FYP Msc Biotch Report

www.final-yearproject.com | www.finalyearthesis.com

A DISSERTATION PRESENTED TO THE DR.BHIM RAO

AMBEDKAR UNIVERSITY IN PARTIAL FULFILLMENT OF

MASTER OF SCIENCE

IN

BIOTECHNOLOGY

(2010-2012)

Dissertation under the supervision of

Mr. Shudhanshu Sir

H.O.D. of Microbiology

Windlass Healthcare Pvt ltd.

Submitted by:- Submitted To:-

Gavendra Mr. Sudeep Tiwari ( H.O.D)

Department Of Biotechnology Internal Supervisor

I.I.M.T Institute (Training / Project Incharge)

Aligarh (U.P.) Dept. of Biotechnology / Microbio.

1

http://www.finalyearthesis.com/

http://www.final-yearproject.com/


ACKNOWLEDGEMENT

I would like to take this opportunity to thank all the people who have helped me directly or

indirectly in my training programme. My time with this great and coveted organization was

an experience that will go a long way in molding my corporate behavior.

I feel always happy to pursue this project in a reputed firm like ). I am highly thankful to ,

(H.R. Manager) and (Quality Control Manager) who allowed me to join the plant to

perform this project work.I wish to express to my heartfelt thanks and immense gratitude to

( H.O.D. microbiology section), and Mr. Arun kumar( Lab attendant) for their expert

guidance, encouragement and inspiration throughout this work.

I would like to express my thanks to Mr. Pankaj Mahalwar (Chairman of I.I.M.T

Institute Of Management & Science Aligarh. I owe a deep debt of gratitude to Mr.Sudeep

Tiwari (Head of the department Life science) whose constant guidance and suggestion

helped me throughout my M.Sc. Programme.

I have no words to express my sincere thanks and sense of reverence to my project guide Mr.

Younish Khan (Sr.Lecturer of Biotechnology). He helped me a lot to compile this whole

project and deleted simultaneously all my mistakes. I also thankful to faculity of

Biotechnology Miss Raka Bhatiya, Miss Sandhya sharma, for their moral guidance

throughout this project

I express my gratitude to my parents without their bless and love this project cannot be

completed. I would like to dedicate this work to my parents who are always with me in light

and dark phase of my life.

Gavendra Pratap Singh

2




ABBREVIATIONS

1. BGA - Brilliant green agar.

2. BSA - Bismuth sulphite agar.

3. CA - Citramide agar.

4. DM - Demineralized water.

5. FDA - Food and drug administration.

6. FTM - Fluid thioglycollate .

7. LAF - Laminar air flow.

8. LAL - Limulus amoebocyte lysate.

9. MCA - MacConkey agar medium.

10. MLT - Microbial limit test.

11. MSA - Mannitol salt agar medium.

12. MVD - Maximum valid dilution.

13. SCDA - Soyabean casein digest agar.

14. SCDM - Soyabean casein digest medium.

15. SDA - Soyabean dextrose agar medium.

16. WFI - Water for injection.

17. RODAC - Replicate Organism Detection Counting Plate

18. USP – United State pharmacopoeia

19.IP- Indian Pharmacopia

20.BP- British Pharmacopia

3




CONTENTS

S.NO. CHAPTER PAGE NO.

1. Introduction 5 - 7

2. Review of Literature 8 - 18

3. Aims & Objective 19-20

4. Materials & Methods 21 - 41

5. Results 42 - 50

6. Summary 51 - 53

7. Bibliography 54 - 57

8. Appendix 58 - 64

4




5




Microbiology is not only study of diseases but also finds application in industries for the

production of enzymes, antibiotics, vitamins, drugs, processed for fermentation industries.

Pharmaceutical microbiology is a part of industrial microbiology which is concerned with the

production of various drugs for various disease .

All drugs must undergo microbiology testing for the detection of contamination especially

before packing .These test are performed according to USP(United state Pharmacopoeia),IP

(Indian Pharmacopoeia) and BP(British Pharmacopoeia).

The microbiological examination of pharmaceutical product is necessary because they are

directly used by humans .If contaminated product is used by humans it leads to serious

diseases and it may be sometime fatal, so there arises a need to check the quality of the

product.In pharmaceutical industries quality control department is divided into two sections.

Microbiological quality control and chemical quality control.

The project is concerned with microbial quality control. It has been done at AXA

PARENTERALS Pvt Ltd Roorkee (U.A). Axa Parenterals Pvt Ltd has now become a

leading Pharma company of India to help patient leads healthier lives through. Axa

Parenterals Pvt Ltd. have a strong market present in antibiotics, antifungal, gastrointestinal,

antihelminthics, cardiovascular, dermal and erectile dysfunction categories. In microbiology

quality control department, detection of microorganism occurs that may contaminate the raw

material, initial product and final product. During this project work different type of

microbiological tests has been performed. These are Microbial limit test , Sterility test,

Bacterial endotoxin test, and Environment monitoring. The microbial limit test is designed

for the establishment of numbers of viable aerobic microorganisms present and for detecting

the presence of designed microorganism species in pharmaceutical products. Microbial limit

test is used for the detection of viable aerobic microorganisms from the raw material,

pharmaceutical products and water. This test is also used to determine the total bacterial

count (TBC) and total yeast and mould count (TYMC) of different types of water and

Pharmaceutical products.

6




The test for sterility is used for detection the presence of the viable form of microorganisms

in or on pharmacopial preparations.The working conditions in which the test is performed

should be monitored regularly by sampling in air and surface of the working area and by

carrying out control test.Confirm the sterility of each sterilized batch of medium by

incubating a portion of the batch at the specified incubation temperature fo not less than 14

days.

Bacterial Endotoxin is also called as LimulusI Amoebocyte Lysate Test (LAL). In

Pharmaceutical company it is done to detect the endotoxin. Endotoxin is part of the outer cell

wall of bacteria. Endotoxin is invariably associated with Gram-negative bacteria as

constituents of the outemembrane of the cell wall. Endotoxin is released when the

microorganism is lysed. Some also released during bacterial multiplication. Toxicity of

lipopolysaccharide is associated with the lipid component (Lipid A) and immunogenicity is

associated with polysaccharides component. LPS elicit a variety of inflammatory responses

by the alternative pathway, it is often part of the pathology of gram negative bacterial

infection (Hurley JC 1995). Bacterial endotoxin also known as pyrogen for its fever

producing effect is heat stable and with stand during the process of autoclave.

Endotoxin is harmless to humans except when it comes in direct contact with the circulating

blood. In the bloodstream, endotoxin are extremely toxic at levels as small as a few hundred

nanogram, they can cause fever, shock, hemorrhage and even death. For this reason

medicines and medical equipment, which are used in intravenous therapy must be free of

significant endotoxin contamination.

Environment monitoring is done for the detection of bio-burden of the aseptic area or

controlled environment. The purpose of this is to understand the various issues that relate to

establishment, maintenance and control of the microbiological quality of the controlled

environment. If the bioload of the sterile area exceeds the limit there is a contamination in the

environment of the sterile area.

7




8



www.final-yearproject.com | www.finalyearthesis.com In Pharmaceutical Industry, Quality control is a process to ensure a certain level of quality of

product. Pharmaceutical products are used by humans in daily life .These substance used for

the prevention, treatment and diagnosis of disease contain a wide variety of in gradients and

chemicals.(Sudershan Kumari)

As the topic of the project is “Microbiological Analysis of Pharmaceutical product” means to

test the pharmaceutical product along with their raw material and environment monitoring. If

the quantity of the microorganism in the product exceed these may induce harmful effects on

patients. So during the manufacturing process from raw material to desired end product,

proper care must be taken regarding the contamination by microorganisms. So all area in the

plant are kept neat and clean. The concentration of the microbes are decreased to a level

which is not so harmful or dangerous in the production point of view. The microbial

population is decreased by applying the different types of disinfectant in the different types

of works. Different areas in the pharmaceutical plant are regularly monitored for the presence

of any microbial population.The process is known as “Environmental Monitoring”. In this

case total bioburden or microbial load is calculated to check, weather the microbial load in

particular area is under the limit or it exceed.

In pharmaceutical plant, water (Pharmaceutical water) is an essential raw material for the

pharmaceutical production. It must comply with very stringent quality specification and be

available for use by the production plant 24 hours a day. Therefore, modern pharmaceutical

water production and distribution facilities feature a high degree of automation

and process instrumentation technologies .variation in water quality, or even of water

delivery failures, can cause the pharmaceutical production to stop immediately (Greenberg

A.E.et al). So it should also be free from any type of microbial contamination and also free

from any type of harmful chemicals.

While many different grades of water are needed for pharmaceutical manufacturing, the two

grades that are required most, and require treatment, are purified water (PW) and water for

injection ( WFI ). Purified water is also called demineralized water. It is prepared by means

of ion exchange.

During production and subsequent storage appropriate measures are taken to ensure that the

total viable aerobic count is adequately controlled and monitored..Under normal condition,

total viable count is 100 cfu / ml. This is determined by membrane filtration or pour plate

method.

