Microbiology 2
Lecture 23
Dua’a Tantawi
 Sterility assurance:
This lecture we will talk about sterility assurance
A product to be labelled ‘sterile’ must be free of viable microorganisms. To
achieve this, the product, or its ingredients, must undergo a sterilization process
of sufficient microbiocidal capacity to ensure a minimum level of sterility
assurance. It is essential that the required conditions for sterilization be
achieved and maintained through every operation of the sterilizer.
Historically, the quality control of sterile products consisted largely, or in some
cases, even exclusively, of a sterility test, to which the product was subjected at
the end of the manufacturing process.
However, a growing awareness of the limitations of sterility tests in terms of
their ability to detect low concentrations of microorganisms has resulted in a
shift in emphasis from a crucial dependence on end testing to a situation in
which the conferment of the status ‘sterile’ results from the attainment of
satisfactory quality standards throughout the whole manufacturing process.
A sterility test is essentially a test which assesses whether a sterilized
pharmaceutical or medical product is free from contaminating microorganisms
by incubation of either the whole or a part of that product with a nutrient
medium. It thus becomes a destructive test and is of questionable suitability for
testing large, expensive or delicate products or equipment. Furthermore, by its
very nature such a test is a statistical process in which part of a batch is
randomly sampled and the chance of the batch being passed for use then
depends on the sample passing the sterility test. (Random sampling should be
applied to products that have been processed and filled aseptically. With
products sterilized in their final containers, samples should be taken from the
potentially coolest or least sterilant accessible part of the load.)
A further limitation is that which is inherent in a procedure intended to
demonstrate a negative. A sterility test is intended to demonstrate that no viable
organisms are present, but failure to detect them could simply be a
consequence of the use of unsuitable media or inappropriate cultural
conditions. To be certain that no organisms are present it would be necessary to
use a universal culture medium suitable for the growth of any possible
contaminant and to incubate the sample under an infinite variety of conditions.
Clearly, no such medium or combination of media is available and, in practice,
only media capable of supporting non-fastidious bacteria, yeasts and moulds
are employed.
Furthermore, in pharmacopoeial tests, no attempt is made to detect viruses,
which on a size basis, are the organisms most likely to pass through a
sterilizing filter.
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Microbiology 2
Lecture 23
Dua’a Tantawi
To sum up >> limitations of sterility assurance test as it is:
-
Taking random sample which may give a false result!!
Using a non-suitable media for the microorganism, which means its
existence but it would never show up!
No attempt to detect viruses
In other words, the quality is ‘assured’ by a combination of process monitoring
and performance criteria; these may be considered under four headings:
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Bioburden determinations
Environmental monitoring
Validation and in-process monitoring of sterilization procedures
Sterility testing
 Bioburden determinations
The term ‘bioburden’ is used to describe the concentration of microorganisms
in a material; this may be either a total number of organisms per millilitre or
per gram, regardless of type, or a breakdown into such categories as aerobic
bacteria or yeasts and moulds. Bioburden determinations are normally
undertaken by the supplier of the raw material, whose responsibility it is to
ensure that the material supplied conforms to the agreed specification, but they
may also be checked by the recipient. The maximum permitted concentrations
of contaminants may be those specified in various pharmacopoeias or the levels
established by the manufacturer during product development.
The level of sterility assurance that is achieved in a terminally sterilized
product is dependent upon the design of the sterilization process itself and upon
the bioburden immediately prior to sterilization
The level of sterility assurance that is achieved in a terminally sterilized
product is dependent upon the design of the sterilization process itself and upon
the bioburden immediately prior to sterilization. However, the adoption of high
standards for the quality of the raw materials is not, in itself, a strategy that will
ensure that the product has an acceptably low bioburden immediately prior to
sterilization. It is necessary also to ensure that the opportunities for microbial
contamination during manufacture are restricted, and that those organisms that
are present initially do not normally find themselves in conditions conducive to
growth. It is for these reasons that manufacturing processes are designed to
utilize adverse temperatures, extreme pH valuesand organic solvent exposures
in order to prevent an increase in the microbial load.
