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Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
Microbial limit test
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Microbial limit test

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    • 1. SCHOOL OF STUDIES IN MICROBIOLOGY, VAGDEVI BHAVAN, VIKRAM UNIVERSITY,UJJAIN (M.P.) “MICROBIAL LIMIT TEST” A DISSERTATION REPORT ATA DISSERTATION REPORT AT IPCA LABORATORIES LTD.IPCA LABORATORIES LTD. RATLAM (M.P.)RATLAM (M.P.) SUBMITTED BY ASHISH DIWAKAR M.Sc. IV SemesterM.Sc. IV Semester MICROBIOLOBYMICROBIOLOBY YEAR 2009- 2010YEAR 2009- 2010
    • 2. INTRODUCTIOIN TO IPCAINTRODUCTIOIN TO IPCA • It’s genesis in 1949.It’s genesis in 1949. • Company has visible progress in 1975, under theCompany has visible progress in 1975, under the chairmanship ofchairmanship of Mr. Ajitabh BachachnMr. Ajitabh Bachachn and ableand able direction ofdirection of Mr. Premchand Godha & Mr. M. R.Mr. Premchand Godha & Mr. M. R. Chandurkar.Chandurkar. • IPCA manufactures- Antimalarial, Antibiotics,IPCA manufactures- Antimalarial, Antibiotics, Analgesics and Cardio-Care products. And it is aAnalgesics and Cardio-Care products. And it is a brand leader in antimalarial drug.brand leader in antimalarial drug. • IPCA’s formulation manufacturing units areIPCA’s formulation manufacturing units are located at Mumbai, Ratlam, Athal(Silvassa),located at Mumbai, Ratlam, Athal(Silvassa), Kandla(Gujrat),Indor, and Sidhpur(Ahmedabad).Kandla(Gujrat),Indor, and Sidhpur(Ahmedabad).
    • 3. • It export the product to over 60 countries both developIt export the product to over 60 countries both develop and developing countries including- Canada, Australia,and developing countries including- Canada, Australia, Ethopia, Germany, Italy, Japan, Srilanka, U.K. etc.Ethopia, Germany, Italy, Japan, Srilanka, U.K. etc. • For products development and process IPCA have R&DFor products development and process IPCA have R&D department at Mumbai, Ratlam and Indore.department at Mumbai, Ratlam and Indore. • IPCA receive life time achievement award for the yearIPCA receive life time achievement award for the year 2002-03 from CHEMEXIL for export promotion ovefr the2002-03 from CHEMEXIL for export promotion ovefr the year.year. • Forbes, a leading US business magazine selected IPCA asForbes, a leading US business magazine selected IPCA as “Best Under A Billion Company” for the second time“Best Under A Billion Company” for the second time concequantly in 2003-04.concequantly in 2003-04.
    • 4. Ipca Laboratories Ltd. Ratlam (M.P.)
    • 5. INTRODUCTION TO MICROBIAL LIMIT TESTINTRODUCTION TO MICROBIAL LIMIT TEST • The Microbial Limit Tests are designed to perform theThe Microbial Limit Tests are designed to perform the qualitative and quantitative estimations of specific viablequalitative and quantitative estimations of specific viable microorganisms present in samples.microorganisms present in samples. • It includes tests for total viable count (bacteria and fungi)It includes tests for total viable count (bacteria and fungi) and Pathogen (and Pathogen (Escherichia coli, Salmonella,Escherichia coli, Salmonella, Pseudomonas aerugenosaPseudomonas aerugenosa andand Staphylococcus aureusStaphylococcus aureus).).
    • 6. Microbial Limit TestMicrobial Limit Test Introduction: -Introduction: -This test is performed for the estimation of theThis test is performed for the estimation of the number of viable microorganisms present in sample.number of viable microorganisms present in sample. Principle:Principle: --This test is based on the principle that theThis test is based on the principle that the microbiological quality of non-sterile pharmaceuticalmicrobiological quality of non-sterile pharmaceutical materials can be controlled by the adoption of both thematerials can be controlled by the adoption of both the standards.standards. 1. The first is a limit on the total viable count.1. The first is a limit on the total viable count. 2. The second is the exclusion of specific pathogens.2. The second is the exclusion of specific pathogens.
