FYP MSC Biotch Report

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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:Gavendra Department Of Biotechnology I.I.M.T Institute Aligarh (U.P.) 1
Submitted To:Mr. Sudeep Tiwari ( H.O.D) Internal Supervisor (Training / Project Incharge) Dept. of Biotechnology / Microbio.
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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
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
CONTENTS
S.NO. 1. 2. 3. 4. 5. 6. 7. 8. CHAPTER Introduction Review of Literature Aims & Objective Materials & Methods Results Summary Bibliography Appendix PAGE NO. 5 - 7 8 - 18 19-20 21 - 41 42 - 50 51 - 53 54 - 57 58 - 64
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.
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.
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.
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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 grapelike 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
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 coagulasenegative. (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).
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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/a2zs.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 million
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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 of 14
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 crabs 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
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) -glucanFactor c Activated factor c Activated factor G Factor GFactor B Activated factor BProclotting 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 media. 16
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:i. Plate setteling methods. (Passive sampling) 17
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 plates 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 5. In this case particle counts are recorded as the number of 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 . 18
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)
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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 monitoringa) By the Air sampler method. b) By the Settle plate method. c) By the Swab method.
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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.
A) Agar used in MLT:-
MICROBIAL LIMIT TEST
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 paper, cotton plugs, slides, Bunsen burner and cover slips. Instruments used:Autoclave, laminar air flow, incubator, Refrigerator and microscope plate, test tubes, micropipettes, measuring cylinder, conical flask, volumetric flask, inoculation loop, aluminium foil, butter
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B)
BACTERIAL ENDOTOXIN TEST
Material & Equipments:1 Vortex mixture 2 Heating block 3 Diluents 4 Micropipettes 5 Micro tip 6 Test tube : temp 370 C : pH 3.0 KH2PO4 : 20-200 l : 200 l, : 12x75 mm 100-1000l 1000 l 10x75 mm
(Dehydrogenated &individually packed) 7 Reagent : Limulus amoebocyte lysate(LAL-R)
C) Media Used:-
STERILITY TEST
1) SCDM (Soyabean casein digest medium):Preparation Method: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 121 C at 15 lb/ in 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) 23
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 121 C at 15 lb/ in 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 2.5C for about 2 days and SCDM vials in BOD incubator at 22.5 2.5C 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 Disodium hydrogen phosphate Sodium chloride Peptone (meat or casein) Water 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. 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 121 C at 15 lb/ in for 15 mins. And after preincubation for 48 hrs are transfer to the sterility room through the pass box for the further testing - 3.0g - 7.2g - 4.3g - 1.0g - 1000ml
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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), Saborauds 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.
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METHODS
A) 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. 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. 26 MICROBIAL LIMIT TEST
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. 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 1520ml of sterile R2A agar medium. 27
Mix
the
conten
properly
by
gently
rotating
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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. 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. 28
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 VogelJohnson 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.
2.Detection And Identification Of Escherichia coli Primary Test for E.coli: 29
Transferred 1ml sample from turbid SCDM into 100ml sterile MacConkey broth. Incubated at 42-44C 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 Blairs agar & incubate at 30350c for 24-48 hrs. Simultaneously carry out the positive control by streaking a growth of Salmonella sp.on the surface of Wilson Blairs agar medium.Invert & incubate the plates at 30-350c for 24-48 hrs. 30
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 Blairs 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 C for 18-72 hrs. If upon examination colonies having green in colour it indicates the presence of Pseudomonas aeruginosa.
Confirmatory Test:Pigment test: 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. 31
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 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. indicates the pesence of Pseudomonas
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.
B)
BACTERIAL ENDOTOXIN TEST
32
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) Dilute 0.05 ml of dilution (1) with 1.95 of LWR.dilution (2)
33
Using dilution (2) prepare a series of tow fold dilution to give concentration of 2,,0.5 &0.25 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 &0.25 i.e.0.25, 0.125, 0.06 &0.03 EU/ML respectively. Where = 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 3710 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:Geometric mean end point concentration antilog e/f. 34 2, , 0.5
Where e 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 0.5 and not more than 2 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 3710 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. 1 2 3 4 5 6
Description WNC WNC Test Tube Test Tube PPC PPC
Sample --50 50 50 50
LWR 100 100 50 50 ---
CSE ----50 50
Lysate 100 100 100 100 100 100
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)
Preparation before test:-
STERILITY 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 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 0.45 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 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. removed the filter paper from the membrane filter assembly with the help of the sterile forceps and cut it switch off UV light of
laminar air flow. All the required materials were transferred to the testing room from
37
Filter papers were completely submerged in their respective completion along with media lot number.
media and the tubes
were marked with the sample number, batch number, date of testing and date of 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 30-35C and all tubes of SCDM were incubated at 20-25C 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. 1 2 3 4 GRADES Grade A Grade B Grade C Grade D AREAS Filling machine / LAF. Filling room / sterile corridor. Ceiling room. Change room II / washing area.
Alert limit and action limit of different areas are:S. No. 1 2 3 4 GRADES Grade A Grade B Grade C Grade D Alert limit <1 3 20 50 Action limit 1 5 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

