Sterilization physical methods

1. STERILIZATION BY
PHYSICAL METHODS
Dr.T.V.Rao MD
12/2/2012 Dr.T.V.Rao MD 1

2. Why we need Sterilization
• Microorganisms capable of causing
infection are constantly present in the
external environment and on the human
body.
• Microorganisms are responsible for
contamination and infection.
• The aim of sterilisation is to remove or
destroy them from materials or from
surfaces.
12/2/2012 Dr.T.V.Rao MD 2

3. How can microorganisms
be killed?
Denaturation of proteins (e.g. wet heat, ethylene
oxide)
Oxidation (e.g. dry heat, hydrogen peroxide)
Filtration
Interruption of DNA synthesis/repair (e.g.
radiation)
Interference with protein synthesis (e.g. bleach)
Disruption of cell membranes (e.g. phenols)
12/2/2012 Dr.T.V.Rao MD 3

4. Classification
There are two types of 2. Chemical sterilization
sterilization: physical and includes:
chemical.  Alcohols
1. Physical sterilization  Aldehydes
includes:
 Phenolics
 heat
 Oxidizing agents
 radiation
 Quaternary ammonium
 filtration compounds
 ethylene oxide gas
 Others
12/2/2012 Dr.T.V.Rao MD 4

5. Definitions:
Sterilisation :
– It is a process by which an article, surface or medium is
made free of all microorganisms either in vegetative or
spore form.
Disinfection :
– Destruction of all pathogens or organisms capable of
producing infections but not necessarily spores.
– All organisms may not be killed but the number is
reduced to a level that is no longer harmful to health.
12/2/2012 Dr.T.V.Rao MD 5

6. Antiseptics :
Antiseptics :
– Chemical disinfectants which can safely
applied to living tissues and are used to
prevent infection by inhibiting the growth
of microorganisms.
Asepsis :
– Technique by which the occurrence of
infection into an uninfected tissue is
prevented.
12/2/2012 Dr.T.V.Rao MD 6

7. Factors that influence efficacy of
disinfection/sterilization
Contact time
Physico-chemical environment (e.g. pH)
3 Presence of organic material
4 Temperature
5 Type of microorganism
6 Number of microorganisms
7 Material composition
12/2/2012 Dr.T.V.Rao MD 7

8. Uses of sterilisation:
1. Sterilisation of materials, instruments
used in surgical and diagnostic
procedures.
2. Sterilisation of Media and reagents
used in the microbiology laboratory.
3. Food and drug manufacturing to
ensure safety from contaminating
organisms.
12/2/2012 Dr.T.V.Rao MD 8

9. Understanding the Terminology
• a suffix indicating that the antimicrobial agent will kill or
• destroy a certain group of microorganism
• suffix “cide” – meaning to kill
• viricide – destroys virus
• fungicide – destroys fungi
• bactericide – destroys bacteria
•
• Suffix “static/stasis” – meaning to stand still
• a suffix indicating that the agent will prevent the growth or
• multiplication of the type of organism but are not killed outright
•
• bacteriostatic - prevents the growth of bacteria
• fungi static – prevents the growth of fungi
12/2/2012 Dr.T.V.Rao MD 9

10. Relative Resistance of Microbial
Forms
Highest resistance Moderate resistance Least resistance
bacterial endospore protozoan cyst most bacterial vegetative cells
(Bacillus & Clostridium) some fungal spores ordinary fungal spores & hypae
some naked virus enveloped virus
vegetative bacteria that Yeasts
have higher resistance Trophozoites
( M. tuberculosis, S.aureus,
Pseudomonas)
12/2/2012 Dr.T.V.Rao MD 10

11. What to sterilize?
• It is mandatory to sterilize :
– all instruments that penetrate soft tissues and bone.
– Instruments that are not intended to penetrate the
tissues, but that may come into contact with oral
tissues.
• If the sterilization procedure may damage the
instruments, then, sterilization can be replaced
by Disinfection procedure
12/2/2012 Dr.T.V.Rao MD

