The document discusses various methods of sterilization and disinfection, focusing on their mechanisms, applications, and effectiveness against microorganisms. It outlines techniques such as heat, radiation, filtration, and chemical agents, while also defining terms like sterilization, disinfection, and antisepsis. Additionally, the document highlights the importance of monitoring sterilization processes and the characteristics of different disinfectants.

1. STERILIZATION
BY- SANCHIT DHANKHAR

2. 2

3.  Killing or removing all forms of microbial
life (including endospores) in a material or
an object.
 Mainly due to: oxidation of cell component,
denature proteins, nucleic acids, RNA and
loss of membrane permeability.
3

4. Sterilization
Heat
Radiation
Gases
Chemicals
Filtration
Methods of Sterilization
4

5. • Killing or removal of all
living organisms in or on a
substance
Sterilization
• Killing of most microorganisms
on a substance (Inanimate
Objects)
Disinfection
•Reduction or Inhibition of
microbes found on LIVING
TISSUE
Antisepsis
5

6.  Procedure carried out in the
absence of living things
 Procedures performed in a way to
prevent contamination with infectious
microorganisms
 Used to prevent contamination of
surgical instruments, medical personnel,
and the patient during surgery
6

7. Other
Terms
7

8. Other
Terms  Bacteriostatic Agent: Agent that inhibits
the growth of bacteria, but does not
necessarily kill them.
 Suffix stasis: To stop or steady.
 Germicide: Agent that kills certain
microorganisms.
Bactericide: Agent that kills bacteria.
Most do not kill endospores.
Viricide: Agent that inactivates viruses.
Fungicide: Agent that kills fungi.
Sporicide: Agent that kills bacterial
endospores or fungal spores.
8

9. Microbial Control Methods
9

10. 10

11. 11

12. Factors
Nature of
Heat
(dry & moist)
Temperature
Time
Type of
Material
Characteristics
of organisms
Number of
Organisms
Factors Affecting Sterilization Process
12

13.  Heat in Bunsen flame till red hot
2500C – 3000C
 Destroy vegetative cells by
oxidation
 Points of forceps & Inoculation
loops, scalpel blade, glass slides,
mouths of test tubes or flasks
Physical
Methods
HEAT
DRY
HEAT
Dry heat -
FLAMING
13

14. Dry Heat: Incineration
 Incineration
 Oxidation of microbes
to ashes and gas
 incineration of loops
and needles using a
Bunsen burner,
Common practice in
microbiology lab-
 Tabletop infrared
incinerators can also
be used
14

15.  8700C - 9800C
 Complete Burning to ASHES
 Used for soiled dressings,
animal carcasses,
pathological material,
disposables, non-reusable
soiled bedding
15

16.  Dry heat - HOT AIR OVEN
 Used for glassware, forceps, swabs, water
impermeable oils, waxes & powders
 Holding temp & time: 1600C for 1 hr
16

17.  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
 To allow free circulation of air between
the material
USE of HOT AIR OVEN
Important Notes
17

18. Central Dogma of
Life
Protein
DNA RNA
18

19. One of the Key Cell Constituents
Examples
Cell membrane, other cell
organelles
Enzymes
Proteins
Structural Functional
19

20. Lethal effect due to
DENATURATION
&
coagulation of proteins
Temp below 1000C
Temp at 1000C
Temp above 1000C
Physical
Methods
HEAT
MOIST
HEAT
20

21. cell membrane Enzymes
21

22. Structure of Proteins
22

23. Moist heat: Temp below 1000C
 Pasteurization
630C for 30 min (Holder method)
720C for 15-20 sec (Flash method)
1320C for 1 sec (Ultra high temp)
 Vaccine baths: 600C for 60 min
For vaccines of non-sporing bacteria
 Water bath: 560C for 60 min (for 3 days)
For serum/body fluids containing coagulable proteins
 Inspissation:
80 to 850C for 30 min (for 3 days)
 For media containing egg or serum
23

24. HEAT
Moist Heat
 Fluids are heated at temperatures below boiling
point to kill pathogenic microorganisms
without altering the fluid’s palatability.
 Conditions: 62℃, 30min or 71.7℃, 15sec
 Significance: kills vegetative pathogens
 Applications: milk, Juice
Pasteurization
24

