Points to be covered in this topic

1. INTRODUCTION
2. MEMBRANE FILTRATION
3. DIRECT INOCULATION FILTRATION
4. EQUIPMENTS EMPLOYED IN LARGE
SCALESTERLIZATION
5. STERLITY INDICATOR
a INTRODUCTION
Sterility test is defined as Microbiological test applied to sterile product to show are products
manufactured and processed under specification guided by cGMP, Or to confirm the products
either sterile or nonsterile.
Evaluation of efficiency of sterilization / Sterility testing Sterility tests can be carried out
using following methods:
Method A: Membrane filtration.
Method B: Direct Inoculation.
Membrane filtration
 First clean the membrane filter unit and
sterilize the unit and membrane filter
separately by moist heat sterilization.
 Transfer the unit on laminar air flow
bench or aseptic area and place the
membrane filter in the unit.
 Pass all the solution through filter under
vacuum
 After filtration, the mem rane is removed
aseptically and cut into two parts using
sterile scissors.
 One half part of membrane filter is placed
in 100ml of fluid thioglycolate medium
(FTM) and incubate at 30-35º C for not
less than 7 days.
 Other half part of membrane filter is
placed in soyabean casein digest medium
(SCDM) and incubate at 20-25ºC for not
less than 7 days.
Observe the turbidity in the medium by comparing with the standard tube. If it has no
turbidity in fluid thioglycollate medium and soyabean casein digest medium means it is free
from microorganisms and suitable for use.
Direct inoculation filtration
Cotton or prefilled syringe is transferred directly to culture media using
sterile instruments such as sterile forceps.
Incubate sample containing fluid thioglycollate medium (FTM) at 30-35ºC for not less than 7
days and soyabean casein digest medium (SCDM) at 20-25ºC for not less than 7 days.
Observe the turbidity in the medium by comparing with the standard tube.
If it has no turbidity in fluid thioglycollate medium and soyabean casein digest medium
means it is free from microorganisms and suitable for use.

Thermocouples:
An autoclave's temperature can be measured using thermocouples. A particular autoclave
temperature is deemed to be the appropriate temperature for sterilization,
Brown Tubes:
Along with the articles, these tubes go into the autoclave. When the temperature inside the
autoclave reaches 121ºC, these tubes turn green which helps determine whether the articles
are properly sterilized.
Bacillus Stereo thermophilus Spores
These spores must be exposed to 121ºC for 12 minutes in order to be killed. Within the
envelope are placed paper strips containing 106 spores before being placed in the autoclave.
Inoculated with culture media, these strips are autoclaved and then inoculated. It is
determined if these spores are not growing in the culture media it means sterilization has been
properly performed.

Autoclave tape
In the autoclave, the product changes color when heated to 121ºC due to lead carbonate. To
check whether the sterilization process has the ability to kill bacteria (microorganisms),
and then to ensure that all microorganisms have been killed.
• This can be measured by the following three factors:
D value (decimal reduction time):
The D value indicates the time in minutes it takes to destroy 90% of viable microorganisms at
a constant(defined) temperature. The less the value of D decreases (less).
Then the more efficient sterilization will be. This is because D value is the time required to
destroy microorganisms. So less time (D value) results in killing more bacteria, so
sterilization becomes very efficient.
Z value:
 This represents the amount of temperature change required to decrease one point in
D- value.
 Change in temperature is the Z- value
 As the temperature rises, more bacteria are killed and the D- value decreases and the
Z- value increases.
 As Z value is proportional to the D- value and a lower D value will
 result in a more efficient sterilization.
F- value:
Efficacy of a sterilization method is determined by the number of minutes
it takes to kill bacterial spores through heating. It is possible to calculate the probability of
survivors remaining from a load using the F value as follows;

Where,
No = initial population volume original population number
N = number of final residents volume of units
D= D the organism's temperature at 121 degree Celsius.
EQUIPMENTS EMPLOYED IN LARGE SCALE STERILIZATION
List of equipment's employed in large scale sterilization:
1. Autoclave
2. Hot air oven
3. Microwave
4. HEPA filter

1. AUTOCLAVE
Principle
The higher pressure also ensures the rapid penetration of heat into the
deeper parts of equipment. The moisture present in the steam causes
coagulation of proteins of microbes causing irreversible loss of their activity and functions
Construction
The autoclave is made of following components-
1. Vessel or pressure chamber
2. Lid or door
3. Pressure gauge
4. Pressure releasing unit (whistle)
5. Safety valve
6. Electrical heater
The laboratory autoclave or pressure cooker type
autoclave consists of a vertical or horizontal
cylinder of gun metal or stainless steel in a
supporting frame or case.

