INTRODUCTION

INTRODUCTION
The Microbiology (Quality Control) department contributes a major role in bringing
safe product to the market. They are involved in critical decisions regarding
manufacturing environment, formulation and manufacturing process development that
can prevent microbial contamination of pharmaceutical products. These tests include
Bioburden test, Microbiological Enumeration test, Bacterial endotoxins test, Sterility
test and Microbial testing of water (total microbial count test and test for pathogen).
In this project report, the appropriate level of involvement of microbiologists in
establishing the quality, purity, efficacy, and safety of pharmaceutical and over-the-
counter drug products is discussed, and areas of future challenge to the
pharmaceutical microbiologist are explored.

The development of many vaccines and other medicines that have been so crucial to
the improvement in world health has been the result of large investment in research by
the major international pharmaceuticals becoming one of the most consistently
successful and important industries in many countries, not only in traditional
strongholds of North America, Western Europe and Japan but increasingly in Eastern
Europe, the Indian subcontinent and far East.

The growth of pharmaceutical industry in recent decades has been paralleled by rising
standards for the product quality and more rigorous regulation of manufacturing
procedures. In order to receive a manufacturing license, a modern medicine must be
shown to be effective, safe and good quality. Most medicine consist of active
ingredients that is formulated with a variety of other materials (excipient) that are
necessary to ensure that the medicine is effective, and stable, palatable and safe
during storage and use.

Analytical chemist and pharmacists take lead responsibility for ensuring that the
components of medicine are present in the correct physical form and concentration,
but quality is not judged solely on physicochemical properties of product:
microorganisms also have the potential to influence efficacy and safety. It is obvious
that medicines contaminated with potentially pathogenic microorganisms can in
addition to initiating infections, cause product spoilage by chemically discomposing
the active ingredient or excipient. This may lead to product being under-strength,
physically or chemically unstable or possibly contaminated with toxic materials.
Thus, this is clear that Pharmaceutical microbiology must encompass the subjects of
sterilization and preservation against microbial spoilage, and with responsibility for
the safe, hygienic manufacture. In this respect the Pharmaceutical microbiology have
very important role. Commercial antibiotic production has created a great importance
of Pharmaceutical microbiologist in pharma industry.

The microbiology quality of pharmaceutical products is influenced by the

environment in which they are manufactured and by the materials used in their
formulation with the exception of preparation which is terminally sterilized in. Their
final container, the micro flora of the final product may represent the contaminants
from the raw materials, from the equipment with which it was made from the

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atmosphere. From the person operating the process or from the final container into
which it was packed . Some of these contaminants may be pathogenic, while others
may grow even in the presence of preservative and spoil the product.

Air is not a natural environment for the growth and reproduction of microorganisms
since it does not contain the necessary amount of moisture and nutrients in a form that
can be utilized. A lowermost sample of untreated air contains suspended matter
including bacteria, yeasts and moulds but in order to survive, microorganisms must be
able to tolerate desiccation and the dry state. The spore forming bacteria, bacillus
species and clostridium species and the non-sporing bacteria, staphylococcus species,
streptococcus species, the mucor species and the yeast rhodotorula species are all
commonly isolated from air.

The microbial content of air may be increased during the handling of contaminated
materials during dispensing, blending and their addition to formulations. In particular,
the use of starches and sugars in dry state increases the mould count. Some packaging
components for example card and paperboard have a microbial flora of both moulds
and bacteria, and this is often reflected in high air counts around cartooning and
packaging machines.

The microbial ecology of water is of great importance in the Pharmaceutical industry

since it is used in the formulation of new products as well as for various washing and
cleaning processes. Two main aspects are involved; the quality of raw water and any
processing it receives and the distribution system. Both should be taken into
consideration when reviewing the hazards to the finished products or at any critical
control point.

A test was undertaken with following objectives:

A) Bioburden testing of Prefilltered bulk solution.

B) Microbiological enumeration testing of soluble and insoluble
nonsterile products.
C) Sterility test.
D) Microbiological testing of water.
E) Bacterial endotoxin test.

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AIM AND OBJECTIVE

Microbiological analysis of water and pharmaceutical products is achieved through

following objectives:

Microbial Limit Test: This test is performed to estimate the number of viable aerobic
microorganism present in water sample.

Sterility Test: The test is performed to determine that the product is sterile or not.

Bacterial Endotoxin Test: The test is performed to determine the presence or absence
of endotoxin in Gentamycin with the help of LAL test.

Environmental monitoring: The test is performed to estimate the number of viable

aerobic microorganism present in environment.

 To study different methods of Sterilization for different pharmaceutical

preparations.

 To study of Sterility testing of different pharmaceutical preparations.

 To study Validation methods

 To study the principle and methods involved in different microbiological

testing.

 To study disinfectants, antiseptics, fungicidal and virucidal agents

 To study the mechanism of action of the above

 To study of nutritional requirements, growth and cultivation of bacteria and

virus

 To study of different Media required for the growth of aerobic and anaerobic
bacteria and fungi.

 To learn methods available to maintain laboratory cultures

 To study of different isolation & identification techniques

 To study counting techniques of bacteria

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REVIEW OF LITRATURE
The microbiological tests are designed to estimate the number of viable aerobic
organisms present in pharmaceutical and raw materials. The test is also used to
determine whether the articles harbor designated microbial species which are
pathogenic in organisms called for in the USP include Pseudomonas aerginosa,
Salmonella sp., Staphylococcusaueus.
Microbiological testing of raw material as well as finished pharmaceutical products
can help to determine whether the product complies with the requirements of the BP,
Ph. Eur. or USP.
Microbiologocal Tests are designed to perform the qualitative and quantitative
estimations of specific viable microorganisms present in samples. It includes tests for
total viable count (bacteria and fungi) and Escherichia coli. The most care must be
taken in performing the tests, so that microbial contamination from the outside can be
avoided. When test samples have antimicrobial activity or when they include
antimicrobial substances, these antimicrobial properties must be eliminated by
dilution, filtration, neutralization, inactivation, or other appropriate means.

Total viable aerobic count :

This test is to determine mesophilic bacteria and fungi which grow under aerobic
conditions. Psychrophilic, thermophilic, basophilic, and anaerobic bacteria, and
microorganisms which require specific ingredients for growth may give a negative
result, even if they exist in a significant number. There are four methods for this test:

* Membrane Filtration Method.

* Pour Plate Method.

* Spread Plate Method.

* Serial Dilution Method (most probable number method).
An appropriate method should be taken from among these four, depending on
purposes. Different media and incubation temperature are required for the growth of
bacteria and fungi (molds and yeasts). The serial dilution method is applicable only to
bacteria.
Preparation of test fluids:

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To dissolve or dilute the sample, use phosphate buffer (pH 7.2), sodium chloride
peptone buffer solution, or fluid medium used for the test. Unless otherwise specified,
use 10 g or 10 ml of the sample. However, a different weight or volume of the sample
should be used, depending on the nature of the sample. Adjust the test fluid to pH 6-8.
Use the test fluid within one hour after preparation.
Fluid samples or soluble solid samples:
Take 10 g or 10 ml of the sample and mix with the buffer or fluid medium given
above to make 100 ml. Use this mixture as the test fluid. For a fluid sample including
insoluble substances, shake well just before mixing make a homogeneous suspension.
Insoluble solid samples Take 10 g of the sample, grind to a fine powder, and suspend
it in the buffer or fluid medium given above to make 100 ml. Use this suspension as
the sample fluid. A larger volume of the buffer or fluid medium than specified here
may be used to make a suspension, depending on the nature of the sample. If
necessary, a blender may be used to disperse the insoluble particles well in the
suspension.
Fatty samples:
For semisolid samples and liquids consisting mainly of lipid, take 10 g or 10 ml of
the sample, emulsify the sample in the buffer or fluid medium given above using a
surfactant such as polysorbate 20 or polysorbate 80, and make to 100 ml. Use this
emulsified sample as the sample fluid. If necessary, warm at a temperature not
exceeding 45 to emulsify the sample. Avoid warming for not longer than 30 minutes.
Pathogen testing:
Pathogenic organisms are those that can cause illness in humans either by infections
such as Salmonella, pathogenic Ecoli or in toxications such as Bacillus cereus,
Staphylococcus aureus or Clostridium botulinum, But in case of pharma industry we
test the following pathogens:
01. Staphylococcus aureus.
02 Bacillus subtilis.
03. Escherichia coli.
04. Salmonella sps
05. Clostridium sporogenes
06. Candida albicans.
07. Aspergillusniger.
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CLEAN ROOM DESIGN:

Microbiological testing should be performed under aseptic conditions, which are

preferably consistent with the standard of clean room required for the aseptic
manufacture of pharmaceutical products. Premises and equipment should be qualified
according to the relevant principles of Installation Qualification and Operational
Qualification.

