Lecture for microbiology

1. “Practice of Sterilization”
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2. Sterilization
 The process of killing or removing bacteria and all other
forms of living micro-organisms and there spares from
preparation.
 Essential concept in the preparation of sterile
pharmaceutical products.
 Its aim is to provide a product that is safe and eliminates
the possibility of introducing.
 The sterilization of microbiological materials is necessary
to minimize the health hazard associated with these articles.
 So, Sterilization refers a great commercial value in this
pharmaceutical companies.
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3. Sterilization : 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.
Disinfectants: These are antimicrobial agents that are applied to the surface of
non-living objects to destroy microorganisms that are living on the objects.
Antiseptics : Chemical disinfectants which can safely be 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.
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4. Survivor curves
When exposed to a killing process,
populations of microorganisms generally
lose their viability in an exponential
fashion, independent of the initial number
of organisms. This can be represented
graphically with a ‘survivor curve’. Of the
typical curves obtained, all have a linear
portion which may be continuous (plot A),
or may be modified by an initial shoulder
(B) or by a reduced rate of kill at low
survivor levels (C). Survivor curves have
been employed principally in the
examination of heat sterilization methods,
but can equally well be applied to any
biocidal process.

5. Expressions of resistance
D-value:
 D-value refers to the
resistance of an organism to
a sterilizing agent.
 The time taken at a fixed
temperature or the radiation
required to achieve a 90%
reduction in viable cells is
called D-value.
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Figure: Calculation of D-value

6. Expressions of resistance
Z-value:
 Z-value is the increase
in temperature needed to
reduce the D-value of an
organism by 90%.
 Temperature isn’t fixed.
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Figure: Calculation of Z-value

7. Factors that influence efficacy of disinfection/
sterilization:
 Contact time
 Physico-chemical environment (e.g. pH)
 Presence of organic material
 Temperature
 Type of microorganism
 Number of microorganisms
 Material composition
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8. How can microorganisms be
killed?
 Denaturation of proteins
 Interference with protein
synthesis
 Interruption of DNA
synthesis/repair
 Oxidative damage of cell
 Disruption of cell
membranes
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9. Sterility assurance
 There are no degrees of sterility; an item is either
sterile or it is not. So, it is an absolute term.
 The elimination of viable microorganisms are
influenced by-
- The rate & duration of biocidal action.
- The initial microbial contamination level.
 True sterility, represented by zero survivors.
 So, sterility assurance gives a numerical value to
the probability of a single surviving organisms
remaining to contaminate a processed product.
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10. Methods of Sterilization
1. Physical method
-Dry heat sterilization
-Moist heat sterilization
-Sterilization by radiation
-Filtration Sterilization
2. Chemical method
-Gaseous sterilization
-Sterilization by disinfectant
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11. Moist heat sterilization
 An efficient sterilization process that use elevated temperature
(121ºC for 15 minutes & 134 ºC for 3 minutes) with association
of moisture.
 Instrument: Autoclave
 Mode of action:
Moist heat destroys microorganisms by denaturation of
enzymes and structural proteins. The temperature at which
denaturation occurs varies with the amount of water present.
Sterilization in saturated steam thus requires precise control of
time, temperature and pressure.
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12. Application in products
 Processing of many thermostable products and devices.
 Sterilization of
 Dressing
 Sheets
 Surgical & diagnostic equipments
 Aqueous injections
 Ophthalmic operation
 Microbiological media.
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13. Principle of Sterilization by Autoclave
Autoclaves use pressurized steam as their sterilization
agent. The basic concept of an autoclave is to have each
item sterilized whether it is a liquid, plastic ware, or
glassware- come in direct contact with steam at a specific
temperature and pressure for a specific amount of time.
Time, steam, temperature, and pressure are the four main
parameters required for a successful sterilization using an
autoclave.
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14.  When steam enters the sterilization chamber under
pressure;
- It condenses upon contact with cold items.
- This condensation frees heat, simultaneously heating
and wetting all items in the load, thus providing heat and
moisture.
 The amount of time and temperature required for
sterilization depends on the type of material being
autoclaved.
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15. Moist heat sterilization
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(1)
(2)
(3)

