Published on March 26, 2012
Presented By ;- Guided By ; Amit $hah Mr. Jagdish Kakadiya M.Pharm QA Dept. Of P’cology 1st Sem - 03 Dharmaj Degree Pharmacy College , Dharmaj: Presented By ;- Guided By ; Amit $hah Mr. Jagdish Kakadiya M.Pharm QA Dept. Of P’cology 1 st Sem - 03 Dharmaj Degree Pharmacy College , Dharmaj MICROBIOLOGICAL LIMIT TEST CONTENT: CONTENT Objective Preliminary testing Different media Sampling Methods Total aerobic microbial count Membrane filtration Plate count Serial dilution Tests for specified micro-organism Escherichia coli Salmonella species Pseudomonas aeruginosa Staphylococcus aureus Tests for specified organism as per USP Application Bibliography OBJECTIVE: OBJECTIVE  Microbial limit tests are designed to estimate the number of viable aerobic organisms present in pharmaceutical products and raw materials. The microbial limit testing of raw material as well as finished pharmaceutical products can help to determine whether the product complies with requirement of BP, USP or IP. The care must be taken while performing microbial test so that contamination from outside can be avoided. 3 Preliminary Testing : Preliminary Testing  4 Continue: Continue  MODIFICATIONS: If the organisms fail to grow in the relevant medium the procedure should be modified by (a) increasing the volume of diluents with the quantity of test material remaining the same, or (b) incorporating a sufficient quantity of a suitable inactivating agent in the diluents, or (c) combining the above modifications so as to permit growth of the organisms in the media. If inhibitory substances are present in the sample, 0.5% of soya lecithin and 4% of polysorbate 20 may be added to the culture medium. 5 Continue: Continue Alternatively, repeat the test as described using fluid casein digest-soya lecithin- polysorbate 20 medium to demonstrate neutralization of preservatives OR other antimicrobial agents in the test material. 6 Media : Media  Baird – Parker Agar Medium Bismuth Sulphite Agar Medium Brilliant Green Agar Medium Buffered Sodium Chloride-Peptone Solution pH 7.0 Casein Soyabean Digest Agar Medium Cetrimide Agar Medium Desoxycholate-Citrate Agar Medium Fluid Casein Digest-Soya Lecithin-Polysorbate 20 Medium 7 Continue:: Continue: Fluid Lactose Medium Lactose broth Medium Levin Eosin-Methylene Blue Agar Medium MacConkey Agar Medium (culture of enterobacteria) MacConkey Broth Medium Mannitol Salt Agar Medium Nutrient Broth Medium Nutrient Agar Medium Pseudomonas Agar Medium for Detection of Flourescein 8 Continue:: Continue: Pseudomonas Agar Medium for Detection of Pyocyanin Sabouraud Dextrose Agar Medium Sabouraud Dextrose Agar Medium with Antibiotics Selenite F Broth Fluid Soyabean-Casein Digest Medium Tetrathionate -Bile-Brilliant Green Broth Medium Triple Sugar- Iorn Agar Medium Urea Broth Medium Vogel-Johnson Agar Medium Xylose-Lysine-Desoxycholate Agar Medium (selective media for salmonella) 9 Notes: Notes  Where agar is specified in a formula, use agar that has a moisture content of not more than 15%. Where water is called for in a formula, use purified water. The media should be sterilized by heating in an autoclave at 115°c for 30 minutes. In preparing media dissolve the soluble solids in the water, using heat if necessary, to effect complete solution an add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the required pH in the medium when it is ready for use. Determine the pH at 25°c ± 2°c 10 Some common ingredients and its use : Some common ingredients and its use  Agar: A solidifying agent which is a complex polysaccharide derived from marine algae. It has no nutritional value in media. It is bacteriological inert . It is stable at different temperature used for incubation. Peptones: Protein is large, relatively insoluble molecules that a minority of organism can utilized directly, but a partial digestion by acid or enzyme reduces protein to shorter chain of amino acids called peptone. These small, soluble fragments can be digested by most bacteria. It should be stored in a tightly closed container as it is hygroscopic in nature. 11 Continue:: Continue: Meat extract: It is prepared from fresh meat by hot water extraction. It contains water soluble constituents of animal tissue that is carbohydrates, organic nitrogen compound, water soluble vitamins and mineral salts. Yeast extract: It is particularly rich in vitamin B. It also contains carbohydrates, amino acids, inorganic salts, growth factors. 