Classification:

 The microbiological test should be conducted within a class A laminar airflow

cabinet located within a class B clean room, or in an isolator that need not be
located within a controlled environment. The test may also be performed
within a class A clean room, if available. Microbiological testing should be
carried out in a work zone that offers sufficient space and material should be
placed in such a way that it does not disrupt the laminar airflow.
 The test environment, which includes the laminar airflow cabinet or isolator,
should be certified at least annually by a competent person for compliance
with the specified standard conditions.

Air supply:
 Air supplied to the environment should be provided through terminal HEPA
filters, which should be fitted with audible and/or visual alarms to indicate any
sustained, out of specification pressure differentials across the HEPA filters.
 There should be a pressure differential of not less than 10 to 15 Pascal
(guidance value) between each of the areas, i.e. ambient/airlock and
airlock/test room. Pressure readings should be taken and recorded from
externally mounted gauges unless a validated continuous monitoring system is
installed. As a minimum, readings should be taken prior to operator entry to
the test suite. Pressure gauges should be labeled to indicate the area served, the
acceptable specification, and whether or not the reading is absolute or
differential.

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AIRLOCK AND ASEPTIC GOWNING

Airlock conditions:

 Entry to the clean room should be via an airlock in which operators are
required to change into clean room garments.
 The airlock should be designed to facilitate movement of the operator between
the relatively unclean and clean areas of the room without compromising the
aseptic gowning procedure. A step-over bench is a suitable division between
these areas.
 The airlock should contain: a full-length wall mirror; gowning instructions;
hand washing, disinfection and drying facilities. If clean room garments are
stored in the airlock, then adequate and appropriate storage facilities should be
provided.

Aseptic gowning:

 The microbiological test operator should change into sterile clean room
garments consisting of a one-piece cover all suit, head cover, beard cover (if
applicable), overshoes, gloves and mask. The use of sanitized garments may
be acceptable if the process has been validated and their use is not routinely
used to justify the performance of repeat sterility tests.
 Protective garments should be changed for each work session or at least once a
day if justified from the results of microbiological monitoring and validation
studies.
 Records should be kept of the sterilization or sanitization of the garments. This
record may be in the form of a certification from an external supplier of sterile
clean room garments provided that they have been approved by the sterility
testing laboratory.
 Each operator should be trained and certified in gowning procedures with
training records maintained.

CLEAN ROOM FITTINGS AND SURFACES:

 All fittings, such as power outlets and light fittings should be flush with the
wall or ceiling surfaces and sealed to prevent entrainment of unclean air.
Surfaces should be smooth and impervious to the cleaning agents used.
 The joints between ceiling/walls/floor should be coved to facilitate cleaning.
 If supplied, intercom or communication systems should be designed to allow
hands-free use, or their design should facilitate disinfection.
 Chairs, storage cabinets and trolleys should be designed to facilitate cleaning
and be suitable for use in a clean room environment.
 There should be no extraneous equipment within the clean room environment.
 Ultraviolet lamps may be fitted within pass-through cabinets only. Where
there is more than one parallel tube, they should be shielded from each other
and checked at least annually for performance, or whenever new lamps are
fitted.

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CLEANING, SANITISATION AND DISINFECTION:

 Outer surfaces of samples and equipment entering the testing suite should be
disinfected, preferably with a sporicidal agent. Disinfection of surfaces and
sample containers should be carried out in such a way as to avoid adventitious
contamination of the samples by the chemical agent. Therefore, disinfectants
should be free of microbiological contamination, which may be achieved
through aseptic filtration or use of a product-compatible terminal sterilization
method.
 Surfaces and operators' gloved hands should be disinfected regularly during
the test session.
 There should be protocols to cover all daily, weekly, and periodic cleaning,
sanitization, disinfection and fumigation procedures used in the testing
environment. If an isolator is used, the method of disinfection or sterilization
should be specified.
 Prior to implementation, all cleaning, sanitizing and disinfecting procedures
should be validated from a microbiological perspective with respect to
minimum disinfectant contact times and efficacy. Cleaning and disinfecting
agents should be purchased to agreed and documented specifications.
 Records should be retained in respect of routine preparation of cleaning and
disinfecting agents, directions for their use, and validation of their efficacy.

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INSTRUMENTS USE IN MICROBIOLOGICAL

LABORATORY

Digital colony counter:

It is designed for quick and accurate counting of bacterial and mould colonies in petri
dishes. Feature packed & easy to use, this is an indispensable bench top tool for the
busy microbiologist. It is designed for rapid and accurate counting of bacterial and
mould colonies. Simply place the petri dish on the illuminated pad and touch the dish
with the pen provided to mark each colony in turn. This causes a count to be
registered on the digital display and an audible tone confirms each count made.
Marking the dish with the pen avoids missing colonies or double counting. The digital
count on the display cab be reset manually any time by pressing the RESET key.
Optimum viewing of colonies is aided by peripheral glare-free illumination. An
integral magnifying glass provides for easier counting of small colonies.

pH meter:
A pH meter is an electronic instrument used for measuring theph(acidity or alkality)
of a liquid (though special probes are sometimes used to measure the pH of semi-solid
substances). A typical pH meter consists of a special measuring probe (a glass
electrode) connected to an electronic meter that measures and displays the pH
reading.

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Analytical balance:
An analytical balance is used to measure mass to a very high degree of precision and
accuracy. The measuring pan(s) of a high precision (0.1 mg or better) analytical
balance are inside a transparent enclosure with doors so that dust does not collect and
so any air currents in the room do not affect the balance's operation. The use of
a vented balance safety enclosure, which has uniquely designed acrylic airfoils,
allows a smooth turbulence-free airflow that prevents balance fluctuation and the
measure of mass down to 1 μg without fluctuations or loss of product. Also, the
sample must be atroom temperature to prevent natural convection from forming air
currents inside the enclosure, affecting the measure of mass.

The 3 place manifold:

The stainless steel manifold for 3 or 6 filtration units is fitted with stainless steel units.
The apparatus can be autoclaved and sterilized by dry heat at up to 180°C. Suitable
only for vacuumoperation. If flushing tubes are used, do not exceed 1.3 bar (300 mbar
over-pressure).

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Particulate or biological contamination analysis via vacuum filtration. Includes

grounding set for use with flammable liquids. Filter holder is not autoclavable with
filter in-place.

Incubators:

Incubator is a device used to grow and maintain microbial culture or cell cultures. The
incubator maintains optimal temperature, humidity and other conditions such as the
carbon dioxide (CO2) and oxygen content of the atmosphere inside. Incubators are
used to culture both bacterial as well as eukaryotic cells.

Incubators also include a timer, some can also be programmed to cycle through
different temperatures, humidity levels etc. Incubators can vary in size from tabletop
to units the size of small rooms.

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Laminar flow cabinet:

A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully

enclosed bench designed to prevent contamination of semiconductor wafers,
biological samples, or any particle sensitive device. Air is drawn through
a HEPA filter and blown in a very smooth, laminar flow towards the user. The cabinet
is usually made of stainless steel with no gaps or joints where spores might collect.

Such hoods exist in both horizontal and vertical configurations, and there are many
different types of cabinets with a variety of airflow patterns and acceptable uses.

Laminar flow cabinets may have a UV-C germicidal lampto sterilize the shell and
contents when not in use.

Steam heat sterliser (SHS):

Steam/Moist heat sterilization technology is the most widely used given its versatility

and effectiveness for heat resistant products. Moist heat kills the organisms by
coagulating and denaturing their enzymes and structural protein. Sterilization by
moist heat of the most resistant spores generally requires 121 °C for 15-30 minutes.
Moist heat is used for the sterilization of culture media, and all other materials
through which steam can penetrate.

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Vortex:

It is the variable speed mixer to eliminate hand mixing. Holding tube against vibrating
rubber cup does rapid mixing of contents. It is offer with speed regulator which
controls the degree of vibration. These Cyclomixer has the unique touch feature
operates the unit when the tube is pressed on the rubber cup. Interchangeable adaptors
greatly enhance the use of Cyclo Mixer for mixing in different applications

Water Bath:

Temperature range from few degree above ambient temperature to 100°C.

Temperature is controlled by Electronic Digital Temperature Controller-Cum-
Indicator with an accuracy of plus minus 0.5°C. Double walled inside Stainless Steel
304 Qlty. and outside Mild Steel Sheet Painted in epoxy powder coating. Bath
consists two pilot lamps, On-Off switch. To work on 220/230 Volts A.C. Stirrer with

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1/20 H.P. high speed motor with stainless steel stirring rod and blade provided with
speed regulator. Lid of water bath is made of Stainless Steel 304 Qlty.

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BUFFER SOLUTIONS AND MEDIA

Introduction:
Use the buffer solutions and media, given below, for the Microbiological Test. Other
media may be used if they include similar nutritive ingredients and have similar
selectivity and growth-promoting ability toward the microorganism to be tested.