16. Procedure (Autoclave)
 Water is added on the bottom of the autoclave and articles to
be sterilized are placed in a perforated shelf.
 The lid is closed, discharge tap is opened and safety valve is
adjusted to the required pressure.
 When the air bubbles stop emitting from the discharge tap it
indicates all the air from inside the autoclave has been
removed.
 At this stage, the discharge tap is closed.
 Steam pressure rises inside and when it reaches the desire set
level (15 psi) the safety valve opens and excess steam
escapes.
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17. Procedure (Cont.)
 From this point the holding time (15 mins) is counted.
 When the holding time is over, the heating is stopped and
autoclave is allowed to cool till pressure gauze indicates that
the inside pressure has reached to the atmospheric pressure
 The discharge tap is opened slowly and air is allowed to
removed from the autoclave.
 The lid is opened and the sterilized articles are removed.
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18. Advantage:
 Works at lower temperature;
 Penetration power is very high;
 Assure sterility;
 Kills vegetative forms of bacteria, fungus, all forms of
viruses and endospores.
Disadvantage:
 Can’t be used for heat sensitive particles;
 Glass wares can’t be sterilized by this way;
 Ineffective against organisms in material impervious to
steam.
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19. Dry heat sterilization
 Dry heat sterilization usually employs higher temperatures in
the range 160-180ºC and requires exposure time of up to 2
hours depending upon the temperature employed.
 Instrument: Oven
Mode of action:
Dry heat sterilization is carried out in a hot air oven. It takes
higher temperature and time than moist heat sterilization. Dry heat
does most of the damage by oxidizing molecules. The essential
cell constituents are destroyed and organism dies. The temperature
is maintained to kill the most difficult of the resistant spores.
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20. Hot Air Oven
• Hot air ovens are electrical devices used in sterilization.
• The oven uses dry heat to sterilize articles.
• Generally, they can be operated from 50 to 300 C (122 to
572 F) .
• There is a thermostat controlling the temperature.
• This is the most widely used method of sterilization by
dry heat.
• Items:
Glassware, forceps, scissors, scalpels, all-glass syringes,
swabs, liquid paraffin, dusting powder etc. can be sterilized
by this.
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21. 21
Fig: Hot Air Oven

22. Advantage
 Can be used for substances that would be harmed by moisture;
 Provide sufficient time for penetration;
 Can kill bacterial endotoxin (pyrogen);
 Less damaging to glass and metal equipment than moist heat.
Disadvantage
 Require high temperature;
 Long exposure time;
 Unsuitable for surgical dressing;
 Slower and time consuming process.
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23. Gaseous sterilization
 The compound that are used for gaseous
sterilization are-
 Ethylene oxide (CH2)2O.
 Formaldehyde (methanol, HCHO).
 Ethylene oxide sterilization are for more commonly used
than those formaldehyde.
Application in products:
 Re-usable surgical instruments
 Certain medical, diagnostic and electrical equipments
 The surface sterilization of powders.
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24. Mode of action
 Alkylation of sulphydryl, amino, hydroxyl, carboxyl groups on protein and
imino groups of nucleic acids .
Ethylene oxide
 Highly explosive gas
 For the reduction of explosion hazard of ethylene oxide gas following
methods are used:
 It is supplied as a 10% mix with carbon dioxide;
 An 8.6% mixture with HFC-124 (2-chloro1,1,1,2 tetrafluoroethane).
 Pure ethylene oxide gas can be used at below atmospheric pressure in
sterilizer chamber.
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25. Sterilizer design and operation
An ethylene oxide sterilizer consist of a leak proof and explosion
proof steal chamber normally of 100-300 liter capacity.
This can be surrounded by a hot water jacket to provide uniform
chamber temperature.
Successful operation of the sterilizer requires removal of air
from the chamber.
Absorption of ethylene oxide by the load is enhanced by the
introduction of excess gas at the beginning or by the addition of
more gas as the pressure drops during the sterilization process.
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26. After treatment, the gases are evacuated either directly to
the outside atmosphere or through the special exhaust
system.
Filtered sterile air is then admitted either for a repeat of the
vacuum or for air purging until the chamber is opened.
In this way safe removal of the ethylene oxide is achieved
reducing the toxic hazards to the operator.
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Sterilizer design and operation