12. Ideal sterilization/disinfection process
• Highly efficacious
• Fast
• Good penetrability
• Compatible with all materials
• Non-toxic
• Effective despite presence of organic material
• Difficult to make significant mistakes in process
• Easily monitored
12/2/2012 Dr.T.V.Rao MD 12

13. Control of Microbial Growth:
Rate of Microbial Death
When bacterial populations are heated or treated
antimicrobial chemicals, they usually die at a
constant rate.
12/2/2012 Dr.T.V.Rao MD 13

14. Figure 9.1 A plot of microbial death rate
Number of living microbes
90% die Constant percentage
of the extant population
1 min is killed each minute
90% die
1 min
Time (min)
12/2/2012 Dr.T.V.Rao MD 14

15. Methods
1.Physical
methods
2.Chemical
methods
12/2/2012 Dr.T.V.Rao MD 15

16. Physical methods:
• Physical
methods:
1.Sunlight
2.Heat
1.Dry heat
2.Moist heat
3.Filtration
4.Radiation
12/2/2012 Dr.T.V.Rao MD 16

17. Chemical methods
• Chemical methods:
1. Alcohols
2. Aldehydes
3. Phenols
4. Halogens
5. Oxidizing agents
6. Salts
7. Surface active agents
8. Dyes
9. Vapor phase disinfectants
12/2/2012 Dr.T.V.Rao MD 17

18. Physical Methods
12/2/2012 Dr.T.V.Rao MD 18

19. How to Sterilize
Materials Method
1 Inoculating wires and loops Red heat
2 Glass ware- syringes, petridishes, testtubes, flasks Hot –air oven
etc.
3 Disposable syringes, and other disposable items Gamma radiation
4 Culture media Autoclaving
5 Culture media containing serum and egg Tyndallisation
6 Toxin , serum, sugar, and antibiotic solutions Filtration
7 Cystoscope and endoscope Glutaraldehyde
8 Infected soiled dressings Incineration
9 Skin Iodine, alcohol
12/2/2012 Dr.T.V.Rao MD 19
10 Milk Pasteurisation

20. Physical Methods of Microbial Control
Dry Heat:
Direct Flaming: Used to sterilize inoculating
loops and needles. Heat metal until it has a
red glow.
Incineration: Effective way to sterilize disposable
items (paper cups, dressings) and biological waste.
Hot Air Sterilization: Place objects in an oven.
Require 2 hours at 170oC for sterilization. Dry heat is
transfers heat less effectively to a cool body, than
moist heat.
12/2/2012 Dr.T.V.Rao MD 20

21. Physical Methods of Microbial Control
• Heat-Related Methods
– Moist heat
• Pasteurization
– Used for milk, ice cream, yogurt, and fruit juices
– Not sterilization
» Heat-tolerant microbes survive
– Pasteurization of milk
» Batch method
» Flash pasteurization
» Ultrahigh-temperature pasteurization
12/2/2012 Dr.T.V.Rao MD 21
© 2012 Pearson Education Inc.

22. Physical Methods of Microbial Control
Moist Heat (Continued):
Pasteurization: Developed by Louis Pasteur to
prevent the spoilage of beverages. Used to
reduce microbes responsible for spoilage of
beer, milk, wine, juices, etc.
 Classic Method of Pasteurization: Milk was
exposed to 65oC for 30 minutes.
 High Temperature Short Time Pasteurization
(HTST): Used today. Milk is exposed to 72oC for 15
seconds.
12/2/2012 Dr.T.V.Rao MD 22

23. Inspissation:
1. Inspissation:
 Heating at 80-85°C for half an hour daily on
three consecutive days
 Serum or egg media are sterilised
2. Vaccine bath:
 Heating at 60°C for an hour daily in vaccine
bath for several successive days.
 Serum or body fluids can be sterilised by
heating at 56°C for an hour daily for several
successive days.
12/2/2012 Dr.T.V.Rao MD 23