9




Pharmaceutical water and product can be contaminated with microorganism during their

storage manufacturing and consequent rejection of material may bring death. The main

culprits are usually bacteria mainly four Staphylococcus, Escherichia coli, Salmonella and

Pseudomonas and fungi. Bacteria may form colonies in or on the product but are not

generally visible as mould. (Mehta R.M 3rd edition )

Microbial contamination of pharmaceutical product may have serious consequences for the

patients. Patients are often immune compromised and exposure to bacteria and fungi by

taking contaminated pharmaceutical products may result in serious infection .The route of

administration is important consideration .There is more risk arises when the route is

intravenous or intramuscular. (Kuby 6th edition )

Microbiological testing of pharmaceutical products for quality control falls in three

categories:-

1. QUALITATIVE TEST

This test provides presence or absence of microorganism contamination.

For example – Sterility test.

2. QUANTITATIVE TEST:-

This test provides the numerical value for microbial content of a sample.

For example – Microbial limit test.

Environment monitoring.

Air Sampler Method .

Sattle Plate Method.

Swab Test Method.

3. IDENTIFICATION TEST:-

These tests provide a name or at least description of organism. Before release of

pharmaceutical product into market ,there are many tests done to check the product for

microbial examination .These tests may show that the products are good and they may used

by the people.These tests include:

A) Microbial limit test.

B) Endotoxin test

C) Sterility test

D) Environment monitoring.

10




A) MICROBIAL LIMIT TEST

Microbial limit test is done for the detection of number of viable aerobic microorganism. The

detection of presence of microorganism is done from raw material to the finished product.

Under microbial limit test two type of test occurs.

1)USP (United state pharmacopoeia) Microbial limit test: This test is designed to

determine total aerobic microbial ,yeast and mold count .This test demonstrate that the

product is free from Staphylococcus ,Escherichia coli ,Pseudomonas aeruginosa ,Salmonella

.This test takes 7-8 days.

2) Confirmatory Test For MLT: To perform the presence and absence Of Staphylococcus

aureus ,Escherichia coli ,Pseudomonas aeruginosa ,Salmonella microbial acidification is

performed .They can be identified finally by Coagulase test, oxidase test and some other

biochemical tests. Mainly 4 types of bacteria are major contamination in Pharmaceutical

company causes various side effect. These bacteria are:-

1) Staphylococcus aureus

2) Escherichia coli

3) Salmonella spp

4) Pseudomonas aeruginosa

5) Shigella

1) Staphylococcus aureus:-

Staphylococcus (from the Greek: staphyle, "bunch of grapes" and kokkos, "granule") is a

genus of Gram-positive bacteria. Under the microscope they appear round (cocci), and form

in grape-like clusters. (Ryan, Ray 2004). Staphylococci were first observed in human

pyogenic lesions by Recklinghausen in 1871.It was Sir Alexander Ogston, a Scottish

surgeon, who established conclusively the causative role of coccus in abscesses and other

suppurative lesions (1880). (Ananthnarayan and Panikar, 2005)

Staphylococcus genus includes just 30 species (Holt. 1994). Most are harmless and reside

normally on the skin and mucous membrane of humans and other organismsAbout 20% of

the people are long term carrier of S.aureus (Kluytmans J, van Belkum A, Verbrugh H, July

1997).Staphylococcus a facultatively anaerobic Gram positive coccus It is however most

virulent of staphylococci.

11



www.final-yearproject.com | www.finalyearthesis.com S.aureus is catalase positive and able to convert hydrogen peroxide to water and oxygen,

which make the catalase test useful to distinguish the Staphylococcus , which appears as

grape like cluster when viewed in microscope and has large round golden yellow colonies.

After with haemolysis when grow on blood agar plates. forming. S. aureus is primarily

coagulase-positive (meaning that it can produce "coagulase", a protein product, which is an

enzyme) that causes clot formation while most other Staphylococcus species are coagulase-

negative. (Ryan KJ, Ray CG 2004) It is Catalase positive and able to convert Hydrogen

peroxide(H2O2) to water and oxygen which makes the catalase test useful to distinguish the

Staphylococcus from Enterococcus and Streptococcus.

S. aureus can cause a range of illnesses from minor skin infections, such as pimples,

impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome and

abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis,

endocarditis, toxic shock syndrome (TSS), bacteremia and sepsis. It is still one of the five

most common causes of nosocomial infections, often causing postsurgical wound infections.

2) Escherichia coli:-

Escherichia coli is a Gram negative bacterium that is commonly found in the lower intestine

of warm-blooded animals. Most E. coli strains are harmless, but some, such as serotype

O157:H7, can cause serious food poisoning in humans. E. coli normally colonizes an infant's

gastrointestinal tract within 40 hours of birth, arriving with food or water or with the

individuals handling the child. In the bowel, it adheres to the mucus of the large intestine. It

is the primary facultative anaerobe of the human gastrointestinal tract.

The harmless strains are part of the normal flora of the gut, and can benefit their hosts by

producing vitamin K2, (Bentley, Meganathan, 1982) or by preventing the establishment of

pathogenic bacteria within the intestine. (Hudault S, Guignot J, Servin AL, July 2001). This

genus is named after Theodor Escherich who was the first to describe the colon bacillus

under the name Bacterium coli commune in 1885. (Ananthnarayan and Panikar, 2005)

Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal

meningitis. In rare cases, virulent strains are also responsible for hemolytic-uremic syndrome

(HUS), peritonitis, mastitis and septicemia. E. coli are not always confined to the intestine,

and their ability to survive for brief periods outside the body makes them an ideal indicator

organism to test environmental samples for fecal contamination. (Thompson, Andrea, 2007).

12



www.final-yearproject.com | www.finalyearthesis.com The IMViC is ++-- for E. coli; as its indole positive (red ring) and methyl red positive (bright

red), but VP negative (no change-colorless) and citrate negative (no change-green color).

Tests for toxin production can use mammalian cells in tissue culture, which are rapidly killed

by shiga toxin (Paton JC, Paton AW 1998).

3) Salmonella:-

The genus salmonella was named after Daniel Elmer Salmon, an American veterinary

pathologist. While Theobald Smith was the actual discoverer of the typebacterium

Salmonella enteric var. Choleraesuis in 1885. Salmon was the administrator of the USDA

research program and thus the organism was named after him. (http://www.cfsan.fda.gov/

~dms/a2z-s.html.)

Salmonella is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly

motile enterobacteria with flagella which project in all directions (i.e. peritrichous). They are

aerobe and facultative anaerobes; temperature ranges 15-41oC (R. Cruickshank); Salmonella

possess the following antigens based on which they are classified:

- Flageller antigen H,

- Somatic antigen O,

- Surface antigen Vi. (Ananthnarayan and Panikar, 2005)

They obtain their energy from oxidation and reduction reaction using organic sources and are

facultative anaerobes; most species produce hydrogen sulfide, which can readily be detected

by growing them on media containing ferrous sulfate, such as TSI. They cause illness in

humans and many animals such as typhoid fever, paratyphoid fever and food borne illness

salmonellosis (Ryan and Ray CG, 2004).

Enteritis Salmonella (e.g., Salmonella enterica subsp. enterica serovar Enteritidis) can cause

diarrhea. However, people at risk such as infants, small children, the elderly, Salmonella can

become very serious, leading to complications. If this is not treated, HIV patients and those

with suppressed immunity can become seriously ill. Children with sickle cell anemia who are

infected with salmonella may develop osteomyelitis.

It is estimated that every fifth person in Germany is a carrier of Salmonella. In the USA,

there are approximately 40,000 cases of Salmonella infection reported each year. (Centers for

Disease Control and Prevention). According to the World Health Organization, over 16

13



http://www.cfsan.fda.gov/

www.final-yearproject.com | www.finalyearthesis.com million people worldwide are infected with typhoid fever each year, with 500,000 to 600,000

of these cases proving to be fatal.

3) Pseudomonas aeruginosa:-

Pseudomonas is a genus of gamma proteobacteria, belonging to larger family of

pseudomonad. Pseudomonad literally means 'false unit', being derived from the Greek

pseudo ('false') and monas (‘a single unit'). The term "monad" was used in the early history

of microbiology to denote single-celled organisms. Pseudomonas is widely distributed in

nature (soil, water, plants, and animals). It may colonize healthy humans without causing

disease, but is also a significant opportunistic pathogen, and a major cause of nosocomial

infections.

It is slender gram negative bacillus, actively motile by a polar flagellum. It is non capsulated

but many strains have mucoid slime layer P.aeruginosa produces a number of pigments, the

best known being pyocyanin (blue-green), fluorescein (yellow-green and fluorescent, now

also known as pyoverdin). Pyocyanin is a virulence factor of the bacteria and has been

known to cause death in C. elegans by oxidative stress. However, research indicates that

salicylic acid can inhibit pyocyanin production. (Prithiviraj et.al 2005).

Pseudomonas aeruginosa is increasingly recognized as an emerging opportunistic pathogen

of clinical relevance. Several different epidemiological studies indicate that antibiotic

resistance is increasing in clinical isolates. ( J. van Eldere.) An opportunistic pathogen of

immunocompromised individuals, P.aeruginosa typically infects the pulmonary tract, urinary

tract, burns, wounds, and also causes other blood infections. It is the most common cause of

infections of burn injuries and of the external ear, and is the most frequent colonizer of

medical devices (e.g., catheters).

