A

PROJECT REPORT
ON
MICROBIOLOGICAL &QUALITY
ANALYSIS OF
dairy PRODUCTS
CARRIED OUT AT
MILK SPECIALITIES LIMITED CHANDIGARH(P.B.)
Submitted In Partial fulfillment of the requirement
For the award of Degree of
MASTER OF SCIENCE
In
MICROBIOLOGY
Submitted by
KALASH KUMAR
Department of Microbiology
Kunti naman


institute of pharma



ACKNOWLEDGEMENT
Words are very poor comforters to express the deep debt of gratitude which one
feels in ones corner of the Thank when one is helped to achieve the ultimate goal, in
this boundless & endless field of project work.
I would like to express my sincere gratitude and indeptness to
Prof (Dr.) NAVNEET, Head of Botany & Microbiology Department, Gurukul Kangri
University, Haridwar (Uttarakhand) whose constant guidance and suggestion helped
me throughout my M.Sc. Program and also my dissertation work.
I am Thankful to PSYCHOTROPICS INDIA LIMITED for permitted me for project
work at HARIDWAR Plant-I as a part of my M.Sc. Microbiology (IV Sem.).
I wish to take this opportunity to thank the following who have assisted with the
completion for selecting me as a candidate to complete my project work
I offer my cordial thanks to Mr. Suneel Chaudhari (H.R. Manager) and Mr. S.P.
Singh Chauhan (Q.C. Manager) for suggesting me to undergo training in Q.C.
Department.
I express my sincere thanks to Mr. Azad Singh (Sr. Executive Microbiologist)
and Miss. Neha Tyagi (Microbiologist) for their immense help and kind inspiration.
I offer my cordial thanks to Mr. Nitin Baliyan, Mr. Dharmendra Verma, Mr.
Neelesh Babu, Miss Diksha Juyal and Mr.Manikant for their constant encouragement
and co-operations throughout my project work.
I deeply convey my homage with regards to all Q.C. Staff members for their
constant encouragement, and valuable criticism throughout the course of my work and
beyond that.


(NEETU TYAGI)



















DECLARATION

I hereby declare that project entitled Microbiological Analysis of
Pharmaceutical Products undertaken by me is an authentic record of my own
work carried out at Psychotropics India Limited, SIDCUL, Haridwar,
Uttarakhand as a requirement of Internship Project for the award of degree of
M.Sc. (Microbiology) in Gurukula Kangri Vishwavidyalaya, Haridwar during
Jan to April 2014

DATE: Neetu tyagi
PLACE: Haridwar M.Sc. (Microbiology)














CONTENTS
An Overview Of The Company

Visit To PIL

Microbiological Abbreviation

Review Of Project

Basic Rules And Regulation

List Of Media Used In Microbiology

Instrumentation

Microbial Limit Test

Water Used For Pharmaceutical Purpose
1. Raw water
2. Purified water

Environment Monitoring
Discussion
Conclusion
Appendix
Reference



AN OVERVIEW OF COMPANY

COMPANY: Psychotropics India Limited
(PIL)

LOCATION: Plot No.46&49, Sector-6A, IIE,
SIDCUL, Haridwar,
Uttarakhand-249403
(INDIA)

MANUFACTURING: (BLOCK-1)
Solid Oral Dosage Forms-Capsules
And Tablets in general category.
Liquid Orals.

(BLOCK-2)
Medicated and Cosmetic Soaps.
Pet care soaps and shampoos.
External applications- lotions and
Creams.

MANAGMENTS: Mr. NAVDEEP CHAWLA
(Managing Director)
Mrs. AMITA CHAWLA
(Director)
Mr. SIDDHARTH CHWLA
(Director)
Mr. S.B. NAUTIYAL
(Technical Director)












COMPANY PROFILE:

Established in 1987, PIL was born out of the vision to create a
healthcare company that would address to the compassionate healthcare needs.
Psyhotropics is derived from our initial focus on psychiatry and tropical products, which
over the years have grown into various therapeutic segments like cardiovascular,
gynecology, dermatology, anti diabetic, antibiotics & anti invectives, antihistamine,
analgesics, acute pain killers, OTC products, pet care products & medicated soaps. Our
commitment and derive have boosted our growth from a single manufacturing facility at
Faridabad, Haryana to range of three more multi formulation manufacturing facilities in a
pollution free & lush green surrounding at Indias best industrial area at SIDCUL,
Haridwar, Uttarakhand, India.
With 800 plus highly motivated and skilled employees and a
world class manufacturing infrastructure the company is poised to establish itself as one
of the top Indian pharma company.
Due to our strong emphasis on product quality, PIL is an
approved vendor of some of our products to Government Institution like Govt. Health
Departments, Defence services, CDS canteens, Indian Railways and other public sector
undertakings hospitals in India. We at PIL are committed to develop new technologies.
We have been able to launch products with enhanced release profiles to ensure better
treatment compliance and dosage convenience. PIL has developed cinnarzine,
dexchlorphenramine maleate, metoprolol tartrate and metformin tablets in
sustained release form. After the success in domestic market, PIL has now ventured into
the overseas markets & is currently exporting its products to various international
destinations in West Africa, Middle East, Philippines, Vietnam and steadily increasing
our global presence.




VISIT TO PIL

I visited to psychotropics India Limited (PIL) which is located at
plot no. 46 & 49, sector-6A, IIE, SIDCUL Industrial area, Ranipur, Haridwar,
Uttarakhand.
After the security checking, we entered in Psychotropics India
Limited, as per rules, we covered our hands, shoes and wore aprons.
We learned the processing of preparing the medicines in the
different sections of the pharmaceutical plant such as Raw material section, tablet
coating section, compression section, granulation section, soap section, with QA and QC
departments. But some points observed in Psychotropics India Limited (PIL), are as
follows:-

The mobile phones are strictly prohibited.
Movement of material is unidirectional.
They maintain all Atmospheric conditions like Temperature (generally 25-
30 c) Pressure, Humidity etc.
All the processes were done by semi-automatic and automatic machines.
They were very careful about contamination.
They check the quality of material at all the stages.
They have well maintained documentation of each batch.



MICROBIOLOGICAL ABBREVIATION

Various microbiological abbreviation used in pharmaceutical industry.