25. Moist Heat - Temp at 1000C
 Boiling: 1000C for 10 min
 Kills vegetative bacteria, hepatic virus & some
spores
 Use deionized or distilled water
 2% sodium bicarbonate promotes the process
 Steaming (free steam): 30-60 min in Arnold
/Koch steamer
 For heat labile media
 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 25

26. Moist Heat: Temp above 1000C
 Autoclave (steam under pressure): 1210C, 15
min, 15 lbs
 Uses: rubber articles, dressings, sharp instruments, infectious medical waste,
culture media
Sterilization control (to check whether the equipment is
working properly)
Thermocouples
Chemical Control: Browne’s tube (Color change
from red to green)
Bowie & Dick tape (white-brown)
26

27. Steam Jacketed Horizontal Autoclave
27

28. Moist vs. Dry Heat
28

29.  Sterilization controls: to check whether the equipment is
working properly
Biological controls: paper strips containing 106
spore of B. stearothermophilus and Incubate at
550C for 5 days
paper strips containing 106 spores of
Clostridium tetani
Place strips in oven along with other material
for the sterilization
Later culture the strips in thioglycollate broth
at 370C for 5 days
Growth in medium indicates failure of
sterilization 29

30. Browne Sterilizer Control Tubes
Multi-variable indicators for Dry Heat and Fluid
sterilization
 Test critical parameters of Time and Temperature
 For Dry Heat Sterilizers & Steam autoclaves
 Colour change from red (unprocessed) to green (pass).
Black Spot (Type 1) for 121°C - 15 min Steam Fluid
Yellow Spot (Type 2) for 134°C - 3 min Steam Fluid
Green Spot (Type 3) for 160°C - 60 min Dry Heat
Blue Spot (Type 4) for 180°C - 12 min Dry Heat
White Spot (Type 5) for 180°C - 35 min Dry Heat
30

31. For steam sterilization
The easy to interpret; black stripes prove
successful sterilization.
Tape securely sticks to non woven, paper, board,
metal, plastic and glass
Manufactured from treated crepe paper and coated
with high performance cross linked heat sensitive
adhesive.
Autoclave
Tape
31

32. 32

33.  Filtration: Removal of microbes by passage of
a liquid or gas through a screen like material with
small pores.
 Used to sterilize heat sensitive materials like
vaccines, enzymes, antibiotics, and some culture media,
serum, carbohydrates solution, filtrates of toxins &
bacteriophages, in water bacteriology, in examination of
Schistosoma eggs
 Earthenware candles
 Unglazed ceramic & diatomaceous earth filters
 e.g. Chamberland filters, Doulton filters
 Asbestos filter: Seitz, Carlson, Sterimat
 Sintered glass filter
33

34.  Membrane Filters: cellulose nitrate, cellulose acetate,
polycarbonate, polyester filters with Uniform pore size.
Used in industry and research. Different sizes:
 0.22 and 0.45um Pores: Used to filter most bacteria. Don’t retain
spirochetes, mycoplasmas and viruses.
 0.01 um Pores: Retain all viruses and some large proteins.
34

35. 35

36. HEPA Filtrers
 HEPA: high-efficiency particulate air
filters
 Advantages: No thermal damage
 Used in operating rooms and burn units to
remove bacteria from air.
 Disadvantages: viruses not eliminated
and must be either liquid or gas
Filter
Sterilized
fluid
(a)
Vacuum
Pump suction
Filter
Liquid
Pore
(b)
b: © Fred Hossler/Visuals Unlimited
36

37. 37

38. RADIATION
Ionizing
Gamma, X ray
Canulas, Plastics, Syringes, Oil
(X-ray for Large packages and
pallet loads of medical devices
Non-ionizing
Infra Red radiation
(mass sterilization of
syringes)
Ultra Violet radiation
(In closed areas)
38

39. 39

40. Ultraviolet
Radiation
 Wavelength:
 200-300 nm
 Poor penetrating power
 Pyrimidine dimers
 Mutations induced
 Disinfection rather than
sterilization
 Hospital rooms, operating
rooms, schools, food prep
areas, dental offices
 Treat drinking water
purify liquids
40

41. Modes of Action of Ionizing Versus Non-
ionizing Radiation
 Ionizing: ejects electron, causing ions to form
41

42.  Wavelength ranges from 1 millimeter to 1
meter.
 Heat is absorbed by water molecules.
 May kill vegetative cells in moist foods.
 Bacterial endospores, which do not contain
water, are not damaged by microwave
radiation.
 Solid foods are unevenly penetrated by
microwaves.
Microwave Radiation
42

