A

PROJECT REPORT
ON
MICROBIOLOGICAL TESTING TECHNIQUES
OF PHARMACEUTICAL DRUGS
AT
Tirupati Medicare Ltd., Paonta Sahib (HP)
Submitted to
Kurukshetra University, Kurukshetra

In partial fulfillment of the requirement of

Masters of Science (Microbiology)
SESSION: 2016-17

Submitted to : Submitted by :
Dr. Neena Puri Parminder Kaur
Department of Microbiology M.Sc. (Microbiology)
G.N.K. College, Roll no. 16311602
Yamuna Nagar

DEPARTMENT OF MICROBIOLOGY
GURU NANAK KHALSA COLLEGE
YAMUNA NAGAR 135001
(Affiliated to Kurukshetra University, Kurukshetra)
 -: ABSTRACT:-
Microbiological testing technique is the part of Pharmaceutical drug analysis. In
Pharma industry microbial testing play important role.

Mostly drug have need of microbial testing for their quality assurance and also safe
from the microbial contamination. A Microbiologist have experienced to maintain the
quality and stability of drugs by do the testing of pathogens. As well as Drug analysis
and also water analysis is proceed by him in Microbiology lab.

For This testing I.P.(Indian Pharmacopeia) and U.S.P(United State of

Pharmacopeia) is used and studies.
Many technique is used for detection of microbes for prevent the medicine injury and
make the effect able for human life.
The result were found to be shown satisfactory and specified limit.
 ACKNOWLEDGEMENT

The superior of science & technology has through about a great revolution in
this modern world. Project work including thing in detailed study due to which they
go deep in our mind & hence increase our knowledge to greater extent.
I am specially greatful to Dr. Mandeep Singh (Principal), Guru Nanak
Khalsa College for providing me all the facilities in the department.
I cannot forget to mention the contribution of my guide Dr. Neena Puri
(H.O.D. Industrial Microbiology Deptt.) for their constant guidance, thoughtful
criticism, untiring help.
Gratefully thanks are extended to Sh. Rajneesh Arya (Executive
Supervisor), Mr. Amit Shandiiya( Quality Control Manager), Mr. Mohan Negi,
of Tirupati Medicare Ltd., Paonta Sahib, HP, for providing me this opportunity to
undergo training at Indian Herbs and made me available the required laboratory.
I would also like to thanks to our lab attendant Mr. Phool Chand for his
immense co-operation & from whom I have learned so much that I was able to do
satisfactory work during my training.

Parminder Kaur
 ABBREVIATION
% Percentage

BP British Pharmacopeia

IP Indian Pharmacopeia

CA Cetramide Agar
\
CFU Colony Forming Unit

DCA Deoxychocolate Agar

DI Water De Ionized water

DM Water De Mineralised water

E.Coli Escherichia coli

HEPA High Efficiency Liquid

HPLC High Performance Liquid Chromatography

LAF Laminor Air Flow

Ml Mili liter

MPN Most Probable Number

NA Nutrient Agar

RW Raw Water

FP Finish Product

BSA Bismuth Sulphide Agar

EMB Eosin methylene blue agar

SCDA Soyabean Casein digest agar

SDA Saboured dextrose agar
USP United State Pharmacopeia

TBC Total Bacterial Count

TFC Total Fungal Count

BPW Buffer Pepton Water

 * CANDIDATES DECLARATION *

I, Parminder Kaur student of M.Sc. Microbiology(IV Semester), hereby declare

that the work entitled Microbiological Testing Technique of Pharmaceutical
Drugs done in Tirupati Medicare Ltd., Paonta Sahib, HP, submitted to Guru
Nanak Khalsa College, Yamunanagar in partial fulfillment of the requirement for
the award of degree Masters of Science in Microbiology, is a record of the original
copy work done by me. All the information and knowledge furnished during my
learning period is accurate and without a manipulation and the certificate issued by
the Institute is fully authentic. This had not been submitted anywhere for the award of
degree , if any information found to be false, I shall be fully responsible.

Date :
Place : Yamuna Nagar Parminder Kaur
M.Sc. Microbiology (Final)
G.N.K. College,
Yamuna Nagar
 : TABLE OF CONTENT :

S.No. Title Page No.

Title Page (i)
Abstract (ii)
Acknowledgement (iii)
Candidates Declaration (iv)
Table of Contents (v)
List of Table (vi)
List of Figure (vii)
List of Symbol and Abbreviation (viii)
Chapter (ix)
1. OVERVIEW OF THE COMPANY 1-2

2. INTRODUCTION 3-5

3. AIM & OBJECTIVE 6

4. 7-16
MATERIALS &METHODS:-
INSTURUMENTATION
3.1 Autoclave-
3.1.1Principle
3.1.2Uses
3.2 Laminar Air Flow-
3.3 Oven
3.4 Incubator
3.5 Oven
3.6 Vortex Mixer
3.7 PH meter
3.8 Colony Counter
3.9 Micropipette

5. SAMPLING PROCEDURE 17-19

5.1Sampling of Water-
5.1.1Precaution
5.1.2Procedure
5.2 Sampling of Raw Material
5.1.1Procedure
5.2.2Shifting of container into sampling area

6. GROWTH PROMOTION TEST 20-22

6.1 Procedure
6.2 Preparation of media

7. MEDIA PREPARATION 23-24

 7.1Media
7.2 Procedure of preparation media
7.3Name of Media Used In Pharmaceuticals

8. MICROBIAL LIMIT TEST 25-32

8.1 Procedure
8.1.1 Membrane filtration method.
8.1.2 Pour plate method.
8.1.3 Spread plate method
8.1.4 Most probable number (MPN).

9. PATHOGEN TESTING 33-45

9.1 Pre Enriched Medium
9.1.1 E.coli
9.1.2 Salmonella
9.1.3 Staphylococcus aureus
9.1.4 Pseudomonas aeruginosa

10. ENVIRONMENT MONITORING 46-52

10.1- Settle plate method.
10.2- Air sampling.
10.3- Contact plate.
10.4- Surface testing by swab method.

11. PYROGEN TEST Or BACTERIA ENDOTOXIC TEST 53-56

11.BET
11.1 Test Procedure

12. STERILITY TESTING 57-59

12.1 Procedure

13. OBSERVATION 60-61

14. RESULT 62

REFRENCES 63

APPENDIX 64-67
LIST OF TABLE :
TABLE No. Table Name Page No.

1. Showing the GTP of Different Media 22

2. Name of Media Used in Pharmaceuticals 24
3. Detection of S.aureus. 42
4. Detection of P.aeruginosa 43
5. CFU In Settle Plate 47
6. CFU in Air Sampling 49
7 .CFU in Swab Testing 52
8. CFU in Water 53
9. Bet for Test 56
LIST OF FIGURE :
Figure No. Figure Name Page No.

4.1 Structure Of Autoclave 8

4.2 Structure of Laminar Air 9
Flow
4.3 Structure of Incubator 10
4.4 Structure of Oven 12
4.5 Vortex Mixer 13
4.6 PH meter 14
4.7 Colony Counter 15
4.8 Micropette 16
6.1 Bacterial Colony grow in 21
Petriplates
8.1 M.F.M Procedure 26-27
8.2 Procedure of pour plate Method 30
8.3 Spread Plate Method 31
9.1 Enriched Medium SCDM 34
9.2 Primary test of E.coli 35
9.3 Showing the red non mocoid 36
Colony of E.coli
9.4 Two Subculture 37
9.5 Colony Zone of S.aureus 39-41
in MSA petridish
9.6 Blue pigmented colony appear in 43
P.aeruginosa
10.1 Bacterial and fungus colony 47
10.2 Contact Plate 51
10.3 Swab Test tube 52
11.1 Horse shoe crap CSE 55
 OVERVIEW OF TIRUPATI
MEDICARE LTD.:-

NH- 72, Nahan Road, Paonta Sahib,

Dist. Sirmour,
Himachal Pradesh - 173025

Tel : +91 9805 967 721 / 722

Fax : +91 1704 222 882
Email : info@tirupatigroup.co.in

Tirupati Medicare Limited is a state-of-art formulation manufacturing facility

approved by WHO-GMP and is compliant with International Regulatory Standards.
Our portfolio boasts of manufacturing capabilities of products in Solid, Semi-Solid
(Internal & External preparation), Liquid Oral & Powder Doses form manufacturing
of Pharmaceuticals and Ayurvedic formulations.