24. Sun light:
• Sun light:
– Active germicidal
effect due to its
content of ultraviolet
rays .
– Natural method of
sterilisation of water
in tanks, rivers and
lakes.
12/2/2012 Dr.T.V.Rao MD 24

25. Heat :
• Factors influencing:
• Nature of heat
• Temperature and duration
• Characteristic of organism and
spores
• Type of material
12/2/2012 Dr.T.V.Rao MD 25

26. Heat effectively kills Majority of
Microbes
Heat :
• Principle:
– Dry heat kills the organism by
• denaturation of the bacterial proteins,
• oxidative damage
• toxic effect of elevated levels of
electrolytes.
12/2/2012 Dr.T.V.Rao MD 26

27. Heat :
• Dry heat:
1.Red heat
2.Flaming
3.Incineration
4.Hot air oven
12/2/2012 Dr.T.V.Rao MD 27

28. Dry-Heat Sterilization
• Involves heating at atmospheric pressure
and often use a fan to obtain uniform
temperature by circulation.
• Heat at 180º for half hour , 170º for 1 hr.,
or 160º C for 2 hrs.
• Times are the periods during which
object is maintained at the respective
temp.
12/2/2012 Dr.T.V.Rao MD

29. Dry heat:
• Dry heat:
1. Red heat:
Materials are held
in the flame of a
bunsen burner till
they become red
hot.
» Inoculating
wires or loops
» Tips of forceps
» Needles
12/2/2012 Dr.T.V.Rao MD 29

30. Dry heat:
• Dry heat:
2. Flaming: Materials are
passed through the
flame of a bunsen
burner without
allowing them to
become red hot.
» Glass slides
» scalpels
» Mouths of culture
tubes
12/2/2012 Dr.T.V.Rao MD 30

31. Incineration:
• Materials are
reduced to ashes by
burning.
• Instrument used was
incinerator.
• Soiled dressings
• Animal carcasses
• Bedding
12/2/2012 • Pathological material
Dr.T.V.Rao MD 31

32. Dry-Heat Sterilization
Disadvantages
• Disadvantages:
–Less reliable than autoclaving
–Large temp difference may arise within
device.
–sharp instruments get dulled
–Many materials do not tolerate dry heat
12/2/2012 Dr.T.V.Rao MD

33. Hot air oven:
• Most widely used method
• Electrically heated and fitted with a fan to
even distribution of air in the chamber.
• Fitted with a thermostat that maintains
the chamber air at a chosen temperature.
• Temperature and time:
» 160 C for 2 hours.
» 170 C for 1 hour
» 180 C for 30 minutes.
12/2/2012 Dr.T.V.Rao MD 33

34. Uses of Hot Air Oven
– Sterilisation of
1.Glassware like glass syringes, petri
dishes, pipettes and test tubes.
2.Surgical instruments like scalpels,
scissors, forceps etc.
3.Chemicals like liquid paraffin, fats
etc.
12/2/2012 Dr.T.V.Rao MD 34

35. – Precautions :
1. Should not be overloaded
2. Arranged in a manner which allows free
circulation of air
3. Material to be sterilized should be perfectly dry.
4. Test tubes, flasks etc. should be fitted with
cotton plugs.
5. petridishes and pipetts should be wrapped in
paper.
6. Rubber materials and inflammable materials
should not be kept inside.
7. The oven must be allowed to cool for two hours
before opening, since glass ware may crack by
12/2/2012 sudden cooling. Dr.T.V.Rao MD 35

36. Sterilisation controls :
• Sterilisation controls
1.Spores of
Bacillus
subtilis
subsp. niger
2. Thermocouples
3.Browne’s
tube
12/2/2012 Dr.T.V.Rao MD 36