The lid is fastened by screw clamps and rendered airtight by a asbestos

gasket.
The autoclave has on its lid or upper side a discharge tap for air and
steam a pressure gauge and a safety valve that can be set to blow off at any desired pressure.
Working
Steps in Autoclave cycle-
1. Boiling phase: The electric heat causes boiling of water and generate the steam. The
produced steam replaces the trapped air by displacement.
2. Rising temperature phase: The temperature rises and reaches up to the set level i.e. 121ºC.
3. Sterilization time: This is the time when microbes are killed.
4. Release the pressure: The entrapped pressure is released by opening the valve.
The steam circulates within the jacket & is supplied under high pressure to closed inner
chamber where articles for sterilization are kept.
One fifth of the cylinder is filled with water, materials to be sterilized are kept inside, lid
closed & heater is put on. Safety valve is adjusted to required pressure.
The boiling of water inside the chamber after sometime results in steam which is allowed to
escape with air mixture fill the cylinder becomes air free.
The discharge tap is closed and the desired pressure inside in chamber is allowed to rise to the
one chosen for autoclaving for a fixed time Is thus complete sterilization achieved.
Standard operating procedures for autoclaving is: 115ºC, half an hour. 121ºC, 15 minutes.
Advantages
Rapid and effective
Effective for sterilizing cloth surgical packs and towel packs.
Less time consuming.
Applicable for both thermoplastic and thermolabile components.
Disadvantages
Items sensitive to heat cannot be sterilized
It tends to corrode carbon steel bum and instruments .
It is costly technique.
Applications
It is used to sterilize anything, which is not injured by steam and high temperature of
sterilization. These includes, Aqueous parenteral solutions e.g. distilled water, saline solutions
Aqueous liquid media e.g. liquid media with or without carbohydrate and gelatin.
Surgical dressings and fabrics.
Plastic and rubber closures.
Metal instruments.
Glass apparatus and containers.
2. HOT AIR OVEN
Principle: It kills microorganisms by destructive oxidation of essential cell constituents.
Construction
The modern hot air ovens consist of a double walled chamber of aluminium or stainless steel
separated from the outer case by a thick layer of insulation made of fiberglass.
Insulation is also filled in the hollow flanged
door, which carries an asbestos jacket that
provides a tight seal.
Heating is affected by electrical heating elements
and thermo-stats automatically control
temperature.

Working
Oven is an electrical devise that works on Heat convection principle. It
consist of electrical heating coil that produces dry heat. In the chamber, the
produced dry heat air displaces the cooled air forming heat gradient.
The use of fan allows to the uniform distribution of heat.

Application
• Used to sterilize the equipment's like glassware, forceps, scissors, spatula, swabs, some
pharmaceutical substances such as glycerin, fixed oil, paraffin propylene.
• Used in many industries for drying and baking and curing process.
• Used to sterilize powders and other non volatile compounds.
Advantages
1. It is Eco-friendly
2. It is most efficient method to degrade microbial endotoxins.
3. It is safer than autoclave.
4. It is the ideal instrument for sterilizing oil and powders.
Disadvantages
1. It is time consuming because the dry heat penetrate slowly as compared to moist
heat.
2. It may not be efficient to degrade prions.
3. The heat sensitive or heat labile material cannot be sterilized.
4. It cannot be operated without electricity.
3. HEPA FILTER
Air purifiers reduce air pollutants by removing
particulate matter (HEPA), or
High-Efficiency Particulate Absorbers. An example
of a filter with 99.97
percent efficiency would be one that can trap
particles smaller than 93
microns. During the pass of a HEPA filter, particles
are emitted in four
directions :

1. Direct impaction - large contaminants like dust,

mold, pollen, and mold adhere to fiber In a straight
line and travel directly through it.
2. Sieving - When an air particle moves between two fibers, ifs bigger than the gaps, and
thus becomes ensued .
3. Interception - The air is capable of rerouting around fibers, but due to inertia, particles
continue on their path and remain attached to the fibers.
4. Diffusion - It is more likely that ultrafine particles will hit and stick to fibers because
they move more erratically than larger ones.