Pseudomonas can, in rare circumstances, cause community-acquired pneumonias, (Fine et.

al.;1996) as well as ventilator-associated pneumonias, being one of the most common agents

isolated in several studies. (The Diekema et.al 1999). P.aeruginosa may aiso can be a cause

14



www.final-yearproject.com | www.finalyearthesis.com of ‘’Hot-tub rash” (dermatitis), caused by lack of proper periodic attention to water quality.

The organism is also associated with the skin lesion ecthyma gangrenosum. With plant P.

aeruginosa induces symptoms of soft rot with Arabidopsis thaliana (Thale cress) and Letuca

saliva (Lettuce) (Rahne et. al 1995).The association of virulence factor are the same for

vegetal and animal infections (Rahme et. al 2000). Most common test is done for the

confirmation of Pseudomonas. Pseudomonas is oxidase positive.For this readymade oxidase

strips are available.

B) BACTERIAL ENDOTOXIN TEST:-

Bacterial Endotoxin is also called as LimulusI Amoebocyte Lysate Test (LAL). In

Pharmaceutical company it is done to detect the endotoxin. Endotoxin is part of the outer cell

wall of bacteria. Endotoxin is invariably associated with Gram-negative bacteria as

constituents of the outer membrane of the cell wall. Endotoxin is released when the

microorganism is lysed. Some also released during bacterial multiplication. Toxicity of

lipopolysaccharide is associated with the lipid component (Lipid A) and immunogenicity is

associated with polysaccharides component. LPS elicit a variety of inflammatory responses

by the alternative pathway, it is often part of the pathology of gram negative bacterial

infection (Hurley JC 1995). Bacterial endotoxin also known as pyrogen for its fever

producing effect is heat stable and with stand during the process of autoclave.

Endotoxin is harmless to humans except when it comes in direct contact with the circulating

blood. In the bloodstream, endotoxin are extremely toxic at levels as small as a few hundred

nanogram, they can cause fever, shock, hemorrhage and even death. For this reason

medicines and medical equipment, which are used in intravenous therapy must be free of

significant endotoxin contamination.

History and background:-

Frederick Bang observe that bacteria caused intravascular coagulation in the American horse

shoe crab, Limulus polyphemus. (Ref, U.S.P 2003). In collaboration, Levin and Bang found

that the agent responsible for the clotting phenomenon reside in the crab’s amoebocyte, as

circulating blood cells and the pyrogen (bacterial endotoxin) produce a gelatinous reaction of

amoebocyte lysate by enzymatic process. In 1970 the FDA approved LAL for testing drugs,

15



www.final-yearproject.com | www.finalyearthesis.com products and devices that come in contact with the blood. Prior to that date, much slower and

more expensive tests on rabbits had been used for this purpose. (Segukuchi, Koichi, 1988).

Mechanism:-

Endotoxin→(1,3) – β-glucan→Factor c →Activated factor c→ Activated factor G→ Factor

G→Factor B→ Activated factor B→Proclotting enzyme clotting enzyme(Co-agulase)→Gel

coagulin coagulogen.

1. Endotoxin activates a proenzyme of limulus amoebocyte lysate having a high molecular

weight of 15,000 Daltons.

2. The activated proenzyme co-agulase react with low molecular weight protein fraction

(coagulase) contained in the LAL substance.

3. Low molecular fraction called coagulogen is cleaved by the proenzyme into a soluble and

insoluble unit. The insoluble unit appears as a solid clot precipitate or a turbid solution

depending on the amount insoluble coagulogen by product formed.

C) STERLITY TEST:-

Sterility Test is regulatory requirement for release of drug product that is labled as sterile.

The sterility test intended for detecting the presence of viable forms of bacteria, fungi and

yeast in or on pharmacopoeia preparations. The sterility test is based upon the principle that

if bacteria or fungi are placed in a medium which provides nutritive material and water,

incubated at a favorable temperature, the organisms will grow and their presence can be

indicated by turbidity in the originally clear medium.

The test must be carried out under sterile conditions specifically designed to avoid accidental

contamination of the product during the test Sterility test is done in the sterile room and done

with totally sterile wears and dress. It is done with membrane filter technique. In which

membrane filter used are 50mm in diameter and having pore size 45 um. Sterility test is most

specific method for checking the contamination in pharmaceutical products. The sterility test

is carried out by membrane filtration assembly. For sterility test we require two types of

16



www.final-yearproject.com | www.finalyearthesis.com media. Soyabean casein digest agar, Fluid thioglycollate medium. The working condition in

which the tests are performed should be monitored regularly by sampling the air and surface

of the working area.

D) Environment monitoring.

Environment monitoring is done for the detection of bio-burden of the aseptic area or

controlled environment. The purpose of this is to understand the various issues that relate to

establishment, maintenance and control of the microbiological quality of the controlled

environment. If the bioload of the sterile area exceeds the limit there is a contamination in the

environment of the sterile area. A large proportion of products are manufactured by the

processing, relies on the exclusion of microorganism from the process stream and the

prevention of microbial bioburden of the manufacturing environment are important

factor relating to these product.

Contamination is a process or acts that cause entry/presence of the undesirable

microorganisms (Bacteria and Fungus) in the environment of the sterile area. The

environment monitoring in the sterile area is done to know the microorganisms in the sterile

area presenting the air, attached to some surface of equipments, walls, roof or floor etc. the

microorganisms are also spread by the person working in the sterile manufacturing area.

Therefore the environment monitoring in the sterile area is done by different methods to

know the microorganisms present in the different sites and location in the sterile

manufacturing area. Some of the major methods used for doing the environment monitoring

are:-

17



www.final-yearproject.com | www.finalyearthesis.com i. Plate setteling methods. (Passive sampling)

ii. Air sampling method. (Active sampling)

iii. Personal monitoring method.(Rodec)

Plate setting method is done to check out presence of any microorganisms in the air of the

sterile area which act as the contamination the sterile area. In caseof setting plate method in

the biomass present in the air is down on the Petri plate’s contains media for the growth of

microorganism.

Air sampling method, air sampler is used which suck 1000 liters of the air per cubic meter.

The air sucked through the lid with uniform pore.The bio-load present in the air attach to

same particle, is sucked along with the air and blow over the plate containing SCDM

medium.

Personal monitoring method of environment is used to know the hygienic condition of the

person working in the sterile manufacturing area. The hygienic condition of the person

working in the sterile manufacturing area is checked by using rodac (replicate organism

detection and counting) plate containing SCDA medium. Environmental monitoring allows

pharmaceutical manufacturers to continually monitor the effectiveness of control measures

and also allows the any threats to the products being manufactured to be identified. A formal

system biocontamination control should be established, implemented and maintained within

clean rooms and the associated environment. A poorly designed or badly managed

programme may result in misleading data in relation to manufactured or assessment of

products. Purpose of environment monitoring is determine the microbial and particulate

count of clean room air and surface, highlights the conditions contribuction to excessive

microbial and particulate level due to ineffective cleaning or personnel/equipment issues alert

to conditions exceeding the classification, proactive tool for quality assurance.

Contamination sources are people~75%, ventilation~15%, room structure~5%,

equipment~5%. In environment monitoring non viable air born particles and viable air born

particles are counted. A calibrated laser particle counter is used to know the non viable air

born particles. It is used to sample a define volume of air. It can measure a variety of particle

size most commonly 0.5 and 5μ. In this case particle counts are recorded as the number of

18



www.final-yearproject.com | www.finalyearthesis.com particles per volume of the air sampled. Viable air born particles are measured by settling

plate method. (passive air monitoring)

Alert limit is a count (microbial and particle) that exceeds normal operating level but does

not adversely affect product quality. It serve to alert appropriate official to a potential to a

control .

Action limit is a count or trend that exceeds normal operating level that could adversely

affect product quality and require action on the part of the firm. (Wilson, James 1997)

19




AIMS AND OBJECTIVES

AIM: To check the microbial contamination of pharmaceutical products, sterility test,

bacterial endotoxin test and environment monitoring.

OBJECTIVES:-

The objectives of present study are:-

1) To perform the microbial limit test for food products.

2) To perform Bacterial endotoxin test.

3) To perform sterility test by filter membrane technique.

4) Environment monitoring-

a) By the Air sampler method.

b) By the Settle plate method.

20



www.final-yearproject.com | www.finalyearthesis.com c) By the Swab method.

21




MATERIALS

To performed different tests different media has been prepared. The successful result of any

testing depends upon the materials which are used during the performance of the test.


Agar used in MLT:-

Soya bean casein digest agar (SCDA), Sabouraud dextrose agar medium (SDA), Baired

parker agar (BPA), Mannitol salt agar medium (MSA), MacConkey agar (MCA), Brilliant

green agar (BGA), Bismuth sulphite agar medium (BSA), Xylose lysine dextrose agar

(XLDA), Cetrimide agar (CA), GN Broth,

Requirement:-

Membrane filter having pore size of 0.45mm, Two sterile hand gloves pair. One set of sterile

dress, Aluminium foil., IPA 70%, Test tube stand, Petri plate, test tubes, micropipettes,

measuring cylinder, conical flask, volumetric flask, inoculation loop, aluminium foil, butter

paper, cotton plugs, slides, Bunsen burner and cover slips.