USP = United State Pharmacopeia

BP = British Pharmacopeia

IP = Indian Pharmacopeia

AHU = Air Handling Unit

PVC = Poly Vinyl Chloride

RO = Reverse Osmosis

EDI = Electro Deionization

MGF = Multi Grade Sand Filter

MLT = Microbial Limit Test

WFI = Water for Injection

PWU = Purified Water Unit

PTW = Potable water

IPA = Isopropyl Alcohol

LAF = Laminar Air Flow

TBC = Total Bacterial Count

TFC = Total Fungal Count

CFU = Colony Forming Unit.









REVIEW OF PROJECT

INDIAN PHARMACOPOEIA:
The origin of Indian pharmacopoeia is concern to the
publication of Bengal pharmacopoeia in 1884, generally known as the Bengal
pharmacopoeia.
The pharmacopoeia was prepared by William Brook O
Shaughnessy and ambushed by the order of government. Its main focus was on the
indigenous drugs although it includes some products imported from Europe.
The Indian pharmacopoeia list 1946 was prepared to serve as
an India supplement to British pharmacopoeia 1932.
After independence an Indian pharmacopoeia committee was
constituted in 1948, which prepared the pharmacopoeia of India 1955. A supplement to it
was published in 1960. In this pharmacopoeia of India 1985 and 1991, tradition drug
were not included as publication of pharmacopoeia of traditional system drugs was taken
up separately and only those herbs included which supporting definitive quality control
standard.

GOOD MANUFACTURING PROCEDURE:
GMP is a system for ensuring that products are consistently
that produced and according to quality standards. It is designated to minimize the risk
involved in any pharmaceutical production that cannot eliminated through testing the
final products the main risk are unexpected contamination of products causing damage
to health or even death; incorrect labeling of container, which would mean that patient
receive the wrong medicines; insufficient or too much active ingredients, resulting in
infective treatment or adverse effects.
GMP covers all aspects of production from the starting
materials, premises and equipment to the training and personal hygiene of staff. Detailed
written procedures are essential for each process that could affect the quality of the
finished products. WHO has established detailed guidelines for good manufacturing
practice. Many countries have formulated their own requirements for GMP based on
WHO GMP.

QUALITY ASSURANCE:
Quality assurance is a wide ranging concept covering all
matters that individually or collectively influence that the quality of the arrangements
made with the object of ensuring that pharmaceuticals are the good quality required for
their intended use. Quality assurance there in corporate GMP and other factors.

QUALITY CONTROL:
Quality control is the part of GMP concerned with the sampling,
specification and testing with the organization, documentation and release procedure
which ensure that the necessary and relevant steps are actually carried out and that
materials are not released for use products released for safe or supply, until their quality
has been judged to be satisfactory.
Quality control is not confirm to the laboratory operations but
also involved in all the decisions covering the quality of the product. The independence
of quality control from production is considered fundamental.

PRESENT STATUS:
PIL is a good rank in pharmaceutical products business and
among the top in total market in India and abroad. Its products are being accepted in
greasingly by specialist worldwide.



BASIC RULES AND REGULATION

There are some rules and regulations which must be observed for the successful
completion of the laboratory exercise, personal safety during when
students/researchers/microbiologist/scientists working with micro organisms.
A microbiology laboratory is a place for working with a variety of microorganism
since several culture media are prepared and organic materials are present, the chances
exist for the presence of high spectrum of microbial community. Secondly, while working
with pure culture one should always follows the microbiological rules so that neither the
experiment should be unsuccessful nor any hazard may occur. If a large number of
students are working in a microbiology laboratory, they should be aware what to do and
what not? What are the apparatuses/instruments/equipments present in the laboratory
and what is there functioning? What are the chemical solutions and stains and how to
handle? How should the students enter in the microbiology lab and now should they
work? Therefore, the fresher such as students, microbiologists, teachers, laboratory
assistants and helpers must follow the following guidelines
1. Always wear an apron before entering the microbiology laboratory to protect from
microbial contamination and laboratory hazards. At regular intervals get the
apron washed.
2. Cut nails regularly.
3. Tie long hairs back to avoid contamination and fire hazard.
4. Keep your working laboratory bench clean of everything. Nothing should be
leaving on the bench.
5. Never keep books, purses, bags, etc on the working bench.
6. Always wash your hands with soap and disinfectant before and after the work.
7. Clean your working bench with ethanol (70%) or Phenol (1:100)
8. Never spit and smoke in the laboratory.
9. Dont put anything of the laboratory (e.g. Pencil, thread, labels, inoculations
needle, pins etc) in your mouth, ears, nose and eyes.
10. Dont put your finger in your eyes, ears, mouth. It may facilitate the chance of
infection by pathogenic microorganism.
11. Dont eat or drink or talk while working with microorganisms.
12. Dont mishandle the chemical solutions, stains, sprit lamp, UV light,
instruments/apparatus or electricity.
13. Always maintain aseptic condition while working with microorganisms.
14. Always use flame sterilized inoculation needle/loop.
15. Dont open the culture tubes/plates directly and never inhale them nor observe
with naked eyes.
16. Open the culture tubes/plates near the vicinity of flame of the burner.
17. While working with broth cultures dont such the suspension with mouth. Always
use pipette sucker.
18. After completion of work always label the cultures with names, code and date of
work.
19. Never leave your cultures on working bench or seat.
20. Clean the working bench/seat when the work is completed.
21. Clean lenses of objective with tissue paper.
22. Keep the stains, reagents, stock culture to their respective places when the work
is completed.
23. After completion of work keep your slide/pipette/culture tubes/plates in container
and steam sterilize before washing.
24. Record your result at time.







LIST OF MEDIA USED IN MICROBIOLOGY

Soyabean Casein Digest Agar (SCDA)
Soyabean Casein Digest Medium (SCDM)
Sabouraud Dextrose Agar (SDA)
MacConkey Agar (MCA)
MacConkey Broth (MCB)
Levine Eosin-Methylene Blue Agar
Tetrathionate Bile Brilliant Green Blue Broth
Bismuth Sulphite Agar
Brilliant Green Agar
Deoxycholate Citrate Agar
Cetrimide Agar (CA)
Pseudomonas Agar (Flouroscence)
Pseudomonas Agar (pyocynin)
Vogel Johnson Agar
Mannital Salt Agar (MSA




INSTRUMENTATION

List of equipment / apparatus used in microbiology laboratory:

1. Autoclave,
2. Incubator,
3. Hot air oven,
4. Inoculating loop,
5. Vortex mixer/shaker,
6. Water bath,
7. Hot plate with magnetic stirrer,
8. pH meter,
9. Bunsen Colony counter,
10. Microscope,
11. Refrigerator,
12. Burner,
13. Spirit lamp,
14. Balance (Digital and 4-beam),
15. Thermometer,
16. Membrane filter set

DESCRIPTION:
Autoclave
It is a robust, electrically heated steam vessel meant for sterilizing thermo stable
culture media, glassware, and other materials that are not spoiled by moist heat.
Autoclave runs on the principle of pressure cooker. The moist heat (steam) has a very
good penetrating power. Microorganisms/ cells are killed as a result of denaturation of
cellular constituents (protein and nucleic acids). In routine process, sterilization can be
achieved by operating the autoclave at 121C (15 psi) for 15 min.
In its simplest form, the equipment has a removable lid for the delivery of
materials to be sterilized. It is necessarily equipped with a gasket, pressure-cum-
temperature gauge, a vent for letting out air or excess pressure, a safety valve and a
drain. The figure of a portable autoclave is given in Fig.1.