Buffer solutions:

(i)Phosphate buffer (pH 7.2) Stock solution:

Dissolve 34 g of monopotassium phosphate in about 500 ml of water, add about 175

ml of sodium hydroxide (4.3 → 100) to adjust to pH 7.1－7.3, and add water to make
1,000 ml. Use this solution as the stock solution. Autoclave and cool this solution.
Store it at a cool place. For use, dilute the stock solution with water to 800 times its
original volume and autoclave for 15－20 minutes at 121℃.
(ii) Sodium chloride-peptone buffer (pH 7.0):
Mix all the ingredients and autoclave for 15－20 minutes at 121℃. Its pH becomes
6.9－7.1 after autoclaving. 0.1－1.0%w/v polysorbate 20 or polysorbate 80 may be
added.

Media:
Media used in Bacteriological testing:
 MacConkey agar:

MacConkey agar is a culture medium designed to grow Gram-negative

bacteria and stain them for lactose fermentation. The medium was developed by
Alfred Theodore MacConkey while working as a bacteriologist for the Royal
Commission on Sewage Disposal.

It contains bile salts, crystal violet dye, neutral red  dye, lactose  and peptone.

There are many variations of MacConkey agar depending on the need. If the
spreading or swarming of Proteus species is not required, sodium chloride is omitted.

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Crystal violet at a concentration of 0.0001% (0.001 g per litre) is included when

needing to check if Gram-positive bacteria are inhibited.

Acting as a visual pH indicator, the agar distinguishes those Gram-negative bacteria

that can ferment the sugar lactose (Lac+) from those that cannot (Lac-).

This medium is also known as an "indicator medium" and a "low selective medium".
Absence of electrolytes serves to inhibit swarming by Proteus species.

 Mannitol salt agar:

Mannitol salt agar or MSA is a commonly used growth medium in microbiology. It

encourages the growth of a group of certain bacteria while inhibiting the growth of
others. This medium is important in medical laboratories by distinguishing pathogenic
microbes in a short period of time. It contains a high concentration (~7.5%-10%) of
salt (NaCl), making it selective for gram positive bacterium Staphylococci (and
Micrococcaceae) since this level of NaCl is inhibitory to most other bacteria. It is also
a differential medium for mannitolfermentors, containing mannitol and the indicator
phenol red.

 Columbia agar:
Columbia Agar is used with or without blood for the isolation and cultivation of a
wide variety of fastidious microorganisms.

This medium contains a mixture of peptones, which provides a blend of nitrogenous

compounds and amino acids to enhance growth. Columbia Agar is used for the
cultivation of fastidious microorganisms and as a base for the preparation of various
specialized culture media to which supplements can be added. Columbia Agar can be
supplemented with 5 - 10% sheep, rabbit, or horse blood for use in isolating,
cultivating, and determining hemolytic reactions of fastidious pathogenic
microorganisms.

 Cetrimide agar:

Cetrimide agar is a type of agar used for the selective isolation of the gram-

negative bacterium, Pseudomonas aeruginosa. As the name suggests, it

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contains cetrimide, which is the selective agent against alternate microbial

flora. Cetrimide also enhances the production of Pseudomonas pigments such as
pyocyanin and fluorescein, which show a characteristic blue-green and yellow-green
colour, respectively.

 Xylose lysine deoxycholate agar (XLDA):
Xylose lysine deoxycholate agar (XLD agar) is a selective growth medium used in
the isolation of Salmonella and Shigella species from clinical samples and from
food. It has a pH of approximately 7.4, leaving it with a bright pink or red appearance
due to the indicator phenol red. Sugar fermentation lowers the pH and the phenol red
indicator registers this by changing to yellow. Most gut bacteria,
including Salmonella, can ferment the sugar xylose to produce acid; Shigella colonies
cannot do this and therefore remain red. After exhausting the xylose
supply Salmonella colonies will decarboxylate lysine, increasing the pH once again to
alkaline and mimicking the red Shigella colonies.

 Soybean-Casein Digest Agar:

Soybean-Casein Digest Agar Medium (TSA), USP is a highly nutritious versatile

medium which is recommended for general laboratory use in the preparation of agar
plates. Due to the inclusion of both Tryptone and Soy Peptone, the medium will
support growth of many fastidious organisms without the addition of serum, etc.

 Sabouraud agar:

Sabouraud agar  is a type of agar containing peptones. It is used to cultivate

dermatophytes and other types of fungi.

 EEBM:
Enterobacteria Enrichment Broth Mossel (EEBM) is used test for bile-tolerant gram-
negative bacteria. Culture medium for testing non-sterile products.

 Rappaport-Vassiliadis Broth:
Rappaport-Vassiliadis Broth is a selective media used for the enrichment of
Salmonella species. A selective media for subculturing,such as Xylose Lysine
Desoxycholate (XLD) agar, is used to cultivate and indicate Salmonella.

Reinforced clostridial media:

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Reinforced Clostridial Media is used for the cultivation and enumeration of

Clostridium species, lactobacilli, other anaerobes,and other facultative organisms
from clinical and food specimens.

VRBG AGAR:

Bile Glucose Agar containing crystal violet and neutral red (VRBG Agar) was used
by Mossel for the detection and enumeration of enterobacteria in dairy products, meat,
prepared pork products and other food products.

 Fluid Thioglycollate Medium:

Fluid Thioglycollate Medium is primarily intended for the culture of

anaerobic bacteria. However, it will also detect aerobic bacteria. 
 3. Soybean Casein Digest Medium:

This medium is used for culturing fungi and aerobic bacteria.

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MATERIALS AND METHOD

As per the objectives to complete the project work the materials and methods which
have been applied, are as under.

Bioburden testing of Prefilltered bulk solution

INTRODUCTION:

Bioburden is the quantitative estimation of the number of viable microorganisms in a

solution and on a surgical instrument or medical device before decontamination and
sterilization. Almost all products built within a manufacturing environment will have
some microorganisms that are naturally present on the product before any sterilisation
processing occurs. These contaminants might be borne from the materials used, for
example many natural fibres and organic-based compounds, or they might simply be
introduced by the workforce during handling.

Materials Requirements:

 Manifold assembly (Sartorius)

 Magnetic flask (Sartorius)
 Sterilized 0.45 micron, 47mm membrane filter (Sartorius stedim)
 Agar Plates (SCDA)
 Rinsing fluid (Fluid-A)
 Forceps
 Laminar air flow
 Vacuum pump
 Incubators
 Isopropyl alcohol (70% IPA)
 LPG line with burner/sprit lamp

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Procedure:

Sample Collection:

 Collect 100ml sample of prefiltered solution in a presterilized bottle after

the 1.2 micron filter in the filtration series in the controlled area.
 In case of saturated or super saturated solution such as Arginine, Sodium
carbonate maintained at higher temperature during dissolution, sample
should be diluted immediately to avoid any re-precipitation.
 In such case of sampling, 100ml of the solution to be diluted with 100ml
of pre-sterilized fluid-A (0.1% peptone water) after sampling at
production facilities.
 Bring the samples to the microbiology laboratory for the testing.

TEST PROCEDURE:

 Assemble the poly propylene unit consisting of a closed reservoir and a

receptacle between which a properly supported membrane is placed, whole set
should be sterilized and testing done under aseptic conditions.
 Assemble the sterilized filter set to the filtration unit in the laminar air flow
station.
 Aseptically place the sterilized or pre sterilized membrane, mount the top
portion of the filtration cup.
 Outer surface of the sample bottle shall be dis infected using filtered 70%IPA.
 Transfer little amount of sterilized fluid-A to the membrane and filter using
the vacuum pump for wetting purpose.
 Transfer 100mL of the pre filtered sample (200mL in case of saturated or
super saturated solution) and simultaneously filter the sample with the help of
the pump immediately without any hold-up of the solution in the filtration
unit.
 Transfer 8×100mL of sterilized fluid-A to the membrane. This volume is for
rinsing the membrane for neutralization.
 Filter it with the help of the vacuum pump.
 After rinsing of the membrane, remove the filtration assembly.

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 Incinerate the forceps and remove the membrane and transfer the membrane
onto the surface of the pre incubated SCDA plates.
 Negative control: 100mL of pre-sterilized fluid-A shall be filtered and
membrane shall be transferred on the surface of the pre incubated SCDA plate.
 All the test and negative control plates shall be incubate in upright position
initially at 20-25 °c for 72 hours and further shall be incubated for 72 hours at
30-35 °c.

Observation and interpretation of result:

 Observe all the incubated plates after the incubation period.

 The microbial counts observed on the membrane shall be recorded as total
viable aerobic counts per 100mL of sample.

Report of Total Viable aerobic microbial Counts:

Test Observation after 3-6 Action level

days
SCDA plates- 01 Nil 100 CFU/g
SCDA plates- 02 Nil 100 CFU/g
Average Nil 100 CFU/g
Dilution factor Nil 100CFU/g
Total aerobic microbial Nil 100 CFU/g
counts/g of the product
Negative control of the Nil _
media
Negative control of the Nil _
Diluent

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 INTRODUCTION

RESULT:
The sample complies because there is no growth found.

Microbiological enumeration testing of soluble and insoluble

nonsterile products

Introduction:
Microbiological enumeration Test is performing the qualitative and quantitative
estimations of specific viable microorganisms present in samples. It includes tests for
total viable count bacteria and fungi. The tests are designed for the estimation of the
number of viable aerobic micro-organisms present and for detecting the presence of
designated microbial species in pharmaceutical substances.