12 TERMS: TERMS  Culture medium: A nutrient material prepared for growth of micro-organism in a laboratory is called culture medium. Culture: The microbes can grow and multiply in or on a culture medium are referred to as a culture. Nutrient broth: if the complex media is in liquid form , it is called nutrient broth. Nutrient agar: when agar is added to media, it is called nutrient agar. 13 TYPES OF CULTURE MEDIA: TYPES OF CULTURE MEDIA  Selective: suppression of unwanted microbes and encouraging desired microbes . Differential: differentiation of colonies of desired microbes from others. Enrichment: similar to selective but designed to increase numbers of desired microbes to detectable levels. 14 SAMPLING: : SAMPLING  : Use 10 ml or 10 g specimens for each of the tests specified in the individual monograph. PRECAUTION: The microbial limit tests should be carried our under conditions designed to avoid accidental contamination during the test. The precautions taken to avoid contamination must be such that, they do not adversely effect any micro organism that should be revealed in the test. 15 PowerPoint Presentation: 16 METHODS:: METHODS  : 1.TOTAL AEROBIC MICROBIAL COUNT: Unit: cfu/ml or gm  colony-forming unit (CFU or cfu) is a measure of viable bacterial or fungal numbers . Unlike direct microscopic counts where all cells, dead and living, are counted, CFU measures viable cells. For convenience the results are given as CFU/ml (colony-forming units per milliliter) for liquids, and CFU/g (colony-forming units per gram) for solids. This technique allows the determination of the number of CFU per ml in the sample, and thus the microbiological load and the magnitude of the infection in humans or animals, or the degree of contamination in samples of water, vegetables, soil or fruits, and in industrial products and equipment. 17 Water soluble products: : Water soluble products: Dissolve 10 g / 10 ml of the preparation being examined in buffered sodium chloride peptone solution pH 7.0 OR any other suitable medium shown to have no antimicrobial activity and adjust the volume to 100 ml with the same medium. If necessary, adjust the pH to about 7. 18 Products insoluble in water (non-fatty) : : Products insoluble in water (non-fatty) : Suspend 10 g or 10 ml of the preparation in buffered sodium chloride-peptone solution pH 7.0 or any other suitable medium shown to have no antimicrobial activity adjust the volume to 100 ml with the same medium. If necessary, divide the preparation being examined and homogenize the suspension mechanically. A suitable surface-active agent such as 0.1% w/v of polysorbate 80 may be added to assist the suspension of poorly wettable substances. 19 Fatty products : : Fatty products : Homogenize 10 g or 10 ml of the preparation with 5g of polysorbate 20 or polysorbate 80. If necessary heat to not more than 40°c Add 85 ml of buffered sodium chloride-peptone solution pH7.0 or any other suitable medium shown to have no antimicrobial activity under the conditions of the test. Maintain this temperature for the shortest time necessary for formation of an emulsion and in any case for not more than 30 minutes. If necessary adjust the pH to about 7. 20 Membrane filtration method : Membrane filtration method Use membrane filters 50 mm in diameter and having a nominal pore size of not greater than 0.45 μm the effectiveness of which in retaining bacteria has been established for the type of preparation being examined. Eg. Cellulose nitrate membrane filter Sterilize and assemble the filtration apparatus. Transfer 10 ml or a quantity of each dilution containing 1 g of the preparation being examined to each of two membrane filters and filter immediately. If necessary, dilute the pretreated preparation so that a colony count of 10 to 100 may be expected. 21 Membrane filtration assembly and A sterile screw-capped container : Membrane filtration assembly and A sterile screw-capped container  22 Continue:: Continue: Transfer one of the membrane filters, intended for the enumeration of bacteria, to the surface of a plate of casein soyabean digest agar and the other, intended for the enumeration of fungi, to the surface of a plate of Sabouraud dextrose agar with antibiotics. Incubate the plates for 5 days, unless a more reliable count is obtained in shorter time, at 30° to 35°c in the test for bacteria and 20°c to 25°c in the test for fungi. Count the number of colonies that are informed. Calculate the number of micro-organisms per g or per ml of the preparation being examined, if necessary counting bacteria and fungi separately. 