AsxasaWS
ADVANTAGES
In addition to sterilizing thermolabile medications, such as blood products, insulin, and
enzymes, the method is also suitable for sterilizing blood plasma.
During the preparation, all types of bacteria are removed including those that are alive
and those that are dead.
The sterilization process is performed simultaneously with the clarification process.
A parenteral solution in a small quantity can be rapidly supplied in an emergency with
this method.
DISADVANTAGES
Sterility testing is necessary because the method is not reliable.
With this method, it is not possible to sterilize suspensions and oily preparations.
Filters may allow medicaments to be absorbed from a solution.
There are no immediate indications when the media is defective. Aseptic techniques must
be used .
Staff must be highly trained.
APPLICATIONS
Parenteral solution containing thermolabile medicines can be sterilized using this method
without decomposition, such as insulin, blood stream, and other products containing
protein matter, such as heat sensitive injections and biological products.
STERLITY INDICATORS
It is essential that strict controls are carried out on products to be labeled
'sterile.' Such controls must then ensure, the absence of viable microorganisms from these
products.
There are basically two types of controls:
1. Controls on the process of sterilization i.e. sterilization monitors or sterilization
indicators.
2. Sterility testing of the products.
3. Monitoring of the sterilization process can be achieved by the use of physical, chemical
or biological indicators of the sterilization performance.
PHYSICAL INDICATORS
1. Moist heat:
A master process record (MPR) is prepared as part of the validation
procedure for a particular autoclave and for each specified product and load
configuration. The MPR should be checked at annual intervals and whenever
significant changes occur in the BPR when compared with the MPR
2. Dry heat:
In dry heat sterilization processes, a temperature record chart made of each sterilization
cycle and is compared against a master temperature record.
3. Gaseous:
For gaseous sterilization procedures, elevated temperatures are monitored for each
sterilization cycle by temperature probes and routine leak. tests are performed to ensure
gas-tight seals.
Gas concentration is measured independently of pressure rise, often by reference to the
weight of gas used. Pressure and humidity measurements are recorded.
4. Filtration:
• Bubble point pressure test is a technique employed for determining the pore size of
filters and may also be used to check the integrity of certain types of filter devices
immediately after use.
The principle of the test is that the filter is soaked in an appropriate fluid and pressure is
applied to the filter.
 • The pressure difference when the first bubble of air breaks away from the filter is
equivalent to the maximum pore size. When the air pressure is further increased slowly,
there is general eruption of bubbles over the entire surface. The pressure difference is
equivalent to the mean pore size.
CHEMICAL INDICATORS
Chemical monitoring of a sterilization process is based on the ability of heat,
steam, sterilant gases and ionizing radiation to alter the chemical or
physical characteristics ofa variety of chemical substances.
1. Browne tubes
Each tube consists of a sealed glass tube which contains a red fluid (an ester and acid -
base indicator) that changes to yellow, brown and finally green on heating(the ester
undergoes heat hydrolysis to form an acid alcohol. The acid will change the color of the
indicator.
2. Witness tubes:
Witness tubes consist of single crystalline substances of known melting
point contained in glass tubes e.g. Sulphur (115ºC), succinic anhydride
(120ºC), benzoic acid (121ºC) etc. A dye may be included to show more
clearly that the crystals have melted.
Such a device only indicates that a certain temperature has been reached. Exposure time
can be calculated by putting the crystals in one end of an 'hour-glass' tube, the volume of
the crystals and the diameter of the constriction of the tube being adjusted so that the time
for transfer of the melt is the same as that required for the sterilization at the required
temperature
3. Royce sachet:
The Royce sachet is a chemical indicates for ethylene oxide sterilization. This consists of a
polythene sachet containing magnesium chloride, HCl and a bromophenol blue indicator. A
given concentration-time exposure to ethylene oxide results in the formation of ethylene
chlorohydrin and a color change from yellow to purple.
BIOLOGICAL INDICATORS
1. Biological indicators consist of a suitable organism deposited on a carrier and are
distributed throughout the sterilizer load.
2. At the end of the sterilization process, the units are recovered and cultured to determine the
presence or absence of survivors. The biological indicator measures sterilization processes
directly and is able to integrate all sterilization parameters.
3. The selected organism should possess high and reproducible resistance to the sterilizing
agent, should be genetically stable, readily characterizable and non-pathogenic.
4. The viability of the organisms, the storage conditions before use and the incubation and
culture conditions after sterilization must be standardized for the results. The organisms used
as biological indicators are usually resistant bacterial spores.