Materials Requirements:

 Soyabean digest agar (SDA) media plates

 Sabouraud dextrose agar (SDA) media plates
 Soyabean casein digest agar (SCDA) media plates
 Soyabean casein digest medium (SCDM) 100mL
 Sabouraud dextrose medium (SDM) 100mL
 Entero bacteria enrichment broth mossel (EEBM)
 Macconkey broth
 Rappaport vassiliadis salmonella (RVS) enrichment broth
 Reinforced medium for clostridia
 Mannitol salt agar (MSA) plates
 Centrimide agar (CA) plates
 Xylose-lysine deoxycholate agar (XLDA) plates
 Macconkey agar (MCA) plates
 Violet red bile glucose agar (VRBG) plates
 Columbia agar plates
 Laminar air flow station
 Sterilized 0.45µ 47mm, hydrophilic and MOC of cellulose nitrate/PVDF
 Sterilized fluid-A (0.1%peptone water) in 100mL quantities

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 INTRODUCTION

 Inoculation loop
 Incubators
 LPG line with burner/sprit lamp

METHODS:
Microbial Counts:

Membrane Filtration method for Water soluble product :

 Dissolved 1g of the sample being examined in 100ml of sterilised Fluid

A.

 Prepare a suitable glass or stainless steel unit consisting of a closed

reservoil and receptable between which a properly supported
membrane is placed, whole set should be sterilised and testing done
under aseptic conditions.

 Use the filter paper having pore size not greater than 0.45µ diameter
approximately of 47mm, hydrophilic and MOC of Cellulose
nitrate/PVDF.

 Filter 100ml of sterilised fluid A through the membrane as a pre

wetting step.

 Dissolve 1g of the sample being examined in 100ml of sterilised fluid

A and transfer the counts of the membrane filter and filter immediately.
Two such sets to be filtered. One filtered membrane for bacterial count
and the other filtered membrane for yeast/Mold counts.

 Rinse each membrane by filtering through it by 8×100ml of sterilised

fluid A.

 Transfer the filtered and rinsed membrane filter intended for

enumeration of bacteria, to the surface of the plate of Soyabean casein
Digest Agar and the membrane intended for the enumeration of
Yeast/Mold to the surface of the plate of Sabouraud Dextrose Agar.

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 INTRODUCTION

 Incubate the SCDA membrane plate at 30-35°C for 3-5 days incubate
the Sabouraud Dextrose Agar membrane plates at 20-25°C for 5-7days
along with the controls. Do not invert the plates during the incubation.

 For negative control media, incubate one SCDA plate at 30-35°C for 3-
5 days and one SabouraudDextrase Agar plate at 20-25°C for 5-7 days.

 For negative control of diluents filter 100ml of sterilised fluid A and

transfer the membrane filter to the surface of the plate of Soyabean
Casein Digest Agar and incubate at 20-25°C for 3- days followed by
30-35°C for 2-days.

 After the incubator period, Count the number of bacteria and number
of yeast/Molds in per gram of the tested sample.

Pour plate method for Water insoluble product Insoluble

Product:

 Suspend 10g of the sample being examined, in 100ml of sterilized fluid

casein Digest medium in 250ml flask and homogenized the suspension
by manual vortexing.

 Surface active agents such as 1ml per L of polysorbote 80 shall be

added to assist the suspension of poorly wettable substances.

For bacterial enumeration:

 Add to each sterilised 90-100mm petri dish, 1mL of the supernatant of

the enriched sample and aseptically add.

 20-25mL of sterilised Soyabean Casein Digest Agar at not more than

45°c, perform it in duplicates and allow it to solidify.

 For negative control of medium, add 20-25mL of SCDA to one

sterilized 90-100mm petri plate and for negative control of sterilized
soyabean casein Digest medium (SCDM) aseptically transfer 1mL of
sterilized SCDM to one sterilized plate and pour 20-25mL of SCDA
media.
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 INTRODUCTION

 After solidification, incubate all the plates in an inverted condition at

30 to 35°c for 3-5 days along with the test controls.

 After the incubation period, count the number of CFU in the plates.

 Calculate the results by expressing the average for the two plates and
report the total aerobic microbial count/g by multiplying the dilution
factor taken for testing.

For Yeast/Mold enumeration:

 Add to each sterilized 90/100mm petri dish, 1ml, of the supernatant of the
enriched sample and aseptically add 20-25ml of sterilized Sabourand Dextrose
Agar at not more than 45°c. Perform it in duplicates and allow it to solidify.
 For negative control of medium, add 20-25ml of SDA to one sterilised 90-
100mm petri plate and for negative control of sterilized Soyabean Casein Digest
Medium(SCDM) aseptically transfer 1ml of sterilized SCDM to one sterilised
plate and pour 20-25ml of SDA media.
 After solidification, incubate all the plates in an inverted condition at 20-25°c for
5-7days.
 After incubation period, count the number of CFU in the plates.
 Calculates the results by expressing the average for the two plates and report the
total combined molds yeast count by multiplying the dilution factor taken for
testing.

Spread Plate Method:

 Place 0.05-0.2 ml of the test fluid on the solidified and dried surface of the
agar medium and spread it uniformly using a spreader.

 Proceed under the same conditions as for the Pour Plate Method, especially
about petri dishes, agar media, incubation temperature and time, and
calculation method.

Serial Dilution Method (Most Probable Number Method):

 Use 12 test tubes: 9 containing 9 ml of soybean-casein digest medium each

and 3containing 10 ml of the same medium each for control.

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 INTRODUCTION

 Prepare dilutions using the 9 tubes. First, add 1 ml of the test fluid to each of
three test tubes and mix to make 10-times dilutions. Second, add 1 ml of each
of the 10-times dilutions to each of another three test tubes and mix to make
100-times dilutions. Third, add 1 ml of each of the 100-times dilutions to
each of the remaining three test tubes and mix to make 1,000-times dilutions.

 Incubate all 12 test tubes for at least 5 days at 30 - 35℃. No microbial growth
should be observed for the control test tubes. If the determination of the result
is difficult or if the result is not reliable, take a 0.1ml fluid from each of the 9
test tubes and place it to an agar medium or fluid medium, incubate all media
for 24－72 hours at 30－35℃, and check them.
 Calculate the most probable number of microorganisms per ml or gram of
the sample.

Pathogen testing:
Introduction:

Pathogenic organisms are those that can cause illness in humans either by infections
such as Salmonella, pathogenic Ecoli or in toxications such as Bacillus cereus,
Staphylococcus aureus or Clostridium botulinum, But in case of pharma industry we
test the following pathogens:

Procedure:

Test for Escherichia Coli:

 Suspend/dissolve 1g of the sample being examined in 100ml of

sterilised Soyabean Casein Digest Medium in a 250ml flask and
homogenize the suspension by manual vortexing and incubate at
30-35°c for 18 to 24 hours.
 Transfer 1 mL of the enriched SCDM to 100mL of MacConkey
broth and incubate at 42 to 44°c for 24 to 48 hours.
 Observe for growth if growth observed proceed for the following,
by means of an inoculation loop, streak a portion from the
enrichment broth on the surface of MacConkey agar medium.
Incubate the streaked plates at 30-35°c for 18 to 72 hours.
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 INTRODUCTION

 Growth of colonies indicates the possible presence of [Link],

confirm by Mini API identification.
 The product complies with the test if no colonies are present or if
the identification tests are negative.

Test for Salmonella:

 Suspend/ Dissolve 10g of the sample being examined in 100ml of

sterilised Soyabean Casein Digest medium in a 250mL flask and
homogenize the suspension by manual vortexing and incubate at 30
to 35°C for 18 to 24 hours.

 Transfer 0.1ml of the enriched SCDM to 10ml of

RapportedVassaliadis Salmonella Enrichment brorth and incubate
the tubes at 30-35°c for 18-24 hours.

 Observe for growth, if growth observed proceed for the following,

by means of an inoculation loop, streak a portion from the
enrichment broth on the surface of Xylose LysinDeoxycholate
Agar Medium. Incubate the streaked plates at 30-35°c for 18-48
hours.

 The possible presence of Salmonella is indicated by the growth of

well developed, red Colonies with or without black center. Confirm
by Mini API identification.

 The product Complies with the test if no colonies are present or if

the identification tests are negative.

Test for Staphylococcus aereus:

 Suspend/dissolve 1g of the sample being examined in 100ml of

sterilised Soyabean Casein Digest Medium in a 250ml flask and
homogenize the suspension by manual vortxing and incubate at 30
to 35°C for 18 to 24 hours.

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 INTRODUCTION

 Observe for growth, if growth observed proceed for the following.

By means of an inoculation loop, streak a portion from the
enrichment broth on the surface of Mannitolsalt Agar Media.
Incubate the streaked plates at 30-35°c for 18-72 hours.