43. 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 many bacteria
43

44. 44

45.  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
Disinfection
45

46. SHOULD
BE
Broadly
active
Killing pathogens
Not
Harmful
Good
Penetration
No damage to
non-living
material
Easily to
work with
Stable &
Resistant to
inactivation
46

47. Microbe type
(e.g., Mycobacteria,
spores, and certain
viruses) very resistant
Nature and Number of
Cells
Vegetative cells in log
phase, Easy to kill
Substrate effects
(high organic content
interferes with
disinfection)
Density
It’s easier (and faster) to
kill fewer microbes than
many microbes
Dependents on: Conc., length (time),
temperature Chemical reaction 2x increase in
rate with each 10°C
47

48. 48

49. Categories of disinfectants
1. Alcohol
2. Aldehyde
3. Ethylene oxide
4. Dyes
5. Halogens
6. Phenolics
7. Surface active agents
8. Metallic salts
9. Diguanides
10. Amides 49

50. Chemical control of microorganisms
50

51.  Act as surfactants
 Cationic detergents are more
effective because the positively
charged end binds with negatively
charged bacterial surface proteins
 Soaps are weak microbicides but
gain germicidal value when mixed
with agents such as chlorhexidine or
iodine
Chemicals with Surface
Active Action: Detergents
51

52. SOAPS and SURFACE ACTIVE AGENTS
 Soaps & surfactants DISRUPT cell membrane
 Soaps are Na- or K- salts of fatty acids, a natural product
 There are 4 main groups of surface Active Agents
I. Anionic surfactants: strong detergent action, weak antimicrobial action
II. Non-ionic surfactants
III. Cationic surfactants: Quaternary ammonium compounds: cetrimide,
benzalkonium chloride: bacteriostatic
IV. Amphoteric surfactants: both detergent & antimicrobial properties
52

53.  Quats are cationic detergents that act by
disrupting lipid bilayers
 Benzalkonium chloride
Cetylpyridinium chloride
53

54.  Quats are bactericidal, fungicidal, viricidal
(enveloped), and amoebicidal
 Most effective against Gram-positive
bacteria, less effective against gram-
negative bacteria.
 Quats do not kill endospores, Mycobacteria
spp., nor non-enveloped viruses
 Disadvantages: rapidly inactivated by
organics including cotton, soap and anionic
detergents
 Advantages: Strong antimicrobial action,
colorless, odorless, tasteless, stable, and
nontoxic. 54

55. ALCOHOL
Ethanol, isopropyl alcohol
 Skin antiseptics at 70%
 Kills bacteria and fungi
 Less effective on spores (Less sporicidal activity) & non-enveloped viruses
(Less virucidal activity)
 Denature bacterial proteins and dissolves lipids
 Flammable; also may damage rubber, plastic, etc.
 Methyl alcohol: to treat cabinets / incubators affected by fungal spores
 Others: benzyl alcohol, chlorbutol, phenylethanol
55

56.  Denature bacterial proteins and dissolves lipids
56

57. ALDEHYDE
Inactivate proteins by forming covalent crosslinks
with several functional groups
 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 strong smell, irritant to skin, eyes, mucus
membrane & toxic when inhaled
 Formaldehyde gas:
 Excellent disinfectant.
 Commonly used as formalin, a 37% aqueous solution.
 Formalin was used extensively to preserve biological
specimens and inactivate viruses and bacteria in
vaccines. 57

58. 58

59.  Glutaraldehyde: less toxic, less irritant
 Endotracheal tubes, metal instruments, polythene tubing
 Less irritating and more effective than formaldehyde.
 One of the few chemical disinfectants that is a sterilizing
agent.
 A 2% solution of glutaraldehyde (Cidex) is:
 Bactericidal, tuberculocidal, and viricidal in 10 minutes.
 Sporicidal in 3 to 10 hours.
 Commonly used to disinfect hospital instruments.
 Also used in mortuaries for preserving.
 Βeta-propiolactone (BPL): condensation product of ketane & formaldehyde
 More efficient for fumigation but is carcinogenic
 0.2% generally used 59

60. 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
60

61. Halogens
 Iodine: commonly used as an antiseptic
against all microbes, fungi, and viruses
 Iodine: It inhibits protein synthesis and
oxidizes –SH groups of amino acids
 Chlorine: Used as a disinfectant (10%
bleach)
 Chlorine: Hypochlorous acid (HOCl) is a
product, formed in water, that is the active
form of the disinfectant
Cl2 + H2O ------> H+ + Cl- + HOCl
Hypochlorous acid
61