    • 7. RequirementsRequirements • Sterilized glass waresSterilized glass wares-- Glass plates,test tubes, pippets flaskGlass plates,test tubes, pippets flask etc.etc. • Incubators-Incubators- BOD and Bacteriological for 20-25°C, 30-BOD and Bacteriological for 20-25°C, 30- 35°C & 40-45°C.35°C & 40-45°C. • Laminar Air Flow-Laminar Air Flow- Horizontal LAF.Horizontal LAF. • Dry Heat Sterilizer-Dry Heat Sterilizer- About 200°C.About 200°C. • Filter Paper Disc-Filter Paper Disc- It is made up of cellulose nitrate andIt is made up of cellulose nitrate and have 0.45um pore size.have 0.45um pore size. • Other Materials-Other Materials- Sterilized media, weight machine,Sterilized media, weight machine, filtration unit etc.filtration unit etc.
    • 8. Culture Media:-Media are substance used to provide nutrients for the growth and multiplication of microorganism. Now a day, dehydrated media containing all the ingredients in powdered form are available. There are three types of media are required- Enrichment Media- Soyabean Casien Digest Media. Selective Media- MacConkey Agar for E.coli. Differential Media- Sabourud Chloramphenicol Agar for fungi.
    • 9. REQUIRED MEDIAREQUIRED MEDIA • BRILLIANT GREEN AGARBRILLIANT GREEN AGAR • BISMUTH SULPHITE AGARBISMUTH SULPHITE AGAR • CETRIMIDE AGARCETRIMIDE AGAR • EOSINE METHYLENE BLUE AGAREOSINE METHYLENE BLUE AGAR • MACCONKEY AGARMACCONKEY AGAR • MANNITOL SALT AGARMANNITOL SALT AGAR • PSEUDOMONAS AGARPSEUDOMONAS AGAR • SABOURAUD CHLORAMPHENICOL AGARSABOURAUD CHLORAMPHENICOL AGAR • SOYABEAN CASEIN DIGEST AGARSOYABEAN CASEIN DIGEST AGAR • TRIPLE SUGAR IRON AGARTRIPLE SUGAR IRON AGAR
    • 10. • BUFFERED PEPTONE WATERBUFFERED PEPTONE WATER • FLUID SELENITE CYSTEINE BROTHFLUID SELENITE CYSTEINE BROTH • MACCONKEY BROTHMACCONKEY BROTH • PEPTONE WATERPEPTONE WATER • SOYABEAN CASEIN DIGEST MEDIUMSOYABEAN CASEIN DIGEST MEDIUM • TETRATHIONATE BRILLINT GREEN BILETETRATHIONATE BRILLINT GREEN BILE BROTHBROTH
    • 11. METHODS OF MICROBIAL LIMIT TESTMETHODS OF MICROBIAL LIMIT TEST There are two types of method of microbial limit test- 1. Direct inoculation- In this method sample is directly inoculated into the media and that are incubated. 2. Membrane filtration method- In this method sample solution is filter with filter membrane and then this filter membrane is transfer to the freshly prepared sterilized media. Above methods are performed for- I. Total Bacterial Count II. Total Fungal Count III. Pathogen testing
    • 12. DIRECT INOCULATION METHODDIRECT INOCULATION METHOD TEST FOR TOTAL BACTERIAL COUNTTEST FOR TOTAL BACTERIAL COUNT Preparation of Sample: - 10 Gms of substance/10ml of liquid/10 tablets is added to 100ml of buffered sodium chloride peptone solution [pH-7.0]. In case of lumpy on material nature it is kept at 30~35°C for 30 minutes. PROCEDURE- · With the help of sterile pipette, 1.0 ml. Of sample is aseptically transfer to petridish. · Now add 20.0 ml. Of liquefied , sterilized SA medium. · Now the plate swirled to mixing and allow to solidify for about 1.0 hour. · Then plates incubated in inverted position in incubator at 35-37 C for 5 days.