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Microbiology 2
Lecture 23
Dua’a Tantawi
 Environmental monitoring
The levels of microbial contamination in the manufacturing areas are
monitored on a regular basis to confirm that the numbers do not exceed
specified limits. The concentrations of bacteria and of yeasts/moulds in the
atmosphere may be determined either by use of ‘settle plates’ (Petri dishes of
suitable media exposed for fixed periods, on which the colonies are counted
after incubation) or by use of air samplers which cause a known volume of air
to be passed over an agar surface. Similarly, the contamination on surfaces,
including manufacturing equipment, may be measured using swabs or contact
plates (also known as Rodac replicate organism detection and counting plates)
which are specially designed Petri dishes slightly overfilled with agar, which,
when set, projects very slightly above the plastic wall of the dish. This permits
the plate to be inverted onto or against any solid surface, thereby allowing
transfer of organisms from the surface onto the agar.
Less commonly, environmental monitoring can extend also to the operators in
the manufacturing area whose clothing, e.g. gloves or face masks, may be
sampled in order to estimate the levels and types of organisms that may arise as
product contaminants from those sources.
 Validation and in-process monitoring
of sterilization procedures:
There are several definitions of ‘validation’ but, in simple terms, the word
means demonstrating that a process will consistently produce the results that it
is intended to. Thus, with respect to sterile products, validation would be
necessary for each of the individual aspects of the manufacturing process, e.g.
environmental monitoring, raw materials quality assessment, the sterilization
process itself and the sterility testing procedure. Of these, it is the sterilization
process that is likely to be subject to the most detailed and complex validation
procedures, and these will be used to exemplify the factors to be considered.
A typical validation procedure for a steam sterilization process is likely to
incorporate most, or all, of the following features:
• The calibration and testing of all the physical instruments used to monitor the
process, e.g. thermocouples, pressure gauges and timers.
• Production of evidence that the steam is of the desired quality (e.g. that the
chamber temperature is that expected for pure steam at the measured pressure).
• The conduct of leak tests and steam penetration tests using both an empty
chamber and a chamber filled with the product to be sterilized in the intended
load conformation.
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Microbiology 2
Lecture 23
Dua’a Tantawi
• The use of biological indicators either alone or in combination with bioburden
organisms to demonstrate that the sterilization cycle is capable of producing an
acceptable level of sterility assurance under ‘worst case’ conditions.
• The production of data to demonstrate repeatability of the above (typically for
three runs).
• Comprehensive documentation of all of these aspects
In the UK, biological indicators are used primarily in validation rather than
routine monitoring of heat sterilization processes, although their use in routine
manufacturing may be required in other countries.
Chemical indicators of sterilization are more convenient to use than biological
indicators, but as they provide no direct measure of the efficacy of the process
in terms of microbial killing they are considered to be less useful. Physical
measurements of temperature, pressure, time, relative humidity, etc. are of such
fundamental importance to the assurance of sterility that records of these
parameters are retained for each batch of sterilized product.
 Physical indicators
In heat sterilization processes, a temperature record chart is made of each
sterilization cycle with both dry and moist heat (i.e. autoclave) sterilizers; this
chart forms part of the batch documentation and is compared against a master
temperature record (MTR).
Which means keeping an eye on parameters of sterilization !!
It is recommended that the temperature be taken at the coolest part of the
loaded sterilizer. Further information on heat distribution and penetration
within a sterilizer can be gained by the use of thermocouples placed at selected
sites in the chamber or inserted directly into test packs or bottles. 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. Pressure and humidity measurements are recorded.
Gas concentration is measured independently of pressure rise, often by
reference to weight of gas used. In radiation sterilization, a plastic (often
perspex) dosimeter which gradually darkens in proportion to the radiation
absorbed gives an accurate measure of the radiation dose and is considered to
be the best technique currently available for following the radiosterilization
process.
Sterilizing filters are subject to a bubble point pressure test, which 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 device (membrane and
sintered glass) immediately after use.
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Microbiology 2
Lecture 23
Dua’a Tantawi
The principle of the test is that the wetted filter, in its assembled unit, is
subjected to an increasing air or nitrogen gas pressure differential. The pressure
difference recorded when the first bubble of gas breaks away from the filter is
related to the maximum pore size. When the gas pressure is further increased
slowly, there is a general eruption of bubbles over the entire surface. The
pressure difference here is related to the mean pore size. A pressure differential
below the expected value would signify a damaged or faulty filter. A
modification to this test for membrane filters involves measuring the diffusion
of gas through a wetted filter at pressures below the bubble point pressure
(diffusion rate test); a faster diffusion rate than expected would again indicate a
loss of filter integrity. In addition, a filter is considered ineffective when an
unusually rapid rate of filtration occurs.