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:39
Prepare the SCDA plates after autoclaving the media. Incubate these plates in inverted position for 48 hrs. at 30-35C 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 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. 40
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 30-35C 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 Then put these plates in sterile container and bring back to lab. Incubate these plates in the bacteriological incubator at 30-35C for 48 hrs for the bacterial count. 41
After taking the bacterial count, Incubate these plates in the BOD incubator at 20-25C 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
A) MICROBIAL LIMIT TEST

Table shows the detection of the microorganism from the differrent products. PRODUCT NAME NSM MANGO AFRESH LEMON MULTIVITAMIN ACTIVATED FIBRE CELL ACTIVATOR ALOE PLUS ESTER 500 IG 6 SPIRULLINA AFRESH ELAICHI TBC 11000 cfu/g
(NMT 10000CFU/G)
1 2 3 4 5 6 7 8 9 10
TFC 310 cfu/g

(NMT 300CFU/G)
PATHOGEN present absent absent absent absent absent absent absent absent absent
REMARK fail pass pass pass pass pass pass pass pass pass
<10
(NMT 10000CFU/G)
<10
(NMT 300CFU/G)
10
(NMT 10000CFU/G)
<10
(NMT 300CFU/G)
<10
(NMT 10000CFU/G)
<10
(NMT 300CFU/G)
<10
(NMT 10000CFU/G)
<10
(NMT 300CFU/G)
<10
(NMT 10000CFU/G)
<10
(NMT 300CFU/G)
<10
(NMT 10000CFU/G)
<10
(NMT 300CFU/G)
<10
(NMT 10000CFU/G)
<10
(NMT 300CFU/G)
40
(NMT 50000CFU/G)
<10
(NMT 500CFU/G)
30
(NMT 10000CFU/G)
<10
(NMT 300CFU/G)
Result of mango shake was found to be unsatisfactory Because Number of colonies are more than their limit and pathogen are also present. Where as Results of other products are under limits and satisfactory.
44
B) RESULTS OF BACTERIAL ENDOTOXIN TESTS
Table shows the result of bacterial endotoxin test:S. No. 1 2 3 4 Product Name Ramadole inj. Gentamycin sulphate inj. Positive control Negative control Gel Formation No No Yes No Result Negative Negative Positive 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 Date of Sampling Batch No./ Lot no. Date of incubation Rinsing Fluid : Membrane Filtration : 14/5/11 : RC013 : 17/5/11 : ------Media . : SCDM , FTM 23 bottles 3000 ltr April 2013 14 Days : :
Sample Quantity : Batch Size Date of Expiry
Date of Manufacturing : May 2011
Day of incubation :
Settle plate under LAF in nos.<1cfu/plt
OBSERVATION:Fluid Thioglycollate Days Date Medium at 32.5 2.5oC TEST NEGATIVE POSITIVE Soyabean Casein digest Medium at 22.5 2.5oC TEST NEGATIVE POSITIVE Analysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14
15/5 16/5 17/5 18/5 19/5 20/5 21/5 22/5 23/5 24/5 25/5 26/5 27/5 28/5
-ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve
No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth
Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth
-ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve
No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth No growth
Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth
OK OK OK OK OK OK OK OK OK OK OK OK OK 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 2 3 4 5 6 7
Under LAF Filling Machine Sterile corridor Filling room Ceiling room 6 Change room Washing room
A A B B C D D
<1 <1 <3 <3 < 20 < 50 < 50
2 2 2 2 2 2 2
<1 <1 <1 <1 < 15 < 41 < 42
<1 <1 <2 <1 <9 < 36 < 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 2 3 4 5 6 7
Under LAF Filling Machine Sterile corridor Filling room Ceiling room 6 Change room Washing room
A A B B C D D
<1 <1 <3 <3 < 20 < 50 < 50
1 1 2 2 2 2 2
<1 <1 <1 <1 < 10 < 35 < 41
Nil Nil <1 <1 <8 < 37 < 40
Result were found within the limit.
Table shows the results of personal monitoring of workers working in sterile area, by RODAC plate method. 48
S. No. 1 2 3 4 5 6 7 8 9
Name Tapender Tapender Tapender Mahesh Mahesh Mahesh Sarvjeet Sarvjeet Sarvjeet
Garments F. Hood Sterile Dress Booties F. Hood Sterile Dress Booties F. Hood Sterile Dress Booties
Grade B B B B B B B B B
Colony Observed 4 3 5 2 1 2 1 3 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
Fig. E.coli showing green metallic sheen coloured on MacConkey agar plate.
Fig. E.coli Showing pink colonies on EMB agar plate.
50
Fig. Pseudomonas aeroginosa showing green coloured colony on Citramide Agar plate
Fig. S. aureas produce yellow coloured colonies on Mannitol Salt Agar
51
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 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.
53
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 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.
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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: 174178. 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. 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 EndotoxinChallenged 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): 24951. 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, Dziel 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.