Tirupati Medicare Limited is a knowledge based healthcare company, fully

supported by qualified and highly experienced teams of professionals including
production managers, dedicated quality units and logistical support; working
continuously towards the multidimensional growth of the company.
Our purpose is to improve community health by offering safe, efficacious and quality
products.

Vision & Mission

Vision
To accomplish, uphold and emerge as a Premiere Research based Product
Development and Manufacturing Healthcare Company; constantly enhancing quality
through derivation of technological efficacy. To serve globally as a dedicated, ethical
and socially responsible business group by building long term synergistic
associations; for aiding a healthcare driven tomorrow.

Mission
To strive and accomplish on creating newer business avenues through infrastructure
development. To stand committed to developing and delivering a range of research
based quality products by adhering to cGMP standards for an enhanced healthcare,
happier living and a healthy society.

MANUFACTURING

Tirupati Group provides the advanced manufacturing capabilities and processes that
creates quality specialty to offer Contract Manufacturing Facilities. We helps to
deliver innovative solutions for companies from discovery to development to
commercialization of the product.

Our expert manufacturing facilities are spread amidst the lush green fields of the
Himachal Pradesh. We have multi-location manufacturing facilities in Paonta sahib
spread over 15 acres of land. These associations of Tirupati are specialized in different
healthcare services such as Pharmaceuticals, Nutraceuticals and Ayurvedic
preparations. All facilities have independent infrastructure and have different Quality
Control and Quality Assurance functions to meet GMP and Regulatory Requirements.

With many years of experience, state-of-the art facilities (which adhere to stringent
specifications of GMP) with an experienced team, we provide flexible, cost-effective
drug development and substantial manufacturing solutions to our customers with
value added services.
We are offering different formulation ranges such as Tablets, Capsules, Powders,
Granules, Ointments, Creams, Gels, Pastes, Oral solutions, Syrups and Suspensions.

OUR STRENGTHS:
o Dedicated R&D facilities
o Provide solutions from discovery to development to commercialization of the
product.
o Larger manufacturing capacity for different dosage forms.
o Technology and expertise to develop specialty formulations
o Huge range of diversified product portfolio.

We can confidently claim that as far as contract manufacturing in India goes, we are
the company of choice for parties looking for such kind of associations.
 INTRODUCTION :-
Microbiology is the study of living organism of microscopic size.which includes
bacteria, fungi, protozoa, algae, and the infectious agents at the borderline of life
that called virus. These are called Microbes
It is devided in two types;
1. Unicellular (Single Cell)
2. Multicellular (Cell Colony or group of cell)
The instruments that is use for see it called Microscopy and the person who do study of
these microbes called Microbiologist. Microbiologist have the interest in the
physiology and morphology of microbes.

The first work of microbiology done by a Roman philosophers Lucretius in

98 55B.C. than he gave a view that certain Organism are present in the
environment that are responsible for the disease in Human. He could not view
those micro organism.

The Second Main contribution was of Francesco. He develop a microscope

by the help of Galileo. He view different flies under the microscope. However
he was the first person to observe and describe the micro organism Antony
Von Lewenhock of Depth in Netherland.

He construct a single microscope composed of double convex lenses held b/w two
silver plates. He could magnified the micro organism about 3o-50 times. Beginning in
1673 lewenhock sent a detail later describing his discovery to the Royal society
London and the work was published in 1680 it was clear his description that he saw
bacteria and protozoa. So he get the Nobel prize and Called the FATHER OF
MICROBIOLOGY.
Then many Scientist gave their many theories about microbes-

Spontaneous generation Era

Germ theory
Pasturization
Modern microbiology is a large discipline with many different field. It have great
impact on various field such as Medicine , Agriculture, Foodscience Biochemistry,
Genetics, etc.

Pharmaceutical microbiology is an applied branch of microbiology. it involves the

study of micro organisms associated with the manufacture of Pharmaceuticals e.g.
minimizing the no. of microbes in a process environment excluding microbes and
microbial products like exotoxin and endotoxin from water and other starting material.
Other aspects of pharmaceuticals microbiology include the research and development
of anti-infective agents, the use of microbes to detect Mutagenic and Carcinogenic
activity in prospective Drugs, and the use of micro organism in the manufacture of
Pharmaceuticals products like Insulin and Human Growth Hormone.

In this Pharmaceutical Microbiology has an important role in product development,

manufacturing process development, ensuring control of the microbes in the
environment and Raw material product testing in Pharma industry. The involvement of
experienced microbiologist in each stage of the product life cycle is important to
maintain product quality. Specification- setting decisions that can prevent microbial
contamination of Pharmaceuticals products.

The Quality control unit must have access to and equate testing laboratory to aid in
the approval of the materials under its control. One of these laboratory facilities would
be suitable equipped and staffed to conduct microbiological testing. The Quality control
unit need not manage the microbiology laboratory. The laboratory could be run by
quality control, research and development, even manufacturing or could be a contract
testing laboratory. All not a regulatory requirement , it is industry practice to use the
audit process as a tool to ensure that the Micro. Lab. meets all regulatory requirements
and internal company policies.

Microbiologist need to understand the requirement of the quality control and

ensure that the unit is supplied with quality test result in a timely and cost
effective manner. There need to be a laboratories batch of drug product to determine
that the product conforms to specification, including the identity and strength of each
active ingredient, before release. Where Sterility and Pyrogen testing are conducted
on specific batches of short-lived radiopharmaceuticals, such batch may be released
before completion of this testing is completed as soon as possible. Each batch of drug
product required to be free of objectionable micro organisms.

The Microbiologist in product development plays a major role in brings safe products
to the market. The role of microbiologist is to develop an validate the microbial test
that may be applied to the new pharmaceutical products to confirm that they are not
contaminated with an excessive no. of microbes or objectionable microbes that may
infect patients or degrade the quality of the product during its self life.

These test include compendia test for Microbial limits, Sterility, Bacterial endotoxins,
and Antimicrobial effectiveness. Drug development of the manufacturing process, an
experienced microbiologist should be consult as to the potential for microbial
contamination of product. The ability of the manufacturing steps to control microbial
contamination, the validation of sterilization process, the cleaning and sanitization of
process equipment, the adequacy of the water system, the holding times for
intermediates, the training of personal.
 Aim & Objectives :

In this project we study the about all microbiological testing of Pharmaceuticals.

The Aim of this project is to study the All method of testing of pharmaceutical
drugs which are commonly used in microbiological testing in Pharmaceuticals
Industry. These are the important part of Pharma industry for stabilization the
medicine and safe from the microbial contamination. Instrumentation is also
play a important role in this because all testing is proceed by these instruments.

Oven, Autoclave, Incubator, Shaker, HPLC, PH meter etc. there are many
instruments that is used.

By this we isolate the microbes and also get the microbial growth. We study
these microbes and do testing for prevent this microbial growth for the maintain
& increase the Quality of Drugs.

OBJECTIVES :
1. Growth promotion test
2. Microbial limit test
3. Pathogen testing
4. Environmental monitoring
5. Pyrogen test or bacteria endotoxic test
6. Sterility test
 MATERIALS & METHODS:-

INSTRUMENTATION:
Instrumentation is a important part of Pharma industry.because mostly all
process is also proceed by these instruments.

There are both three part as Production, Quality control and Microbiology department
is depend on instruments. Without it there is something not.