Instruments used:-

Autoclave, laminar air flow, incubator,

22



www.final-yearproject.com | www.finalyearthesis.com Refrigerator and microscope

B) BACTERIAL ENDOTOXIN TEST

Material & Equipments:-

1 Vortex mixture

2 Heating block : temp 370 C

3 Diluents : pH 3.0 KH2PO4

4 Micropipettes : 20-200 µl 100-1000µl

5 Micro tip : 200 µl, 1000 µl

(Dehydrogenated &individually packed)

6 Test tube : 12x75 mm 10x75 mm

7 Reagent : Limulus amoebocyte lysate(LAL-R)

C) STERILITY TEST

Media Used:-

1) SCDM (Soyabean casein digest medium):-

Preparation Method:-

23



www.final-yearproject.com | www.finalyearthesis.com Dissolve 30.0 gm media in 1000 ml of WFI and transfer into the 100ml glass vials with

rubber bunk and aluminium seals.Autoclave the sealed vials at 121º C at 15 lb/ in² for15

mins. It is used for fungal detection as well as aerobic bacteria. It is used for sterility testing

of biologicals and for the cultivation of aerobic and anaerobic organisms.

2) FTM (Fluid Thioglycolate Medium)

Preparation Method:-

Dissolve 29.75 gm of FTM in 1000 ml of WFI. Boil to dissolve the medium completely and

transfer into the glass vials and seal the glass vials with rubber bunks and aluminium seals.

Sterilized the media vials by autoclaving the sealed vials at 121º C at 15 lb/ in² for 15 mins. It

is used for bacterial detection as well as anaerobic bacteria.

Put these two media vials in the incubator ( FTM vias in bacteriological incubator at 32.5 ±

2.5ºC for about 2 days and SCDM vials in BOD incubator at 22.5 ± 2.5ºC for about 2 days).

If any growth shows in the media, discard it while the other vials are transfer to the sterility

room through the pass box for the further testing.

It is a pre – enrichment media used for increasing the recovery of the injured Salmonella spp.

From foods and other materials, prior to the selective enrichment and isolation. It is also used

as a diluent for carrying out the various tests from clinical and non- clinical specimens.

3) Buffered Sodium Chloride-Peptone solution ph7.0 Composition

Potassium dihydrogen phosphate - 3.0g

Disodium hydrogen phosphate - 7.2g

Sodium chloride - 4.3g

Peptone (meat or casein) - 1.0g

Water - 1000ml

4) Peptone Water:-

It is a general purpose media used for the cultivation of a wide variety of the

microorganisms. SCDM is recommended for the st erility testing of fungi, moulds and lower

bacteria.

24



www.final-yearproject.com | www.finalyearthesis.com Preparation Method:-

Dissolve 1.0 gm of peptone in 1000ml of WFI and pour in the 500ml glass vials. Seal the

vials containing the peptone water with rubber bunks and aluminium seals. Sterilized the

peptone water vials by autoclaving the sealed vials at 121º C at 15 lb/ in² for 15 mins. And

after pre- incubation for 48 hrs are transfer to the sterility room through the pass box for the

further testing

Requirements:-

Membrane filtration assembly kit, Membrane filters having the pore size of 0.45mm,

Sterilized surgical scissors and forceps, Sterile syringes assembled with sterile needle, Sterile

dress and gloves, Butter paper and aluminium foil, 70%IPA, Vacuum flask, Vaccum pump.

D) Environment Monitoring

REQUIREMENT : -

Centrifugal Air Sampler & Sampler Strips, Petri Plates of 90 to 100 mm diameter,

Contact Plates & Swab sticks, Soyabean Casein Digest Agar (SCDA), Saboraud’s

Chloramphenicol agar (SCA), RODAC (Replicating organism detection) plates,

Normal saline, Incubators & Laminar air flow and Colony counter, I.P.A. 70%,

Cotton, Aluminium Foil, Mask, Gloves, Hair cap.

25




METHODS


Sampling:-

1ml of liquid sample and 1 g of solid sample was taken to perform this test. The solid sample

is distinguished into three groups and then examined.

(a) Water soluble products:-

1 g of product was dissolved in a buffer sodium chloride peptone solution having Ph 7.0.

(b) Product insoluble in water:-

1gm of the sample that is insoluble in product was dissolved in buffer sodium chloride

peptone solution having Ph 7.0.

Microbial test including two tests:-

1) Total microbial count

2) Test for pathogen

1) Total microbial count by filtration method:-

Total microbial count has been done with the help of membrane filtration method.

Membrane filtration method. In this method membrane filter was used having

diameter 50mm and the pore size is 0.45 m used for retaining bacteria.

26



www.final-yearproject.com | www.finalyearthesis.com Without disturbing the filter,place the funnel on top of the filter holder base and

transfer 1ml in case of purified water & 200ml in case of WFI to each separate

membrane filters & filter immediately.

After completion of filteration process,shut off the vacuume with the help of vacuume

control key.

Transfer the membrane filter,to the surface of the sterile petri plate contaning R2A

agar media.

For negative control filter 100ml of sterile WFI &transfer the membrane to the

surface of sterile R2A agar.

Invert & incubate all petri plates at 20-25cfor first three days for fungi & 30-35c for

last two days for bacteria with negative control.

After completion of incubation, count the number of cfu formed on the plates with the

help of colony counter.

Pharmaceutical water was also tested by Total plate count method (Aerobic plate

count) this includes the:-

Pour Plate Method:-

a) Total Bacterial Count

Pipette out 1-1ml of water sample from each sample bottle corresponding to different

sampling point(for SP01 TO SP04D) and transfer aseptically to different petriplates

under LAF in duplicates.

Pour aseptically approx.15-20ml of sterile liquefied cooled R2A agar media to each

plate.Mix the content properly by gently rotating the petri plates clockwise &

anticlockwise .

Allow media in petriplates to solidify under LAF. After solidification of the media,

incubate all petri plates at 20-25c for first three days for fungi & 30-35c for last two

days for bacteria.

27



www.final-yearproject.com | www.finalyearthesis.com Simultaneously run Growth Promotion Test by inoculating 10-100cfu(approx) of

Pseudomonas aeruginosa & Bacillus subtilis into a sterile petri plates and add 15-

20ml of sterile R2A agar medium.

Incubate all petri plates at 30-35c for not more than three days in inverted position.

For negative control add 1ml of sterile WFI into a sterile petri plate and then add 15-

20ml of sterile R2A agar medium.

Mix the conten properly by gently rotating the plates clockwise and

anticlockwise.Allow to solidify the plates under LAF.

Invert and incubate all the plates at 20-25c for first three days for fungi & 30-35c for

last two days for bacteria.

After completion of incubation,count the number of cfu formed on the plates with the

help of colony counter.

2) Test for Bacterial pathogen

Mainly four types of pathogens were tested:

1. Staphylococcus aureus

2. Escherichia coli

3. Salmonella

4. Pseudomonas

Pre-enrichment:-

Aseptically connect the rubber tube of sterile mannifold to receiver tank rubber pipe

to vacuume pump.

Using sterile smooth tip foreceps,place a 47 mm diameter 0.45 sterile membrane filter

on the center of the filter support screen.

Without disturbing the filter,place the funnel on top of the filter holder base and

transfer 100ml quantity of each sample to each separate membrane filters and filter

immediately.

28



www.final-yearproject.com | www.finalyearthesis.com After completion of filteration process,shut off the vacuume with the help of the

vacuume control key.

Transfer the membrane filter to a tube contaning 100ml of sterile SCDM for

enrichment of pathogens.

Incubate the tubes at 30-350c for 18-24hr.

For negative control filter 100ml of sterile WFI & transfer to a tube contaning 100ml

of sterile SCDM.

Observe the tubes for growth,by means of turbidity.If the growth observed in sample

tube & absent in negative control,then proceed for identification for pathogens.

1. Detection And Identification Of Staphylococcus aureus

Primary Test for Staphylococcus aureus :

Streak a portion of the enriched medium i.e. SCDM on the surface of Mannitol Salt

Agar medium.

Simultaneously carry out the positive control by streaking a loopful growth of

Staphylococcus aureus on the surface of agar medium.

Invert & incubate all the plates at 30-350c for 18-72 hrs.

Upon examination if colony are yellow or white colony with yellow zone,It indicate

the presence of Staphylococcus aureus.

Confirmatory Test

If test is positive in Mannitol Salt Agar Medium,subculture on the surface of Vogel-

Johnson Agar.

Simultaneously carry out the positive control by streaking a loopful growth of

Staphylococcus aureus on the surface of agar medium.

Invert and incubate all the plates at 30-350c for 18-72hr.

Upon examination if colony are black surrounded by yellow zone,it indicate the

presence of Staphylococcus aureus.

29




2.Detection And Identification Of Escherichia coli

Primary Test for E.coli:

Transferred 1ml sample from turbid SCDM into 100ml sterile MacConkey broth.

Incubated at 42-44ºC for 24-48 hrs.