Incubator
This an insulated, electrically heated cabinet meant for providing microorganisms
with optimum temperature for growth. The cabinet is insulated and thermostatically
controlled. For routine purposes, the temperature is maintained at 30-35C for bacteria,
about 20-25C for moulds, and 35-37C for mesophilic bacteria. A temperature as high
as 100C can also be maintained for extremely thermophilic organisms
(stereothermophiles).A very common laboratory incubator is shown in Fig.2

Hot air oven
This is similar to incubator in make except that it can operate at temperatures up
to 200C and has a fan for circulating hot air. Hot air oven is used for sterilization of
glassware and materials that are spoiled by moist heat.
The death of cells occurs due to the oxidation of cellular constituents by the dry
heat.
For routine purpose, sterilization can be achieved by running the equipment at
180C for 1.5 hours. Hot air oven is less effective than autoclave. Fig.3 shows a typical
hot air oven popular in microbiology laboratories.

Inoculating loop
This is a tool for transferring and streaking cultures. It consists of a thin nichrome
wire whose one end is twisted into a small loop while the other end is fixed to a thermo
set plastic handle. Sometimes, the looped end is straightened out to form what is called
inoculating needle. Inoculating needles are used for preparing stab cultures. Fig. 4
shows inoculating loops and needles.



Vortex mixer
This equipment is used for mixing liquids kept in a test tube. It has one or more
cup-like depressions at the top to receive the bottom of the test tube. The machine is
electrically powered. When actuated, the machine moves the bottom of the test tube in a
gyratory motion, thereby affecting a thorough mixing of the solution. The speed of the
mixer can be varied. Fig.5 shows a typical vortex mixer.


Water bath / Boiling water bath
Water bath is used for heating and melting of media, solutions, samples etc. at
temperatures below 100C. It can also be used to maintain constant temperature that is
required in microbiology lab work. Several models and types of water bath are available.
Fig. 6 shows a typical water bath commonly used in laboratories. It is electrically heated
and thermostatically controlled.

Heating mantle
It is an electrically heated and thermostatically controlled unit used to heat or melt
samples and reagents. The inner lining is made of asbestos and therefore gives an
indirect heat to the materials to be heated. Fig. 7 shows a typical heating mantle being
used for heating water in a beaker.

Hot plate with magnetic stirrer
This is an electrically powered equipment performs the dual function of heating
and agitation. The agitation occurs by magnetic arrangement. Any type of glassware can
be used for the heating and agitation. Magnetic beads are used for the agitation.





pH meter
pH meter is an electrical instrument used for measuring hydrogen ion
concentration of solutions and mixtures (Fig. 11). In microbiology lab, it is used for
maintaining pH of the medium and diluents. The pH meter must be standardized with
buffer solutions before operation. Since the instrument is very sensitive, it must not be
used for stirring and it must not be dipped in hot or very cold solutions. The electrodes
must always be kept immersed in suitable solutions. Read the manual carefully before
using the instrument.


Colony counter
It is used for counting microbial colony (bacterial and yeast). The instrument is
equipped with a backlight source, gridlines and a magnifying lens. It also has a sensor
for digitally registering the number of colonies counted (Fig.12).

Microscope
It is an instrument for observing microscopic items such as cells, crystals and cell
organelles. It has the dual function of magnification and resolution. For routine
microbiological works, bright field compound microscope with oil immersion objective is
adequate. A compound microscope is shown in Fig. 13.


Refrigerator
This is a common household equipment for keeping foods and beverages cool.
This equipment is used in microbiology laboratory for storing / preserving cultures,
media, and many sensitive materials (Fig. 14). The equipment is electrically powered
and uses ammonia as the refrigerant.


Bunsen burner
Bunsen burner is a common tool used in science lab (Fig. 15). In microbiology
lab, it is used for sterilizing inoculating loop, plating out cultures, transferring cultures,
heat-fixing of smears and creating a sterile zone for aseptic operation.



Spirit lamp
The function of spirit lamp is the same as the Bunsen burner but is portable. It uses
rectified spirit as the fuel (produces smoke-free flame). The lamp must be covered with a
lid when not in use to prevent loss of spirit (Fig.16).



Balance
Balance is needed in microbiology lab for weighing chemicals, samples, media,
etc. Digital balances are fast tonwork with but needs frequent calibration (Fig. 18).
The triple-beam and 4-beam balances are robust equipment that need little care
and maintenance. Beam balances run on mechanical principles while the principles on
which electronic balances run is quite complicated (Fig. 19).

Thermometer
Thermometers are required to ensure the heating equipment is running at the
correct temperature. The temperature of the medium, incubator, etc., need to be
frequently checked. Mercury in glass thermometers are standard.



Coli form membrane filter
This glass equipment is used for the testing of coli forms in water (Fig. 20). 100
ml of test water is poured in the funnel and filtered through a special Millipore filter
through external application of suction. The filter retains the microorganisms. The filter is
then aseptically transferred to a selective-cum-differential semisolid medium kept in a
Petri dish. If there are coli forms, they will appear as pink dots after incubation at 35C
for 22 hrs.















MICROBIAL LIMIT TEST

The Microbial Limit 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 tests for specific
microorganisms (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus &
Salmonella).
The most care must be taken in performing the tests, so that microbial
contamination from the outside can be avoided.
This procedure is applicable for microbiological test of Raw material & finished
products to microbiology lab of quality control department.