    • 13. TEST FOR TOTAL FUNGAL COUNTTEST FOR TOTAL FUNGAL COUNT • Same procedure is used.Same procedure is used. • Instead of SA, SD medium is used.Instead of SA, SD medium is used. • Plates incubated at 20-25 C for 3 days.Plates incubated at 20-25 C for 3 days. • After the complition of incubation period colonies areAfter the complition of incubation period colonies are counted and multiply by dilution factor to get count percounted and multiply by dilution factor to get count per gm.gm.
    • 14. TEST FOR PATHOGENTEST FOR PATHOGEN Initial process:- 1. Firstly sample are prepare as mention above. 2. Now aseptically transfer 10ml of prepare sample into 100ml of Soyabean Casecin Digest media (SCDM). 3. Incubate at 35-37°C for 18-48 hours. 4. Observe after incubation, if growth is present carry out the pathogen testing.
    • 15. TEST FORTEST FOR Escherichia coliEscherichia coli • PRIMARY TEST: -PRIMARY TEST: - • 1.0 ml of enrichment culture is added to 10 ml Mac1.0 ml of enrichment culture is added to 10 ml Mac conkey broth (MB) containing inverted Durham’sconkey broth (MB) containing inverted Durham’s tube and then incubated at 40 ~ 45°C for 18 ~ 24tube and then incubated at 40 ~ 45°C for 18 ~ 24 hours.hours. • If the tube content shows acid & gas formation, thenIf the tube content shows acid & gas formation, then confirmatory test is carried out. Acid production isconfirmatory test is carried out. Acid production is indicated by change in colour of the broth from purpleindicated by change in colour of the broth from purple to yellow and gas production is indicated byto yellow and gas production is indicated by accumulation of gas at the top of Durham’s tube.accumulation of gas at the top of Durham’s tube.
    • 16. SECONDARY TEST: - • 0.1ml enrichment culture is inoculated to 5 ml of peptone water, then it is incubated at 35 ~ 37 °C for 18 ~ 24 hours. • Simultaneously a loop full of culture is streaked on Mac conkey agar. These plates are incubated at 35 ~ 37 °C for 18 ~ 72 hrs. Indole Test: - After incubation of peptone water tubes, 0.5 ml. Of Kovac’s reagent is added into each of them and shacked well and allowed to stand for one minute. If red ring appears in reagent layer then indole is confirmed.
    • 17. Then the colonies transfer by streaking on the surface of Levine Eosine-Methylene Blue agar (EMBA) on petridish. The plate incubated at 35 ~ 37 °C for 18 ~ 72 hrs. If the colonies exhibit metallic shine under reflected light and blue- black appearance, confirms the presence of E.coli.
    • 18. TEST FORTEST FOR SALMONELLASALMONELLA PRIMARY TEST: -1.0ml of enrichment culture is inoculated in 10 ml of Tetrathionate Brilliant Green Bile Broth and 10ml.of Fluid Selenite Cystine broth and then incubated at 41-43°C for 18 ~ 24 hrs. SECONDARY TEST:-If growth is observed, then sub culturing is done from this culture on Brilliant green agar, (BGA), Bismuth sulphite Agar (BSA).This plates are incubated at 35 ~ 37 °C for 18 ~ 72 hrs. Then the plates are observed for any colonies confirming to the description given below. BGA- Small, transperant, colorless or pink colonies with pink or red zone. BSA- Black and green colonies.
    • 19. CONFIREMATORY TEST:- Colony subculture onto Triple sugar Iran Agar (TSIA) slants by inoculating the surface of slope first then stabbing. Also inoculate into Urea Broth and incubated at 35 ~ 37 °C for 18 ~ 72 hrs. The presence of salmonella is confirmed if in the deep culture but not the surface, there is a change of colour from red to yellow and usually formation of acid and gas in stab culture with or without production of H2 S in the agar and by the absence of red color in the urea broth.