Subcultureon the plate of MacConkey Agar & incubate at 30-350cfor 18-72hrs.

Simultaneously carry out the positive control by streaking a growth of E.coli on the

surface of MacConky agar plate.Invert & incubate the plates at 30-350c for 18-72hrs

Upon examination if colony are pink,non mucoid,it indicate the presence of E.coli.

Confirmation Test:

Suspected colony was transferred to the surface of Levine eosin-methylene blue agar

medium & perform Gram staning.

Simultaneously carry out the positive control by streaking a growth of E.coli on the

surface of Levine eosin-methylene agar medium.

Invert & incubate all the plates at 30-350c for 18-72hrs.

Upon examination if colonies exhibit both a characteristic metallic sheen under

reflected light blue black appearance under transmitted light.it indicates the presence

of E.coli.

3. Detection And Identification Of Salmonella

Primary Test for Salmonella

Transfer .1ml sample from turbid SCDM into 10ml of Rappaport Vassiliadis

Salmonella Enrichment Broth & incubate at 30-350c for 24-48 hrs.

After completion of incubation if growth appears in Rappaport Vassiliadis Salmonella

Enrichment Broth subculture on the plate of Wilson and Blair’s agar & incubate at

30-350c for 24-48 hrs.

30



www.final-yearproject.com | www.finalyearthesis.com Simultaneously carry out the positive control by streaking a growth of Salmonella

sp.on the surface of Wilson Blair’s agar medium.Invert & incubate the plates at 30-

350c for 24-48 hrs.

Upon examination if colonies are green with black centre which turns uniformly

black in 48 hrs. and also colonies surrounded by a dark zone & metallic sheen

indicates possibility of the presence of Salmonella.

Confirmatory Test

If test positive in Wilson Blair’s agar,subculture on Xylose-lysine-deoxycholate agar

& also perform Gram staning.

Simultaneously carry out positive control by streaking a loopfull growth of

Salmonella sp.on the surface of the XLDA medium.

Invert & incubate all the plates at 30-350c for 24-48hrs.

Upon examination if colonies are red with or without black centers,it indicates the

presence of Salmonella.

4. Detection And Identification Of Pseudomonas

Primary Test for Pseudomonas:

Streak a portion of the enriched medium i.e SCDM on the surface of cetrimide agar

medium.

Simultaneously carry out the positive control by streaking a loop full growth of

Pseudomonas aeruginosa on the surface of cetrimide agar.

Incubated at 30-35 ºC for 18-72 hrs.

If upon examination colonies having green in colour it indicates the presence of

Pseudomonas aeruginosa.

Confirmatory Test:-

Pigment test:

31



www.final-yearproject.com | www.finalyearthesis.com Streak representative suspect colonies from the agar surface of cetrimide agar on the

surface of Pseudomonas agar medium for the detection of fluorescein &

Pseudomonas agar medium for detection of Pyocyanin.

Cover & invert the inoculated plates & incubated at 30-350c for 18-72 hrs.

Examine the streaked surface area under UV light & determine if the colonies give

yellowish fluorescence in UV light,it indicates the pesence of Pseudomonas

aeruginosa.

Oxidase test:

If growth of suspect colonies occur,place 2 or 3 drops of a freshly prepared 1% w/v

solution N –tetramethyl-4-phenylenediamine dihydrochloride on filter paper & smear

with the suspect colony.

If there is no development of pink color,changing to purple, the sample meets the

requirements of the test for absence of Pseudomonas aeruginosa.

5.Detection and Identification of Shigella:

Transfer 1ml sample from SCDM into 100ml of GN Broth & incubate at 30-350c for

24-48 hrs.

After incubation if growth appears,subculture on a plate of Xylose Lysine

Deoxycholate Agar medium,invert & incubate at 30-350c for 24-48 hrs.

Simultaneously carry out the positive control by streaking a loopfull growth of

Shigella on XLDA agar medium,incubate the plates at 30-350c for 24-48 hrs.

Upon examination if none of the plate contains colonies having the characteristic red

coloured translucent colony without black centre,the sample meets the requirements

for freedome from shigella,Such type of colonies should be observed in positive

control.

If such type of colonies are found in sample,confirm then by Gram staning.

32




B) BACTERIAL ENDOTOXIN TEST

Sample preparation :-

Depyrogenate all the glassware required for testing by heating in dry heat sterilizer

(oven) at 250 C for 30 min.

Start the heating block around 1 hr before starting the test to get the required

temperature to be stabilized at the time of testing.

Maximum valid dilution:-

Where the endotoxin limit cone is specified in term of volume (EU/ML)

Endotoxin limit conc. in EU/Ml

MVD = ---------------------------------------------

Labeled sensitivity of lysate in EU/ML

Where the endotoxin limit cone is specified in term of weight or of units of active

drug(in EU/ML or in EU/units units)

Endotoxin limit conc. in EU/Ml or in EU/unit units x potency

MVD = -----------------------------------------------------------------

Labeled sensitivity of lysate in EU/ML

The MVD factor so obtained is the limit dilution factor for the preparation for test to

be valid.

Preparation of CSE dilution series :-

Reconstitute 10 mg / Vail Endotoxin standard with 5.0 ml of LWR and mix for not

less than 30 min ……………..dilution (1)

33




Dilute 0.05 ml of dilution (1) with 1.95 of LWR……….dilution (2)

Using dilution (2) prepare a series of tow fold dilution to give concentration of

2ℷ,ℷ,0.5ℷ &0.25ℷ i.e.0.25,0.125, 0.06 &0.03 EU/ML respectively. Mix each dilution

for not less than one minute just before proceeding to the next.

Positive control tubes: - gel formation

Negative control tubes: - no gel formation

Preparation of LAL reagent:-

Reconstitute the LAL-r vail LRW as mentioned on LR vial swirl gently but

thoroughly. Do not shake or vortex.

Test for conformation of labeled LAL reagent sensitivity:-

Confirm the labeled sensitivity of each new lot prior to use in the test. Prepare a series

of two fold dilution of the CSF in LRW to give concentration of 2ℷ, ℷ, 0.5ℷ &0.25ℷ

i.e.0.25, 0.125, 0.06 &0.03 EU/ML respectively.

Where ℷ = labeled sensitivity of LR in EU/ML

Perform the test on the four standard concentration in quadruplicate and include

negative control.

Mix a volume of the LAL-R with an equal volume (0.1 ml) one of the standard

solutions in each tube.

Incubate the reaction mixture for 60± 2 min. at 37±10 C by avoiding vibration.

To test the integrity of the gel take each tube in turn directly from the incubator and

invert it through about 1800C in one smooth motion. If a firm gel is formed that

remain in place upon inversion , record the result as positive .

This test is not valid unless lowest concentration of the standard solution shows a

negative result in all replicate tests.

The end point is last positive test in the series of decreasing concentration of

endotoxin.

Calculate the mean value of the end point concentration and then the antilog of the

mean value using the following equation:-

34




Geometric mean end point concentration antilog Σe/f.

Where Σe is the sum of the log end point concentration of dilution series used and f

is the no of replicate test tube used.

Acceptance criteria :-

The geometric mean end point concentration is the measured sensitivity of the LR (in

EU/ML). if it is not less than 0.5λ and not more than 2 λ the labled sensitivity in confirmed

and is used in test performed with the lysate.

Method of analysis for sample

Reconstitute and dilute the sample vail with LRW as MVD.

Follow table (1) for quantity of dilute sample LRW, CSE and lysate used in final

testing.

Incubate the reaction mixture for 60± 2 min. at 37±10 C avoiding vibration.

To test the integrity of gel take each tube in directly from the incubator and invert it

through about 1800 C in one smooth motion.

If a firm gel is formed which remain in place upon inversion record the result as

positive result is negative if a intact gel is not formed.

Acceptance criteria:

The preparation being examined complies if negative result in sample & LRW and

positive result in PPC.

The preparation being examined does not comply if the result is negative in PPC,

positive result in sample negative in both sample and PPC and positive in both sample

&PPC.

35




Quantity of dilute sample, LRW, CSE and lysate used in testing

Tube no. Description Sample ½ LWR CSE Lysate

1 WNC -- 100 -- 100

2 WNC -- 100 -- 100

3 Test Tube 50 50 -- 100

4 Test Tube 50 50 -- 100

5 PPC 50 -- 50 100

6 PPC 50 -- 50 100

WNC = Water negative control

PPC = Product positive control

General Precautions:-

Vortex standard endotoxin for not less than 30 min.

Vortex all endotoxin dilution and sample for not less than one minute.

Do not allow dilution of endotoxin for more than 10 min without revotexing.

Incubate tube in heating block kept on stable surface.

Do not transfer or disturbs the tube at any stage of incubation.

Read result only once gel are fragile & may break when handled.

36




C) STERILITY TEST

Preparation before test:-

The sterilization of sterility room was done over night.

Membrane filtration assemblies were washed with distilled water and were dried

properly.

Wet membrane filter paper was inserted in the holder of each membrane filtration

assemblies.

Membrane filtration assemblies then wrapped with aluminium foil and sterilized by

autoclaving.

Switch on the laminar air flow and UV light for 15 minutes.