PROCEDURE:
TOTAL VIABLE AEROBIC COUNT:
It determines the mesophilic bacteria & fungi that grow under aerobic conditions.
Psychrophilic, thermophilic, basophilic & anaerobic bacteria which require specific
nutrients for growth, may give negative result.
Total Bacterial Count
Fungal Count (Yeast and Mold)

TEST FOR SPECIED MICROORGANISMS:
Escherichia coli
Salmonella
Pseudomonas aeruginosa
Staphylococcus aureus


PRETREATMENT OF SAMPLE:-
Water Soluble Products:

Dissolve or suspend 10gm/ml of sample buffered sodium chloride peptone solution
pH 7.2 or soyabean casein digest broth or any other suitable medium shown to have
antimicrobial activity adjust the volume to 100ml with same diluents (solution A). A
surface active agent such as 1g/l of polysorbate 80 may be added to assist the
suspension of poorly wettable substance. If necessary, adjust pH 6 to 8.
Non Fatty Products Insoluble in Water:
Dissolve or suspend 10gm/ml of sample buffered sodium chloride peptone
solution pH 7.2 or soyabean casein digest broth or any other suitable medium
shown to have antimicrobial activity adjust the volume to 100ml with same
diluents (solution A). A surface active agent such as 1g/l of polysorbate 80 may
be added to assist the suspension of poorly wettable substance. If necessary,
adjust pH 6 to 8.
Fatty Products:
Suspended 10gm or 10ml of sterile polysorbate 80 and heated if
necessary to a temperature not exceeding 45 C. Add 85ml of Buffered sodium
chloride peptone solution pH 7.0 or phosphate buffer solution 7.2 or soyabean
casein digest broth or any other suitable medium to have no antimicrobial activity
adjust the volume to 100ml with same diluent (solution A).

TOTAL VIABLE AEROBIC COUNT (By pour plate method):
Total Bacterial Count:

Media Used: SCDA
Incubation Temperature: 30-35C
Incubation Time: 5 days
Sample taken: 10ml/gm
Media Quantity: 15-20ml

Petridishes of 90mm in diameter. Add to each Petridish 15-20ml SCDA that has
been previously melted and cooled at 45C. If larger petridishes are used increase
quantity of agar medium accordingly. Cover the petridishes and mix the sample with
agar by rotating the plates. Allow the contents to solidify at room temperature. Invert the
petridishes and incubate at 30-35 C for 5 days.

Total Fungal Count:

Media Used: SDA
Incubation Temperature: 20-25C
Incubation Time: 5 days
Sample taken: 10ml/gm
Media Quantity: 15-20ml

Pipette 1ml of solution A into two sterile petridishes of 90mm in diameter. Add to
each Petridish 1520ml SDA should be increased accordingly. Cover the petridishes
and mix the sample with agar by rotating the plates. Allow the contents to solidify at
room temperature. Invert the petridishes and incubate at 20-25 C for 5 days.


Pouring of media with SDA/SCDA
TEST FOR SPECIED MICROORGANISMS:
Test for Escherichia coli:
Transfer 10 ml of solution A corresponding to 1gm or ml of the sample to 90ml of
SCDM and incubate at 30-35C for 18-24 hours (solution B). After complete the
incubation shake the container and transfer 1ml of enriched medium solution B to 100ml
of MacConkey broth. Incubate at 42-44C for 24-48 hours.
If acid and gas produced then subculture on a plate of MacConkey agar and
incubate at 30-35C for 18-48 hours. Growth of red, generally non mucoid colonies of
Gram-negative rods, sometimes surrounded by a reddish precipitation zone, indicates
the possible presence of E.coli.
Confirm the presence of E.coli by inoculating the suspended colonies into 5ml of
peptone water and incubating at 43.5-44.5 C for 24 hours in water bath.
To test for indole, add 0.5ml of Kovacs reagent. Shake well and allow standing
for 1 minute. If a red colour is produced in the reagent layer, indole is present. The
product passes the test if such colonies are not seen or if the confirmatory biochemical
tests are negative.




Colonies of E.coli on different media


Gram stain of E.coli




Indole Test (Positive) Indole Test (Negative)
Test for Salmonella:
If specified in monograph/specification Salmonella should be absent per gram of the
sample.
Transfer 10ml of solution A corresponding to 1gm or ml of the sample to 90ml of
SCDM and incubate at 30-35 C for 18-24 hours.
Primary Test:
Shake the container and transfer 0.1ml of the desired dilution i.e. solution B or
solution C to 10ml of Rappaport Vassiliadis Salmonella Enrichment Broth and incubate
at 30 C for 18-24 hours. If any colony confirming to the description in Table-1 are
produced carry out the secondary test.

Table-1
Medium Description of colony
Xylose lysin deoxycholate agar Red with or without black centers

Secondary Test:
Subculture any colony showing characteristics given in Table-1 on Triple
Sugar Iron Agar using surface and deep inoculation. Incubate at 35-37 C for 18-24
hours. The presence of Salmonella is previously confirmed if, in the deep culture but in
surface culture. There is the change of colour from red to yellow accomplished by gas
formation with or without blackening. Precise confirmation may carry out by appropriate
biochemical and serological tests. The preparation being examined passes the test, if, in
the primary test, cultures of the type description do not appear or if, in the secondary
test, the confirmatory biochemical and serological test are negative.


Salmonella on TSIA Salmonella on XLDA

Test for Pseudomonas aeruginosa:
Subculture solution B on a plate of Cetrimide agar and incubate at 30-35
C for 18-72 hours. If any colony confirming to the description in Table-2 are
produced, carry out pigment test and oxidase test. If no growth of microorganism
is detected, the preparation being examined passes the test.
Table-2

Medium Characteristic Fluorescence in
UV light
Gram stain Oxidase
Cetrimide Agar Generally
greenish
Greenish -ve rods Purple
P. Agar
(Fluoroscein)
To yellowish Yellowish -ve rods Purple
P. Agar
(Pyocyanin)
Generally
greenish
Blue -ve rods Purple

Oxidase Test:
Place 2 to 3 drops of a freshly prepared 1% solution of NNNN
tetramethyl-p-phenylene diamine dihydrochloride on a piece of filter paper (Whattman
No.1 is suitable) and smear the suspect colony on these papers.
If a purple colour is produced within five to ten second, the test
is positive, which indicates the presence of P. aeruginosa. Oxidase disc available from
HI-MEDIA or by other supplier may be used.
Pigment Test:
Streak out representative suspect colonies given in Table-2 from the agar
surface of Cetrimide agar medium for detection of fluoscein and Pseudomonas agar
medium for detection of pyocyanin contained in petridishes. Incubate at 35-37 C for not
less than 3 days. Examined the streaked surfaces under ultra violet light presence of
colonies confirming to the description in Table-2 indicated the presence of P.
aeruginosa.


