This document summarizes a dissertation report submitted by Ashish Diwakar on microbial limit testing conducted at IPCA Laboratories Ltd. in Ratlam, Madhya Pradesh, India. It provides an introduction to IPCA and the principles and requirements of microbial limit testing. Methods for total bacterial count, total fungal count, and testing for pathogens like E. coli, Salmonella, P. aeruginosa, and S. aureus are described. Both direct inoculation and membrane filtration methods are covered. Requirements include various culture media, glassware, and equipment. Test procedures and observations are outlined.

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=