Test procedure:-

Entered the sterility testing room by wearing sterile gown and switch off UV light

of laminar air flow. All the required materials were transferred to the testing room

from change and were kept under LAF. IPA was sprayed on hands.

Vaccum flask was connected to vaccum pump and the membrane filtration assembly

was joined with the vaccum flask.

IPA was sprayed on the outer surface of the sample container and dried them under

LAF.

Sufficient amount of sample was taken in a sterile syringe and filtered through 0.45μ

filter paper in filter assembly.

The membrane filter was then washed by filtering of peptone water.

After filtration vacuum pump was switched off and aseptically removed the filter

paper from the membrane filter assembly with the help of the sterile forceps and cut

it aseptically into two equal pieces.

Aseptically one half of the membrane filter was transferred into 100ml of sterile

FTM near gas burner. Similarly the other membrane filter half was transferred into

100ml of sterile SCDM.

37




Filter papers were completely submerged in their respective media and the tubes

were marked with the sample number, batch number, date of testing and date of

completion along with media lot number.

Negative control was set up in which no sample was added and contain only the

filtered peptone water.

All the tubes of FTM were incubated at 30-35ºC and all tubes of SCDM were

incubated at 20-25ºC for 14 days.

If microbial growth is detected and no negative control is contaminated, the result

of the test is indeterminate and repeat test is carried out using same number of

container.

If no growth is observed during the repeat test. The product passes the test of sterility.

38




D)ENVIRONMENT MONITORING

ENVIRONMENT MONITORING:-

Environment monitoring in the sterile area is done to know the bioburden

of the environment of the sterile area. The sterile areas are

divided into 4 grades and having different alert limit and action limit.

S. No. GRADES AREAS

1 Grade A Filling machine / LAF.

2 Grade B Filling room / sterile corridor.

3 Grade C Ceiling room.

4 Grade D Change room II / washing area.

Alert limit and action limit of different areas are:-

S. No. GRADES Alert limit Action limit

1 Grade A <1 1

2 Grade B 3 5

3 Grade C 20 50

4 Grade D 50 100

Environmental monitoring of microbiological areas shall be done by

following methods:

1. Plate settling method

2. Air sampling method

3. Personal monitoring method

1. Environment Monitoring using Settle Plate Method

39



www.final-yearproject.com | www.finalyearthesis.com Environment monitoring in sterile area using settle plate method is to check out the presence

of any microorganism in the air of the sterile area which acts as contamination in the sterile

area. In case of settling plate ethod the biomass present in the air is settling down on the Petri

plates containing media for the growth of the microorganism.

METHOD:-

Prepare the SCDA plates after autoclaving the media.

Incubate these plates in inverted position for 48 hrs. at

30-35°C for the purpose of pre-incubation in bacteriological incubator

Discard the contaminated plate.

Take the pre-incubated plates under LAF & mark them with date, area and location of

plates to be exposed.

Put the sterile dress and gloves & carry the plates in closed container to the marked

location.

Exposed the plates in the sterile area for about 4hrs.

After the exposure time is completed collect these plates and place them in the

container and bring back to the microbiology lab

Incubate all these plates in the bacteriological incubator at 30-35oC for 48 hrs.

After 48hrs observe the plates for the growth of the microorganisms (Bacteria).

Further incubate all these plates in the B.O.D. incubator at 20-25oCfor 72 hrs. for

fungal count.

Inform the concerned production department, if the count exceed the limits.

2. Environment Monitoring using Air Sampler (Active Sampling

Method)

In this method of environment monitoring air sampler sucked 1000Lts. of air per cubic meter.

The bio load present in the air is present attach to some particle, is sucked along with the

air .The air is sucked through the lid with uniform pressure.

METHOD:-

Prepare SCDA plates and pre-incubate the agar plates at 30-35oC for 24 hours to

check for any type of contaminant

40



www.final-yearproject.com | www.finalyearthesis.com Mark the agar plates with date of exposure and area to be sampled.

Expose plates with the help of air sampler.

Fit the plate in the air sampler unit and locked it with the S.S Filter (lid) having

uniformly distributed pores which was sterilized by autoclaving.

Adjust the volume of the air sampler i.e.1000 litters and switch on the sampler.

Remove the Petri plate from the air sampler and put in the S.S. Container and bring to

the micro lab aseptically.

Incubate all the plates sampled in the bacteriological incubator position at 30-35°C

for 48 hrs for bacterial count.

Further incubate the plates in the BOD incubator at 20-25oC for 72 hrs for fungal

count.

Inform the concerned production department, if the count exceeds the limits.

3. Environment Monitoring using Personal Monitoring Method (Rodac

Method)

In this method of the environment monitoring the hygienic condition of the persons working

in the sterile manufacturing area is checked by using RODAC-plates containing SCDA (Soya

Bean Casein Digest Agar) Medium.

METHOD:-

RODAC-plates is used in the personal monitoring method of the environment

monitoring.

Pre-incubate these plates for 48 hrs. in the bacteriological incubator.

Take sterile Container and clean it by using 70% IPA.

Put the plates in the container and take them to the sterile area.

In the sterile area before analysis make sure that the surface to be analyzed is dry and

free from any disinfectant.

Open the lid and gently press the convex surface of the plate containing SCDA media

on the area of the person to be tested (such as hands, forearm, forehead, shoe cover)

without twisting or sliding.

Close the lid and marked each plate with name of the person body part sampled and

date of analysis

41



www.final-yearproject.com | www.finalyearthesis.com Then put these plates in sterile container and bring back to lab.

Incubate these plates in the bacteriological incubator at 30-35°C for 48 hrs for the

bacterial count.

After taking the bacterial count, Incubate these plates in the BOD incubator at 20-

25°C for 72 hrs for the Fungal count.

After 72 hrs incubation observe the plates for the growth of the fungus and record the

count in the format provided.

42




43





Table shows the detection of the microorganism from the differrent products.

PRODUCT NAME TBC TFC PATHOGEN REMARK

1 NSM MANGO 11000 cfu/g

(NMT 10000CFU/G)

310 cfu/g

(NMT 300CFU/G)

present fail

2 AFRESH LEMON <10

(NMT 10000CFU/G)

<10

(NMT 300CFU/G)

absent pass

3 MULTIVITAMIN 10

(NMT 10000CFU/G)

<10

(NMT 300CFU/G)

absent pass

4 ACTIVATED

FIBRE

<10

(NMT 10000CFU/G)

<10

(NMT 300CFU/G)

absent pass

5 CELL ACTIVATOR <10

(NMT 10000CFU/G)

<10

(NMT 300CFU/G)

absent pass

6 ALOE PLUS <10

(NMT 10000CFU/G)

<10

(NMT 300CFU/G)

absent pass

7 ESTER 500 <10

(NMT 10000CFU/G)

<10

(NMT 300CFU/G)

absent pass

8 IG 6 <10

(NMT 10000CFU/G)

<10

(NMT 300CFU/G)

absent pass

9 SPIRULLINA 40

(NMT 50000CFU/G)

<10

(NMT 500CFU/G)

absent pass

10 AFRESH ELAICHI 30

(NMT 10000CFU/G)

<10

(NMT 300CFU/G)

absent pass

Result of mango shake was found to be unsatisfactory Because Number of colonies

are more than their limit and pathogen are also present.

44




Where as Results of other products are under limits and satisfactory.

B) RESULTS OF BACTERIAL ENDOTOXIN TESTS

Table shows the result of bacterial endotoxin test:-

S. No. Product Name Gel Formation Result

1 Ramadole inj. No Negative

2 Gentamycin sulphate inj. No Negative

3 Positive control Yes Positive

4 Negative control No Negative

The results Ramadole inj. and Gentamycin sulphate inj. were satisfactory i.e. there was no

toxin present in these products.

Positive control tubes: - gel formation

Negative control tubes: - no gel formation

45




C) RESULTS FOR STERILITY TEST

Method : Membrane Filtration Media . : SCDM , FTM

Date of Sampling : 14/5/11 Sample Quantity : 23 bottles

Batch No./ Lot no. : RC013 Batch Size : 3000 ltr

Date of Manufacturing : May 2011 Date of Expiry : April 2013

Date of incubation : 17/5/11 Day of incubation : 14 Days

Rinsing Fluid : ------- Settle plate under LAF in nos.<1cfu/plt

OBSERVATION:-

Days Date

Fluid Thioglycollate

Medium at 32.5 ± 2.5oC

Soyabean Casein digest

Medium at 22.5 ± 2.5oC Analysis

TEST NEGATIVE POSITIVE TEST NEGATIVE POSITIVE

1 15/5 -ve No growth Growth -ve No growth Growth OK









46









Result:-

( 1 ) No growth observed in sample bottle or negative control after 7 or 14

days. SAMPLE PASSES STERILITY .

( 2 ) Growth observed in sample bottle DOES NOT PASS.

( 3 ) Growth observed in negative control TEST INVALID.

( 4 ) Growth promotion test shall be positive TEST VALID.

( 5 ) Growth Observed in filter paper contain TEST INVALID

47




D) RESULTS OF ENVIRONMENT MONITORING

Table show the results of different sterile area by settle plate method.