In Micro. lab, All microbiological testing is need seriously and proceed & gives result
with that instrument.
e.g
Autoclave
Laminar Air Flow
Incubator
Oven
Vertex Mixer
PH Meter
Colony Counter
Micropipette
 3.1 Autoclave :
An Autoclave is an instrument used to sterilize equipment and
supplies by subjecting them to high pressure saturated at 121C for
around 15-20 mints. Depending on size of the load and content. It was
invented by Charles Chamberland in 1879, although a precursor
known as the steam digester was created by Denis pippin in 1679.
The name comes from Greek word Auto; ultimately meaning self
and Latin clevis; meaning Key.

3.1.1 Principle:
The principle of autoclave is sterilizing the Using steam and
pressure at very high temperature and pressure. The water content
is absorbed and with absence of water the microbes die thus
sterilizing the material. The principle is basically same as that in an
ordinary pressure cooker. However the temperature and pressure is
higher.

3.1.2 Uses
Sterilization of Media
Sterilization of Microtips, Flask, Petridish
Discard of Media

Fig.-3.1 Structure of Autoclave

 3.2 Laminar Air Flow:-
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 type of cabinet with a variety of airflow patterns and acceptable uses.

Laminar flow cabinet may have a UV-C germicidal lamp to sterilize the shell and
contents when not in use. It is important to switch this light off during use, to limit
exposure to skin and eyes as stray Ultraviolet light emissions can cause cancer and
cataracts.

Fig.-3.2 Structure of Laminar Air Flow

 3.3 INCUBATOR :

An Incubator is instrument used to grow and maintain Microbiological cultures or

Cell cultures.

The incubator Maintains Optimal temprature , humidity and other condition succh as
the Carbon di oxide and Oxygen content of the atmosphere inside.

The Simplest incubators are insulated boxes with an adjustable heater, typically going
up to 60 to 655C, though some can go slightly higher(no more then 100 C). The Most
Commonly used temperature both for bacteria such as the frequently used E. coli as
well as for mammalian cells is approximately 37 C, as these organisms grow well
under such condition.

Fig. -3.3 Structure of Incubator

In Micro. lab., When water analysis and Drug analysis is done then two types Petri
plate prepared ;

TBC (Total bacterial count)

TFC (Total fungal Count)

Now For Incubation,

TBC plates is incubate in Bacteriological Incubator have temp. from 30 to 35C
for 5 days. It is because bacteria is mostly grow at 30 to 35C.
e.g. Salmonella typhi.

TFC Plates is incubate in BOD incubator have temp. from 20 to 25C for 5 days.
It is because of Fungi grows at 25 to 25C.
e.g. Pseudomonas aeruginosa.
 3.4 OVEN :
Hot air ovens are electrical device which use dry heat to
sterilize.
They were originally developed by Pasture.

Generally they can be operated from 50 to 300 C using a

thermo state to control the temp.

Their double walled insulation keeps the heat in and converse

energy. The Inner layer being a poor conductor and outer layer
being metallic.

An air circulating fan helps in uniform distribution of the heat.

Temp. Sensitive tapes or biological indicators using bacterial
spores can be used as controls, to test for the efficiency of the
device during use.

Fig.-3.4 Oven
 3.5 VORTEX MIXTURE :
Vortex is useful for gentle mixing for the responding pellets the unit has a circular
orbit rather than elliptical one. This allows a overtaxing motion to be achieved at even
low speed a task that cant be accomplished with elliptical orbit units.

Many vortex mixtures prevent excessive vibration with added weight or suction
feed which hold the unit in position. Vortex mixture which hold the unit in
position.

Vortex mixture utilizes an optimized counter balance system to minimize vibration

during operation and prevent the unit from walking across the bench event as high
speed.

The vortex mixture can be operated in touch or continuous made, when in touch
mode the motion is activated by depressing the sample head & stop by removing
the pressure.

Fig. 3.5 Vortex Mixer

 3.6 pH METER :

A pH meter is an electronic instrument used for measuring the pH (acidity or

alkality) of a liquid.

A typical pH meter consist of a special measuring probe glass electrode connected to

an electronic meter that measure & displace the pH reading.

The pH probe major pH as the activity of hydrogen cations surrounding the thin wall
glass bulb at its tip. The probe Produce a small voltage that is measured & displayed
as pH units by the meter.

3.6.1 Uses;-
For Measure the PH of Chemical Solution & Media

For Maintain the PH of any Liquid Solution in Lab.

Fig. 3.6 PH Meter

 3.7 COLONY COUNTER :-
Colony counter is a instrument by which colony of bacterial culture count in a Petri
plates and observe normal CFU.
The purpose of plate counting is to estimate the no. of cells present based on their
ability to give rise to colonies under specific condition of nutrient medium, temp and
time. Theoretically one viable cell can give rise to a colony through replication.

A Colony forming unit(CFU) is a unit used to estimate the no. of viable(the

ability to multiply via binary fission under the controlled condition) bacteria or
fungal cells in a sample.

Fig.3.7 Colony Counter

Counting with Cfu requires culturing the microbes and counts
only viable cells. In contrast with Microscopic examination
which counts all cells, living or dead. Cfu will in most cases undercount
the no. of living cell present in a sample for these reasons. This because the
counting of CFU assumes that every colony is separate and founded by a single
viable microbial cell.
The plate count is linear for E. coli over the range of 30 300 CFU on a
standard sized petri dish.
 3.8 Micropipette :
It is used to take and transport a accurate amount of reagent or samples for biological
experiment.

First micropipette was patented in 1957 by Dr. Heinrich Schnitger in Germany.

The founder of company Eppendorf , Dr. Heinrich netheler.]

Fig.- 3.8 Micropipette

Pipette comes in Several designs for various purpose with different level of accuracy
and precision.
Air displacement micropipette are a type of adjustable micropipette that measured
volume between about 0.1l to 1000 l(1ml) used in Microbiology lab for the
micro.testing e.g.
Water analysis
Drug analysis

These Pipette require disposable tips that come in contact with the fluid. These
pipettes operate by piston driven air displacement. A vaccum is genrated by the
vertical travel of a metal or ceramic piston with an airtight sleeve. As the piston
moves upward, driven by the depression of the plunger,a vaccum is created in the
space left vacant by the piston.The liquid around the tip moves into this vaccum and
can then be transported and released as necessary.
 * SAMPLING:
Sampling is a common process that is proceed in pharma industry. It have a
important role for testing procedure of Drugs.

4.1 Sampling of water :

Sample for microbial examination shall be collected in clean sterilized, narrow
mounted neutral glass bottle of 100ml to 1000ml capacity.

The bottle shall be sterilized in hot air oven at 180 degree celcius for 1hrs. or in
autoclave at121degree celcius for 30 minutes.

4.1.1 Precautions
Surgical gloves, face mask, clean lab coat shall be worn at the time of
sampling.

Sampling bottle shall not be touched by bare hand and the bottle shall be held
from the base.

Sampling bottle shall be kept in Bucked which is wrap with Aluminum foil
because of protect from UV light.

4.1.2 PROCEDURE
The sampling points and gloves shall be sanitized by spraying 70% IPA(ISO
PROPYL ALCOHAL).
The sampling shall done after draining out of water for at least 2 minutes from
the sampling point to permit clearing of service line.

Collect the sample in the container without rinsing and immediately close it,
and do not open the cap of bottle till the time of sampling.

Sample shall collect up to the brim of the bottle , and fit the cap immediately
to avoid contamination and proper sealed with aluminum foil.

Analysis shall be performed within 2 hours from the time of sampling for
microbiological analysis.
 All sample legibly mark with the source of the sample, date, time of
collection, Batch No. and the name and designation of the person collection
the sample.

4.2 SAMPLING OF RAW MATERIAL:-

All the Raw material and Final Gathering is store in Ware House, and from this
place RM and FG will be distributed to outside for marketing and inside for testing
and production.

4.2.1 Procedure:-
One copy of goods receipt note (GRN) shall be received from ware house by
Executive QC shall record the detail of GRN in Raw material GRN registers.