 The possible presence of Staphylococcus aereus is indicated by the

growth of yellow or white colonies surrounded by a yellow zone.
Confirm by Mini API identification.

 The product complies with the test if colonies are not present or if
the confirmatory identification test is negative.

Test for Pseudomonas aeruginosa:

 Suspend/dissolve 1g of the sample being examined in 100ml of

sterilised Soyabean Casein Digest Medium in a 250ml flask and
homogenize the suspension by manual vortxing and incubate at 30
to 35°C for 18 to 24 hours.

 Observe for growth, if growth observed proceed for the following,

by means of an inoculation loop, streak a portion from the
enrichment broth on the surface of Cetramide Agar Medium.
Incubate the streaked plates at 30-35°c for 18-72 hours.

 Growth of colonies indicates the possible presence of pseudomonas

aeruginosa. Confirm by Mini API identification.

Test for Bile tolerant gram –ve Bacteria:

 Suspend/dissolve 1g of the sample being examined in 100ml of

sterilised Soyabean Casein Digest Medium in a 250ml flask and
homogenize the suspension by manual vortxing at 20-25°C. Test the
sample within 2-5 hours while kept at 20 to 25°C.

 Transfer the entire 100kL broth to 100mL of Enterobacteria

Enrichment broth Mossel in a 250mL flask and incubate at 30 to 35°C
for 18 to 24 hours.

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 INTRODUCTION

 Observe for growth, if growth observed proceed for the following. By

means of an inoculation loop, streak a portion from the enrichment
broth on the surface of Violet Red Bile Glucose Agar Media. Incubate
the streaked plates at 30-35°c for 18-24 hours.

 The possible presence of Bile tolerant gram –ve Bacteria. Confirm by

Mini API identification.

 The product complies with the test if colonies are not present or if the
confirmatory identification test is negative.

Test for Clostridia:

 Suspend/dissolve 1g of the sample being examined in 100ml of

sterilised Soyabean Casein Digest Medium in a 250ml flask and
homogenize the suspension by manual vortxing.

 Take two equal portions. Heat one portion at 80°C for 10 minutes and
cool rapidly. Do not heat the other portion.

 Transfer 10mL from the portion to two separate 100mL of reinforced

medium for Clostridia and incubate under anaerobic condition at 30 to
35°C for 48 hours.

 Observe for growth, if growth observed proceed for the following. By

means of an inoculation loop, streak a portion from the enrichment
broth on the surface of Columbia Agar Media. Incubate the streaked
plates under anaerobic conditions at 30-35°c for 48 hours.

 The Occurrence anaerobic growth of rods (with or without

endospores) giving a negative catalase reaction indicates the presence
of Clostridia. Confirm by Mini API identification.

 IF no anaerobic growth of microorganisms is detected on Columbia

Agar or the Catalase test is positive, the product complies with the
test.

Test for Candida albicans:

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 INTRODUCTION

 Suspend/dissolve 1g of the sample being examined in 100ml of

sterilised SabouraudDextrase broth Medium in a 250ml flask and
homogenize the suspension by manual vortxing and incubate at 30 to
35°C for 3 to 5 days.

 Observe for growth, if growth observed proceed for the following. By

means of an inoculation loop, streak a portion from the enrichment
broth on the surface of SabouraudDextrase Agar Media. Incubate the
streaked plates at 30-35°c for 24-48 hours.

 Growth of Water Colonies may indicate the presence of Candida

albicans. Conform by Mini API identification.

Test Observation after 3-5 Action level

days
SCDA plates- 01 Nil 100 CFU/g
SCDA plates- 02 Nil 100 CFU/g
Average Nil 100 CFU/g
Dilution factor Nil 100CFU/g
Total aerobic microbial counts/g of the Nil 100 CFU/g
product
Negative control of the media Nil _
Negative control of the Diluent Nil _
Results:

Report of Total aerobic microbial Counts:

Test Observation after 3-5 Action

days level
SDA/SCA plates- 01 Nil 50 CFU/g
SDA/SCA plates-02 Nil 50 CFU/g
Average Nil 50 CFU/g
Dilution factor Nil 50CFU/g
Total Combined molds and yeast counts/g of Nil 50 CFU/g
the product
Negative control of the media Nil _

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 INTRODUCTION

Negative control of the Diluent Nil _

Report of Total Combined molds and yeast counts:

Report for Pathogen (Staphylococcus, Pseudomonas, and Candida,) analysis of

Sample- 1:

Pathogen Enrichment Presumptive Confirmatory Remarks

Test Test Test
Staphylococcus NO Growth Not applicable Not applicable
Complies
aureus
Pseudomonas NO Growth Not applicable Not applicable Complies
aeruginosa
Candida NO Growth Not applicable Not applicable Complies
Report for Pathogen (Escherichia Coli, Salmonella sps., Clostridia, and Bile
tolerant Gram-ve bacteria) analysis of Sample- 1:

Pathogen Enrichm Presumptive Presumptive Confirmato Remarks

ent Test Test-1 Test-2 ry Test
Escherichia Coli No NA NA NA Complies
growth
Salmonella sps. No NA NA NA Complies
growth
Clostridia No NA NA NA Complies
growth
Bile tolerant No NA NA NA Complies
Gram-ve growth
Bacteria

Report for Pathogen (Staphylococcus, Pseudomonas, and Candida,) analysis of

Sample- 2:

Pathogen Enrichment Presumptive Confirmatory Remarks

Test Test Test
Staphylococcus NO Growth Not applicable Not applicable Complies
aureus
Pseudomonas NO Growth Not applicable Not applicable Complies
aeruginosa

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 INTRODUCTION

Candida NO Growth Not applicable Not applicable Complies

Report for Pathogen (Escherichia Coli, Salmonella sps., Clostridia, and Bile
tolerant Gram-ve bacteria) analysis of Sample- 2:

Pathogen Enrichm Presumptive Presumptive Confirmato Remarks

ent Test Test-1 Test-2 ry Test
Escherichia Coli No NA NA NA Complies
growth
Salmonella sps. No NA NA NA Complies
growth
Clostridia No NA NA NA Complies
growth
Bile tolerant g- No NA NA NA Complies
ve Bacteria growth

Results:

Pathogen CFU in Sample 1 CFU in Sample 2 Limit

Staphylococcus aureus Nil Nil Should be absent
Pseudomonas aeruginosa Nil Nil Should be absent
Escherichia Coli Nil Nil Should be absent
Salmonella sps. Nil Nil Should be absent
Clostridia Nil Nil Should be absent
Candida Nil Nil Should be absent
Bile tolerant Gram-ve Nil Nil Should be absent
Bacteria
*Remarks: Complies

TEST FOR STERILITY

INTRODUCTION

The sterility test is used to detect the presence of viable forms of bacteria, fungi and
yeast in or on pharmacopoeial preparations. The working conditions in which the tests
are performed should be free from any microbial form and should be monitored
regularly by sampling the air and surfaces of the working area and by carrying out
control tests.

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 INTRODUCTION

PRINCIPLE

The tests are based upon the principle that if bacteria or fungi are placed in a medium
which provides nutritive material and water, and kept at a favorable temperature, the
organisms will grow and their presence can be indicated by turbidity in the originally
clear medium.

Equipments Required:
 LAF
 Manifold holder assembly
 Vacuum pump.
 Forceps
 Scissor

Material Required:
 Sample for sterility testing
 Sterile 0.45µ Membrane filter
 Sterile FTGM
 Sterile SCDM
 Sterile 0.1% w/v Peptone water.
 70% sterile IPA Solution.
 Gas burner
 Sterile Filtration assembly
 Sterile SCDA plates
 Sterile Swab

Membrane Filtration Method

Procedure:
 Collect the samples to be tested for sterility as per SOP. And keep in a clean
S.S trays marked with Product Name, Batch No and Lot No, and then transfer
the samples to the sterility room through clean pass box for performing
sterility.
 Prepare the media tubes 100-100 mlquantity (FTM and SCDM) as per the
SOP for preparation of culture media.
 Pre-incubate the media tubes at appropriate temperature i.e. SCDM tubes at 20
to 250C whereas FTGM tubes at 30 - 35ºC for 24 - 48 hrs before subjecting
them for sterility operations.