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http://www.cgmh.org.tw/chldhos/intr/c4a00/academy/bugs/salchole.html(www.millipo re.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.Retrievedon200711-30. www.bd.com/ds/technicalcenter/citramide_agar_bare.pdf. www.bd.com/ds/technicalcenter/citramide_agar_bare.pdf. Microorganism in the environment. http:/www.waksmanfoundation.org/lab/Isu/omni.html Omnipresence of
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MEDIA USED
1) Soya bean casein digest agar (SCDA) Pancreatic digest of casein Sodium chloride Agar Water to pH after sterilization 15.0g 5.0g 15.0g 1000ml 7.3 0.2 Papain digest of soyabean meal 5.0g
2) Sabouraud dextrose agar (SDA) Peptone Dextrose Agar Distilled water pH after sterilization 10.0g 40.0g 15.0g 1000ml 5.60.2
It is used to cultivate dermatophite and other fungi 3) Baired parker agar (BPA) Pancreatic digest of casein Beef extract Yeast extract Lecithin chloride Agar Glycine 10.0g 5.0g 1.0g 5.0g 20.0g 20.0g 60
Sodium pyruvate Distilled Water
10.0g 1000ml
It is selective medium for growth of coagulase +ve Staphylococcus.
4) Mannitol salt agar medium (MSA) Pancreatic digest of casein Peptic digest of animal tissue d-mannitol Sodium chloride Agar Phenol red Distilled water 5.0 g 5.0g 10.0g 75.0g 15.0g 25mg 1000ml
It is selective medium for Staphylococcus aureus. 5) MacConkey agar (MCA) Pancreatic digest of gelatin Peptone Lactose NaCl Bile salt Agar Neutral red Crystal violet Distilled Water pH after sterilization 17.0g 3.0g 10.0g 5.0g 1.5g 13.5g 30.0mg 1.0mg 1000ml 7.10.2
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 coliforms from non lactose fermenting salmonella and dysentery group (Robert Cruickshank)
61
6) Brilliant green agar (BGA) Peptone Yeast extracts Lactose Sucrose Sodium chloride Phenol red Brilliant green Agar Distilled Water 10g 3.0g 10g 10g 5.0g 80.0mg 12.5g 12.0g 1000ml
An enriched a highly selective medium for the isolation of Salmonella species.
7) Bismuth sulphite agar medium (BSA) Beef extract Peptic digest of animal tissue Dextrose Agar Ferric citrate Brilliant green Disodium phosphate Distilled water pH of carbon. (Atlas, R.M 2004) 8) Xylose lysine dextrose agar (XLDA) Xylose L-lysine Sucrose Sodium chloride Yeast extract 3.0g 5.0g 7.5g 5.0g 3.0g 62 6.0g 5.0g 5.0g 15.0g 6.4g 10 mg 4.0g 1000ml 7.6
Bismuth sulphite agar media used to isolate Salmonella spp. Dextrose is used at prime source
Phenol red Agar Sodium deoxycholate Sodium thiosulphate Ferric ammonium citrate Distilled Water
80mg 13.5g 2.5g 6.8g 800mg 1000ml
It is selective medium for Salmonella.
9) Cetrimide agar (CA) Pancreatic digest of gelatin Magnesium chloride Potassium sulphate Cetrimide Agar Glycerin Water aeruginosa. 10)Selenite F broth Peptone Lactose Disodium orthophosphate Sodium hydrogen selenite Distilled water 11) Triple sugar iron agar medium Beef extract Yeast extract Peptone Lactose Sucrose 3.0g 3.0g 20.0g 10.0g 10.0g 63 5.0g 4.0g 10.0g 4.0g 1000ml 20g 1.4g 10g 0.3 g 13.6 g 10g 1000ml
Citramide agar is used as selective isolation of Gram negative bacteria, Pseudomonas
D-glucose monohydrate Iron (II) sulphate Sodium chloride Phenol red Agar Distilled water
1.0g 0.2g 5.0g 24.0 mg 13g 1000ml
12)Peptone Peptone Sodium Chloride water 13)Glucose phosphate media Peptone Dipotassium hydrogen phosphate Glucose Distilled water 7.0g 5.0g 5.0g 1000ml 10g 5g 1000ml
14) Substrate utilization media (Simmons citrate medium) Sodium chloride Magnesium sulphate Ammonium Dihydrogen phosphate Dipotassium hydrogen phosphate Sodium citrate Agar Bromothymol blue Distilled water 5.0g 0.2g 1.0g 1.0g 2.0g 15.0g 0.08g 1000ml
15) SCDM (Soyabean casein digest medium):Pancreatic Digest of casein - 15 gm 64
Papaic Digest of Soybean Sodium chloride Agar Distilled water pH after sterilization
- 5.0 gm - 5.0 gm - 15 gm - 1000 ml - 7.3 0.2
16) FTM (Fluid Thioglycolate Medium) Casien Enzymic Hydrolysate Yeast Extract Dextrose Sodium Chloride L - Cystine Sodium Thioglycolate Resazurin Sodium Agar Water pH - 15 gm - 5.0 gm - 5.5 gm - 2.5 gm - 0.5 gm - 0.5 gm - 1 ml - 0.75 gm - 1000ml - 7.1 0.2
16) Buffered Sodium Chloride-Peptone solution ph7.0 Potassium dihydrogen phosphate Disodium hydrogen phosphate Sodium chloride Peptone (meat or casein) Water 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 121 C at 15 lb/ in for 15 mins. And after preincubation for 48 hrs are transfer to the sterility room through the pass box for the further testing - 3.0g - 7.2g - 4.3g - 1.0g - 1000ml
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www.final-yearproject.com | www.finalyearthesis.