Requirement for sample:

Sampling Rod, spatula for solid material.
Glass tube for liquid material.
Sampling Bag/glass bottle/plastic container
Label to be pasted on containers.
Label to be pasted on sample bag/bottle.

Executive QC shall inter the sample room to check and verify the sampling room and
cleaned it before sampling. And switch on the Reverse laminar air Flow (RLAF)
unit before 15 min. of sampling.

Executive QC also checks the room temperature, humidity, RLAF magnetic gauge
record, balance record.

4.2.2 Shifting of container into sampling

area
First sample are taken from the warehouse department. RM containers shall be
brought near to the sampling room on clean SS trolley.
Executive QC shall verify the following detail of GRN against container

Material description (physical appearance)

Specification
Mfg. date/ Expire date.
Batch no.
Inspection lot no.
 Manufacture name.
Packing of container

4.2.3 Sampling of material

Open the container carefully after breaking the seal under RLAF which contain 3
filter and they are:-

Pre filter.
Intermediate filter.
HEPA filter (high efficiency particulate air).

When we start this instrument the air comes from HEPA filter then it sent
into pre filter after it sent to the intermediate filter again the purified air sent
into the HEPA filter. In this instrument the simple technique is revolution of
air. The container must be opened in presence of RLAF because of to control
the contamination.

Before sampling, person doing proper mixing of material . In case of poly bag
sampling shall be done from top, middle and bottom to form a homogenous
sample.

Sample will be taken from top, middle and bottom layer of container using
sampling SS rod and transferred to clean butter paper.
[NOTE;-Before sampling stainless steel rod is sanitized with 70% IPA]

Sample will put into plastic container/ polybag . And remaining sample shall
not be put back into container.

Now sample will be send to Quality control department for chemical and
microbiological analysis.

Cleaning of sample booth is required after completion of sampling of RM.

Change the uniform while sampling of Raw material.

 * GROWTH PROMOTION TEST :
The purpose of the Growth promotion test is to determine the suitability of culture
media. The medium is challenged with a small number of microorganisms to assure
the nutritive properties.

5.1 PROCEDURE
Take clean, dried glass conical flask of desired volume as quantity of media.
Use separate spatula for separate media to ovoid cross contamination.
Clean the balance in between to successive weighing.
Use purified water for media preparation.

5.1.1 Preparation of media.

Take clean dried conical flask as per the requirement of media. Transfer the
half of the volume of required quantity of the conical flask. Weight the
quantity of the dehydrated as per volume required, transfer in conical flask
and re hydrated it as per manufacture instruction. Makeup the volume with
remaining quantity of water.
Check the ph of media using calibrated ph meter, adjust the PH using 0.1M
HCl/0.1M NaOH if required.
Dispense the medium in an individual container as per requirement plug the
containers with cotton plug or screw cap.
Sterilized the media for 20 minutes at 121 degree celcius and 15psi or for
validated time period.

Growth promotion test of sterilized

media
Select the quantified microbial culture for the growth promotion test.
For growth promotion test of the agar medium transfer microbial culture to
sterile petridishes in duplicate aseptically and pour 15 to 20 ml of agar
medium.
Incubate the petriplates for 24-48 hours for bacterial count at 30-35 degree
celcius and 5 to 7 days for fungal count at 20-25 degree celcius. After
completion of incubation period observe the growth of microorganisms as
turbidity
Recovery of microbial count of the agar medium should be more then 70% of
the count added to the medium.
 The medium passes in GPT can be use for analysis of sample. Always run
negative control of medium to check the sterility of medium.

% Recovery = mean Cfu observed *100

Inoculated CFU

Fig.5.1 Bacterial Colony grow in Petriplates

Name of media Microorganisms Property

Macconkey broth E.coli Growth promoting
Macconkey agar E.coli Growth promoting
Macconkey broth S.aureus Inhibitory
Tetrathionate brilliant Salmonella typhimurium Growth promotion
green broth(TTBG)
TTBG Salmonella abony Geowth promotion
TTBG S.aures Inhibition
Xylose lysine Salmonella typhimurium Growth promotion
deoxycholate agar(XLDA)
XLDA Salmonella abony Growth promotion
Cetrimide agar Pseudomonas Aeruginosa Growth promotion
Cetrimide agar E.coli Growth promotion
Mannitol salt agar S.aures Growth promotion
Mannitol salt agar E.coli Inhibitory
Sauvorant dextrose agar C.albicans Growth promotion
Soyabean casein digest B.Subtilis Growth promotion
medium(SCDM)
Fluid thioglycollate Pseudomonas Aeruginosa Growth promotion
medium(FTM) S.aures
Growth promotion
R2A agar Staphylococcus aures Growth promotion
R2A agar P.Aeruginosa Growth promotion
Peptone water E.coli Growth promotion
Soyabean casein digest Staphylococcus aures Growth promotion
agar
Vogel johnsan agar E.coli Inhibitory
Vogel johnsan agar Staphylococcus aures Growth promotion
Pseudomonas agar Staphylococcus aures Inhibitory
Pseudomonas agar P.Aeruginosa Growth promotion

* MEDIA PREPARATION :
6.1 MEDIA
Media is a solid and liquid preparation use to grow, transport & store Micro organism.
These are a medium contain all nutrient for micro organism required for growth.

Many types of media are used

Synthetic media
Complex Media
Supportive media
Enriched media
Selective media
Differential media

Media are used in the pharma industry for microbiological testing of Drugs and Water
that is very important. For every analysis media is necessary in micro. Lab.
All microbial growth & also contamination is observed by media in the drug for the
medicine quality & stability measurement.

6.2 Procedure of media preparation

1. Firstly Take Required quantity of dehydrated media shall
be in a clean butter paper using an analytical balance.
2. If the container is new/from different batch, Growth
Promotion test shall be perform for Media growth promotion
test.
3. Weighed media shall be dispensed in a glass contain
containing desired volume of purified water.
4. PH of the media shall be checked and adjusted with 0.1 M
NaOH or 0.1M HCl
5. This media shell be distributed into glass flasks/bottles.
6. Each glass flask shall plugged with non-absorbent cotton and
wrap by using butter paper with rubber band.
7. In case of bottle slightly loosen the cap /screw caps, so that
steam may enter the bottle.
8. Prepared media shall be loaded in autoclave and the
autoclave shall be operated at 15PSI pressure (121 degree
celcius) for 15 minute to sterilize the media.
 9. Sterilized media shall be UN loaded and cooled up to approx
45-50 degree celcius for agar media and room temperature
for liquid media respectively.
10. Now media shall be prepared for analysis.

6.3 Name of Media Used In

Pharmaceuticals
Media Full Name of Media Use for testing of

R2A Agar Medium Water Testing

SCDA Soyabean casein digest agar TBC(Total Bacterial Count)

SDA Sabourads dextrose agar TFC(Total Fungel Count)

MCB Macconkey Broth E.Coli

MSA Mannitol salt agar Streptococcus Aureus

RVSB Ravapport vassiliadis Salmonella

CA Cetramide agar P.aueruginosa

SCDM Soyabean casein digest Enrichment water and Sample

medium
XLDA Xylose Lysine Deoxycholate Salmonella & Shigella
Agar
 * MICROBIAL LIMIT TEST (MLT) :
Microbial limit test are used for estimation of the no. of Viable aerobic
microorganism present and for detecting the process of microbial species in the
pharmaceuticals products (RM, and Water).It includes test for Total viable count
(Bacterial and Fungal) and pathogen testing.

7.1 Procedure
There are 4 methods for detection of total microbial count (MLT).

7.1.1 Membrane filtration method.

7.1.2 Pour plate method.

7.1.3 Spread plate method

7.1.4 Most probable number (MPN).

7.1.1 Membrane Filtration Method:-

Membrane filtration method has been a common preferred method of evaluating the
microbiological analysis of water. This procedure shall be followed for isolating
bacterial and fungi.

Pre-incubated agar media and sterile 0.45 filter paper shall be transferred to
horizontal laminar air flow.
Sterilized membrane filtration assembly / Millipore membrane apparatus shall be
transferred to horizontal laminar air flow.