Efficiency testing of HEPA filters used for the supply of sterile air to aseptic
workplaces is normally achieved by the generation upstream of
dioctylphthalate (DOP) or sodium chloride particles of known dimension
followed by detection in downstream filtered air. Retention efficiency is
recorded as the percentage of particles removed under defined test conditions.
Microbiological tests are not normally performed.
Note: they measure the flow rate of the air, and they calculate it as a percentage
 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 and/or
physical characteristics of a variety of chemical substances.
Chemical indicators generally undergo melting or colour changes (some
examples are given in, the relationship of this change to the sterilization
process being influenced by the design of the test device . It must be
remembered, however, that the changes recorded do not necessarily correspond
to microbiological sterility and consequently the devices should never be
employed as sole indicators in a sterilization process.
 Biological indicators
Which are non-pathogenic resistant microbes
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. Spores of Bacillus
stearothermophilus in sealed ampoules of culture medium are used for steam
sterilization monitoring, and these may be incubated directly at 55°C; this
eliminates the need for an aseptic transfer.
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Microbiology 2
Lecture 23
Dua’a Tantawi
Note: the species of bacteria used vary according to the sterilization method to
be used
Aseptic transfers are also avoided by the use of self-contained units where the
spore strip and nutrient medium are present in the same device ready for
mixing after use. 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. Resistance is adjudged from
the spore destruction curve obtained upon exposure to the sterilization process;
recommended BI spores and their decimal reduction times (D-values)
Filtration sterilization requires a different approach from biological monitoring,
the test effectively measuring the ability of a filter to produce a sterile filtrate
from a culture of a suitable organism. For this purpose, Serratia marcescens, a
small Gram-negative rod-shaped bacterium (minimum dimension 0.5 µm), has
Brevundimonas diminuta (formerly Pseudomonas diminuta) having a minimum
are defined as those capable of completely removing Brev. diminuta from
suspension. In this test, using this organism, a realistic inoculums level must be
adopted, as the probability of bacteria appearing in the filtrate rises as the
number of Brev. diminuta cells in the test challenge increases; a standardized
inoculum size of 10^7 cells cm is normally employed. The extent of the
passage of this organism through membrane filters is enhanced by increasing
the filtration pressure. Thus, successful sterile filtration depends markedly on
the challenge conditions. Such tests are used as part of the filter manufacturer’s
characterization and quality assurance process, and a user’s initial validation
procedure. They are not employed as a test of filter performance in use.
 Sterility testing:
In this test we are looking for the absence of bacteria
In addition to limitations that we have mentioned earlier we have one more
limitation which is the high financial cost of the sterility test !!
Nevertheless, the sterility test does have an important application in monitoring
the microbiological quality of filter-sterilized, aseptically filled products and
does offer a final check on terminally sterilized articles. In the UK, test
procedures laid down by the European Pharmacopoeia must be followed; this
provides details of the sample sizes to be adopted in particular cases.
So sometimes even if sterility was checked step by step, it won't be enough,
and we have to
And we have to know that performing it by itself is not enough at all!!!! We
perform sterility test in order to support the other measures
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Microbiology 2
Lecture 23
Dua’a Tantawi
 Methods
There are three alternative methods available when conducting sterility tests.
1)The direct inoculation procedure involves introducing test samples directly
into nutrient media. The European Pharmacopoeia (2002) recommends two
media: (i) fluid mercaptoacetate medium (also known as fluid thioglycollate
medium), which contains glucose and sodium mercaptoacetate (sodium
thioglycollate) and is particularly suitable for the cultivation of anaerobic
organisms (incubation temperature 30–35°C); and (ii) soyabean casein digest
medium (also known as tryptone soya broth), which will support the growth of
both aerobic bacteria (incubation temperature 30–35°C) and fungi (incubation
temperature 20–25°C). Other media may be used provided that they can be
shown to be suitable alternatives. Limits are placed upon the ratio of the weight
or volume of added sample relative to the volume of culture medium so as to
avoid reducing the nutrient properties of the medium or creating unfavourably
high osmotic pressures within it.