23 Laminar air flow unit: Laminar air flow unit  24 Plate count method : Plate count method  For bacteria Using Petri dishes 9 to 10 cm in diameter, add to each dish a mixture of 1 ml of the pretreated preparation and about 15 ml of the liquefied casein soyabean digest agar at not more than 45°. Alternatively, spread the pretreated preparation on the surface of the solidified medium in a Petri dish of the same diameter. If necessary, dilute the pretreated preparation as described above so that a colony count of not more than 300 may be expected. Prepare at least two such Petri dishes using the same dilution and incubate 30° to 35° for 4 days , unless a more reliable count is obtained in a shorter time. 25 Plate showing the colony colony counter : Plate showing the colony  colony counter  26 Continue:: Continue: Count the number colonies that are formed. Calculate the results using plates with the greatest number of colonies but taking 300 colonies per plate as the maximum consistent with good evaluation. For fungi – Proceed as described in the test for bacteria but use Sabouraud dextrose agar with antibiotics in place of casein soyabean digest agar and incubate the plates at 20° to 25° for 5 days , unless a more reliable count is obtained in a shorter time. Calculate the results using plates with not more than 100 colonies. 27 Serial Dilution Method (Multiple tube method) : Serial Dilution Method (Multiple tube method)  Use 12 test tubes : 9 containing 9 ml of soybean-casein digest medium each and 3 containing 10 ml of the same medium each for control. Prepare dilutions using the 9 tubes. First, add 1 ml of the test fluid to each of three test tubes and mix to make 10- times dilutions. Second, add 1 ml of each of the 10-times dilutions to each of another three test tubes and mix to make 100-times dilutions. Third, add 1 ml of each of the 100-times dilutions to each of the remaining three test tubes and mix to make 1,000- times dilutions. 28 Continue:: Continue: Incubate all 12 test tubes for at least 5 days at 30 - 35°c. No microbial growth should be observed for the control test tubes. If the determination of the result is difficult or if the result is not reliable, take a 0.1ml fluid from each of the 9 test tubes and place it to an agar medium or fluid medium, incubate all media for 24-72 hours at 30°-35°c, and check them for the absence or presence of microbial growth. Calculate the most probable number of microorganisms per ml or gram of the sample 29 TESTS FOR SPECIFIED MICRO ORGANISMS : TESTS FOR SPECIFIED MICRO ORGANISMS As per IP Escherichia coli Salmonella Pseudomonas aeruginosa Staphylococcus aureus As per USP Escherichia coli Salmonella Pseudomonas aeruginosa Staphylococcus aureus Candida albicans clostridium sporogenes 30 Escherichia coli is a Gram negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2 and by preventing the establishment of pathogenic bacteria within the intestine. : Escherichia coli  is a Gram negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K 2 and by preventing the establishment of pathogenic bacteria within the intestine. 31 As per IP Escherichia coli : As per IP Escherichia coli  Place the prescribed quantity in a sterile screw-capped container, add 50 ml of nutrient broth, shake, and allow standing for 1 hour and shaking again. Loosen the cap and incubate at 37°c for 18 to 24 hours. Primary test: Add 1.0 ml of the enrichment culture to a tube containing 5 ml of Mac Conkey broth . Incubate in water-bath at 36° to 38° for 48 hours. If the contents of the tube shows acid and gas carry out secondary test. 32 Continue:: Continue: Secondary test: Add 0.1 ml of the content of the tubes containing (a) 5 ml of MacConkey broth for acid and gas and (b) 5 ml of peptone water for indole. Incubate in a water-bath at 43.5° to 44.5° for 24 hours Test for indole , add 0.5 ml of Kovac’s reagent , shake well and allow to stand for 1 minute; if a red color is produced in the reagent layer indole is present. The presence of acid and gas and indole in the secondary test indicates the presence of Escherichia coli. 33 Continue:: Continue: Carry out a control test by repeating the primary and secondary tests adding 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 Escherichia coli organisms, prepared from a 24 hour culture in nutrient broth, to 5 ml of MacConkey broth. The test is not valid unless the results indicate that control contains Escherichia coli. Kovac's reagent  : A reagent used to detect the presence of indole which is used in identification of bacteria. Indole test  :The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to split indole from the amino acid tryptophan. 