P.aeruginosa on cetramide agar Gram stain of P.aeruginosa

Test for Staphylococcus aureus:
Subculture on preincubated Mannital Salt Agar and incubated
at 30-35 C at 18-72 hours. If any colony confirming to the description in Table-3 are
produced, carry out the coagulate test. If no growth of microorganism is detected the
preparation being examined passes the test.
Table-3
Medium Characteristics Gram stain
Mannital salt agar Yellow colonies with
yellow zone
Positive rods


Coagulate Test:
Transfer representative suspected colonies given Table-3 two individual
tubes, each containing 0.5ml of Mammalian plasma under LAF. Incubate in a
water bath at 35-37 C, examining the tubes at 3 hours and subsequently at
suitable intervals up to 24 hours. If coagulation in any degree is observed, the
test is positive.


S.aureus on Mannital salt agar Gram stain of S.aureus












WATER USED FOR PHARMACEUTICAL PURPOSE

Water is the most widely used substances, raw material or ingredients in
the production, processing & formulation of compendial article. Control of
microbiological quality of these water is important because proliferation of
microorganisms. Ubiquitous to water may occur during the proliferation, storage
& distribution of the substances. If water is used in the final product, these
microorganisms or their metabolic product may eventually cause adverse
consequence.
TYPES OF WATER:
Raw Water
Purified Water

Raw Water:
Water is one of the main substances for any pharmaceutical product. It may
provide by municipal supply or by any local agency. Sometimes private well are main
source of water. The water as in its natural form is termed as the raw water. Raw water
(drinking water) used in manufacturing in the pharmaceutical products. This water is
pretreated for its transfer in purified water by multi-grade filtration or Quartz filtration,
softening of water chlorination and many other dosing by chemical such as SMBS as per
specification.

Purified Water:
Purified water is used an excipients in the production of official preparation in the
pharmaceuticals application such as cleaning of certain equipments & in the preparation
of some bulk pharmaceutical chemicals. Purified water must meet the requirements for
ionic & organic chemical purity & must be protected from microbial proliferation. It is
prepared using drinking water as feed water & is purified using unit operation that
includes deionization, distillation ion-exchange, reverse osmosis, filtration or other
suitable procedure. Purified water system must be validated. Purified water system that
produce store &circulate water under ambient condition are susceptible to the
establishment of Tenacious biofilm microorganism, which can be the source of
undesirable levels of viable microorganisms or end toxin in the effluent water. These
systems require frequent sterilization & microbiological monitoring to ensure water of
appropriate microbiological quality at the point of use.

ANALYSIS OF WATER (Raw, Potable and Purified water):
Collect the 250ml of raw, purified and potable water from predetermined location
in sterilized bottle.

Test for Total Viable Aerobic Microbial Count:

Detection of Bacteria:
Media Used: SCDA/R
2
A
Incubation Temperature: 30-35C
Incubation Time: 5 days
Sample taken: 1ml
Media Quantity: 15-20ml
Detection of fungi:

Media Used: SDA/R
2
A
Incubation Temperature: 20-25C
Incubation Time: 5 days
Sample taken: 1ml
Media Quantity: 15-20ml

By pour plate method (For Raw, Potable and Purified Water):
1. Pour 1.0ml of raw and potable water in four plates, two for fungi and two
for bacteria.
2. Pour 15-20ml of previously cooled (NMT 45C) SCDA and SDA for bacteria and
fungi.
3. Cover petridishes, mix the sample by rotating disc clockwise & anticlockwise and
then allow the content to solidify at room temperature.
4. Incubate the petridishes in invert position for 5 days at 30-35C and 20-25C.
5. After incubation count the colony and record the results.













Pouring of Media

By Membrane Filtration Method (For Purified Water):

1. Add 1.0ml of purified water sample in sterile flask in duplicates containing 100ml
of cooled sterile purified water, under aseptic condition.
2. Mix it properly & filter it immediately through sterilized filtration assembly having
membrane filter of porosity 0.45, diameter 47nm rinsing the membrane twice
with sterile purified water.
3. Prepare blank preparation by filtering 100ml of cooled sterile purified water used
for dilution and rinsing through membrane in sterile filtration assembly.
4. Place filter paper aseptically on preincubated R
2
A agar plate press the filter
paper slightly with the help of sterile forcep.
5. Label the plates properly and incubate it at 30-35C in inverted position for 5
days. After incubation count the colony and record the results.

Calculation:

Total Viable Aerobic Count = Average Count/2


Membrane Filtration Method

Diagrammatic sketch with a Disposal Presterilized Plastic Filtration Assembly

Pathogen Testing For Raw, Purified and Potable Water:
i. Transfer 100ml water to be tested in sterilized filtration assembly and filter
it immediately through sterilized membrane rinse the assembly with 100ml
sterile purified water and filter it through the same filter paper.
ii. After filtration, aseptically transfer the filter paper to 100ml SCDM for
enrichment purpose and incubate the tube for 24-48 hours at 30-35C
(solution A).
Test for Escherichia coli:
1. Transfer 10 ml of solution A corresponding to 1gm or ml of the sample to 90ml of
SCDM and incubate at 30-35C for 18-24 hours (solution B). After complete the
incubation shake the container and transfer 1ml of enriched medium solution B to
100ml of MacConkey broth. Incubate at 42-44C for 24-48 hours.
2. If acid and gas produced then subculture on a plate of MacConkey agar and
incubate at 30-35C for 18-48 hours. Growth of red, generally non mucoid
colonies of Gram-negative rods, sometimes surrounded by a reddish precipitation
zone, indicates the possible presence of E.coli.
3. Confirm the presence of E.coli by inoculating the suspended colonies into 5ml of
peptone water and incubating at 43.5-44.5 C for 24 hours in water bath.
4. To test for indole, add 0.5ml of Kovacs reagent. Shake well and allow to stand
for 1 minute. If a red colour is produced in the reagent layer, indole is present.
The product passes the test if such colonies are not seen the confirmatory
biochemical tests are negative.

