Sterilization- Pharmaceutical Microbiology

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.
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
Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms)
Degerming: Mechanical removal of microbes from limited area. e.g., Alcohol swab on skin, washing of hands with soap
Sepsis: Bacterial contamination
Antisepsis: Reduction or Inhibition of microbes found on LIVING TISSUE

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. •Killingorremovalofall
livingorganismsinorona
substance
Sterilization
•Killing of mostmicroorganisms
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 patientduring surgery
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7. Other Terms
7

8. Other Terms
Bacteriostatic Agent: Agent that inhibits
the growth of bacteria, but does not
necessarily killthem.
Suffix stasis: To stopor steady.
Germicide: Agent that killscertain
microorganisms.
Bactericide: Agent that killsbacteria.
Most do not kill endospores.
Viricide: Agent that inactivatesviruses.
Fungicide: Agent thatkills fungi.
Sporicide: Agent that killsbacterial
endosporesor fungal spores.
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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
250
0
C –300
0
C
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. 870
0
C -980
0
C
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 forglassware, forceps, swabs, water
impermeable oils, waxes & powders
Holding temp & time:160
0
C 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 loadthe oven
To allow free circulationof 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 100
0
C
Temp at 100
0
C
Temp above 100
0
C
Physical
Methods
HEAT
MOIST
HEAT
20

21. cell membrane Enzymes
21

22. Structure of Proteins
22

23. Moist heat: Temp below 100
0
C
Pasteurization
63
0
C for 30 min (Holder method)
72
0
C for 15-20 sec (Flash method)
132
0
C for 1 sec (Ultra high temp)
Vaccine baths:60
0
C for 60 min
For vaccines of non-sporingbacteria
Water bath:56
0
C for 60 min (for 3 days)
For serum/body fluids containing coagulableproteins
Inspissation:
80 to 85
0
C for 30 min (for 3 days)
For media containing egg or serum
23

24. HEAT
Moist Heat
Fluids are heated at temperatures below boiling
pointto 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 100
0
C
Boiling: 100
0
C for 10 min
Kills vegetative bacteria, hepatic virus & some
spores
Use deionized or distilled water
2% sodium bicarbonate promotesthe process
Steaming (free steam): 30-60 min in Arnold
/Koch steamer
For heat labile media
Tyndallisation(intermittent sterilization): 100
0
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
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26. Moist Heat: Temp above 100
0
C
Autoclave (steam under pressure): 121
0
C, 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 redto green)
Bowie & Dick tape (white-brown)
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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 10
6
spore of B. stearothermophilusand Incubate at
55
0
C for 5 days
paper strips containing 10
6
spores of
Clostridium tetani
Place strips in oven along with other material
for the sterilization
Later culture the strips in thioglycollatebroth
at 37
0
C 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 Timeand Temperature
For Dry Heat Sterilizers & Steamautoclaves
Colourchange from red(unprocessed) to green(pass).
Black Spot (Type 1) for 121°C -15 min Steam Fluid
YellowSpot (Type 2) for 134°C -3 min Steam Fluid
GreenSpot (Type 3) for 160°C -60 min Dry Heat
BlueSpot (Type 4) for 180°C -12 min Dry Heat
WhiteSpot (Type 5) for 180°C -35 min Dry Heat
30

31. For steam sterilization
The easy to interpret; black stripesprove
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
Schistosomaeggs
Earthenware candles
Unglazed ceramic & diatomaceous earth filters
e.g. Chamberlandfilters, Doultonfilters
Asbestos filter: Seitz, Carlson, Sterimat
Sintered glass filter
33

34. Membrane Filters:cellulose nitrate, cellulose acetate,
polycarbonate, polyesterfilters 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.
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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
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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)
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39. 39

40. Ultraviolet
Radiation
Wavelength:
200-300 nm
Poorpenetratingpower
Pyrimidinedimers
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
microorganismsbut 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),
temperatureChemical reaction 2x increase in
rate with each 10°C
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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
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50. Chemical control of microorganisms
50

51. Act as surfactants
Cationic detergentsare more
effective because the positively
charged end binds with negatively
charged bacterial surface proteins
Soapsare weak microbicidesbut
gain germicidal value when mixed
with agents such as chlorhexidineor
iodine
Chemicals with Surface
Active Action: Detergents
51

52. SOAPS and SURFACE ACTIVE AGENTS
Soaps &surfactants DISRUPT cell membrane
Soapsare 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:Quaternaryammonium compounds: cetrimide,
benzalkoniumchloride: bacteriostatic
IV.Amphoteric surfactants:both detergent & antimicrobial properties
52

53. Quatsare cationic detergents that act by
disrupting lipid bilayers
Benzalkoniumchloride
Cetylpyridiniumchloride
53

54. Quatsare bactericidal, fungicidal, viricidal
(enveloped), and amoebicidal
Most effective against Gram-positive
bacteria, less effective against gram-
negative bacteria.
Quatsdo 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%
Killsbacteria and fungi
Less effective on spores (Less sporicidalactivity) & non-enveloped viruses
(Less virucidalactivity)
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. solnis virucidal, bactericidal, sporicidal
Used to fumigatewards, 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
specimensand 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 viricidalin 10 minutes.
Sporicidalin 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% in70% 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: Itinhibits protein synthesis and
oxidizes –SH groups of amino acids
Chlorine: Used as adisinfectant(10%
bleach)
Chlorine: Hypochlorousacid (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, hexachlorophaneless 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(NO
3)
2, CuSO
4 , ZnCl
2, HgCl
2
Oligodynamicaction: exert antimicrobial activity
React with sulfhydral(–SH) groups of proteins,
denaturing proteins
Silver nitrate is used to treat Ophthalmia
neonatorumin newborns as caused by Neisseria
gonorrhoeae(gonorrheal eye infections)
Copper salts:antifungal, antialgae–water
reservoirs, swimming pools
65

66. Oligodynamicaction of Heavy Metals
66

67. Heavy Metals or Metallic Salts
Mercury: Organic mercury compounds like
merthiolateand mercurochromeare 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 H
2O
2
Used as an antiseptic.
Bacteria and body tissues contain enzymes (catalase) that inactivate hydrogen
peroxide
Oxygen released upon inactivation can help oxygenatedeep wounds and thus
kill strict-anaerobe contaminants, e.g., Clostridium tetani
Sporicidalat 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. PeraceticAcid
One of the most effective liquid sporicides
available.
Sterilant:
Kills bacteria and fungi in less than 5 minutes.
Kills endosporesand 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-positivebacteriaand various fungi
Yellow acridinedyes(acriflavine,
euflavineand 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 (C
2H
4O)
72

73. ETHYLENE OXIDE
Ethylene oxide Used to sterilize heat-or
moisture-sensitive items
Not corrosive, not damaging to delicate
instruments,
Goodpenetration of materials, and
Diffuses rapidly from material
Highly inflammable, mixed with inert gases
(CO
2)
But is costly, toxic, carcinogenic,
explosive , and relatively lengthy process
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74. THANKYOU
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