34 Salmonella is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly motile enterobacteria. Flagella which project in all directions (i.e. peritrichous). They cause illnesses like typhoid fever, paratyphoid fever, and the food borne illness. : Salmonella  is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly motile enterobacteria. Flagella which project in all directions (i.e. peritrichous). They cause illnesses like typhoid fever, paratyphoid fever, and the food borne illness. 35 Salmonella : Salmonella  Transfer a quantity of the pretreated preparation being examined containing 1 g or 1 ml of the product to 100 ml of nutrient broth in a sterile screw-capped jar , shake, allow to stand for 4 hours and shake again. Loosen the cap and incubate at 35º to 37º for 24 hours. Primary Test: Add 1.0 ml of the enrichment culture to each of the two tubes containing (a) 10 ml of selenite F broth and (b) Tetrathionate bile-brilliant green broth and incubate at 36º to 38º for 48 hours. From each of these two cultures subculture on at least two of the following four agar media : bismuth sulphite agar, brillinat green agar, desoxycholate-citrate agar and xylosetysine desoxycholate agar. 36 Test for salmonella:: Test for salmonella: 37 Continue:: Continue: Incubate the plates at 36ºc to 38ºc for 18 to 24 hours. Upon examination, if none of the colonies conforms to the description given in Table, the sample meets the requirements of the test for the absence for the genus Salmonella. If any colonies conforming to the description in Table are produced, carry out the secondary test. Secondary test: Subculture any colonies showing the characteristics given in Table in triple sugar- iron agar by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle , and at the same time inoculate a tube of urea broth . Incubate at 36ºc to 38ºc for 18 to 24 hours. 38 Continue:: Continue: The formation of acid and gas in the stab culture and the absence of acidity from the surface growth in the triple sugar iron agar , together with the absence of a red color in the urea broth , indicates the presence of salmonellae. Carry out the control test by repeating the primary and secondary test using 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 salmonella organisms , prepared form a 24-hour culture in nutrient broth. The test is not valid unless the results indicate that the control contains Salmonella. 39 Pseudomonas aeruginosa : Pseudomonas aeruginosa  It is a gram negative bacterium which can cause disease in humans and non-human animals. It is found in soil, water, skin flora , and most man-made environments throughout the world. It uses a wide range of organic material for food; in animals, the versatility enables the organism to infect damaged tissues or people with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis . If such colonization occur in critical body organs such as the lungs , the urinary tract , and kidneys , the results can be fatal. 40 Continue: Continue  P. aeruginosa strains produce two types of soluble pigments , the fluorescent pigment pyoverdin and the blue pigment pyocyanin . The latter is produced abundantly in media of low-iron content and functions in iron metabolism in the bacterium. Pyocyanin refers to "blue pus" , which is a characteristic of infections caused by Pseudomonas aeruginosa. 41 Continue: Continue  Pretreat the preparation being examined as described above and inoculate 100 ml of fluid soyabean-casein digest medium with a quantity of the solution, suspension or emulsions, thus obtained containing 1 g or 1 ml of the preparation being examined. Mix and incubate at 35º to 37º for 24 hours. Examine the medium form growth is present, streak a portion of the medium on the surface of cetrimide agar medium , each plated on Petri dishes. Cover and incubate at 35º to 37º for 18 to 24 hours. If upon examination, none of the plates contains colonies having the characteristics listed in Table for the media used, the sample meets the requirement for freedom from Pseudomonas aeruginosa. If any colonies conforming to the description in Table are produced, carry out the oxidase and pigment tests. 