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 INTRODUCTION

 Enter in sterility room as per the Entry / Exit procedure for Sterility Room.
 Start the LAF as per SOP for operating Instruction for LAF.
 Wipe out all samples to be tested for sterility with 70% IPA solution.
 Connect the Filtration manifold holder assembly with the S.S. reservoir
properly with pipe and place sterilized S.S. cups in the sterile receptacle under
Laminar airflow unit. Check the Manometer reading of working LAF and
check the temperature as well as humidity of the sterility room
 Switch ON the vacuum pump but close the vacuum of the manifold holder
assembly with the help of vacuum control key, present on the base of
individual manifold holder. Now place 0.45m sterile membrane filters
between filtration cup and receptacle with the help of sterilized forceps.
 Wet the membrane filter by adding approx 15 ml of sterilized Fluid A (0.1%
peptone water) to filter holder, and filter the fluid by employing vacuum.
 Cut the tip of bottle/vial or ampoule with sterile SS blade in front of the gas
burner and immediately transfer not less than half of the contents for LVP and
whole content of the vial for SVP to membrane.
 Immediately filter the solution with the aid of vacuum and wash the membrane
three times with 100 ml of sterilized fluid A (Washing with fluid A is not
required in case of sterile water for injection).
 After complete filtration, stop the vacuum of manifold with the help of
manifold vacuum control key.
 Lift the membrane carefully with the help of sterile forceps, aseptically cut the
membrane filter into two halves with sterile SS scissor and transfer one half to
FTGM and one half to SCDM tubes by unplugging in front of gas burner only.
 Label both the tubes with product name, B. No, lot No., date of testing,
Completion date & Tested by.
 Simultaneously prepare a negative control by filtering 100 ml of 0.1% peptone
water instead of product sample, cut the membrane into two halves with sterile
SS scissor and transfer one half to FTGM and one half to SCDM and label
both the tubes as Negative control.
 Simultaneously prepare a chamber control during the sterility take two tubes,
one is SCDM & other one is FTGM tube, unplug the cotton plug of the tube
and expose in LAF during sterility, after completion of sterility replug the
tubes and then incubate the tubes as a chamber control.
 Incubate the FTGM tubes at 300C – 350C and SCDM tubes at 200C – 250C for
14 days. Incubation period for terminally sterilized products is not less than 7
days and 14 days for aseptically filled products as per IP. If the Product is as
per USP, BP, incubation period is 14 days for both terminally sterilized as
well as for aseptically filled products.

Positive and Negative Controls:

Aseptically transfer 1 x 100 ml of diluting fluid to the filtration assembly.

After filtering the volume, cut the membrane into four parts, two of them in two
separate Fluid Thioglycollate (FTGM) tubes and the other in two separate Soybean
Casein Digest (SCD) tubes. Incubate these four tubes along with the sample tubes.
After inoculating one of the FTGM tubes with a mixed culture of organisms-
 Staphylococcus aureus
 Pseudomonas aeruginosa
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 INTRODUCTION

 Cl. Sporogenesand
One of the SCD medium tubes with
 Bacillus subtilis
 Candida albicans
 A. niger

The two incubated tube are of positive control to check growth promotion property of
the media and the other two are negative control to check the aseptically accessories
and working conditions of the test.

Observation and Interpretation of Results:

 Visually examine the media tubes daily to its conclusion for macroscopic
Evidence of microbial growth.
 If no evidence of growth observed in any of the tube the product to be
examined for the test complies with the test for sterility.
 The test is not valid unless the Negative control shows negative till at end of
incubation, and positive control shows growth within specified incubation
period.
 Destroy all the positive controls after confirmation of growth in both FTGM
and SCDM tubes (Generally growth will observed in 24 to 48 hrs).
 If evidence of microbial growth is found in any of the tube, investigate the
Cause of its failure as per SOP for Sterility test failure Investigation, SOP. If
investigation report concludes that test is invalid, repeat the test with 20 units.
 If no evidence of growth is found in the repeat test the product examined
complies with the test for sterility. If evidence of microbial growth is found in
the repeat test the product examined does not comply with the test for
sterility.

Precautions:

 Sample bottles / vials should be wiped properly with filtered 70% IPA before
transferring them to sterility room and also inside the sterility room before
sterility operations. Hatch box should also be cleaned properly with filtered
70% IPA solution.
 Any material used in the sterility operation or inside sterility room should be
autoclaved properly.
 The tip of sample bottles / vials should be heated properly in front of gas
burner before cutting with sterile heated cutting blade.

OBSERVATIONS:

Incubatio FLUID THIOGLYCOLLATE SOYABEAN CASEIN

n Day MEDIUM DIGEST MEDIUM Remarks
Test Positive Negativ Test Positive Negativ
Control e Control e
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 INTRODUCTION

Control Control
I II III I II III
1. -ve +ve +v +v -ve -ve +ve -ve -ve -ve O.K.
e e
2. -ve +ve +v +v -ve -ve +ve -ve -ve -ve O.K.
e e
3. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
4. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
5. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
6. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
7. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
8. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
9. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
10 -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
11 -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
12. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
13. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
14. -ve +ve +v +v -ve -ve +ve +v +v -ve O.K.
e e e e
Note: + Ve = growth present - Ve = growth absent

Application For bacterial detection For Fungal Detection

Fluid Thioglycollate Soyabean Casein Digest
Medium Medium
o
Incubation Temp. 30 – 35 C 20 – 25oC
Incubation Period 14 Days 14 Days
Positive Control 1. Staphylococcus aureus 1. Bacillus subtilis.
2. Pseudomonas 2. [Link]

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 INTRODUCTION

aeruginosa 3. A. niger
3. Cl. sporogenes
Result: The above sample complies the sterility test.

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 INTRODUCTION

MICROBIOLOGICAL TESTING OF WATER

INTRODUCTION:

Water is one of the most widely and abundantly used substances in pharmaceutical
manufacturing. It is required for a variety of purpose ranging from manufacturing
processes to the preparation of the final dosage forms. The quality of water therefore
assumes considerable importance.

Many factors of which the most important is the variability of the basic source viz.
municipal or any other water governs the control of the chemical and microbiologist
quality of water for pharmaceutical use. The starting material for most forms of water
is drinking water, which should normally to be subject to municipal or any other local
regulations or is drawn from a private well or reservoir. Water prepared from other
starting material may have to be processed to meet drinking water standards. Drinking
water itself may be used in the manufacture of drug substances but not in the
preparation of dosage forms, or in the preparation of reagent and test solutions.

TYPES OF WATER

 Raw Water
 Soft Water
 Purified Water
 Treated water
 Water for injection (WFI)
 Clean condense pure steam (CCPS)

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 INTRODUCTION

These water are: (flow chart):

Ground water (Raw water)

Storage tank on the top

( Here sodium hypchlorate is added to kill bio load)

MGF(multigrade filters)

(Bigger particles like sand etc. are removed here but it still contains magnesium,
calcium and another ions.)
Softner (resins)
( Hardness is removed )

(soft water) Cardrige filter

Reverse osmosis 1

Reverse osmosis 2

(treated water)

EDI ( Electrode de ionization)

Ultrafiltration
(To decrease conductivity)
(To remove endotoxins & bio load)

(purified water)
Storage tank

Water level is always>40%

WFI PURE STREAM PURIFIED

WATER

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METHODS:

Total viable aerobic Count Determination of water sampling by

Membrane filtration technique for treated, purified water, WFI and
pure steam condensate

Requirement:
 Prepare the following media such as R2A agar media plates

 Filtration assembly

 Membrane filtration cups

 Laminar air flow station

 Sterilised/ Pre-sterilized 0.45µ, 47mm membranes and MOC- Cellulose

acetate/ nitrate

 Forceps and Incubators.

Procedure:
Total viable aerobic microbial counts by Membrane filtration for WFI
and pure steam condensate:

 Prepare a suitable glass/stainless steel/polypropylene unit consisting of a

closed reservoir and a receptacle between which a properly supported
membrane is placed, whole set should be sterilised and testing done under
aseptic conditions.
 Use the filter paper having pore size not greater than 0.45µ, hydrophilic
membrane and diameter approximately of 47mm and MOC of cellulose
acetate/nitrates.
 Assemble the sterilised filter set to the filtration unit in the laminar air flow
station.
 Aseptically place the sterilised or pre sterilised membrane using the forceps in
the base of the filtration cup.

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 After fixing the sterilised/pre sterilised membrane cup or use filtration system
with pre sterilised membrane cups with membrane.
 Transfer 200ml of the sample being, examined to the membrane filter and
filter immediately.
 Apply Vacuum and filter the sample and ensure that the sample is completely
filtered.
 Flame the Forceps, allow it to cool and then transfer the filtered membrane to
the surface of the plate R2A agar by slowly placing the membrane from the
edge to the complete portion in the center of the agar surface,
 Ensure that no air bubble is trapped inside the membrane and entire surface of
the membrane should be in contact with the agar surface.
 Incubate the R2a membrane plates at 30-35°C for 5 days for 48-72 hours
along with the test controls. Do not invert the plates during incubation.
 Negative control of media also incubates at same temperature.
 After the incubation period, count the number of CFU and divided the CFU
value by the factor of 2. Report the total number of CFU for 100mL of the
sample quantity.
 Also check the control plates for the growth.

Total Viable Aerobic microbial counts by membrane filtration for purified water
and Treated Water:

 Closed reservoir and a receptacle between which a properly supported

membrane is placed, whole set should be sterilised and testing done under
aseptic conditions.
 Use the filter paper having pore size not greater than 0.45µ, hydrophilic
membrane and diameter approximately of 47mm and MOC of cellulose
acetate/nitrates.
 Assemble the sterilised filter set to the filtration unit in the laminar air flow
station.
 Aseptically place the sterilised or pre sterilised membrane using the forceps in
the base of the filtration cup.
 After fixing the sterilised/pre sterilised membrane cup or use filtration system
with pre sterilised membrane cups with membrane.

