

In aq. soln is virucidal, bactericidal, sporicidal
Used to fumigate wards, sic...
ETHYLENE OXIDE
• Highly inflammable, mixed with inert gases
– CO2, N
• Especially for heart lung machines,
respirators, su...
DYES
• Combine with nucleic acids
• Aniline dyes
– Brilliant green, malachite green, crystal violet

• Acridine dyes
– Pro...
HALOGENS
• Kills by oxidation
• Iodine – 2.5% in 70% alcohol, Skin antiseptic
• Iodophores (iodine + non-ionic surface act...
PHENOLICS
• Carbolic acid – 2-5%
– Powerful microbicidal, very corrosive
– General purpose disinfectant in hospital

• Cre...
SURFACE ACTIVE AGENTS
• Disrupt cell memb, 4 main groups
• Anionic surfactants – strong detergent
action, weak antimicrobi...
METTALIC SALTS
• Mercuric salts – ointments
• Silver salts – AgNO3 – to prevent infection
of burns, ophthalmia neonatorum
...
DIGUANIDES
• Chlorhexidine – burns, skin disinfection
• Picloxydine –hospital equipment, floors
AMIDES
• Propamide
• Dibromopropamide
• Antiseptic cream, eye ointments
TESTS FOR DISINFECTANTS
• To determine efficacy of disinfectants
– Phenol Co-efficient method
• Rideal Walker test
• Chick...
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

PRIONS
BACTERIAL SPORES
Bacillus subtilis
Clostridium sporogenes
MYCOBACTERIA

Sterilis...
Categories
• Sterilisation
• High level disinfection (HLD) kills all microorganisms except
high number of bacterial spores...
Plasma technology
• Glow discharge or low temperature
plasmas
• Plasma has sufficient energy to disrupt
molecular bonds
• ...
A known HIV positive patient is admitted in an
isolation ward after an abdominal surgery following
an accident. The reside...
Heat labile instruments for
use in surgical procedures
can be best sterilized by
•
•
•
•

Absolute alcohol
Ultra violet ra...
Sterilizing agents include
–
–
–
–

Cyclohexidene
Ethylene oxide
Diethyl ether
Gallamine
Sharp instruments are not sterilized
by
•
•
•
•

Dry heat
Boiling
2% glutaraldehyde
Autoclaving
Which is a form of cold sterilization
•
•
•
•

Gamma rays
Beta rays
IR rays
Autoclave
Sterilization of serum containing
media
•
•
•
•

Autoclaving
Filtration
Gamma radiation
Incineration
Sterilizing agents include
•
•
•
•
•

Ether
Alcohol
Chlorhexidine
Dry heat
Ethylene oxide
Sterilization & disinfection
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Sterilization & disinfection