42 Tests for Pseudomonas aeruginosa: Tests for Pseudomonas aeruginosa 43 Oxidase and pigment tests: : Oxidase and pigment tests: Streak representative suspect colonies from agar surface of cetrimide agar on the surfaces of pseudomonas agar medium for detection of fluorescein and pseudomonas agar medium for detection of pyocyanin contained in Petri dishes. Cover and invert the inoculated media and incubate at 33º to 37º for not less than 3 days. Examine the streaked surfaces under ultra-violet light . Examine the plates to determine whether colonies conforming to the description in previous table are present. 44 Continue:: Continue: If growth of suspect colonies occurs, place 2 or 3 drops of a freshly prepared 1% w/v solution of N, N, N1, N1 – tetramethyl-4-phenylenediamine dihydrochloride on filter paper and smear with colony; if there is no development of a pink color, changing to purple , the sample meets the requirements of the test for the absence of Pseudomonas aeruginosa. 45 Staphylococcus aureus  : Staphylococcus aureus  S. aureus are gram positive cocci (in clusters) which can cause a range of illnesses from minor skin infections, such as pimples, scalded skin syndrome, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis , endocarditis , chest pain, bacteremia , and sepsis. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. 46 Staphylococcus aureus : Proceed as described under Pseudomonas aeruginosa. If, upon examination of the incubated plates, none of them contains colonies having the characteristics listed in Table for the media used, the sample meets the requirements for the absence of Staphylococcus aureus. If growth occurs, carry out the coagulase test. Staphylococcus aureus  47 Continue:: Continue: 48 Continue:: Continue: Coagulase test: Transfer representative suspect colonies from the agar surface of any of the media listed in Table to individual tubes , each containing 0.5 ml of mammalian , preferably rabbit or horse, plasma with or without additives. Incubate in water-bath at 37º examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. If no coagulation in any degree is observed, the sample meets the requirements of the test for the absence of Staphylococcus aureus. 49 TEST FOR SPECIFIED ORGANISM AS PER USP : TEST FOR SPECIFIED ORGANISM AS PER USP  Transfer 10 ml of preparation to 90 ml soya bean casein digest broth and shake the container. Incubate at 30-35ºc for 18-24 hour and proceed further for E.coil, S.aureus and P.aureginosa as per follows. For salmonella incubate the preparation at 30-35ºc for 18-24 hour and proceed further. 50 Test for E.coil: : Test for E.coil: After completion of incubation period, transfer 1 ml enriched medium to 100 ml Mac-Conkey΄s broth , with the help of sterile pipette and incubate at 42-44ºc for 24-48 hour. Subculture on the plate of Mac- Conkey agar from above medium and incubate at 30-35ºc for 18-72 hour. Growth of colonies indicates possible presence of E.coil and then performs the confirmatory test. Sample passes the test if colonies not observed as mentioned above and if the growth observed perform the confirmatory test. 51 Test for salmonella: : Test for salmonella: After completion of incubation period, transfer 0.1 ml of above preparation to 10 ml of Rappaport vassilisdis salmonella enriched broth and incubate at 30-35ºc for 18-24 hour. From above enriched broth, streak on the surface of Xylose Lysine Decarboxylate agar and inverted the petri plate and incubate at 30-35ºc for 18-48 hour. The possible presence of salmonella is indicated by the characteristics colony having the following appearance XLD agar : Well developed, red colonies with or with out black centers. Sample passes the test, if no growth as mentioned above. If appearance of colonies found as mentioned above, perform the confirmatory test. 52 Test for pseudomonas aeruginosa: : Test for pseudomonas aeruginosa: After incubation period streak a loopful of above enriched medium on surface of Cetrimide agar plate and incubate at 30-35ºc for 18-72 hour. After incubation period, if there is no growth observed on plates, the sample passes the test and if there is a growth observed in above medium, perform the confirmatory test. 53 Test for staphylococcus aureus: : Test for staphylococcus aureus: After incubation period