Filtration assembly is consist of following part:-

Base
Funnel
Clamps
Suction flask
Vacuum pump
Sterilized 0.45m Millipore filter
7.1.1.1 Advantages of MF Technique

Permits testing of large sample volumes.

Provides presence or absence information within 24 hours.
Effective and acceptable technique. Used to monitor drinking water in
government laboratories.
Reduces preparation time as compared to many traditional methods
Useful for bacterial monitoring in the pharmaceutical, cosmetics, electronics,
and food and beverage industries.
Allows for removal of bacteriostatic or cidal agents that would not be, Spread
Plate, or MPN technique.

7.1.1.2 Procedure
(1.) Collect the sample and make any necessary dilutions.

(2). Select the appropriate nutrient or culture medium.

Dispense the broth into a sterile Petri dish, evenly saturating
the absorbent pad.

(5). Flame the pouring lip of the sample container and pour
the sample into the funnel.

(6). Turn on the vacuum and allow the sample to draw

completely through the filter.
 (7). Rinse funnel with sterile buffered water. Turn on vacuum
and allow the liquid to draw completely through the filter.

(8). Flame the forceps and remove the membrane filter from
the funnel.

(9). Place the membrane filter into the prepared Petri dish.

(10). Incubate at the proper temperature and for the

appropriate time period.

(11). Count the colonies under 10-15 X magnification.

(12). Confirm the colonies and report the results.

Note: Observe aseptic technique throughout the procedure.

Fig.-7.1 M.F.M Procedure

7.1.2 Pour Plate Method

The Pour plate technique can be used to determine the number of microbes/ml or
microbes/gram in a specimen. It has the advantage of not requiring previously
prepared plates, and is often used to assay bacterial contamination of foodstuffs. The
principle steps are to:-

1)Prepare/dilute the sample

2) Place an aliquot of the diluted sample in an empty sterile plate
3) Pour in 15 ml of melted agar which has been cooled to 45o C, swirl to mix well
4) Let cool undisturbed to solidify on a flat table top
5) Invert and incubate to develop colonies.
Each colony represents a "colony forming unit" (CFU). For optimum accuracy
of a count, the preferred range for total CFU/plate is between 30 to 300
colonies /plate.

EQUIPMENT:
15 ml Sterile Plate Count Agar (PCA)*, in capped 16 x 150 mm test
Tubes, melted and cooled to 45oC.
Hot Block, 45oC (or water bath), 3" deep to equal agar depth
Sterile capped 16 x 150 mm test tubes.
0.1, 1.0 and 2.0 ml Pipettes, Sterile Petri dishes, empty and sterile
flame , colony counter with magnifying glass.

7.1.2.1 Step-By-Step Procedures:-

(1). Write out details of preparing

and plating your specimen(s):
Construct a table in your
notebook with a line for each
plate:
 The identity/source of the specimen (notebook entries should be detailed).
the dilution of the specimen expected to contain between 30-300 CFU/0.1-
1.0 ml and how you will prepare it
the volume of diluted specimen you will plate (usually 0.1 to 1.0 ml)
Label the bottom of the empty, sterile plates your initials, seat number, date
and the above data.

(2). Dilute specimen to yield

approximately 30 to 300 CFU per
aliquot to be plated (from 1).

(3). Inoculate labeled empty Petri

dish with the aliquot of diluted
specimen (from 1)

(4). Pour 15 ml of melted Plate

Count Agar (45o C) into the
Inoculated Petri dish.

(5). Cover and mix thoroughly by

gentle tilting and swirling the
dish . Do not slop the agar over the
edge of the Petri dish.

(6). Place on a flat surface undisturbed

for about 10 minutes to allow the
agar to completely gel. In this
illustration, the agar is completely
gelled and the surface is "smooth as
glass."
(7). Invert and incubate at 37o C for
24-48 hours.

(8). Count, record, calculate:

Count all colonies (note that the

embedded colonies will be much
smaller than those which happen to
form on the surface). A magnifying
colony counter can aid in counting
small embedded colonies. Record
the data. Calculate CFU/ml or
CFU/g. Enter results in your table.
CFU/ ml = CFU/plate x dilution factor x 1/aliquot
On the plate shown, milk was diluted 1 to 100 (10 2), 1.0 ml of the dilution was
plated and 40 colonies formed. Therefore the count per ml in the milk was:
40 colonies x 102 x 1/1 = 4 x 103/ml
Fig . -7.2 Fig of Step by Step
Procedure of pour plate Method.
 7.1.3 Spread Plate Method

The Spread plate method is used for the separation of a dilute,

mixed population of micro organism so that individual colonies
can be isolated.
In this technique microorganism are spread over the solidified agar media with
a sterile L-shaped glass rod while Petri dish in spun.
These Petri plates shall be transferred to BOD incubator for 48hr at 30-35
degree celcius for bacteria for 72hr. at 20-25 degree celcius for fungi.
The theory behind this technique is that as the Petri dish spuns at some stage
single cells will be deposited with the bent glass rod on the agar surface.
Some of these cells will be separated from each other by a distance sufficient
to allow the colonies that develop to be free from each other.
Fig.7.3 Spread Plate Method
 7.1.4 Most probable Number
Multiple tube fermentation tests is used to detect coli form bacteria which are
used as indicator of fecal contamination.

The coli form groups include E.coli, Enterobacter , Aerogenes , and Klebsiella
pneumonia.

Coli form are defined as facultative anaerobic Gram- negative, non-endospore

forming, Rod-shaped bacteria that fermenting Lactose with the production of
Acid and Gas within 24-48hr. at 37 degree celcius.

This test is performed sequentially in 3 stages:-

1. Presumptive

2. Confirmed

3. Completed
 * PATHOGEN TESTING :
In Microbiology lab Pathogen testing is also very necessary in Drug analysis &
Water analysis for determine the quality of Medicine.

8.1 PRE ENRICHMENT MEDIUM

Add the 10gm/10ml sample in 90ml of Pre Sterilized SCDM and incubate the flask
for 24hr. at 30-35 degree celcius.
If turbidity is observed after 24hr. In enrichment medium, preceding the broth
medium for pathogen testing.

Negative control and positive control is also proceeding with the test control
/sample control in all pathogen testing.

Fig.8.1 Enrichment medium SCDM

 8.1.1-E.coli:-
Transfer prescribed quantity of sample to a sterile flask containing 50
ml of nutrient broth. Shake, allow to standing for 1 hour (4 hour for gelatin) and
shaking again incubate at 36-38 degree celcius for 18-24 hours.

8.1.1.1 Primary Test:-

Transfer 1ml of enrichment culture from SCDM into tube containing

5ml of sterilized MacConkey broth Durham tube (inverted). Incubate it
at 36-38 degree celcius for 24-48hrs. if the tube acid and gas production
then carry out the secondary test, if not then the sample meets the
requirements of the test for absence of E.coli .
MC broth is a differential media for Enterobacteriaceae and Gram
negative bacilli. The selective action of this medium is by crystal violet
and bile salt, which are inhibitory to most species of Gram positive
bacteria.

Gram negative bacteria usually grow well on this medium and are
differentiated by their ability to ferment lactose. Lactose fermenting strain
grows as red or pink and may be surrounded by a zone of acid
precipitated bile.

Red colour is produced due to production of acid from lactose, colour

change of dye when PH of medium fall below 6.8 .

Observation There is no colour change in MC broth like as

Positive control.

Sample Sample Control Control

Fig.-8.2 Primary test of E.coli
8.1.1.2 Secondary test:-

Take loopful culture from MC broth of pri test and streak into MC Agar which is
selective media for E.coli,
Incubate it at 36-38 C for 24hrs.

MC agar media is used for isolation and differentiation of E.coli and Enterobacter
aerogenes.