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 Dilute 1mL of the sample to be tested in 100mL of sterilised fluid A and

transfer the entire contents to the membrane filter and filter immediately.
 Apply vacuum and filter the sample and ensure that the sample is completely
filtered.
 Flame the Forceps, allow it to cool and then transfer the filtered membrane to
the surface of the plate of R2A agar by slowly placing the membrane from the
edge to the complete portion in the surface of the agar surface.
 Ensure that Incubate no air bubble is trapped inside the membrane and entire
surface of the membrane should be contact with the agar surface.
 Incubate the R2A membrane plates at 30-35 °C for 5 days /48-72hours.
 Negative control of membrane also takes for same temperature and time
period.
 After the incubation period, Count the number of CFU and report the total
number of CFU per mL.
 Also check the control plates for the growth.
 If any growth observed in the plates of R2A for WFI samples, identify the
organism up to the species level.

Total Viable Aerobic microbial counts by determination of water

samples by pour plate method for soft and drinking water:
Requirement:
R2A agar, LAF, Slandered petri plates, Incubators, Calibrated micropipette, Sterilised
micropipette tips.

Procedure:
 Add to each sterilised 90mm petri dish, 1ml of the water sample in duplicates
in the laminar air flow station.
 Aseptically add 20-25ml of sterilised R2A.
 Check the temperature of not more than 45°C using the infrared thermometer.
 Swirl the plates in clockwise and anticlockwise for uniform mixing of the
sample and allow to solidify.
 For negative control of media to one sterilised 90mm petriplate.

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 After solidification incubate all the plates in an inverted condition at 30-35°c

for 48-72hrs for 5 days with test controls.

 After the incubation period, count the number of CFU in the plates.

 Calculate the results by expressing the average for the plates and report the
total aerobic microbial count/ml.

Test for specified microorganisms for Water samples:

Requirements:
Prepare the following media:

 SCDM-100mL,
 Mac-Conkey broth,
 RVS enrichment broth,
 MSA plates,
 CA plates,
 XLDA plates,
 Mac Conkey agar plates.
 LAF,
 0.1% sterilised peptone water in 100mL quantities,
 Inoculation loop,
 Gram staining Kits,
 Incubators,
 LPG set,
 Sterilised/pre-sterilized 0.5µm,47mm membranes and MOC-Cellulose
acetate/nitrate,
 Forceps.

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Procedure:
Filtration of the sample:

 Prepare a suitable glass/stainless steel/polypropylene unit consisting of a

closed reservoir and a receptacle between which a properly supported
membrane is placed, whole set should be sterilised and testing done under
aseptic conditions.
 Filter 100mLb of the sample to be tested through the filter paper having pore
size not greater than 0.45µ, hydrophilic membrane and diameter
approximately of 47mm and MOC of cellulose acetate/nitrate.
 Assemble the sterilised filter set to the filtration unit in the laminar air flow
station.
 Aseptically place the sterilised membrane mount the top portion of the top
portion of the filtration cup or use filtration system with pre-sterilised
membrane membrane cups with membrane.
 Transfer 100mL of the sample being examined to the membrane filter and
filter immediately.
 Apply vacuum and filter the sample and ensure that the sample is completely
filtered.
 Flame the forceps, allow it to cool and then transfer the filtered membrane to
100mL of sterilised SoyabeanCseim digest medium.
 Incubate the enriched SCDM tube at 30-35°C for 18 to 24 hours and proceed
for the specified pathogen test.

Enrichment:

Test for [Link]:

 Transfer 1mL of the enriched SCDM to 100mL of Mac Conkey broth and
incubate at 42-44°C for 24-48hrs.
 Observe growth then streak on MacConkey agar plate and incubate at 30-35°C
for 18-72hrs.
 If Colony grows then confirm by identification test (Mini API).

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Test for Salmonella:

 Transfer 0.1mL of the enriched SCDM to 10mL of RVSE broth and incubate
at 30-35°C for 18-24hrs.
 Observe growth then streak on XLDA plate and incubate at 30-35°C for 18-
48hrs.
 If Colony grows then confirm by identification test (Mini API).

Test for Staphylococcus aures:

 Observe growth in the enriched SCDM, if growth observed proceed for the
following.
 By means of an inoculation loop streak on a portion from the enrichment broth
on the surface of Manitol Salt agar plate and incubate at 30-35°C for 18-72hrs.
 The possible presence of Staphylococcus Aureus is indicated by the growth of
yellow or white colonies surrounded by a yellow zone.
 If Colony grows then confirm by identification test (Mini API).

Pseudomonas aeruginosa:

 Observe growth in the enriched SCDM, if growth observed proceed for the
following. By means of an inoculation loop streak a portion from the
enrichment broth on the surface of Cetramide agar plate and incubate at 30-
35°C for 18-72hrs.
 Growth of colonies indicates the possible presence of Pseudomonas
aeruginosa.
 If Colony grows then confirm by identification test (Mini API).

Results:

Type of Water Quantity in mL Alert level Action level

Raw and Soft Water 1ml in 100mL 200CFU/ml 500CFU/ml
Fluid A
Treated and Purified 1ml in 100mL 80CFU/ml 100CFU/ml
Water Fluid A
WFI and PSG 200ml 5CFU/ml 10CFU/ml
*Media used: R2A
Report of observation of counts:
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Sample (Water) Observation Remarks Media

WFI Sample1 Nil/200ml Complies R2A
WFI Sample 2 Nil/200ml Complies R2A
Purified Sample 1 2/ml Complies R2A
Purified Sample 2 4/ml Complies R2A
Treated Sample 1 13/ml Complies R2A
Treated Sample 2 14/ml Complies R2A
Soft Sample 1 19/ml Complies R2A
Soft Sample 2 14/ml Complies R2A
Raw Sample 1 31/ml Complies R2A
Raw Sample 2 33/ml Complies R2A
Report for Pathogen (Staphylococcus aureus) analysis of Water:

Sample water Enrichment Test Presumptive Test Confirmatory Test

WFI, sample 1 Growth No Growth NA
WFI, sample 2 Growth No Growth NA
Purified, sample 1 Growth No Growth NA
Purified, sample 2 Growth No Growth NA
Treated, sample 1 Growth No Growth NA
Treated, sample 2 Growth No Growth NA
Soft, Sample 1 Growth No Growth NA
Soft, sample 2 Growth No Growth NA
Raw, sample 1 Growth No Growth NA
Raw, sample 2 Growth No Growth NA
*Remarks: Complies

Report for Pathogen (Pseudomonas aeruginosa) analysis of Water:

Sample water Enrichment Test Presumptive Test Confirmatory Test

WFI, sample 1 Growth No Growth NA
WFI, sample 2 Growth No Growth NA
Purified, sample 1 Growth No Growth NA
Purified, sample 2 Growth No Growth NA
Treated, sample 1 Growth No Growth NA
Treated, sample 2 Growth No Growth NA
Soft, Sample 1 Growth No Growth NA
Soft, sample 2 Growth No Growth NA
Raw, sample 1 Growth No Growth NA
Raw, sample 2 Growth No Growth NA
*Remarks: Complies

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Report for Pathogen ([Link]) analysis of Water:

Sample water Enrichment Presumptive Presumptive Confirmatory

Test Test 1 Test 2 Test
WFI, sample 1 Growth No Growth No Analysis NA
WFI, sample 2 Growth No Growth No Analysis NA
Purified,S-1 Growth No Growth No Analysis NA
Purified,S-2 Growth No Growth No Analysis NA
Treated, S-1 Growth No Growth No Analysis NA
Treated, S-2 Growth No Growth No Analysis NA
Soft, S-1 Growth No Growth No Analysis NA
Soft, S-2 Growth No Growth No Analysis NA
Raw, sample 1 Growth No Growth No Analysis NA
Raw, sample 2 Growth No Growth No Analysis NA
*Remarks: Complies

Report for Pathogen (Salmonella) analysis of Water:

Sample water Enrichment Presumptive Test Presumptive Test Confirmatory

Test 1 2 Test
WFI, sample 1 Growth No Growth No Analysis NA
WFI, sample 2 Growth No Growth No Analysis NA
Purified, S-1 Growth No Growth No Analysis NA
Purified, S-2 Growth No Growth No Analysis NA
Treated, S-1 Growth No Growth No Analysis NA
Treated, S-2 Growth No Growth No Analysis NA
Soft, S-1 Growth No Growth No Analysis NA
Soft, S-2 Growth No Growth No Analysis NA
Raw, sample1 Growth No Growth No Analysis NA
Raw, sample2 Growth No Growth No Analysis NA
*Remarks: Complies #NA = Not applicable.

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Fig: Colony grow on membrane

BACTERIAL ENDOTOXIN TEST (BET)

INTRODUCTION:

The test for bacterial endotoxins is used to detect or quantify endotoxins of gram
negative bacterial origin that may be present in or on the sample to which the test is
applied, using amoebocyda lysate from horse shoe crab (Limulus polyphenus) or
(Tachyplustridentatus)
The determination of the reaction end point is made with dilution from the material
under test in direct comparison with parallel dilution of control standard endotoxins
(CSE) of defined potential in endotoxins unit per ml. EU/ml.