37. Sterilizing below100°C
1. temperature
below 100°
Pasteurization of
milk
1.Inspissation
2.Vaccine bath
12/2/2012 Dr.T.V.Rao MD 37

38. Principle of Pasteurization
12/2/2012 Dr.T.V.Rao MD 38

39. A temperature at 100°C
II. A
temperature
at 100°C
1. Boiling
2. Tyndallisation
3. Steam
sterilisation
12/2/2012 Dr.T.V.Rao MD 39

40. Boiling :
1 Boiling for 10 – 30 minutes may kill most of
vegetative forms but spores with stand boiling.
2. Tyndallisation :
 Steam at 100C for 20 minutes on three
successive days
 Used for egg , serum and sugar containing
media.
3. Steam sterilizer :
 Steam at 100°C for 90 minutes.
 Used for media which are decomposed at high
temperature.
12/2/2012 Dr.T.V.Rao MD 40

41. Temperatures above 100°C
III. A temperature
above 100°C
Autoclave :
-Steam above
100°C has a
better killing
power than
dry heat.
-Bacteria are
more
susceptible to
moist heat.
12/2/2012 Dr.T.V.Rao MD 41

42. Components of autoclave:
• Components of autoclave:
– Consists of vertical or horizontal cylinder of
gunmetal or stainless steel.
– Lid is fastened by screw clamps and
rendered air tight by an asbestos washer.
– Lid bears a discharge tap for air and steam,
a pressure gauge and a safety valve.
12/2/2012 Dr.T.V.Rao MD 42

43. Figure 9.6 Autoclave-overview
12/2/2012 Dr.T.V.Rao MD 43

44. Autoclave: Closed Chamber with
High Temperature and Pressure

45. 12/2/2012 Dr.T.V.Rao MD 45

46. Sterilisation conditions
• Sterilisation conditions:
–Temperature – 121 °C
–Chamber pressure -15 lb per square
inch.
–Holding time – 15 minutes
–Others :
• 126°C for 10 minutes
• 133°C for 3 minutes
12/2/2012 Dr.T.V.Rao MD 46

47. Sterilization – instrument Packing
• Often instruments are packed for sterilization to
be stored and handled without being
contaminated.
• Packing depend on the intended shelf life after
sterilization.
• The available packing options are:
– Textile has shelf life of 1 month
– Paper has shelf life of 1 – 6 months
– Nylon, glass, and metal have shelf life of 1 year if
tightly closed
12/2/2012 Dr.T.V.Rao MD

48. Uses of Autoclaves:
• Uses :
1. Useful for
materials which
can not withstand
high temp.
2. To sterilize culture
media, rubber
material, gowns,
dressings, gloves
etc.
12/2/2012 Dr.T.V.Rao MD 48

49. Sterilisation controls:
• Sterilisation controls:
1. Thermocouples
2. Bacterial spores-
Bacillus
stearothermophilus
3. Browne’s tube
4. Autoclave tapes
12/2/2012 Dr.T.V.Rao MD 49

50. Sterility Controls
Yellow medium
means spores are
Cap that allows viable; autoclaved
steam to penetrate objects are not
sterile.
Flexible plastic
vial
Crushable glass Incubation
ampule
Nutrient medium
containing pH
color indicator Red medium
means spores were
After autoclaving, flexible killed; autoclaved
Endospore strip
vial is squeezed to break objects are
ampule and release sterile.
medium onto spore strip.
12/2/2012 Dr.T.V.Rao MD 50

51. Filtration:
• . Filtration:
• Useful for substances which get
damaged by heat.
• To sterilize sera, sugars and antibiotic
solutions.
• To obtain bacteria free filtrates of
clinical samples.
12/2/2012
• Purification of water.
Dr.T.V.Rao MD 51