S No. Part of sterile area Grade Standard limit No. of plate taken Plate 1 Plate 2

1 Under LAF A < 1 2 < 1 < 1

2 Filling Machine A < 1 2 < 1 < 1

3 Sterile corridor B < 3 2 < 1 < 2

4 Filling room B < 3 2 < 1 < 1

5 Ceiling room C < 20 2 < 15 < 9

6 6 Change room D < 50 2 < 41 < 36

7 Washing room D < 50 2 < 42 < 46

Results were found within the limits.

Table show the results of different sterile area by active air sampling method.

S No. Part of sterile area Grade Standard limit No. of plate taken Plate 1 Plate 2

1 Under LAF A < 1 1 < 1 Nil

2 Filling Machine A < 1 1 < 1 Nil

3 Sterile corridor B < 3 2 < 1 < 1

4 Filling room B < 3 2 < 1 < 1

48




5 Ceiling room C < 20 2 < 10 < 8

6 6 Change room D < 50 2 < 35 < 37

7 Washing room D < 50 2 < 41 < 40

Result were found within the limit.

Table shows the results of personal monitoring of workers working in sterile area, by

RODAC plate method.

S. No. Name Garments Grade Colony Observed

1 Tapender F. Hood B 4

2 Tapender Sterile Dress B 3

3 Tapender Booties B 5

4 Mahesh F. Hood B 2

5 Mahesh Sterile Dress B 1

6 Mahesh Booties B 2

7 Sarvjeet F. Hood B 1

8 Sarvjeet Sterile Dress B 3

9 Sarvjeet Booties B 2

The numbers of colony found on the workers working in sterile area were satisfactory.

49




Fig. TSI agar slants for Salmonella Fig. XLD media for Salmonella

50




Fig. E.coli showing green metallic sheen Fig. E.coli Showing pink coloured

colonies on EMB agar plate. on MacConkey agar plate.

Fig. Pseudomonas aeroginosa showing green coloured colony on Citramide

Agar plate

51




Fig. S. aureas produce yellow coloured colonies on Mannitol Salt Agar

52




In Pharmaceutical industries, quality control is a process to ensure a certain level of quality

of a product. In quality control (Q.C) Pharmaceutical product are tested chemically as well as

microbiologically in order to get the reliable and safe product for the use of consumer. In

microbiological quality control department, detection of microorganism was done that may

contaminate the raw material, initial product and final product. During this project work

different type of microbiological tests was performed to check quality of pharmaceutical

products:

1. Microbial limit test.

2. Bacterial endotoxin test (BET).

3. Sterility test.

4. Environment Monitoring.

In order to perform these test different type of media were used, the media were prepared

according to their composition. For the detections of particular pathogen selective media

53



www.final-yearproject.com | www.finalyearthesis.com were prepared. The secondary or the confirmatory tests were also done for the detections of

pathogen, if the primary test were found to be positive. Suitable precautions were also taken

performing these tests.

Microbial limit test was done mainly to detect the presence of pathogenic organism i.e. E.

coli, salmonella, Pseudomonas aeruginosa and Staphylococcus aureus. During this project

work microbial limit test was done for the raw water, water for injection and demineralized

water. WFI (water for injection) did not show positive results for the primary test. So no

further testing was done. But demineralized water showed the positive result and in further

testing and Salmonella was detected.

Bacterial endotoxin test was done to detect the presence of endotoxin, fever producing by

product of the Gram negative bacteria know as pyrogen which might be present in the

pharmaceutical products. It was done by using limulus amoebocyte lysate (LAL) from the

horse shoe crab. LAL test were done the detections of Phermaceutical products. No gel

formations were occurred in both the products. Positive control shows the gel formation but

negative control not shows gel formation.

Sterility test were done to detect the presence of the viable forms of micro organism in or on

pharmaceutical preparations. Two types of media were used FTM and SCDM for the

detections of bacterial and fungus. It was mainly done with the membrane filtrations method.

Phermaceutical products were tested for the related test. Both the product did not show any

growth in the mediums. Positive control shows turbidity.

Environment monitoring was mainly done to check the bio-burden of the sterile area.

Sometime the micro flora in the sterile area will cause contamination to the product and also

cause the contamination in the microbiological lab. So it is necessary to detect the micro flora

of the environment. It was done by many methods like settle plate method, air sampler and

personal monitoring by using RODAC plates. Personal monitoring was done to check the

personal hygiene of the worker working in the sterile area because worker can also

54



www.final-yearproject.com | www.finalyearthesis.com contaminate the products. All the results of the Environment monitoring were found

satisfactory. If the micro flora is increased in the sterile area then the fumigation of sterile

area and cleaning of area with 70% IPA were done to kill the microorganisms.

55




Aton JC, Paton AW (July 1998). "Pathogenesis and diagnosisof Shiga toxin-

producing Escherichia coli infections". Clin.Microbiol. Rev. 11 (3): 450–79.

Anzai Y, Kim H, Park, JY, Wakabayashi H (2000). "Phylogenetic affiliation of the

pseudomonads based on 16S rRNAsequence". Int J Syst Evol Microbiol 50.

'Argenio DA, Gallagher LA, Berg CA, Manoil C (2001). "Drosophila as a model host

for Pseudomonas aeruginosa infection". J. Bacteriol.

56



www.final-yearproject.com | www.finalyearthesis.com Ananthanarayan and Panikers, text book of microbiology, seventh edition 2005.

Atlas, R.M. (2004). Handbook of microbiological media London: crc press. Pp.p.68

Accessed May 03, 2008.

Bentley, R, Meganathan, R., Biosynthesis of Vitamin K (menaquinone) in Bacteria,

Bacteriological Reviews, 1982, 46(3):241-280. Review.Centers for Disease Control

and Prevention

Cornelis P (editor). (2008). Pseudomonas: Genomics and Molecular Biology (1st ed.).

Caister Academic Press.

Cruickshank R: Medical Microbiology, 1968, Churchil Livingstone, Edinburgh.

Diekema DJ, Pfaller MA, Jones RN, et al (1999). "Survey of bloodstream infections

due to gram-negative bacilli: frequency of occurrence and antimicrobial susceptibility

of isolates collected in the United States, Canada, and Latin America for the

SENTRY Antimicrobial Surveillance Program, 1997". Clin. Infect. Dis. 29 (3).

"Escherichia coli O157:H7". CDC Division of Bacterial and Mycotic Diseases.

Retrieved on 2007-01-25.

Fine MJ, Smith MA, Carson CA, et al (1996). "Prognosis andoutcomes of patients

with community-acquired pneumonia. A meta-analysis". JAMA 275 (2): 134–41.

Giannella RA (1996). "Salmonella". in Baron S et al (eds.).Baron's Medical

Microbiology (4th ed. ed.). Univ of Texas Medical Branch..

Holt JG (editor) (1994). Bergey's Manual of Determinative Bacteriology (9th ed.).

Williams & Wilkins.

Hurley JC (April 1995). "Endotoxemia: methods of detection and clinical correlates".

Clinical microbiology reviews 8 (2).

Indian Pharmacopeia (1996).

J. van Eldere. "[Multicentre surveillance of Pseudomonas aeruginosa susceptibility

patterns in nosocomial infections.

Kluytmasns J , Van Belkum A , Verbrugh H (july 1997) “Nasalcarriage of

Staphylococcus aureus : epidemiology, underlyingmachanism and associated links”

Mehta R.M, Drug store and business management, 3rdEdition. Mestrendrea, L.-

PhRMA Env Monitoring group.1997. Microbiological Monitoring of Environmental

conditions for Non sterile Pharmaceutical Manufacturing. Pharm Technol.21:58-74

57



www.final-yearproject.com | www.finalyearthesis.com Mathews KR,Roberson J,Gillespie BE, Luther DA ,Oliver SP 1997).identification

and differentiation of coagulase negative Staphylococcus aureus by polymerase chain

reaction” Journals of food protection.

Mürer, E.H., Levin. J. and Holm, R., 1975. Isolation and studies of the granules of the

ameobocytes of Limulus polyphemus, the horseshoe crab. J. Cell Physiol; 533-542.

Madigan M, Martinko J (editors). (2005). Brock Biology of Microorganisms (11th

ed.). Prentice Hall.

Prithiviraj B, Bais H, Weir T, Suresh B, Najarro E, Dayakar B, Schweizer H, Vivanco

J (2005). "Down regulation of virulence factors of Pseudomonas aeruginosa by

salicylic acid attenuates its virulence on Arabidopsis thaliana and Caenorhabditis

elegans.” Infect Immune 73 (9).

Quality assurance in bacteriological and immunological by Sudarshan kumari, Rajesh

Bhattia, C.C. Heuk.

Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.).

Rahme LG, Stevens EJ, Wolfort SF, Shao J, Tompkins RG, Ausubel FM (1995).

"Common virulence factors for bacterial pathogenicity in plants and animals".

Science 268 (5219).

Rahme LG, Tan MW, Le L, et al (1997). "Use of model plant hosts to identify

Pseudomonas aeruginosa virulence factors". Proc. Natl. Acad. Sci. U.S.A. 94 (24).