streak a loopful of above enriched medium on surface of Mannitol salt agar . Incubate at 30-35ºc for 18-72 hour. After incubation period, there is no typical growth in above medium as mentioned below the sample passes and if there is gram positive cocci, perform the confirmatory test such as coagulation test. The probable presence of S.aureus is indicated by the characteristic of colony having following appearance. Mannitol salt agar : Yellow/white colonies surrounded by yellow zone. 54 Test for Candida albicans : Test for Candida albicans  Candida albicans is a diploid fungus (a form of yeast) and a causal agent of opportunistic oral and genital infections in humans. Systemic fungal infections have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation). Transfer 10 ml preparation to the 100 ml of Sabouraud Dextrose broth and incubate at 30-35ºc for 24 hour. Sub culture from above enriched broth on the plate of Sabouraud Dextrose agar with Chloramphenicol. Invert the Petri plate and incubate at 30-35ºc for 3-5 days. The probable presence of c.albicans is indicated by the growth of white colony. 55 Candida albicans Clostridium sporogenes : Candida albicans  Clostridium sporogenes  56 Test for clostridium sporogenes : Test for clostridium sporogenes  Large Gram positive Straight or slightly curved rods with slightly rounded ends Anaerobic bacilli Spore bearing Causes diseases such as gas gangrene, tetanus, & pseudo-membranous colitis by producing toxins which attack the neurons pathways 57 Continue: Continue  Take 10 ml of preparation in two separate sterile test tubes and marked as set-1 and set-2 . Heat the test tube of set-1 at 80°c for 10 min in water bath and cool rapidly & Do not heat the tube of set-2. Transfer 10 ml from each set to the 100 ml Reinforced medium separately. Incubate both the tubes under anaerobic condition at 30-35⁰c for 48 hour. After incubation, make sub culture from each tube on Columbia agar and incubate under anaerobic condition at 30-35⁰c for 48 hour. Sample passes the test, if anaerobic growth is not observed on Columbia agar. If growth observed above media and it is rod with or with out endospore giving a negative catalase reaction, indicates the presence of cl.sporogenes. 58 Control : Control  Positive control For E.coil, salmonella, s.aureus, p.aeruginosa, c.albican and cl. sporogen carry out the control test by repeating the procedure using 1 ml of inoculum containing <100 organism with out product. Note done the observation of positive control. 59 PowerPoint Presentation: observation No Evidence of growth Evidence of growth Repeat test (RT 1) growth No growth Repeat test (RT 2) Isolate and identify micro organism 60 Pass Retest : Retest  If the sample shows positive result, carry sample as and proceed as per SOP for investigation of microbial limit test failure. For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following their application to a 10.0-g specimen, a retest on a 25-g specimen of the product may be conducted. Proceed as directed for Procedure, but make allowance for the larger specimen size. 61 Bacterial identification: : Bacterial identification: If the test sample shows positive result in retest, finally confirm by Bacterial identification kit as per SOP . Attach the identification report with rejection report of product. 62 APPLICATION : APPLICATION MICROBIOLOGICAL ASSAY OF ANTIBIOTIC DRUGS DISINFECTION EFFICACY TEST OF DISINFECTANTS AND ANTISEPTICS STERILITY TEST OF STERILISED PHARMACEUTICALS TESTS FOR MICROBIAL LIMITS FOR NON-STERILE PHARMACEUTICAL AND BIOLOGICAL PRODUCTS TESTING OF WATER 63 MICROBIOLOGICAL ASSAY OF ANTIBIOTIC DRUGS : MICROBIOLOGICAL ASSAY OF ANTIBIOTIC DRUGS This assay is to determine whether the potency of antibiotics complies with the requirement for label potency and associated limits stipulated in the USP/BP monographs for antibiotics. Specimen required: Antibiotic preparations: Capsules/tablets – 30 nos. Raw material – 2 gm Ampoules (injections) – 6 nos. Ointment tubes – 3 nos. Vials (injections) – 6 nos. Bottles (syrup) – 2 nos. 64 Continue:: Continue: Collect samples of raw materials and light-sensitive antibiotic drugs in screw-capped plastic containers or amber-coloured glass bottles. Do not expose sample to warm temperatures during transport. Method : United States Pharmacopeia (USP)/British Pharmacopoeia (BP) Test results : Pass or fail potency assay limits stipulated in the USP/BP