Validation of sterilization – Pharmaceutical Microbiology Third Semester PDF Notes

 Validation of sterilization


       Validation – meaning and

       Sterilization indicators

      Physical indicators

      Chemical indicators

      Biological indicators

Intended learning objectives

At the
end of this lecture, the student will be able to:

       Explain the significance of
validation in sterilization

       List the sterilization indicators

       Describe the working of
sterilization indicators

Validation- History

The concept of validation was first proposed by
two Food and Drug Administration (FDA) officials, Ted Byers and Bud
Loftus, in the mid-1970s in order to improve the quality of pharmaceuticals

It was proposed in direct response to several
problems in the sterility of large volume parenteral market

 The first
validation activities were focused on the processes involved in making sterile
products, but later on were applied for 
associated processes such as 
environmental control, media fill, equipment sanitization and
purified water production

Validation of sterilization

       Validation means demonstrating that a process
will consistently produce the results that it is intended to

       Validation is
a process of establishing documentary evidence demonstrating that a procedure,
process, or activity carried out in production or testing maintains the desired
level of compliance at all stages. 

       Validation would be necessary for
each of the individual aspects of the manufacturing process,

       Environmental monitoring

       Raw materials quality assessment

       Sterilization process itself

       The sterility testing procedure

Sterilization indicators

                Sterilization procedures should
be monitored through a combination of physical, chemical, and biological
designed to evaluate the sterilizing conditions and the
procedure’s effectiveness

       Physical indicators

       Chemical indicators

       Biological indicators

Physical indicators

the gauges or displays on the sterilizer for

       Assessing the cycle time


       Pressure of sterilization equipment

       Relative humidity


       A temperature record chart is made –
part of the batch documentation

       Compared against a master
temperature record (MTR)

       Information on heat distribution and
penetration within a sterilizer can be gained by the use of thermocouples


       Measurement of elevated temperatures
using temperature probes

       Routine leak tests to ensure
gas-tight seals

       Pressure and humidity measurements
are recorded.

       Gas concentration is measured, often
by reference to weight of gas used


       A plastic (often perspex) dosimeter
is used

gradually darkens in proportion to the radiation absorbed

Integrity testing sterilizing filters

filters have been used successfully for many years to remove yeast, bacteria
and particulate from fluid streams

test the integrity of filters, the following tests are done:

a)      Destructive

b)      Non
– destructive integrity test

a) Destructive test- Destructive challenge
testing is the best way to determine a sterilizing filter’s ability to retain
bacteria. During the bacterial retention test, 0.22 µm filter discs and devices
are challenged with a solution of culture medium containing bacteria (Brevundimonas
 ATCC 19146) at a minimum challenge of 107 per cm2.
The effluent is then passed through a second 0.45 µm filter disc that is placed
on an agar plate and incubated.

 – The filter cannot
be used for filtration purposes again

b) Non – destructive integrity test, may be
done on filters before and after filtration process

-This is done to ensure that the filter meets
specification, is properly installed and intact during filtration, and to
confirm the rating of the filter

– Integrity testing before filtration process monitors
filter integrity, preventing use of a non-integral filter. 
– Integrity testing after a batch has been filtered can detect if the integrity
of the filter has been compromised during the process

There are 3 major tests used to determine the integrity of a
membrane filter: the Bubble Point Test, the Forward Flow Test, and the Pressure
Hold Test

        All three tests are based on the same physics,
the flow of a gas through a liquid-wetted membrane under applied gas pressures

        They differ in which part of the flow/pressure
spectrum they examine

is an indirect method for the measurement of pore size of filter

point pressure test

point test  is based on the fact that
liquid is held in the pores of the filter by surface tension and capillary

        The minimum pressure ( as seen on the pressure
dial) required to force liquid out of the pores is a measure of the pore
The bubble point is expressed as :

k = shape correction factor
Ύ = surface tension
cos ɵ =  liquid solid contact angle
d = pore diameter

of immersing the filter candles in water or filling the funnel with the liquid
in the case of a membrane filter or sintered glass filter

or gas is passed from the bottom of the filter

pressure of Air/ gas is gradually increased until the first bubble is seen at
the filter/liquid interface


       Bubble point pressure test

Chemical indicators

indicators are designed to respond to a characteristic change to one or more of
the physical conditions within the sterilizing chamber

principle is  based on the ability of heat, steam, gases and ionization
radiation to alter the chemical and/or physical characteristics of  chemical substances

       Change should take place only when
satisfactory conditions for sterilization prevail

for monitoring a sterilization process

       Chemical indicators generally undergo
melting or colour changes

       Included in strategically placed
containers or packages

       Monitors  the conditions prevailing at the coolest or
most inaccessible parts of a sterilizer

Dick test:
chemical in a printing ink base impregnated into a carrier material. A
combination of moisture and heat produces a darkening of the ink

: Sealed tubes
partly filled with a solution which changes colour at elevated temperatures;
rate of colour change is proportional to temperature

Consists of a
blue dye in a waxy pellet. At autoclaving temperatures, and in the continued
presence of steam, the pellet melts and travels along a paper wick forming a
blue band the length of which is dependent upon both exposure time and

Thermalog G: Same as Thermalog S. Used for gaseous

Biological indicators

       Consist of standardized bacterial
spore preparations

       Usually in the form either of
suspensions in water or culture medium or of spores dried on paper, aluminium
or plastic carriers.

       As with chemical indicators, they
are usually placed in dummy packs located at strategic sites in the sterilizer

       After the sterilization process, the
aqueous suspensions or spores on carriers are aseptically transferred to an
appropriate nutrient medium, which is then incubated and periodically examined
for signs of growth.

       The bacterial species to be used in
a BI must be selected carefully, as it must be non-pathogenic and should
possess above-average resistance  to the
particular sterilization process.


       Measuring the ability of a filter to
produce a sterile filtrate from a culture of a suitable organism

       Serratia marcescens 
has been
used for filters of 0.45mm pore size,

       Brevundimonas diminuta (formerly Pseudomonas diminuta) having a minimum dimension of 0.3mm is applied to filters of
0.22mm pore size.


       Validation means demonstrating that
a process will consistently produce the results that it is intended to

       Sterilization indicators – Physical
indicators, Chemical indicators, Biological indicators

       Physical indicators validate the
temperature, pressure, radiation dose or filter pore size

       Chemical indicator – Based on the
ability of heat, steam, sterilant gases and ionizing radiation to alter the
chemical and/or physical characteristics of a variety of chemical substances

       Biological indicators – Consist of
standardized bacterial spore preparations

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