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

INTRODUCTIOIN TO IPCAINTRODUCTIOIN TO IPCA

• It’s genesis in 1949.It’s genesis in 1949.• Company has visible progress in 1975, under the Company has visible progress in 1975, under the

chairmanship of chairmanship of Mr. Ajitabh BachachnMr. Ajitabh Bachachn and able and able direction of direction 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 a Analgesics and Cardio-Care products. And it is a brand leader in antimalarial drug.brand leader in antimalarial drug.

• IPCA’s formulation manufacturing units are IPCA’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).

• It export the product to over 60 countries both develop It 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&D For 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 year IPCA receive life time achievement award for the year 2002-03 from CHEMEXIL for export promotion ovefr the 2002-03 from CHEMEXIL for export promotion ovefr the year.year.

• Forbes, a leading US business magazine selected IPCA as Forbes, 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.

  

 

Ipca Laboratories Ltd. Ratlam (M.P.)

 

INTRODUCTION TO MICROBIAL LIMIT TESTINTRODUCTION TO MICROBIAL LIMIT TEST

• The Microbial Limit Tests are designed to perform the The Microbial Limit Tests are designed to perform the qualitative and quantitative estimations of specific viable qualitative 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 aerugenosa Pseudomonas aerugenosa and and Staphylococcus aureusStaphylococcus aureus). ).

Microbial Limit TestMicrobial Limit Test

Introduction: -Introduction: -This test is performed for the estimation of the This 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 the This test is based on the principle that the microbiological quality of non-sterile pharmaceutical microbiological quality of non-sterile pharmaceutical materials can be controlled by the adoption of both the materials 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.

RequirementsRequirements

• Sterilized glass waresSterilized glass wares-- Glass plates,test tubes, pippets flask Glass 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 and It 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.

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.

REQUIRED MEDIAREQUIRED MEDIA

• BRILLIANT GREEN AGARBRILLIANT GREEN AGAR• BISMUTH SULPHITE AGARBISMUTH SULPHITE AGAR• CETRIMIDE AGARCETRIMIDE AGAR• EOSINE METHYLENE BLUE AGAREOSINE METHYLENE BLUE AGAR• MACCONKEY AGAR MACCONKEY 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

• BUFFERED PEPTONE WATER BUFFERED 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 BILE TETRATHIONATE BRILLINT GREEN BILE

BROTH BROTH

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 CountII. Total Fungal CountIII. Pathogen testing

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.

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 are After the complition of incubation period colonies are counted and multiply by dilution factor to get count per counted and multiply by dilution factor to get count per gm.gm.

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.

TEST FOR TEST FOR Escherichia coliEscherichia coli

• PRIMARY TEST: -PRIMARY TEST: -• 1.0 ml of enrichment culture is added to 10 ml Mac 1.0 ml of enrichment culture is added to 10 ml Mac

conkey broth (MB) containing inverted Durham’s conkey broth (MB) containing inverted Durham’s tube and then incubated at 40 ~ 45°C for 18 ~ 24 tube and then incubated at 40 ~ 45°C for 18 ~ 24 hours.hours.

• If the tube content shows acid & gas formation, then If the tube content shows acid & gas formation, then confirmatory test is carried out. Acid production is confirmatory test is carried out. Acid production is indicated by change in colour of the broth from purple indicated by change in colour of the broth from purple to yellow and gas production is indicated by to 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.

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.

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.

TEST FOR TEST FOR SALMONELLASALMONELLAPRIMARY 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.

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 H2S in the agar and by the absence of

red color in the urea broth.

TEST FOR TEST FOR P.aeruginosaP.aeruginosaPRIMARY 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.

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.

TEST FOR TEST FOR S.aureusS.aureusPRIMARY 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.

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.  

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.

OBSERVATIONOBSERVATION- TEST FOR TOTAL VIABLE COUNT- TEST FOR TOTAL VIABLE COUNTSAMPLE 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 of Name of mediamedia

Incubation Incubation conditioncondition

Plate Plate (cfu)(cfu)

Result= No.of Result= No.of colonies x colonies x

dilution factordilution factor

markmark

Total Total Bacterial Bacterial

CountCount SASA 30-35 C 30-35 C for 5 for 5 daysdays

NDND <10 <10 cfu/gm.cfu/gm.

-ve-ve

Total Total Fungal Fungal

CountCount SDSD 20-25 C 20-25 C

for 5 for 5 daysdays

NDND <10 <10 cfu/gm.cfu/gm.

-ve-ve

TEST FOR PATHOGENTEST FOR PATHOGENA. ENRICHMENT:-A. ENRICHMENT:-

Name of Name of organismsorganisms

Smple Smple preparationpreparation

Incubation Incubation conditioncondition

RemarkRemark

E.coliE.coli

SalmonellaSalmonella

S.aureusS.aureus

P.aeruginosaP.aeruginosa

10.0ml.»90.0ml.10.0ml.»90.0ml.SCDMSCDM

30-37 C for 30-37 C for 18-48 hours18-48 hours

PP

P- Growth Observed (Carryout Primary Test).N- No growth Observed (Specified Organism Absent).

B. PRIMARY TEST:-B. PRIMARY TEST:-Name of Name of

OrganismOrganism InoculationInoculation

Incubation Incubation conditioncondition

Charact -Charact -erstic erstic

GrowthGrowth

RemaRemarkrk

E.coliE.coli 1ml. 1ml. Enrichment Enrichment culture culture »10ml.MB»10ml.MB

40-45 C 40-45 C for 18-24 for 18-24

hourshours

Acid & Acid & gas gas productioproductionn

NN

SalmonellSalmonellaa

1ml. Enrich- 1ml. Enrich- ment culture ment culture »10ml.TB »10ml.TB &SB&SB

41-43 C 41-43 C for 18-24 for 18-24

hourshours

Growth Growth observedobserved NN

P.aeruginoP.aeruginosasa

Streak enrich Streak enrich ment culture ment culture on CAon CA

35-37 C 35-37 C for 18-72 for 18-72

hourshours

Greenish Greenish coloniescolonies NN

S.aureusS.aureus Streak enrich Streak enrich ment culture ment culture on MSon MS

35-37 C 35-37 C for 18-72 for 18-72

hourshours

Yellow Yellow colo nies colo nies with with yellow yellow zonezone

NN

SECONDARY TEST FOR SECONDARY TEST FOR SALMONELLASALMONELLA

InoculationInoculation Incubation Incubation conditioncondition

Characterstic Characterstic GrowthGrowth

RemarkRemark

Streak on Streak on BGABGA

35-37 C 35-37 C for 18-72 for 18-72

hourshours

Colorless or Colorless or pink pink

colonies colonies with pink with pink

zonezone

NN

Streak on Streak on BSABSA

35-37 C 35-37 C for 18-72 for 18-72

hourshours

Black or Black or green green

coloniescoloniesNN

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

 

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.

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.

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=