2)Membrane filtration is the technique recommended by most pharmacopoeias
and, consequently, the method by which the great majority of products are
examined. It involves filtration of fluids through a sterile membrane filter (pore
size 0.45 µm); any microorganism present being retained on the surface of the
filter. After washing in situ, the filter is divided aseptically and portions are
transferred to suitable culture media which are then incubated at the
appropriate temperature for the required period of time. Water-soluble solids
can be dissolved in a suitable diluent and processed in this way and oil-soluble
products may be dissolved in a suitable solvent, e.g. isopropyl myristate.
3)A sensitive method for detecting low levels of contamination in intravenous
infusion fluids involves the addition of a concentrated culture medium to the
fluid in its original container, such that the resultant mixture is equivalent to
single strength culture medium. In this way, sampling of the entire volume is
achieved.
With the techniques discussed above, the media employed should previously
have been assessed for nutritive (growth-supporting) properties and a lack of
toxicity using specified organisms (positive controls). It must be remembered
that any survivors of a sterilization process may be damaged and thus must be
given the best possible conditions for growth.
As a precaution against accidental contamination, product testing must be
carried out under conditions of strict asepsis using, for example, a laminar
airflow cabinet to provide a suitable environment.
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Microbiology 2
Lecture 23
Dua’a Tantawi
The European Pharmacopoeia (2002) indicates that it is necessary to conduct
control tests that confirm the adequacy of the facilities by sampling of air and
surfaces and carrying out tests using samples ‘known’ to be sterile (negative
controls). In reality, this means samples that have been subjected to a very
reliable sterilization process, e.g. radiation, or samples that have been subjected
to a sterilization procedure more than once. In order to minimize the risk of
introducing contaminants from the surroundings or from the operator during
the test itself, isolators are often employed which physically separate the
operator from the materials under test. These are designed on the same
principle as a glove box, but on a much larger and more sophisticated scale, so
the operator works inside a sterile cubicle but is separated from the atmosphere
within it by a flexible moulded covering (rather like a space suit) which is an
integral part of the cubicle base
## Notes ##
False negative is dangerous!! Because we will think that the batch is sterile
when it is not!! So it would cause harm for patients who take it because it is
contaminated
False positive is not dangerous however it cause huge financial loss where
there is no reasonable cause
Antimicrobial agents
Where an antimicrobial agent comprises the product or forms part of the
product, for example as a preservative, its activity must be nullified in some
way during sterility testing so that an inhibitory action in preventing the growth
of any contaminating microorganisms is overcome. This is achieved by the
following methods.
Specific inactivation
An appropriate inactivating (neutralizing) agent is incorporated into the culture
media. The inactivating agent must be non-toxic to microorganisms, as must
any product resulting from an interaction of the inactivator and the
antimicrobial agent.
Benzylpenicillin and ampicillin are
-lactamase (from B.
-lactams may also be
-lactamases. Other
antibiotic inactivating enzymes are also known and have been considered as
possible inactivating agents, e.g. chloramphenicol acetyltransferase (inactivates
chloramphenicol) and enzymes that modify aminoglycoside antibiotics.
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Microbiology 2
Lecture 23
Dua’a Tantawi
Dilution
The antimicrobial agent is diluted in the culture medium to a level at which it
ceases to have any activity, for example phenols, cresols and alcohols. This
method applies to substances with a high dilution coefficient, .
Membrane filtration
This method has traditionally been used to overcome the activity of antibiotics
for which there are no inactivating agents, although it could be extended to
cover other products if necessary, e.g. those containing preservatives for which
no specific or effective inactivators are available. Basically, a solution of the
product is filtered through a hydrophobic-edged membrane filter that will retain
any contaminating microorganisms. The membrane is washed in situ to remove
any traces of antibiotic adhering to the membrane and is then transferred to
appropriate culture media.
In well-understood and well-characterized sterilization processes (e.g. heat and
irradiation), where physical measurements may be accurately made, sterility
can be assured by ensuring that the manufacturing process as a whole conforms
to the established protocols for Bioburden determinations, Environmental
monitoring, Validation and in-process monitoring of sterilization procedures. In
this case the process has satisfied the required parameters thereby permitting
parametric release (i.e. release based upon process data) of the product without
recourse to a sterility test
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