Rahme LG, Ausubel FM, Cao H, et al (2000). "Plants and animals share functionally

common bacterial virulence factors". Proc. Natl. Acad. Sci. U.S.A. 97 (16).

Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed. ed.).

McGraw Hill. pp. 362–8.

Reid, G.; J. Howard and B.S. Gan (September 2001). "Can bacterial interference

prevent infection?" Trends in Microbiology 9(9):424-428.

S. cholerasuis pathology. Accessed April 3., 2009Vogt, R.L.; L. Dippold (Mar-Apr

2005). "Escherichia coli O157:H7 outbreak associated with consumption of ground

beef, June-July 2002". Public Health Reports 2: 174–178.

S.; J. Guignot and A.L. Servin (July 2001). "Escherichia coli strains colonizing the

gastrointestinal tract protect germfree mice against Salmonella typhimurium

infection." Gut 49:47-55.

58



www.final-yearproject.com | www.finalyearthesis.com Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor,"

Biology of Horseshoe Crabs, p.334

Sargent, William., The Year of the Crab., W.W. Norton & Company 1987.

Sandven P, Lassenj (November 1999). “Importance of selective media for recovery of

yeast from clinical specimens”. Journal of clinical microbiology 37.

Sandvan P, Lassen J (November1999) “Importance of selective media for recovery of

yeasts from clinical specimens”. Journal of clinical microbiology 37(11). Quigley,

J.P., Corcoran, G., Armstrong, P.B., A Hemolytic Activity Secreted by the

Endotoxin- Challenged Horseshoe Crab: A Novel Immune System Operating at the

Surface of the Carapace. , Biological Bulletin, 193: 273 (October 1997)

"The phylogeny of the genera Chryseomonas, Flavimonas, and Pseudomonas

supports synonymy of these three genera". Int J Syst Bacteriol 47 (2): 249–51. 1997.

Thompson, Andrea (2007-06-04). "E. coli Thrives in Beach Sands". Live Science.

Retrieved on 2007-12-03.

USP 1970 Microbial limit test. USP XVII p 846. Wilson, James. 1997. Setting

Alert/Action Limit for Environment Program.PDA J Pharma Sci Tech. 51:161-162.

Walker TS, Bais HP, Déziel E, et al (2004). "Pseudomonas aeruginosa-plant root

interactions. Pathogenicity, biofilm formation, and root exudation". Plant Physiol.

134 (1).

http://www.cfsan.fda.gov/~dms/a2z-s.html. Retrieved on 2009-02-14.

www.wisegeek.com

www.accugen lab.com/microbial limit test.html.

http://www.cgmh.org.tw/chldhos/intr/c4a00/academy/bugs/

salchole.html(www.millipore.com/processmonitoring/pm3/sterility_testing)

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?

mode=Tree&id=561&lvl=3&lin=f&keep=1&srchmode=1&unlock.Retrievedon2007-

11-30. www.bd.com/ds/technicalcenter/citramide_agar_bare.pdf.

www.bd.com/ds/technicalcenter/citramide_agar_bare.pdf. Omnipresence of

Microorganism in the environment.

http:/www.waksmanfoundation.org/lab/Isu/omni.html

59



http://www.bd.com/ds/technicalcenter/citramide_agar_bare.pdf.%20Omnipresence


MEDIA USED

1) Soya bean casein digest agar (SCDA)

60



www.final-yearproject.com | www.finalyearthesis.com Pancreatic digest of casein 15.0g

Papain digest of soyabean meal 5.0g

Sodium chloride 5.0g

Agar 15.0g

Water to 1000ml

pH after sterilization 7.3± 0.2

2) Sabouraud dextrose agar (SDA)

Peptone 10.0g

Dextrose 40.0g

Agar 15.0g

Distilled water 1000ml

pH after sterilization 5.6±0.2

It is used to cultivate dermatophite and other fungi

3) Baired parker agar (BPA)

Pancreatic digest of casein 10.0g

Beef extract 5.0g

Yeast extract 1.0g

Lecithin chloride 5.0g

Agar 20.0g

Glycine 20.0g

Sodium pyruvate 10.0g

Distilled Water 1000ml

It is selective medium for growth of coagulase +ve Staphylococcus.

4) Mannitol salt agar medium (MSA)

Pancreatic digest of casein 5.0 g

Peptic digest of animal tissue 5.0g

61



www.final-yearproject.com | www.finalyearthesis.com d-mannitol 10.0g


Agar 15.0g

Phenol red 25mg


It is selective medium for Staphylococcus aureus.

5) MacConkey agar (MCA)

Pancreatic digest of gelatin 17.0g

Peptone 3.0g

Lactose 10.0g

NaCl 5.0g

Bile salt 1.5g

Agar 13.5g

Neutral red 30.0mg

Crystal violet 1.0mg


pH after sterilization 7.1±0.2

This is useful media for the cultivation of enteric bacteria. It contain bile salt to inhibit non

intestinal bacteria and lactose with neutral red to distinguish the lactose fermenting

coliform’s from non lactose fermenting salmonella and dysentery group (Robert

Cruickshank)

6) Brilliant green agar (BGA)

Peptone 10g

Yeast extracts 3.0g

Lactose 10g

Sucrose 10g


Phenol red 80.0mg

Brilliant green 12.5g

Agar 12.0g

62



www.final-yearproject.com | www.finalyearthesis.com Distilled Water 1000ml

An enriched a highly selective medium for the isolation of Salmonella species.

7) Bismuth sulphite agar medium (BSA)

Beef extract 6.0g

Peptic digest of animal tissue 5.0g

Dextrose 5.0g

Agar 15.0g

Ferric citrate 6.4g

Brilliant green 10 mg

Disodium phosphate 4.0g


pH 7.6

Bismuth sulphite agar media used to isolate Salmonella spp. Dextrose is used at prime source

of carbon. (Atlas, R.M 2004)

8) Xylose lysine dextrose agar (XLDA)

Xylose 3.0g

L-lysine 5.0g

Sucrose 7.5g


Yeast extract 3.0g

Phenol red 80mg

Agar 13.5g

Sodium deoxycholate 2.5g

Sodium thiosulphate 6.8g

Ferric ammonium citrate 800mg


It is selective medium for Salmonella.

9) Cetrimide agar (CA)

63



www.final-yearproject.com | www.finalyearthesis.com Pancreatic digest of gelatin 20g

Magnesium chloride 1.4g

Potassium sulphate 10g

Cetrimide 0.3 g

Agar 13.6 g

Glycerin 10g

Water 1000ml

Citramide agar is used as selective isolation of Gram negative bacteria, Pseudomonas

aeruginosa.

10)Selenite F broth

Peptone 5.0g

Lactose 4.0g

Disodium orthophosphate 10.0g

Sodium hydrogen selenite 4.0g


11) Triple sugar iron agar medium

Beef extract 3.0g

Yeast extract 3.0g

Peptone 20.0g

Lactose 10.0g

Sucrose 10.0g

D-glucose monohydrate 1.0g

Iron (II) sulphate 0.2g


Phenol red 24.0 mg

Agar 13g


64



www.final-yearproject.com | www.finalyearthesis.com 12)Peptone

Peptone 10g

Sodium Chloride 5g

water 1000ml

13)Glucose phosphate media

Peptone 7.0g

Dipotassium hydrogen phosphate 5.0g

Glucose 5.0g


14) Substrate utilization media (Simmon’s citrate medium)


Magnesium sulphate 0.2g

Ammonium Dihydrogen phosphate 1.0g

Dipotassium hydrogen phosphate 1.0g

Sodium citrate 2.0g

Agar 15.0g

Bromothymol blue 0.08g


15) SCDM (Soyabean casein digest medium):-

Pancreatic Digest of casein - 15 gm

Papaic Digest of Soybean - 5.0 gm

Sodium chloride - 5.0 gm

Agar - 15 gm

Distilled water - 1000 ml

pH after sterilization - 7.3 ± 0.2

16) FTM (Fluid Thioglycolate Medium)

Casien Enzymic Hydrolysate - 15 gm

Yeast Extract - 5.0 gm

Dextrose - 5.5 gm

65




Sodium Chloride - 2.5 gm

L - Cystine - 0.5 gm

Sodium Thioglycolate - 0.5 gm

Resazurin Sodium - 1 ml

Agar - 0.75 gm

Water - 1000ml

pH - 7.1 ± 0.2

16) Buffered Sodium Chloride-Peptone solution ph7.0

Potassium dihydrogen phosphate - 3.0g

Disodium hydrogen phosphate - 7.2g

Sodium chloride - 4.3g

Peptone (meat or casein) - 1.0g

Water - 1000ml

17) Peptone Water:-

Dissolve 1.0 gm of peptone in 1000ml of WFI and pour in the 500ml glass vials. Seal the

vials containing the peptone water with rubber bunks and aluminium seals. Sterilized the

peptone water vials by autoclaving the sealed vials at 121º C at 15 lb/ in² for 15 mins. And

after pre- incubation for 48 hrs are transfer to the sterility room through the pass box for the

further testing

66



FYP Msc Biotch Report

Documents

convert hydrogen

axa parenterals

united state

membrane filtration

laminar air

membrane filtration

tube contaning

membrane filtration