Observation:- there is red non-mocoid colonies is present

Fig.8.3 Showing the red non mocoid colony of E.coli

8.1.2 -Salmonella :
Transfer prescribed quantity of sample to a sterile flask
containing 100 ml of nutrient broth. Shake, allow to standing for 4 hour and
shaking again incubate at 35-37 degree celcius for 18-24 hours.

8.1.2.1 Primary test

Add 1ml of the Enrichment medium (SCDM) to each of the 2 tube containing 10ml
of
Selenite F broth (SF broth)
Tetrathionate brilliant green broth(TTBG)

Incubate at 36-38 C for 48 hrs. For each of these 2 cultures, subcultures on at least 2
of the following media:-

Fig. 8.4 Two Subculture

(a) Bismuth sulphite agar

(b) Brilliant green agar
(c) Deoxycholate citrate agar
(d) Xylose lysine deoxycholate agar.

(a)Brilliant green agar (BGA)-

After subculture-
Now incubate the plates at 36-38 degree celcius for 18-24hrs.BGA used for selective
isolation of salmonella other than salmonella serotypes.
These media contain Brilliant green which inhibit growth of majority of Gram
positive and Gram negative bacteria like that salmonella serotypes, shigella species,
E.coli, Proteus , Staphylococcus aureus.

Lactose non-fermenting bacteria develop opaque white to pinkish red colonies

within 18-24hrs.

Fig.8.5 In BGA showing white to Pinkrish colony

(b) Xylose lysine deoxycholate agar (XLDA) -

After subculture-
Incubate the plates at 36-38 degree celcius for 18-24hrs.
XLDA is selective medium for salmonella serotypes and other species of
salmonella.

Deoxycholate is a selective agent which inhibit Gram positive micro organism.

Xylose is fermented by almost all the enteric bacteria expect shigellae which unable
the differentiation from salmonella.

Salmonella metabolize the Xylose and decarboxylate the lysine and thus change the
PH to alkaline & mimic shigella reaction. However to prevent this reaction, lactose
and sucrose are added in excess to produce acid, hence Lysine is not decarboxylate .
Phenol red is the PH indicator.

Red colour colony observe with or without black center

 Fig.8.6 Showing red colour observe with or without colour center

Observation:- there is absent of specified colony in BGA and XLDA

8.1.2.2 Secondary test:-

Subculture any colony showing characteristics in TRIPLE SUGAR IRON AGAR

SLANT (TSI) by first inoculating the surface of the slope and then making the stab
culture with the same inoculating needle, incubate at 36-38 degree celcius for 18-
24hrs. The formation of acid and gas in the stab culture and the absence of acidity
from the surface growth in TSI slant, indicate the presence of salmonella.

TSI used for the differentiation of Enterobacteriaceae by ability to determine

carbohydrate fermentation and hydrogen sulphide production.Lactose,
sucrose, dextrose is the fermentable carbohydrate.

Sodium thiosulphate and ferric ion make hydrogen sulphide indicator system.
Phenol red is the PH indicator.

TSI slant is a red in colour organism that ferment the glucose produced a
variety of acids. Turning the colour of the medium from Red to Yellow.

More amount of acid is librated in butt (fermentation) then in slant

(Respiration).

Growing bacteria also form alkaline product from the oxidative

decorboxylation of peptone and these alkaline product neutralize the large
amount of acid present in butt. Thus the appearance of an alkaline(Red) slant
and an acid (Yellow) butt after incubation indicate that the organism in a
glucose fermenter but is unable to ferment Lactose/sucrose.

Carbon dioxide is detected by presence of cracks or bubbles in medium.

Uninoculated P.aeruginosa Shigella Salmonella E.Coli
Proteus Sonnie typhi
Control mirab
Fig.8.7- Slant of pathogens
8.1.3 Staphylococcus aureus:-
Transfer a prscribed quantity of a sample in a sterile flask containing 100 ml of fluid
SCDM, shake and incubate at 35 to 37 55 C for 24 to 48 hours.

Take loopful culture from enrichment medium and streak surface on Mannitol
salt agar (MSA). Incubate it at 35-37 C for 48hr.

Staphylococcus aureus grows on this medium and fermented mannitol produce

yellow colonies with yellow zone. The colour of colony and medium is due to
reactivity of phenol-red to the ph of the medium. Phenol-red is red at ph 8.4
and yellow at 6.8

.Staphylococci produces bright yellow zones are pathogenic, while colonies

with red dish purple zones are non-pathogenic.

Fig.8.8 Colony Zone of S.aureus shown in MSA petridish

If upon examination non of the plate contains colonies having the

characteristics listed in table for the media used, the sample meets the
requirements for freedom from S.aureus-

Selective Medium Colony Characteristics Gram Stain

Vogel-Johnson Agar Black colony surrounded by Positive cocci in cluster
yellow zones
Mannitol Salt Agar Yellow colonies with yellow zone Positive cocci in cluster
Baird-Parker agar Black, shiny colonies surrounded Positive cocci in clusters
by clear zone of 2 to 5 mm
Table.- Detection of S.aureus.

Result:- Colony are absent on the plate of test control, so Staphylococcus are
absent in Sample.
8.1.4 Pseudomonas aeruginosa:-
8.1.4.1 Primary test
Take a loopful culture form Enrichment medium and streak on the surface of
Citrimide agar(CA) plates and incubate 35-37 C for 48hrs.
CA is used as selective medium for the isolation of P.aeruginosa.
Also used for determining the ability of an organism to produce Fluorescent
and pyocyanin.
Citrimide (cetyl trimethyl ammonium bromide) is incorporate in the medium
to inhibit bacteria other than P.aeruginosa it acts as quaternary ammonium
compound, cationic detergent which causes nitrogen and phosphorus to be
released from bacterial cells other than P.aeruginosa.
P.aeruginosa colonies may appear pigmented blue, blue green or non-
pigmented.

Fig.8.9 Blue pigmented colony appear in P.aeruginosa

If upon examination non of the plate contains colonies having the

characteristics listed in table for the media used, the sample meets
the requirements for freedom from P.aeruginosa.

Table.Detection of P.aeruginosa-
S.No. Medium Colony Florescence Oxidase Gram
Characteristics in UV light Stain
1. Cetramide Generally greenish Greenish Positive Negative
Agar colonies rods
2. Pseudomona Generally Yellowish Positive Negative
s agar for colourless rods
detection yellowish colonies
fluorescein
3. Pseudomona Generally greenish Blue Positive
s agar for colonies
detection
Pyocyanin
Observation:- there is absence of identified colony on CA plates test control.
8.1.4.2 Secondary test:-
If greenish colony appears on CA plates of test, then take loopful culture from
enrichment medium (SCDM) and streak on 2 different selective media:-

(1)-Pseudomonas Agar for Fluorescent (PSAF):-

After streaking incubate these plates at 35-37 degree celcius for 48hrs.
PSAF is recommended for the detection of fluorescent production which is water
soluble chloroform insoluble fluorescent pigment by pseudomonas sepsis.

The medium enhance the elaboration of fluorescent by pseudomonas and inhibit the
pyocyanin formation. The fluorescent pigment diffuses from the colonies of
pseudomonas into the agar and show Yellow fluorescent coloration. Some
pseudomonas strain produce small amount of pyocyanin resulting in a Yellow green
coloration.

Casein enzyme hydrolysate and protease peptone provide essential nitrogenous

nutrient, dipotassium phosphate buffer the medium, while Magnesium sulphate
provides necessary cation for the activation of fluorescent production.

Fig.8.9 Shown Fluroscein in PSAF

(b)- Pseudomonas agar for pyocyanin(PSAP):-

After streaking incubate this plate at 35-37 degree celcius for 48-72hrs.
On PSAP Blue green coloration appear on plate.
PSAP is recommended for the detection of pyocyanin production which is water
soluble pigment by pseudomonas species.