Colonies of E.coli on different media

Stain of E.coli

Indole Test (Positive) Indole Test (Negative)


Test for Salmonella:
If specified in monograph/specification Salmonella should be absent per gram
of the sample.
Transfer 10ml of solution A corresponding to 1gm or ml of the sample to 90ml of
SCDM and incubate at 30-35 C for 18-24 hours.

Primary Test:
Shake the container and transfer 0.1ml of the desired dilution i.e. solution B or
solution C to 10ml of Rappaport Vassiliadis Salmonella Enrichment Broth and incubate
at 30 C for 18-24 hours. If any colony confirming to the description in Table-1 are
produced carry out the secondary test.

Table-1
Medium Description of colony
Xylose lysin deoxycholate agar Red with or without black centers

Secondary Test:
Subculture any colony showing characteristics given in Table-1
on Triple Sugar Iron Agar using surface and deep inoculation. Incubate at 35-37 C for
18-24 hours. The presence of Salmonella is previously confirmed if, in the deep culture
but in surface culture. There is the change of colour from red to yellow accomplished by
gas formation with or without blackening. Precise confirmation may carried out by
appropriate biochemical and serological tests. The preparation being examined passes
the test, if, in the primary test, cultures of the type description do not appear or if, in the
secondary test, the confirmatory biochemical and serological test are negative

Salmonella on TSIA Salmonella on XLDA


Test for Pseudomonas aeruginosa:
Subculture solution B on a plate of Cetrimide agar and incubate
at 30-35 C for 18-72 hours. If any colony confirming to the description in Table-2 are
produced, carry out pigment test and oxidase test. If no growth of microorganism is
detected, the preparation being examined passes the test.


Table-2
Medium Characteristic Fluorescence in
UV light
Gram stain Oxidase
Cetrimide Agar Generally
greenish
Greenish -ve rods Purple
P. Agar
(Fluoroscein)
To yellowish Yellowish -ve rods Purple
P. Agar
(Pyocyanin)
Generally
greenish
Blue -ve rods Purple
Oxidase Test:
Place 2 to 3 drops of a freshly prepared 1% solution of NNNN
tetramethyl-p-phenylene diamine dihydrochloride on a piece of filter paper (Whattman
No.1 is suitable) and smear the suspect colony on these papers.
If a purple colour is produced within five to ten second, the test
is positive, which indicates the presence of P. aeruginosa. Oxidase disc available from
HI-MEDIA or by other supplier may be used.
Pigment Test:
Streak out representative suspect colonies given in Table-2
from the agar surface of Cetrimide agar medium for detection of fluoscein and
Pseudomonas agar medium for detection of pyocyanin contained in petridishes.
Incubate at 35-37 C for not less than 3 days. Examined the streaked surfaces under
ultra violet light presence of colonies confirming to the description in Table-2 indicated
the presence of P. aeruginosa.

P.aeruginosa on cetramide agar Gram stain of P.aeruginosa

Test for Staphylococcus aureus:
Subculture on preincubated Mannital Salt Agar and incubated
at 30-35C at 18-72 hours. If any colony confirming to the description in Table-3 are
produced, carry out the coagulate test. If no growth of microorganism is detected the
preparation being examined passes the test.

Table-3

Medium Characteristics Gram stain
Mannital salt agar Yellow colonies with
yellow zone
Positive rods

Coagulate Test:
Transfer representative suspected colonies given Table-3 two individual
tubes, each containing 0.5ml of Mammalian plasma under LAF. Incubate in a
water bath at 35-37 C, examining the tubes at 3 hours and subsequently at
suitable intervals up to 24 hours. If coagulation in any degree is observed, the
test is positive.


S.aureus on Mannital salt agar Gram stain of S.aureus





ENVIRONMENTAL MONITORING
(E. M.)
Environmental monitoring is done to check the bioburden of the aseptic
area of the controlled environment. The purpose of this is to understand the
various issues that is related to aseptic processing of bulk drug substance or
finished products (sterile), dose forms, and in certain cases and to
establishments maintenance and control of the microbiological quantity in the
controlled environment.

Methods of E.M.:
1. By Settle Plate Technique
2. By Active Air Sampler
3. By Swab Technique.

Different Type Area for E.M.:
1. Clean Room
2. Rest Clean Room
3. Operation Clean Room
By Settle Plate Technique:
Principle:
It is based on the fact that in the absence of any kind of influence, air
borne microorganism, typically attached to larger particles, will deposit onto open
culture plate.
Requirements:
Incubator
Marker
SCDA Plates
LAF
Colony counter etc.
Procedure:
1. Firstly prepare SCDA media.
2. Media and glassware were sterilized by autoclaving
3. Aseptically pour approximately 15-20ml of sterilized molten, cooled (40c)
SCDA agar in sterile 90mm Petri dishes.
4. Leave the Petri dishes under LAF for about half an hour for solidifying the
media.
5. Incubate these media plates in inverted position at 30-35c for 24 hours.
6. After completion of incubation period examine the media plates for any
contamination .if growth of microorganism and observed on the surface of
plates then discard those plates.
7. Label the plates with following information before sampling e.g. location
no, media name, media batch no. and sampling date etc.
8. After labelling place the Petri plate aseptically in the Petri dish container
and carry it aseptically to the sampling area.
9. Expose the plates containing media at the designated location for 4 hours
and ensure that the surface of agar should be faced towards the
environment.
10. Remove the lid of Petri dishes and place it in the adjacent position.
11. Note down the time of plate exposure.
12. After completion of exposure time, aseptically close the lid of all
plates.
13. Collect aseptically all plates in ss container and note down the time
of plate collection.
14. Aseptically incubate the plates at 20-25c for 72 hours (for fungus)
and further 30-35c for 48hours (for bacteria). After incubation count the
no. of colony formation (cfu) per location with the help of colony counter.
By Active Air Sampler:
Principle:
Microorganisms of different size remain present in the environment. Larger
particles easily deposit on open culture media, while light particles do not settle.
For this purpose air sampler is used. Air sampler take out 1000 liter of air in 6
minute.
Requirements:
Air Sampler
Incubator
Marker
SCDA Plates
LAF
Colony counter etc.