EQUIPMENTS, APPARATUS & REAGENTS:

APPARATUS:

12 X 75 mm glass tubes, 10 X 75 mm glass tubes, 20-200µl micro tips,

(Depyrogenated, individually packed), test-tube stand.

EQUIPMENTS:

Heating block, micropipette (20 - 200µl), micropipette (100 - 1000 µl).

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REAGENTS:
 LAL Reagent of 0.125 EU/ml sensitivity
 LAL Reagent water
Control standard endotoxins:
 Entire content of 1 vial RSE was constituted with 5 ml of LRW
 Mixed intermittently for 30 minutes using a vortex mixer and this concentrate
were used for making appropriate serial dilution.
 Preserved the concentrate in refrigerator for making subsequent dilution for
not more than 14 days.

Endotoxin free 0.1N HCl:

Using an endotoxins free pipette, added 100µl of concentrated HCl into 10 ml

LRW which was contained in dehydrogenated bottle .

Endotoxin free 0.1 N NaOH:

0.1g NaOH was added into 25 ml LRW contained into a depyrogenated

bottle.

Sample preparation :

The sample solution was prepared by dissolving or diluting drugs or extracting

medical devices using LRW. Some substances may be more appropriately dissolved,
diluted, or extracted in other aqueous solution. The pH of solution should be between
6.0 -8.0.

Sample used:

WFI (Water for Injection).

Figure: showing the protocol kit for Endotoxin test.

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Principle:

It is important to have a sensitive test to identify the presence of endotoxins in drugs,

medical devices and body fluids. Materials that have been sterilized may contain
endotoxins, even though no bacteria can be cultured from them. One such lab test
called LAL test, which can detect even minute amount of LAL Test is in accordance
with the latest demand of European pharmacopeia commission for replacements of
animal based test in for our of alternative method where possible. Standard for the test
is prepared from [Link] derived Endotoxin.

Unit of measurement is EU for USP and IU for European Pharmacopoeia & rest of
world Bacterial Endotoxin test can be done by three methods.

 Gel clot method

 Kinetic Chromogenic method.

 Turbidimetric method

METHOD OF ANALYSIS:

Gel Clot Method:

37(± 1) º C

Sample / control standard + LAL reagent observe presence

or
60(± 2) min absence of gel

+ Gel → gel that holds its integrity when tube is inverted 180º

- Gel → clear or viscous liquid, which flows when tube is inverted.

Reconstitution of LAL Reagent:

Lyophilized LAL Reagent water vial was reconstituted as indicated on vial label and
cover the vial with stopper. Vial was rotated gently, till it dissolves. Reconstituted
LAL Reagent was stored below – 200C up to 28 days.

Control Standard Endotoxin Series:

Lyophilized control standard endotoxins vial was reconstituted with the volume of
endotoxins free LAL Reagent water.

Vial was covered with stopper and vortex vigorously for at least 30 min. The
reconstituted CSE was stored at 2-80C up to 4 weeks. The reconstituted CSE vials
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were restored for 1 min. with the help of cyclomixer. The dilution of CSE was
prepared in concentration of 4 λ, 2λ, λ, λ/2, λ/4.

Table 1 showing the CSE dilution.

Tube LRW CSE added from tube Final conc. of CSE

No
01 0.9ml 0.1 ml of 20 EU/ml 20 EU/ml
02 1.2ml 0.4ml of tube no.1 0.5 EU/ml-4λ
03 1.0 ml 01.0 ml of tube no.2 0.25EU/ml-2 λ
04 1.0ml 1.0ml of tube no.3 0.125EU/ml- λ
05 1.0ml 1.0ml of tube no.4 0.06U/ml- λ /2
06 1.0ml 1.0ml of tube no.5 0.03EU/ml- λ /4

Test for labeled claim sensitivity of LAL Reagent:

The label claim sensitivity of each lot of LAL reagent prior to use in test was diseased
as below:

A series of 2 fold dilution of CSE to given conc. of 2λ, λ, λ/2, λ/4 was prepared,
where is labeled sensitivity of LAL reagent in EU/ml the test was performed on four
& standard canc.

Testing Procedure:

 Wiped the work area with 70 % v/v IPA.

 Depyrogenated glassware was used.

 Disinfected the outer surface of sample container with 70 % IPA.

 Prepared the dilutions used in test using water BET as Table I. Sample
dilution was selected b/w non-interfering dilution and MVD to eliminate
interfary factors.

 Wiped the outer surface of vial and container and vial opener with 70% v/m
IPA.

 Calculated the maximum valid dilution for product by a formula.

ENDOTOXIN LIMIT TEST PROCEDURE :

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Material: Purified water/ sterile water for injection (I.P./ B.P./ U.S.P.)

Endotoxin Limit: NMT 0.25Eu/ml

Sensitivity of LAL: 0.125Eu/ml

Calculation of MVD (Maximum Valid Dilution)

MVD = Endotoxin limit (Eu/mg)×potency of Product

Sensitivity of LAL =
(0.25 x 1.0) / 0.125

Negative Control:100µl LRW + 100 µl of Lysate

Test: 50 µl of sample + 50 µl of LRW + 100 µl of Lysate

PPC: 50µl of sample + 50 µl of 4λ + 100µl of Lysate

CSE:

 100µl of each dilution + 100 µl of Lysate

 Gently swirled the bottle to dissolve the content completely and kept it on ice
throughout the test.
 After adding to lysate, test tube was kept for incubation at 37 0C + 10C for 60 +
2 min in calibrated heating block.

Figure: Showing the incubation of sample, PPC, &NPC in BET Incubator.

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OBSERVATIONS:

Table for an Endotoxin Test

TUBES SAMPLES CSE LRW LYSATE OBSERVATION

PPC 50µl 50 µl (CSE - 100 µl Gel formation

(MVD/2) 4λ)
NPC 50µl - 50 µlLRW 100 µl No gel formation
(MVD/2)
NWC - 50 µl LRW 100 µl 100 µl No gel formation

PWC - 50 µl(4λ) 50 µl 100 µl Firm gel

0.5Eu/ µl formation

RESULT:
The sample complies because there is no evidence of considerable amount of
endotoxins.

PRECAUTIONS:

 Vortex standard endotoxins for not less than 30 minutes by taping vial to
vortex mixture.
 Vortex all endotoxins for not less than 1 minute
 Do not allow dilutions of endotoxins to stand for more than 10 minute without
revortexing.
 Reconstitute lysate very gently & mix carefully so as to avoid the formation of
bubbles.
 Incubate tubes in heating block kept on a stable surface, which don’t get
vibrations shake or bump. Do not incubate tube in LAF cabinet which may
vibrate and break fragile gel.
 Don’t transfer or disturb the tubes at any stage of incubation till reading result.
 Read results only once. Gels are fragile and may break when handled.

Application of BET in Pharmaceutical Industry:

1. Validation of Dry Heat Sterilizer (DHS) / Oven or Tunnel for depyrogenation.

2. Monitoring of water system for endotoxins content.

3. In-process control of endotoxins in bulk pharmaceutical chemicals.

4. Detection of endotoxins in Medical Devices.

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5. In-process formulation.

CONCLUSION
Microbiological testing is very important for every pharmaceutical
product because there are several microorganisms which interfere with
these products and their tests are necessary, there are several products
which cannot be detected by chemical methods hence microbiological
assay are very useful for resolving doubts regarding possible change in
potency of these products and their preparation.

All the microbial tests such as sterility, microbial testing of water (total
microbial count test and test for pathogen), bacterial endotoxins test,
microbiological assay of vitamin B12 are within limit as per
pharmacopoeia and the pharmaceutical products manufactured are passed
by microbiological section of quality control division.

Now it is concluded that the quality of Omez Injection, Purified Water,

Becozinc Capsule and WFI are according to standard prescribed by
Indian Pharmacopeia.

Every pharmaceutical product should certified its claim, then it is

launched in market otherwise if it is fail in its claim the product is not
launched in market

Every country has its own pharmacopoeia by which drug and other
products are certified.

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REFERENCES
 Pharmaceutical Microbiology (By Hugo and Russell’s).
 Microbiology for analytical chemist. (By R.K. Dart)
 Blechova R., D Pivodva; LAL Test – an alternative method of
dilution of bacterial endotoxinActa Vet. Brno 2001; 70; 291- 296.
 Cooper JF, Rounding BET related calculations. LAL. Times 5: 3,
1998.
 Danyer S.P and Hodges (NA) (1989) “Theory and Application of
Microbiological Assay” Academic Press London.
 Indian Pharmacopoeia
 Microbiology by Lansing [Link], J. Harley and D. Klein.
 Pharmaceutical Guidelines
 [Link]
 [Link]
 [Link]
 [Link]
 [Link]
 [Link]
 [Link]
 Google Images

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