52. FILTRATION STERILIZATION
 This method is commonly used for  To ensure sterility, the filtration
sensitive pharmaceuticals and protein system must be tested to ensure that
solutions in biological research. the membranes have not been
 A filter with pore size 0.2 µm will punctured prior to or during use.
effectively remove bacteria.  To ensure the best results,
 If viruses must also be removed, a pharmaceutical sterile filtration is
much smaller pore size around 20 nm performed in a room with highly
is needed. filtered air (HEPA filtration) or in a
 Prions are not removed by filtration. laminar flow cabinet or "flowbox", a
device which produces a laminar
 The filtration equipment and the stream of HEPA filtered air.
filters themselves may be purchased
as presterilized disposable units in  HEPA filters are critical in the
sealed packaging, prevention of the spread of airborne
bacterial and viral organisms and,
 or must be sterilized by the user, therefore, infection. Typically,
generally by autoclaving at a medical-use HEPA filtration systems
temperature that does not damage also incorporate high-energy ultra-
the fragile filter membranes. violet light units to kill off the live
bacteria and viruses trapped by the
filter media.

53. Several Types of Filters
• Types of filters:
1. Candle filters
2. Asbestos disc filters
3. Sintered glass filters
4. Membrane filters
5. Air filters
6. Syringe filters
12/2/2012 Dr.T.V.Rao MD 53

54. Filtration
Sterilize solutions
that may be
damaged or
denatured by high
temperatures or
chemical agents.
12/2/2012 Dr.T.V.Rao MD 54

55. The filtering Depends on Pore Size
The pore size for filtering bacteria,
yeasts, and fungi is in the range of
0.22-0.45 μm (filtration membranes
are most popular for this purpose).
12/2/2012 Dr.T.V.Rao MD 55

56. Candle filters
12/2/2012 Dr.T.V.Rao MD 56

57. The roles of HEPA filters in biological flow
Safety glass
Exhaust HEPA viewscreen
filter safety cabinets
Blower
Supply HEPA
filter
Light
High-velocity
air barrier
12/2/2012 Dr.T.V.Rao MD 57

58. Radiations :
• Radiations :
•Ionizing
radiations
•Non -
Ionizing
radiations
12/2/2012 Dr.T.V.Rao MD 58

59. Ionising radiations:
– Ionizing radiations:
1. X rays
2. Gamma rays
3. Cosmic rays
• Gamma radiation
are commercially
used for sterilisation
of disposable items.
(cold sterilisation)
12/2/2012 Dr.T.V.Rao MD 59

60. Physical Methods of Microbial
Control
• Radiation
– Nonionizing radiation
• Wavelengths greater than 1 nm
• Excites electrons, causing them to make new covalent
bonds
– Affects 3-D structure of proteins and nucleic acids
• UV light causes pyrimidine dimers in DNA
• UV light does not penetrate well
• Suitable for disinfecting air, transparent fluids, and
surfaces of objects
12/2/2012 Dr.T.V.Rao MD 60
© 2012 Pearson Education Inc.

61. Forms of Radiation
12/2/2012 Dr.T.V.Rao MD 61

62. Physical Methods of Microbial
Control:
Radiation: Three types of radiation kill
microbes:
Ultraviolet light (Nonionizing Radiation): Wavelength
is longer than 1 nanometer. Damages DNA by producing
thymine dimers, which cause mutations.
Used to disinfect operating rooms, nurseries,
cafeterias.
Disadvantages: Damages skin, eyes. Doesn’t penetrate
paper, glass, and cloth.
12/2/2012 Dr.T.V.Rao MD 62

63. Non-Ionising radiation:
1. Infra red rays
2. Ultraviolet (UV) rays
– Infra red is used for rapid mass sterilisation of
syringes and catheters.
– Ultraviolet radiation is used for disinfecting
enclosed areas such as bacterial laboratory,
inoculation hood, laminar flow and operation
theatres.
12/2/2012 Dr.T.V.Rao MD 63

64. Programme Created by Dr.T.V.Rao MD
for Medical and Paramedical Students
Email
doctortvrao@gmail.com
12/2/2012 Dr.T.V.Rao MD 64