Active Air Sampler
Procedure:
1. Firstly prepare SCDA media.
2. Media and glassware were sterilized by autoclaving
3. Aseptically pour approximately 15 -20ml of sterilized molten, cooled (40c)
SCDA agar in sterile 90mm Petri dishes.
4. Leave the Petri dishes under LAF for about half an hour for solidifying the
media.
5. Incubate these media plates in inverted position at 30-35c for 24 hours.
6. After completion of incubation period examine the media plates for any
contamination .if growth of microorganism and observed on the surface of
plates then discard those plates.
7. Label the plates with following information before sampling .e.g. location
no, media name, media batch no. and sampling date etc.
8. After labeling place the Petri plate aseptically in the Petridis container and
carry it aseptically to the sampling area .Disinfect the cover of the air
sampler at the beginning of each sampling program treating the inside and
outside part with 70%IPA solution.
9. Remove the cover of air sampler by unscrewing it, holding the edge of the
cover .Avoid touching inside and outside of drilled area.
10. Insert a closed pre incubate SCDA plate into the retaining slot and then
remove its lid .avoid contamination from droplets.
11. Replace the sampling head and operate the air sampler.
12. Set the instrument for sampling of 1000 litters of air and start the sampling.
13. Exposure time 6 minutes for thousand litters of air.
14. Note down the time of plate exposure
15. After completion of the air sampling close the lid of all plate.
16. Collect aseptically all plates in ss container.
17. Aseptically incubate the plates at 20-25c for 72 hours (for fungus) and
further 30-35c for 48hours (for bacteria) .After incubation count the no. of
colony formation (cfu) per location with the help of colony count.

By Swab Technique:

Principle:
Surface may become contaminated in a no. of ways e.g. Microorganism settling
out from the environment or from the direct touch by an operator. One of the objectives
of surface sampling is to determine the efficiency of routine cleaning procedures in
removing contamination .therefore sampling should be performed before and after
cleaning to determine the effectiveness of cleaning procedure.

Requirements:
Swab sticks
SCDA plates
Sterile normal saline solution
LAF
Incubator
Autoclave etc.


SWAB
Procedure:
1. Take swab and in 100ml of 0.9% NaCl solution take in a tube and autoclave all
swabs at 121C for 15 minute at 151 psi pressure in a glass beaker.
2. Cool all the swab tubes in a beaker &store in BOD incubator at 20-25C, beaker
covered with aluminum foil, now sterile swab checked before subjecting them for
E.M.
3. Monitor the required equipment or sterile gown surface by swabbing an area
approx 22 (i.e.25cm
2
) area, uniformly and horizontally in one direction. Clean
the swabbed areas with 0.20 filtered 70% IPA solution & allow it to dry.
4. Replace the swab in sterile pp tubes immediately. Repeat similar swabbing
operation for all sampling location. After swabbing a over bring all tubes
containing swab dipped in normal saline to the microbiology lab & transfer to the
LAF for further testing.
5. Now shake the normal saline of each tube reciprocally in such way that any
particle struck on the swab surface should get dispersed properly in normal
saline.
6. Now remove the swab gently under the LAF pour the normal in saline on the
sterile petriplate marked with sampling location. Pour gently approx 20ml of
autoclaved SCDA media on the plate & rotate it in order to mix contents.
7. Allow the plates to solidify under LAF & and after solidification transfer all the
plates for incubation at 20-25C for 72 hours and at 30-35C for 48 hours.
8. Following incubation period, examine all plates under colony counter & count the
no. of colonies.














DISCUSSION


1. In MLT the product meets the requirements with their respective products
released specifications. Therefore sample can be used in pharmaceutical
products like capsules and tables.

2. In MLT for water the products meets the requirements with their respective
products released specifications. Therefore these water samples can be used in
pharmaceutical purpose like equipments washing and other purpose.

3. To detect the aseptic condition in production area, environment monitoring was
products. The number of colonies was less than the alarming rate in aseptic area
was perfect.








CONCLUSION


Study comprises of two things i.e. theory and practical. Theory is incomplete
without doing practical, performing practical in labs is different but industry has got its
own importance. Pharmacy is a profession in which you are dealing with the life of
human being so we can not take risk. Any type of irresponsible behavior can leads to a
major problem.
After performing water chemical testing, microbial limit test, and antibiotic
assay on the given sample of water, it was observed that the sample being tested
has no micro organisms or spores in it.
No growth was observed on selective media plates hence the sample being
tested complies with MLT test and pathogens i.e. E. coli , Salmonella, Pseudomonas
and Staphylococcus aureus are found to be absent in the sample being tested.
Hence, it is concluded that the sample being tested complies with the USP and
IP standards and is considered to be sterile.
The given drug is found to be effective in checking the growth of bacteria as clear
zones are observed in antibiotic assay.
On comparing the results obtained with the standard table, it is found that the given
sample falls within the standard range and hence complies with the standards of Pharma
industry.






APPENDIX

1) Alternative Thioglycollate Medium

L -Cystine 0.5 gm
Sodium chloride 2.5 gm
Dextrose 5.5 gm
Yeast Extract (water soluble) 5.0 gm
Pancreatic Digest of Casein 15.0 gm
Sodium thioglycollate or thioglycolic Acid 0.3 gm
Distilled water 1000 ml.
The pH after sterilization is 7.10.2

2) Baird Parker Agar

Pancreatic digest of casein 10.0 gm
Beef extract 05.0 gm
Yeast extract 01.0 gm
Lithium chloride 05.0 gm
Agar 20.0 gm
Glycine 12.0 gm
Sodium pyruvate 10.0 gm
Water 1000 ml

3) Bismuth Sulphite Agar

Solution 1:
Beef extract 06.0 gm
Peptone 10 gm
Agar 24 gm
Ferric (III) citrate 0.4 gm
Brilliant green 10.0 mg
Water 1000ml


Solution 2:

Ammonium bismuth citrate 03.0 gm
Sodium sulphate 10.0 gm
Anhydrous disodium hydrogen phosphate 5.0 gm
Dextrose monohydrate 5.0 gm
Water 1000 ml
Mix, heat to boil, cool to room temperature, add 1 volume of
solution (2) to 10 volumes of solution (1).