62. 62

63. PHENOLICS
 Carbolic acid: 2 - 5%
 Powerful microbicidal, very corrosive
 General purpose disinfectant in hospital
 Stable, persistant, and especially effective when dealing with disinfecting
materials contaminated with little organics
 Cresol, lysol
 Chloroxylenol, chlorophenol, hexachlorophane less toxic, less irritant, less
active, more readily inactivated by organic matter
63

64.  Affect plasma membrane,
inactivates enzymes, and
denature proteins
 Leave residual films, can
irritate skin, don’t kill
endospores, and are corrosive
to rubber and plastics
 Some phenolics are mild
enough for use as antiseptics
while others are too harsh or
otherwise dangerous to be
employed on living tissue
PHENOLICS
64

65. Heavy Metals or Metallic Salts
Most common: Ag, Cu, Hg (ointment), Ni, Zn,
Ag(NO3)2, CuSO4 , ZnCl2, HgCl2
 Oligodynamic action: exert antimicrobial activity
 React with sulfhydral (–SH) groups of proteins,
denaturing proteins
 Silver nitrate is used to treat Ophthalmia
neonatorum in newborns as caused by Neisseria
gonorrhoeae (gonorrheal eye infections)
 Copper salts: antifungal, antialgae – water
reservoirs, swimming pools 65

66. Oligodynamic action of Heavy Metals
66

67. Heavy Metals or Metallic Salts
 Mercury: Organic mercury compounds like
merthiolate and mercurochrome are used to
disinfect skin wounds.
 Selenium
 Kills fungi and their spores. Used for fungal
infections.
 Also used in dandruff shampoos.
 Zinc
 Zinc chloride is used in mouthwashes.
 Zinc oxide is used as antifungal agent in
paints.
67

68. Peroxygens (Oxidizing
Agents)
A. Ozone
 Used along with chlorine to disinfect water.
 Helps neutralize unpleasant tastes and odors.
 More effective killing agent than chlorine, but
less stable and more expensive.
 Highly reactive form of oxygen.
 Made by exposing oxygen to electricity or UV
light.
 Oxidize cellular components of treated microbes.
 Oxidize DNA & Disrupt membranes and proteins.
68

69. Peroxygens
B. Hydrogen Peroxide H2O2
 Used as an antiseptic.
 Bacteria and body tissues contain enzymes (catalase) that inactivate hydrogen
peroxide
 Oxygen released upon inactivation can help oxygenate deep wounds and thus
kill strict-anaerobe contaminants, e.g., Clostridium tetani
 Sporicidal at higher temperatures.
 Used by food industry and to disinfect contact lenses.
C. Benzoyl Peroxide
 Used in spotted skin medications (an acne treatment)
69

70. D. Peracetic Acid
 One of the most effective liquid sporicides
available.
 Sterilant :
 Kills bacteria and fungi in less than 5 minutes.
 Kills endospores and viruses within 30 minutes.
 Used widely in disinfection of food and
medical instruments because it does not leave
toxic residues.
Peroxygens
70

71. DYES as Antimicrobial Agents
 Combine with nucleic acids
 Aniline dyes (crystal violet and malachite
green, Brilliant green) are very active against
gram-positive bacteria and various fungi
 Yellow acridine dyes (acriflavine ,
euflavine and proflavine) sometimes used
for antisepsis and wound treatment
 Limited applications because they stain and
have a narrow spectrum of activity
71

72.  Denature proteins, by replacing functional
groups with alkyl groups.
 Kills all microbes and endospores, but
requires exposure of 4 to 18 hours.
 Ethylene oxide chambers in hospitals used
to sterilize mattresses and large equipment
 Especially for heart lung machines,
respirators, sutures, syringes, dental
equipments
ETHYLENE OXIDE (C2H4O)
72

73. ETHYLENE OXIDE
Ethylene oxide Used to sterilize heat- or
moisture-sensitive items
 Not corrosive, not damaging to delicate
instruments,
 Good penetration of materials, and
Diffuses rapidly from material
 Highly inflammable, mixed with inert gases
(CO2)
 But is costly, toxic, carcinogenic,
explosive , and relatively lengthy process
73

74. THANKYOU
74