  1. 1. Sterilization & Disinfection Sardar hussain Asst.prof.Biotechnology Gsc, chitradurga Sardar1109@gmail.com
  2. 2. Lecture conducted for I B.Sc,biotechnology (2013-2014 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. 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. 4. HEAT • Factors affecting – 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. 5. DRY HEAT • Kills by oxidation, protein denaturation & toxic effect of elevated levels of electrolyte • Types of processes – Flaming – Incineration – Hot air oven
  6. 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. 7. Flaming
  8. 8. Dry heat - INCINERATION • 8700C - 9800C • Complete burning to ashes • Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable soiled bedding
  9. 9. Incineration
  10. 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. 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 37 0C for 5 days • Growth in medium indicates failure of sterilization
  12. 12. Dry heat – Hot air oven
  13. 13. MOIST HEAT • Lethal effect due to denaturation & coagulation of proteins – Temp below 1000C – Temp at 1000C – Temp above 1000C
  14. 14. Moist heat - Temp below 100 C 0 • Pasteurization • 630C – 30 min (Holder method) • 720C – 15-20 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 – 80-850C – 30 min – 3 days • For media containing egg or serum – LJ, LSS
  15. 15. • Inspissator Water bath
  16. 16. Moist Heat - Temp at 100 C 0 • 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) – 30-60 min in Arnold /Koch steamer – For heat labile media – DCA, TCBS – 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
  17. 17. Moist Heat - Temp above 100 C 0 • Autoclave (steam under pressure) - 1210C, 15 min, 15 lbs – Used for rubber articles, dressings, sharp instruments, infectious medical waste, culture media – Principle – (refer Ananthanarayan & Paniker 7 edn Page 27) th • 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. 18. Moist Heat - Autoclave
  19. 19. Moist Heat - Autoclave
  20. 20. Steam Jacketed Horizontal Autoclave
  21. 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, carbohydrates soln, filtrates of toxins & bacteriophages, in water bacteriology, in examination of Schistosoma eggs
  22. 22. 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: 0.015 – 12 μm • HEPA filters – for large volumes of air Sterilization control – bubble pressure test
  23. 23. • Asbestos Filter holder Earthenware filter
  24. 24. • Sintered glass filter
  25. 25. Membrane filters
  26. 26. RADIATION • 2 types – Non ionising – • 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
  27. 27. STERILANT GASES • Ethylene oxide • Formaldehyde • Betapropiolactone – More efficient than HCHO
  28. 28. 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
  29. 29. 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
  30. 30. Categories of disinfectants 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Alcohol Aldehyde Ethylene oxide Dyes Halogens Phenolics Surface active agents Metallic salts Diguanides Amides
  31. 31. 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
  32. 32. 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
  33. 33. ETHYLENE OXIDE • Highly inflammable, mixed with inert gases – CO2, N • Especially for heart lung machines, respirators, sutures, syringes, dental equipments
  34. 34. DYES • Combine with nucleic acids • Aniline dyes – Brilliant green, malachite green, crystal violet • Acridine dyes – Proflavine, acriflavine, euflavine, aminacrine • Skin & wound antiseptics • Bacteriostatic, more active against GP bacteria
  35. 35. 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
  36. 36. PHENOLICS • Carbolic acid – 2-5% – 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
  37. 37. 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
  38. 38. METTALIC SALTS • Mercuric salts – ointments • Silver salts – AgNO3 – to prevent infection of burns, ophthalmia neonatorum • Copper salts – antifungal, antialgae – water reservoirs, swimming pools
  39. 39. DIGUANIDES • Chlorhexidine – burns, skin disinfection • Picloxydine –hospital equipment, floors
  40. 40. AMIDES • Propamide • Dibromopropamide • Antiseptic cream, eye ointments
  41. 41. 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
  42. 42. • • • • • • • • • • • • • • • • • PRIONS BACTERIAL SPORES Bacillus subtilis Clostridium sporogenes MYCOBACTERIA Sterilisation High Level Disinfection Intermediate Level disinfection M. tuberculosis var bovis NON LIPID OR SMALL VIRUSES Polio virus, Rhino virus FUNGI Trichophyton, Candida, Cryptococcus VEGETATIVE BACTERIA Pseudomonas, Staphylococcus, VRE LIPID OR MEDIUM SIZED VIRUSES HBV, HIV, HSV, HCV, EBOLA, CMV Low Level Disinfection
  43. 43. 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
  44. 44. 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
  45. 45. 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
  46. 46. Heat labile instruments for use in surgical procedures can be best sterilized by • • • • Absolute alcohol Ultra violet rays Cholorine releasing compound Ethylene oxide gas
  47. 47. Sterilizing agents include – – – – Cyclohexidene Ethylene oxide Diethyl ether Gallamine
  48. 48. Sharp instruments are not sterilized by • • • • Dry heat Boiling 2% glutaraldehyde Autoclaving
  49. 49. Which is a form of cold sterilization • • • • Gamma rays Beta rays IR rays Autoclave
  50. 50. Sterilization of serum containing media • • • • Autoclaving Filtration Gamma radiation Incineration
  51. 51. Sterilizing agents include • • • • • Ether Alcohol Chlorhexidine Dry heat Ethylene oxide
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