monograph of the antibiotic drug 65 DISINFECTION EFFICACY TEST OF DISINFECTANTS AND ANTISEPTICS : DISINFECTION EFFICACY TEST OF DISINFECTANTS AND ANTISEPTICS The disinfectants/antiseptics are tested at the use-dilutions or use-conditions prescribed on the product label to determine their efficacy. Specimen required : 100 ml Method : ISO or other standard tests Test results : Pass or fail the standard tests 66 STERILITY TEST OF STERILISED PHARMACEUTICALS : STERILITY TEST OF STERILISED PHARMACEUTICALS Specimen required: Parenteral drugs and administration devices, ophthalmic preparations, surgical dressing, sutures and other sterilized pharmaceutical products, packed in unopened original containers. Generally 10 – 20 sample units (containers) per batch of manufacture. Refer to sample size stipulated in BP for various batch sizes of production. Method : United States Pharmacopeia (USP) /British Pharmacopoeia (BP) Test results : Pass or fail the Tests for Sterility USP /BP 67 TESTS FOR MICROBIAL LIMITS FOR NON-STERILE PHARMACEUTICAL AND BIOLOGICAL PRODUCTS : TESTS FOR MICROBIAL LIMITS FOR NON-STERILE PHARMACEUTICAL AND BIOLOGICAL PRODUCTS 68 Route of adminstration TAMC ( cfu /ml or cfu /gm) TYMC ( cfu /ml or cfu /gm) Specified micro organism Non-aqueous preparation for oral use 10 3 10 2 Absence of E.coli (1 gm or 1 ml ) aqueous preparation for oral use 10 2 10 1 Absence of E.coli (1 gm or 1 ml ) Rectal use 10 3 10 2 - Cutaneous use Nasal use Auricular use 10 2 10 1 Absence of S.aureus (1gm or 1ml) Absence of P.aeruginosa (1gm or 1ml ) Continue:: Continue: 69 Vaginal use 10 2 10 1 Absence of S.aureus, P.aeruginosa, C. Albucans ( 1gm or 1 ml) Transdermal patches (limit for one patch including adhesive layer) 10 2 10 1 Absence of S.aureus, P.Aeruginosa ( in 1 patch) Inhalation use (liquid preparation for nebulization ) 10 2 10 1 Absence of S.aureus , P.Aeruginosa ( 1gm or 1 ml) TESTING OF WATER : TESTING OF WATER This can be done by following two methods: Chemical Analysis Microbial Analysis TOTAL VIABLE AEROBIC COUNT ( cfu /ml): Requirement: Membrane filtration assembly Forceps Vacuum pump Cellulose nitrate membrane filter having porosity of 0.45 μ and 47 mm diameter. Sterile pre-incubated glass plates Sample quantity: 2000 ml of water for injection 70 Procedure: : Procedure: Testing to be done under laminar air flow to avoid accidental contamination of sample to be examined. The filtration unit, membrane and their accessories are sterilized in autoclave. The LAF is cleaned with 70% isopropyl alcohol, then the sterile filtration assembly is arranged with unit and vacuum line is connected. The sterile wet membrane filter is put on holder of the filtration unit with help of sterile forceps with actual sample solution, the valve of assembly is opened and vacuum is applied. The sample is allowed to filter out. The vacuum line is switch off. The membrane filter is transferred aseptically to the surface of sterile agar plate with help of sterile forceps and incubates. 71 Specification of different type of water: Specification of different type of water Specification of potable water: Total viable count (cfu/ml) : Not more then 500 Specification of Raw water: Total viable count (cfu/ml) : Not more then 500 Microbial Limit Test (E.coil, Salmonella, S.aureus, p.aeruginosa) : Should be absent Specification of Water for Injection: Total viable aerobic count Normal level : Not more than 2 Alert level : 3 -10 Action level : more than 10 72 Bibliography: : Bibliography: The Indian pharmacopoeia, IP-1996, volume-2, Indian pharmacopoeia commission, Ghaziabad, India, 1996. Appendix-9.4,A-110:A-117 The United States Pharmacopoeia,USP-25 NF-20, 2002, p 1873-1878 http://en.wikipedia.org http://productimage.tradeindia.com/00530916/s/1/Membrane-Filtration-Assembly-PC-Single-Unit-.jpg http://en.wikipedia.org/wiki/Candida_albicans http://www.google.co.in/image http://www.usp.org/pdf/EN/meetings/asMeetingIndia2009/session2Track2Tirumalai_1.pdf 73 Continue:: Continue: Gerard J. Tortora , Berdell R. Funke , Christine L. Case, Microbiology An Introduction, Eighth Edition, Pearson Education, 2005, p 193-199 http://ffcr.or.jp/zaidan/FFCRHOME.nsf http://en.wikipedia.org/wiki/Indole_test http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Yeast_Plate_Count/07_yeast_0.2mL_plate_P7201181.jpg http://chemicoscientific.com/images/Digital_Colony_Counter.jpg 74
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