The medium enhance the elaboration of pyocyanin but inhibit the formation of
fluorescent pigment. The fluorescent pigment diffuses from the colonies of
pseudomonas into the agar and show Blue coloration. Some pseudomonas strain
produce small amount of fluorescent resulting in a Blue green coloration.
Peptic digest of animal tissue provide nitrogenous growth nutrient, potassium
sulphate and magnesium chloride which enhance the pyocyanin formation and
suppress the fluorescent production.

Results:- There is absence of fluorescent colony on PSAF and Blue green colony
on PSAP of test control.So Pseudomonas areuginosa is absent in sample.
 * Environmental Monitoring
Environmental monitoring is done to check the bioburden of the aseptic area of
controlled environment. The purpose of this is to understand the various issues that
relate to aseptic processing of bulk drug substances or finished products(sterile), dose
forms, and in certain cases, and to establishment, maintanence and control of the
microbiological quantity if the controlled environment.

There are four methods for environmental monitoring;

9.1- Settle plate method.

9.2- Air sampling.
9.3- Contact plate.
9.4- Surface testing by swab method.
 9.1- Settle plate method
Required quantity of Soyabean casein digest a
gar(SCDA) and Sabouraud dextrose agar(SDA) plates shall be prepared for
bacterial and fungal monitoring and shall be pre-incubate.

After completion of pre-incubation period of the media plates, Microbiologist

shall visually check these plates for growth. If no growth is observed, these
plates may be used for environmental monitoring.

Take pre-incubated media plates to the location/area where sampling is to

done, in a SS Petri box and plate will be mark with location code and date of
monitoring with marker.

These plates shall be placed on SS stools: just in front of the return air risers.
The lid of the Petri plates shall be placed under the plate so as to incline them
toward the flow of air.

The Petri plates shall be exposed for 4 hours at each location. After completion
of exposure, these agar media plates shall be transferred to the microbiology
laboratory and incubate, in inverted position, at 30-35 degree celcius for
bacterial growth for 72hrs and 20-25 degree celcius for fungal growth for
120 hrs.

After completion of incubation the media plates shall be observed on colony

counter for the number of count colony characteristic. If the result is out of
the limit immediately inform the QC manager and QA necessary action.

Environmental monitoring shall be done every 15 days in all production

blocks weekly in microbiology laboratory, soft gel area shall be done on
weekly basis.

If colonies having pathogenic morphological characteristics are identified for

the 2 consecutive times, identification of these isolate by Pathogen testing.

Fig.9.1 Bacterial and fungus colony

 Table : CFU In Settle Plate-

Grade Alert limit Action limit Limit

A <1CFU <1CFU <1CFU
B 3 CFU 4 CFU 5 CFU
C 30 CFU 40 CFU 50 CFU
D 60 CFU 80 CFU 100 CFU
 9.2 - Air Sampling:-
In an aseptic parental filling area the assessment of microbial level in the air is of
prime importance.

Procedure;
1. Ensure connect the supply of 230volt./50hz.
2. Power on initial display (0) or last set value.
3. Press up and down key to set sampling time in minutes.
4. Press start/stop switch to start the air sampling after the
desired volume is aspirated. The buzzer will fire an alarm
which indicates elapsed time.
5. Prepare a strip using suitable agar media.
6. Insert the strip in the cup slowly and paste it end with the
adhesive tape.
7.The quantitative result of CFU on the agar strip after appropriate incubation is
then calculated for CFU/m to assign the alert level as per USP/GMP guideline.

Air sampler strip air sampling

Fig.9.1 Air sampling Procedure
Table :CFU Limit in Air Samping:-
Grade Alert limit Action limit Limit
A <1 CFU/m <1 CFU/m < 1CFU/m
B 6 CFU/m 8 CFU/m 10 CFU/m
C 60 CFU/m 80 CFU/m 100 CFU/m
D 120 CFU/m 160 CFU/m 200 CFU/m
 9.3 Contact Plate Method:-
9.3.1 Procedure

Soyabean casein digest agar plates and Sabouraud dextrose agar

plates shall be prepared. Take pre incubated media plates to the
location/area, where sampling is to be done.

Sample shall be taken from microbiologist, who is directly in

contact with product/microbial analysis. Wear surgical gloves
before sampling.

Impression of thumb and four fingers shall be taken on soybean

casein digest agar plates for both right and left hand (which media
is not pressed forcefully).

After taken of finger dabs these petri plates shall be marked with
the details like name of operator, area, name and date of sampling.

These petri plates shall be taken to the microbiology lab and

incubate at 30-35 degree celcius for 72hrs for bacterial growth.

Same procedure shall be followed on the next day on sabouraud

dextrose agar plates for fungal count and these plates shall be
incubate at 20-25 degree celcius for 120hrs.

Finger dab test shall be done once in a month in all production

blocks and in microbiology lab.
 Fig.-9.2 Contact Plate

9.4- Surface Testing By Swab Method:-

This method is used for the microbial contamination observe in
Production area etc.

Procedure:-
Prepare the buffered sodium chloride peptone solution as per
manufacture interactions and sterilize in autoclave at 121
degree celcius temp. For 15min.

Use the sterilize cotton swab and pour the 3-5ml of sterile
buffered sodium chloride peptone solution.

Moist the swab with solution and take the sample by webbing
the area 25cm(place/medium) and transfer it to be a tube.

Vortex the tube for about 1min. so as to liberate the cell from
the swab.

Aseptically remove the cotton swab by gently pressing on inner

side wall of tube.

Take 1ml solution pour sterile petri plate and pour the 20ml
sterilize SCDA media. Allow the agar plates to solidify.

Incubate the plates inverted in incubator at 20-25 degree celcius

for 3 days and 30-35 degree celcius for 2 days.

After incubation count the no. of CFU per plate and record the
result.
 Fig. 9.3Swab Test tube
Table: CFU Limit In Swab Method:-

Grade Alert limit Action limit Limit

A <1 CFU <1 CFU < 1CFU
B 3 CFU 4 CFU 5 CFU
C 30 CFU 40 CFU 50 CFU
D 60 CFU 80 CFU 100 CFU

Table: CFU Limit In Water:-

Grade Alert limit Action limit Limit

Purified water 60 CFU 80 CFU 100 CFU
Raw water 300 CFU 400 CFU 500 CFU
Soft water 50 CFU 100 CFU 300 CFU
Water for NIL NIL NIL
injection
Pure steam NIL NIL NIL
generation
 * BACTERIA ENDOTOXIN TEST Or
PYROGEN TEST *
[Greek Pyro- Fire & gen- beginning]
Endotoxin is part of the outer membrane of cell wall of Gram- negative bacteria.

Endotoxin is invariable associate with Gram-negative bacteria whether the organism

are pathogenic or not. It is properly reversed to refer to the lipopolysaccharide
complex associate with the outer membrane of G-ve pathogen such as E.coli,
salmonella, shigella, Pseudomonas, Neisseria, and vibrio cholera.

Bacteria Endotoxin Consists of:-

Lipid A - toxic
Core polysaccharide - Non toxic
O specific side chain - Non toxic

Effects of Bacterial Endotoxin:-

Fever
Inflammation
Chills
Shock
Hypotension
Hemorrhage
Death

Endotoxin Detection is must in Parenteral Drugs.

 10.1 BET:-
10.1.1 Test (BET) Procedure:-

There are 3 types of techniques for this test.

1. LRW- LAL reagent water (Endotoxin Free water)
2. CSE - control standard endotoxin (kwown endotoxin).
3. LAL- Limulus Amebocyte Lysate is to detect or quantify bacteria endotoxin that
may present in sample and LAL is obtained from aqueous extract of circulating
amebocytes of horse shoe crab which has been prepared and for use as an LAL
reagent.

10.1.2 Reagents supplied and stored condition of LAL and CSE:

1. Lysate is lyophilized and sealed under vacuum and is to be reconstituted with LAL
reagent water. Do not dehydrate until immediately prior to use.

2.Lyopholized (un reconstitute) vial under refrigeration at 2-8 C care should be taken
to avoid exposed the lysate
Bacteria endotoxin test is a sensitive and specific means to detect and measure the
concentration of bacteria endotoxin that may be present in or on the sample which the
test is applied.