A DISSERTATION PRESENTED TO THE DR.BHIM RAO AMBEDKAR UNIVERSITY IN PARTIAL FULFILLMENT OF

MASTER OF SCIENCE IN BIOTECHNOLOGY (2010-2012)

AIMS AND OBJECTIVES

A) Agar used in MLT:-

MICROBIAL LIMIT TEST

BACTERIAL ENDOTOXIN TEST

(Dehydrogenated &individually packed) 7 Reagent : Limulus amoebocyte lysate(LAL-R)

2.Detection And Identification Of Escherichia coli Primary Test for E.coli: 29

BACTERIAL ENDOTOXIN TEST

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

Description WNC WNC Test Tube Test Tube PPC PPC

LWR 100 100 50 50 ---

Lysate 100 100 100 100 100 100

media and the tubes

1. Environment Monitoring using Settle Plate Method

2. Environment Monitoring using Air Sampler (Active Sampling Method)

A) MICROBIAL LIMIT TEST

TFC 310 cfu/g

B) RESULTS OF BACTERIAL ENDOTOXIN TESTS

C) RESULTS FOR STERILITY TEST

Sample Quantity : Batch Size Date of Expiry

Date of Manufacturing : May 2011

Settle plate under LAF in nos.<1cfu/plt

D) RESULTS OF ENVIRONMENT MONITORING

<1 <1 <3 <3 < 20 < 50 < 50

<1 <1 <1 <1 < 15 < 41 < 42

<1 <1 <2 <1 <9 < 36 < 46

Results were found within the limits.

<1 <1 <3 <3 < 20 < 50 < 50

<1 <1 <1 <1 < 10 < 35 < 41

Nil Nil <1 <1 <8 < 37 < 40

Result were found within the limit.

Fig. TSI agar slants for Salmonella

Fig. XLD media for Salmonella

Fig. E.coli Showing pink colonies on EMB agar plate.

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

Sodium pyruvate Distilled Water

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

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

80mg 13.5g 2.5g 6.8g 800mg 1000ml

It is selective medium for Salmonella.

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

1.0g 0.2g 5.0g 24.0 mg 13g 1000ml

15) SCDM (Soyabean casein digest medium):Pancreatic Digest of casein - 15 gm 64

- 5.0 gm - 5.0 gm - 15 gm - 1000 ml - 7.3 0.2

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