    • 20. TEST FORTEST FOR P.aeruginosaP.aeruginosa PRIMARY TEST: - • The enrichment culture is streaked onto cetrimide agar(CA) plates, and incubated at 35 ~ 37 °C for 18 ~ 72 hrs. • If greenish colony with greenish fluorescence under UV light is obtain then secondary test is carried out called pigment test and oxidize test.
    • 21. SECONDARY TEST: - Pigment test:- The suspected colonies are streaked on pseudomonas agar medium for detaection of fluorescein and pseudomonas agar medium for dictation of pyocyanin contained in petridishes and incubated at 35 ~ 37 °C for 18 ~ 72 hrs. Morphological characteristics of P.aeruginosa on selective agar media: - Pseudomonas agar medium for dictation of fluorescent- Generally colourless to greenish with Yellowish Fluorescence. Pseudomonas agar medium for dictation of pyocyanin- Generally greenish with Blue Fluorescence. OXIDASE TEST:- The suspected colonies are smeared on the oxidase test disc (N,N dimethyl p – phenylene diamine oxalate) the test is positive, if purple colour is produced with in 5 ~ 10 seconds.
    • 22. TEST FORTEST FOR S.aureusS.aureus PRIMARY TEST: -The enrichment culture is streaked on Mannitol salt agar (MSA) and incubated at 35 ~ 37 °C for 18 ~ 72 hrs.If Yellow colonies with yellow zone is observed , indicates the presence of Staphylococcus aureus. SECONDARY TEST: -Suspected colonies are transferred to the tube containing 5 ml. Additives plasma(rabbit/Horse) with or without additives and incubated on water bath at 37°C.The tubes examined for 3 hrs. If it coagulates, then it shows presence of S.aureus.
    • 23. MEMBRANE FILTRATION METHODMEMBRANE FILTRATION METHOD Introduction:-This method is applied to the sample which contains antimicrobial substances.Use membrane filters of an appropriate material with a pore size of 0.45 μm or less. Requirements:-Sterilized filter membrane disc (0.45um), membrane filtration unit, 0.1% bacteriological peptone water without tween 80 or 20 (800ml), 0.1%peptone water with tween 20 (3 X 100ml), soyabean casein digest media (SA) and saboured dextrose agar (SD) medium, glass wares etc.
    • 24. Procedure:- 1 Firstly arrange the all requirements and take all precaution before starting work. 2.Now take 1.0 gm of sample and mix into 800 ml of 0.1% of bacteriological peptone water without tween 80. 3 Then filter it with the help of filter membrane disc. 4.Now given them washing with 0.1%of peptone water which contain 1.0 % tween 20, in 3 times of 100ml. 5.After filtration cut the filter membrane disc in two half pieces and transfer on freshly prepared SA and SD plate. 6.Now incubates the plates. SA plates for 5 days at 35-37 °C. and SD plates for 5 days at 20-25 °C. 7.After the completion of the incubation period observe plate & count the No. of colonies.
    • 25. OBSERVATIONOBSERVATION- TEST FOR TOTAL VIABLE COUNT- TEST FOR TOTAL VIABLE COUNT SAMPLE PREPARATIONSAMPLE PREPARATION-10.0gm.sample into 90.0ml.-10.0gm.sample into 90.0ml. Buffered sodium chloride peptone solution.Buffered sodium chloride peptone solution. PerticularsPerticulars Name ofName of mediamedia IncubationIncubation conditioncondition PlatePlate (cfu)(cfu) Result= No.ofResult= No.of colonies xcolonies x dilution factordilution factor markmark TotalTotal BacterialBacterial CountCount SASA 30-35 C30-35 C for 5 daysfor 5 days NDND <10 cfu/gm.<10 cfu/gm. -ve-ve TotalTotal FungalFungal CountCount SDSD 20-25 C20-25 C for 5 daysfor 5 days NDND <10 cfu/gm.<10 cfu/gm. -ve-ve
    • 26. TEST FOR PATHOGENTEST FOR PATHOGEN A. ENRICHMENT:-A. ENRICHMENT:- Name ofName of organismsorganisms SmpleSmple preparationpreparation IncubationIncubation conditioncondition RemarkRemark E.coliE.coli SalmonellaSalmonella S.aureusS.aureus P.aeruginosaP.aeruginosa 10.0ml.»90.0ml.10.0ml.»90.0ml. SCDMSCDM 30-37 C for30-37 C for 18-48 hours18-48 hours PP P- Growth Observed (Carryout Primary Test). N- No growth Observed (Specified Organism Absent).