Principle;
Proenzyme ------endotoxin-------->coagulase

Coagulogen------coagulase-------->coagulin

temperature in excess of 25 C lysate which has been exposed to prolonged period of

temp. Above of 25 C to bright light may turn yellow or become insoluble.

3. Reconstitute lysate may be stored at 2-8 C for 24hrs.

For longer storage reconstitute lysate can be stored below
-10 C. the lysate should be protected from expose to light during storage use eith in
four weeks after reconstitute.

4. CSE vial at 2-8 C prior to reconstitution reconstitute with 5.0ml LRW and vortex
for at least 15min. store reconstitute vial at 2-8 C for up to 4 weeks

10.1.3. SPECIMEN COLLECTION AND PREPARATION:

All material coming in contact with the specimen or test reagent must be endotoxin
free. Clean glassware and material may be endoxin free by heating at 250 degree
celcius for 30min.
 Fig,-10.1Horse shoe crap CSE

Reagents:
Lyophilized Caopecod LAL reagent,
Sensitivity ( EU/ml)
1. Control standard Endotoxin (CSE), for control curve and PPC.
2 .LAL reagent water (LRW) used as Diliuents.
3 .sodium hydroxide 0.1N and hydrochloric acid 0.1N dissolved in LAL reagent water
for ph adjustment of sample if necessary.

ACCESSORIES :-
Vortex mixer.
Heating block.
Depyrogenated Glass test tube for Assay (1075mm)
Depyrogenated glass test tube for Dilution (1275mm).
Micro pipette
Sterile micro pipette tips.
Timer.

10.1.4 Preparation of CSE dilution

Potency of endotoxin 40Eu/ml

1.50l of CSE + 950l of LRW (2Eu/ml)

2.300l of CSE + 300l of LRW (1EU/ml)
3.500l of CSE + 500l of LRW (4)
Table: BET FOR WFI TEST:-

Sr.no. Test LRW in l 4 (CSE) in l Sample Lysate in

l
1 NPC 50 - 50 100
2 PPC - 50 50 100
3 PWC 50 50 - 100
4 NWC 100 - - 100

Where,
NPC= negative product control.
PPC= positive product control.
PWC= positive water control.
NWC= negative water control.
 RESULT:
Positive = firm gel formation.
Negative = no firm gel formation.
 * STERILITY TESTING:-
A sterility test may be defined as A test that critically assesses whether a
sterilized pharmaceutical product is free from contaminating microorganisms.
The Sterility test is used to detect the presence of viable forms of bacteria fungi and
yeast in or on pharmacopoeia preparation.

11.1 Procedure:-

11.2 By Membrane filtration:-

Procedure for sterility room.Start the LAF.

Wipe out all samples to be tested for sterility with 70%IPA solution.

Before starting sterility test, expose the SCDA plates as specific locations.

Connect the filtration manifold holder assembly with SS reservoir properly

with pipe and place sterilized SS cups in the sterile receptor under laminar air
flow units. Check the manometer reading of working LAF and check the temp.
As well as humidity of the sterility room.

Manometer reading of working LAF chamber pressure should be between 08-

15mm of WG. Temperature reading sterility room should be 27 degree celcius
2 degree celcius.

Switch on the vacuum pump but close the vacuum manifold holder assembly with the
help of vacuum control key, present on the base of individual manifold holder. Now
place 0.45m ster
Sample for finished product:-

Collect the sample to be tested for sterility, randomly select 20 articles (as per
pharmacopoeia) of each lot of batch small volume parenterals, large volume
parenterals terminally sterilized product and for aseptic filled product.

Sample for intermediate:- randomly collect 16 pre sterilized bottle samples of

separately in such a way that each cavity of the mould of bottle pack machine.

Transfer the sample to QC department in clean plastic crates.

Wipe the sample article individually with 70%IPA solution and

Keep in clean SS trays marked with product name and transfer the sample to
the sterility room through clean pass box for performing sterility.
 Prepare the medium tube (FTM and SCDM). Sterilized both the media at 121
degree celcius and 15psi pressure for 20min.

After autoclaving label the tube with name of media, media batch no. and pre-
incubate the media tube at appropriate temp. i.e. SCDM tubes at 20-25 degree
celcius whereas FTGM tubes at 30-35 degree celcius for 24-48hrs before
subjecting them for sterility operation.

Autoclave dress, SS cups, scissors and forceps in a SS container at 121 degree

celcius temps. And 15psi pressure for 30 min.

After sterilization cool the contents and aseptically transfer in a SS container

to a cleaned pass box
.
Transfer the pre-incubate sterile media tubes through pass box.

Enter in sterility room as per entry exit ile membrane filter between filtration
cup and receptacle with the help of sterilized forceps.

Wet the membrane filter by adding approx 15ml 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 sterility 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 the membrane.

Immediately filter the solution with the aid of vacuum and wash the membrane
three times with 100ml 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 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
FTM and one half to SCDM tubes by unplugging in front of gas burner only.

Label both the tube with product name, B.no. Date of testing, completion date
and tested by.

Simultaneously prepare a negative control both test tubes (FTGM, SCDM).

Incubate the FTM tubes at 30-35 degree celciusfor 14 days and SCDM tubes
at 20-25 degree celcius for 14 days. As per IP (Indian pharmacopeia).
 OBSERVATION:-
The testing of drug was tested by the microbiological test. There are applying many
test-

MLT:-
Test for E.coli- No acid or gas production was observed in Macconkey broth.
Further no growth of red or non-mucoid colonies with metallic sheen was observed on
EMB plates. Hence the test for E.coli was found to be negative.

Test for Salmonella- No turbidity was observed in selenite f broth . Further,

no growth of black or green color colonies was observed on Bismuth sulphite agar
plate.
No growth of red with or without black center colonies were observed on Xylose
Lysine Dextrose Agar plates. Hence the test for Salmonella were found to be
negative.

Test for Pseudomonas- No turbidity was observed in SCDM tubes and no

formation of Greenish color colonies were observed on cetramide agar plates. Hence,
tests for Pseudomonas were found to be negative.

Test for Staphylococcus aureus- No turbidity was observed in SCDM

tubes. No growth of black, shiny surrounded by clear zone colonies observed on
Baired parker agar plates.

Sterillity Test :-
No turbidity was observed after incubation of filter membrane and incubation at 37 C.
Hence no growth of micro organism is observed.

Firm gel clot formation was observed in sterelity test BET tube, hence the sample
being tested complies with the BET test

The Sample being tested shown negative result for all the pathogens i.e. E.coli ,
Salmonella , Pseudomonas ,and S.aureus . It also show negative result for the test of
Pyrogens and Endotoxin. Negative test is also observed for aerobic and anaerobic
microorganism under sterility test.
All instrument is also calibrated properly there is no any unbalancing in the micro.
testing.
After performing the microbiological testing,; MLT, BET, Sterlity, GPT, Sampling,
testing Of sample Drugs it was observed that the sample being tested has no
microorganism or spore in it.
 RESULT:-

On the basis of Microbiological testing in lab, We conduct that , the work done in
laboratory is with much care and there is no chances of contamination in Pharma
products.

Hence the health of people is continully safe while using any product of Pharmacy.
The Pharma products are checked out at every stage for contamination i.e. Raw
material testing, testing during process and testing of final products, and antimicrobial
activity.The product is packed in a sterile codition under the guidelines of WHO,
FDA, IP, BP, United State Pharmacopeia.

So From My present study on Microbiological Testing Technique of

Pharmaceutical Drugs we observed that the Total Bacterial Count is less than 10
CFU/ml. No growth was observed on selective media plates All the pathogens i.e.
E.coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa were absent.
 REFERENCE:-

(1.)Indian Pharmacopeia (I.P.)

(2.)British Pharmacopeia (B.P.)

(3.)Prescott, Harley, Klein, Microbiology

(4.)U.S.P. (Union State Pharmacopiea)