27. Advantages
 Wider international regulatory acceptance;
 Better gas penetration into plastic and rubber;
 Relatively slow to form solid polymers;
 With long exposure times it is possible to sterilize at ambient
temperatures;
 Very low incidence of product deterioration.
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28. Formaldehyde
 Produced by heating formalin to a temperature of 70-75ºC
with steam;
 Has similar toxicity to ethylene oxide.
Advantages:
 Less hazardous because formaldehyde is not flammable and
is more readily detected by smell;
 Cycle times may be shorter;
 The gas is obtained readily from aqueous solution
(Formalin) which is a more convenient source than gas in
cylinders.
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29. A LTSF sterilizer is designed to operate with sub
atmospheric pressure steam.
Air is removed by evacuation and steam is admitted to the
chamber to allow heating of the load and to assist in air
removal. The sterilization period starts with the release of
formaldehyde by vaporization from formalin
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Sterilizer design and operation:

30. The chamber temperature is maintained by a
thermostatically controlled water jacket and steam and
condensate is removed via drain channel and an evacuated
condenser.
At the end of the treatment period formaldehyde vapor is
expelled by steam flushing and the load is dried by
alternating stages of evacuation and admission of sterile
filtered air.
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Sterilizer design and operation:

31. Disadvantages of Ethylene oxide and
Formaldehyde:
 Mutagenic:
 Carcinogenic;
 Irritation of skin, conjunctiva and nasal mucosa;
 Organism are more resistant to ethylene oxide treatment in a
dried state;
 Formaldehyde has low penetrating power that limits the
packaging materials.
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32. Filtration sterilization
 Filtration means
 Removal of microorganism rather than destroy them;
 Capable of preventing the passage of both viable and
non-viable particles.
 Application in product
 Heat sensitive injection
 Ophthalmic solutions;
 Air and other gases for supply to aseptic areas.
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33. 33
Figure: Filtration

34.  Mechanisms of filtration:
Sieving;
Adsorption;
Trapping.
 Filtration sterilization of liquid
The removal of microorganisms from liquid is done by
filtration with efficiency.
The filters that are used for liquid sterilization are:
Membrane filter;
Sintered filter.
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35. Filtration sterilization of gases
 Composition of filters:
 Pleated sheets of glass microfibres
 Corrugated sheets of Kraft paper or aluminium
 HEPA(High Efficiency Particulate Air) filter
 Remove up to 99.997% of particles greater than 0.3μm
in diameter.
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36. Applications
 Sterilization of displacement air in tissue and
microbial culture;
 Decontamination of air in mechanical ventilators;
 Clarification and sterilization of medical gases;
 Treatment of exhausted air from microbiological
safety cabinet.
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37.  Advantage
Cost effective;
Applicable in heat sensitive substances;
 Disadvantage
Filters with small pores can easily clog;
Creation a tiny little rip in filter may allow bacteria;
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38. Radiation sterilization
 Radiation that kill microorganisms is called sterilizing
radiation.
 Types of radiation:
Radiation sterilization is of two types-
1) Ionizing radiation:
-Gamma rays
-X-rays
-High-energy electron beam.
2) Non ionizing radiation
-Ultraviolet (UV) radiation.
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39. Spectrum of radiation
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Figure: The radiant energy spectrum.

40. Mode of action
 Ionizing radiation:
Ionize the water of cell content .
Produce highly reactive free hydroxyl radical,
peroxides, free hydrogen radical etc.
The above forms cause intracellular damage &
destroy DNA of microbial cell.
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41. Mode of action
 Non ionizing
radiation:
 UV radiation damages
microbial DNA by
causing a bond between
two adjacent pyrimidine
base(thymine).
 leads a formation of
thymic dimer.
 DNA replication can be
inhibited.
 Cell death then occur
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42. Application in product
 Articles in the dried state
 Surgical instruments
 Pharmaceutical & disposable dental and medical supplies
 Plastic syringes
 Catheters etc.
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43. Advantages
 Heat sensitive products can be sterilized;
 High penetration power;
 No aseptic handling is required;
 Some bacterial and viral vaccines can be sterilized without
loss of antigenicity.
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44. Disadvantages
 Capital and replacement costs are high;
 Elaborate and expensive precautions must be taken;
 Damage human eyes,
 Causes skin burn and skin cancer;
 Causes mutation in human cells.
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