4) Brilliant Green Agar

Peptone 10.0 gm
Yeast extract 03.0 gm
Lactose 10.0 gm
Sucrose 10.0 gm
Sodium chloride 5.0 gm
Phenol red 0.08 gm
Brilliant green 12.5 mg
Agar 12.0 gm
Water 1000 ml

5) Cetrimide Agar

Pancreatic digest of gelatin 20.0 gm
Magnesium chloride 01.4 gm
Potassium sulphate 10.0 gm
Cetrimide 0.3 gm
Agar 13.6 gm
Glycerin 10.0 gm
Water 1000 ml


6) Deoxycholate Citrate Agar

Beef extract 05.0 gm
Peptone 05.0 gm
Lactose 10.0 gm
Tri sodium citrate 8.5 gm
Sodium thiosulphate 5.4 gm
Ferric (III) citrate 1.0 gm
Sodium deoxycholate 5.0 gm
Neutral red 0.02 gm
Agar 12.0 gm
Water 1000 ml

7) Eosine Methylene Blue Agar

Tryptone 10.0 gm
Dipotassium phosphate 2.0 gm
Lactose 5.0 gm
Sucrose 5.0 gm
Eosin Y 0.4 gm
Methylene blue 0.065gm
Agar 13.5 gm


8) Fluid Thioglycollate Medium

L cystine 0.5 gm
Sodium chloride 0.5 gm
Glucose monohydrate 5.5 gm
Granular agar 0.75 gm
Yeast extract 5.0 gm
Pancreatic digest of casein 15.0 gm
Sodium thioglycollate 0.5 gm
Resazurin (1.1% fresh solution) 1.0 ml
Water 1000ml

9) Lactose Broth

Beef extract 3.0 gm
Pancreatic digest of gelatin 5.0 gm
Lactose 5.0 gm
Water 1000 ml



10) MacConkeys broth

Peptone 20 gm
Sodium Chloride 5 gm
Sodium taurocholate 5 gm
Water 1000 ml
Bromocresol purple solution 10 ml
Lactose 10 gm


11) Mannitol Salt Agar

Pancreatic digest of casein 5.0 gm
Peptic digest of animal tissue 5.0 gm
Beef extract 1.0 gm
D-mannitol 10.0 gm
Sodium chloride 75.0 gm
Agar 15.0 gm
Water 1000 ml

12) Nutrient agar medium (NAM)

Peptone 5 gm / litre
Beef extract 3 gm / liter
Sodium chloride 5 gm / liter
Distilled water 1000ml
pH after sterilization is 6.9


13) Nutrient Broth

Dextrose 50.0 gm
Tryptone 5.0 gm
Yeast extract 4.0 gm
Mono potassium phosphate 0.55 gm
Potassium chloride 0.425 gm
Magnesium sulphate 0.125 gm
Calcium chloride 0.125 gm
Bromocresol green 0.22 gm
Manganese sulphate 0.0025gm
Ferric chloride 0.0025gm
Water 1000 ml

14) Sabouraud dextrose agar (SDA)

Mycological peptone 10 gm/ liter
Dextrose 40 gm / liter
Agar 15 gm / liter
pH after sterilization is 5.60.2

15) Plate count agar (PCA)

Casein enzymic hydrolysate 5 gm / liter
Yeast extract 2.5 gm / liter
Dextrose 1 gm / liter
Agar 15 gm / liter
pH after sterilization is 7.0 0.2

16) Soybean Casein Digest Agar

Pancreatic digest of casein 15.0 gm
Papaic digest of soyabean 0.5 gm
Sodium chloride 5.0 gm
Agar 15.0 gm
Water 1000 ml
pH after sterilization is 7.30.2



17) Soybean Casein Digest Medium

Pancreatic digest of casein 17.0 gm
Papaic digest of soybean meal 3.0 gm
Sodium chloride 5.0 gm
Dipotassium hydrogen sulphate 2.5 gm
Glucose monohydrate 2.5 gm
Water 1000 ml

18) Selenite F broth

Peptone 5 gm
Lactose 4 gm
Disodium hydrogen Orthophosphate 10 gm
Sodium hydrogen selenite 4 gm
Water 1000 ml

19) Tetrathionate Broth

Beef extract 0.9 gm
Peptone 4.5 gm
Yeast extract 1.8 gm
Sodium chloride 4.5 gm
Calcium carbonate 25.0 gm
Sodium thiosulphate 40.7 gm
Water 1000 ml
20) Triple Sugar Iron agar

Beef extract 3.0 gm
Yeast extract 3.0 gm
Peptone 20.0 gm
Lactose 10.0 gm
Sucrose 10.0 gm
D-glucose monohydrate 1.0 gm
Iron (II) sulphate 0.2 gm
Sodium chloride 5.0 gm
Sodium thiosulphate 0.3 gm
Phenol red 24 mg
Agar 13.0 mg
Water 1000 ml

21) Urea Broth

Potassium dihydrogen orthophosphate 9.1 gm
Anhydrous disodium hydrogen orthophosphate 9.5 gm
Urea 20.0 gm
Yeast extract 0.1 gm
Phenol red 0.01 gm
Water 1000 ml



22) Vogel- Johnson Agar

Pancreatic digest of casein 10.0 gm
Yeast extract 5.0 gm
Mannitol 10.0 gm
Dibasic potassium phosphate 5.0 gm
Lithium chloride 5.0 gm
Glycine 10.0 gm
Agar 16.0 gm
Phenol red 25.0 mg
Water 1000 ml



23) Xylose Lysine Deoxycholate Agar

Xylose 3.5 gm
L-Lysine 5.0 gm
Lactose 7.5 gm
Sucrose 7.5 gm
Sodium chloride 5.0 gm
Yeast extract 3.0 gm
Phenol red 80.0 mg
Agar 13.5 gm
Sodium deoxycholate 2.0 gm
Sodium thiosulphate 6.8 gm
Ferric ammonium citrate 800 mg
Water








REFERENCES

Indian Pharmacopeia (I.P.).

British Pharmacopeia (B.P.).

United States Pharmacopeia (U.S.P.).

Indian drug review (volume XI).

Prescott, Harley, Klein, Microbiology.

Dawson, M.E., Novitsky, T.J. and Gould, M.J. (1988). Microbes and
Water Pharmaceutical Engineering.

Novitsky, T.J. (1984), Monitoring and validation of high purity water
systems.

Medical pharmacology by K.D. Tripathi.

Practical microbiology by R.C. Dubey & Maheshwari.

Experiments in Microbiology, Plant Pathology and biotechnology, 4th
edition by Dr. K.R. Aneja.

Standard Operating Procedure (SOP) of PSYCHOTROPICS INDIA
LIMITED


















CERTIFICATE

This is to certify to Neetu Tyagi of M.Sc. IV semester Microbiology was
assigned to carry out this project work as a part of curriculum at Psychotropics India
Limited, SIDCUL, Haridwar.
This report is being submitted after completion of project training. The certificate
from respective organization is being enclosed.



(Prof. Pragya tyagi)