    • 27. B. PRIMARY TEST:-B. PRIMARY TEST:- Name ofName of OrganismOrganism InoculationInoculation IncubationIncubation conditioncondition CharactCharact -erstic-erstic GrowthGrowth RemaRema rkrk E.coliE.coli 1ml. Enrichment1ml. Enrichment cultureculture »10ml.MB»10ml.MB 40-45 C for40-45 C for 18-24 hours18-24 hours Acid & gasAcid & gas productionproduction NN SalmonellaSalmonella 1ml. Enrich-1ml. Enrich- ment culturement culture »10ml.TB &SB»10ml.TB &SB 41-43 C for41-43 C for 18-24 hours18-24 hours GrowthGrowth observedobserved NN P.aeruginosP.aeruginos aa Streak enrichStreak enrich ment culture onment culture on CACA 35-37 C for35-37 C for 18-72 hours18-72 hours GreenishGreenish coloniescolonies NN S.aureusS.aureus Streak enrichStreak enrich ment culture onment culture on MSMS 35-37 C for35-37 C for 18-72 hours18-72 hours Yellow coloYellow colo nies withnies with yellowyellow NN
    • 28. SECONDARY TEST FORSECONDARY TEST FOR SALMONELLASALMONELLA InoculationInoculation IncubationIncubation conditioncondition CharactersticCharacterstic GrowthGrowth RemarkRemark Streak onStreak on BGABGA 35-37 C for35-37 C for 18-72 hours18-72 hours Colorless orColorless or pink coloniespink colonies with pinkwith pink zonezone NN Streak onStreak on BSABSA 35-37 C for35-37 C for 18-72 hours18-72 hours Black orBlack or greengreen coloniescolonies NN
    • 29. Result:- The products result is observed on the basis of microbial limit. There are different types of products that have different limit given as follow: Types of product Limit of TBC Limit of TFC Liquid NMT 500 cfu/5ml NMT 50 cfu/5ml Tablet NMT 1000 cfu/ml NMT100 cfu/ml Bulk Pharma Compound NMT 100 cfu/ml NMT 10 cfu/ml Raw Material NMT 1000 cfu/ml NMT 10 cfu/ml Pathogen Always should be absent
    • 30. NAME OF SAMPLE- PACIMOL TABLET TOTAL BACTERIAL COUNT- <10 cfu/gm. TOTAL FUNGAL COUNT- <10 cfu/gm. PATHONGENS- ABSENT REMARK-Sample complies the test as per the pharmaceutical limit of MLT.
    • 31. CONCLUSIONCONCLUSION From the above study, it can be concluded that there are different types of pharmaceutical products that are checked by different pharmaceutical procedure. And finally result are observed on the basis of their limit. The pharmaceutical product (Pacimol Tablet) for microbial limit test is complies as per the pharmaceutical limit of microbial limit test.
    • 32. REFERENCEREFERENCE • United State phaemacopoeia NF, Asian edition, vs pharmacopeial convention, 1823-1829. • Europian pharmacopia, edi III quatesnaryforumpublication884-885. • Indian pharmacopocieia, cioveenemtof india, minisry of healt & familly wellfare publication,Delhi vol. I 335-336. • Hi- media manual for microbiology lab practice. Publication-1998. • www.google.co.in/search?=microbial+limit+test=

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