This document describes the microbial limit test, which includes tests to quantify and qualify microorganisms in samples. It involves estimating total viable counts of bacteria and fungi, and detecting specific pathogens. The test is based on culturing samples on various media to support or inhibit growth of target microbes. Methods like membrane filtration, spread plating, and serial dilution are used to quantify microbes, while selective media help identify pathogens like E. coli, S. aureus, P. aeruginosa, and Salmonella. Detailed procedures are provided for quantification, enrichment, and identification of microorganisms in samples.

1. Microbial Limit Test

2. This test is designed to perform:
Total Viable Count (TVC) of bacteria and fungi (Quantitative
estimation).
Enrichment for qualitative estimation:Identification of
microorganisms by cultivating on selective media comparing
ATCC/MTCC culture of pathogen (Qualitative estimation).
The Microbial Limit Tests are designed to perform the qualitative and
quantitative estimations of specific viable microorganisms present in
samples.
It includes tests for total viable count (bacteria and fungi) and Pathogen
(Escherichia coli, Salmonella, Pseudomonas aerugenosa and
Staphylococcus aureus).

3. Principle: -
This test is based on the principle that the microbiological quality of
non-sterile pharmaceutical materials can be controlled by the adoption
of both the standards.
1. The first is a limit on the total viable count.
2. The second is the exclusion of specific pathogens.

4. • 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.

5. • REQUIRED MEDIA BRILLIANT GREEN AGAR BISMUTH
SULPHITE AGAR
• CETRIMIDE AGAREOSINE METHYLENE BLUE
AGARMACCONKEY AGARMANNITOL SALT
AGARPSEUDOMONAS AGARSABOURAUD
CHLORAMPHENICOL AGARSOYABEAN CASEIN DIGEST
AGARTRIPLE SUGAR IRON AGAR
• 6 BUFFERED PEPTONE WATER
• FLUID SELENITE CYSTEINE BROTH MACCONKEY
BROTHPEPTONE WATERSOYABEAN CASEIN DIGEST
MEDIUMTETRATHIONATE BRILLINT GREEN BILE BROTH

6. Quantitative estimation :-
• There is two test carried for quantitative estimation:
• TBC (Total Bacterial Count)
• TFC (Total Fungal count)
• Objective: To determine the population of bacteria/fungi in sample of
pharmaceutical product.
• Requirements:
Sterile petri dish containing solid media,Filtration assembly,
Micropipette, Conical flask ,Laminar air flow hood Filter paper ,Sterile
media (Pro-solidify) ,test tube containing normal saline,BOD
Incubator,IPA 70%Tissue paper role,Forceps,Bunsen burner,Spreader

7. Four Methods are employed for this test:
• Filtration method
• Pour pate method
• Spread plate method
• Serial Dilution Method
• Filtration method.

8. Membrane Filtration Method
• Pretreatment of sample:
• Water soluble product:10 gm./10 ml sample +70 ml buffered normal
saline solution+ make up volume up to 100 ml
• Water insoluble product:10 gm./10 ml sample +70 ml buffered normal
saline solution + make up volume up to % w/v polysorbate 80 (surface
active agent)
• Fatty product:10 gm./10 ml sample +60 ml buffered normal saline
solution+ 5 gm.
• Polysorbate 80 or polysorbate gentle heat + make up volume up to 100
ml
• Solid Like tablets
• Liquid like water

9. Membrane Filtration Method
Procedure:
• Open the filtration assembly and put the filter paper aseptically by the
help of incinerated forcep on the filtration gauge.
• Fix the filtration cup again to rejoin filtration assembly.
• Remove the cotton plug from flask with your left hand’s last two
finger and let remain plug in your hand.
• Pour all treated sample in the filtration cup.
• Wait until all sample is filtered.

10. • Open the filtration assembly and remove the filter paper aseptically by the
help of incinerated forcep from the filtration gauge after all sample is filtered.
Rejoin the filtration assembly again.
• Adequately open the led of plate and put the wet filter paper on media
containing plate (printed side up) with incinerated forceps.
• Press gentle and close the lid and invert the plate.
• Incubate plate in inverted position for 72 hrs. at 22-25ºC and later 24 hrs. at
30-35ºC.
• Observe the plate and count total colony appear by digital colony counter.

11. Pour Plate Method :-
• Procedure:
• Take the treated sample.
• Remove cotton plug with your right hand’s last two finger, take up the flask and
take 1 ml sample by micropipette with your right hand too.
• After taking sample plug the flask again and adequately open the lid of petri plate
with your left hand’s finger and toe and then pour the sample in the plate.
• Now remove the cotton plug of media containing flask as mentioned before and
pour approx. 12 ml of media inside the plate cavity and lid the plate.
• Rotate the plate anti-clockwise and clockwise gently so the media mixed well
with 1 ml of sample.

12. • Be precautious that media should not touch the lid of petri plate.
• Let the plate solidify.
• After solidification invert all the petri plate.
• Incubate all the petri plate in inverted position for 72 hrs.
• Observe the petri plate at regular interval of time.
• After incubation completed then count the colonies with digital colony
counter under SOP.
• Record the colony count and document in report of product.

13. Spread Plate Method :-
• Procedure:
• Take the pre-treated sample.
• Take the solidify media containing plate.
• Open the cotton plug of the flask with your right hand and procure 1 ml of
sample form the flask with the help of micropipette and then plug the flask
again.
• Adequately open the lid of media containing plate and pour the sample and
then close the led of plate.
• Put down the micropipette and pick up the spreader and put the plate at rotating
plate, open it with left hand and apply gently the spreader on the surface of
plate with right hand. Remain in touch with surface until all sample spread well.

14. • Let the plate dry from surface for 1 min.
• After dry invert all the petri plate.
• Incubate all the petri plate in inverted position for 72 hrs at 30-35ºC.
• Observe the petri plate at regular interval of time.
• After incubation completed then count the colonies with digital
colony counter under SOP.
• Record the colony count and documented in report of product.

15. • Serial Dilution Method :-
• Procedure:Take the pre-treated sample.Take the six test tube each
containing 9 ml saline solution.Mark them 10-1 to 10-6.Open the cotton
plug of the flask with your right hand and procure 1 ml of sample form
the flask with the help of micropipette and then plug the flask again.
Adequately unplug the test tube and pour the sample in 10-1 saline tube
containing 9 ml saline and then close the led of plate.
• Vortex the tube 10-1 for ½ minute, unplug the tube and take 1 ml solution
in micropipette. Unplug 102 saline tube and pour the solution of
micropipette inside it.
• Vortex the tube for ½ minute, unplug the tube and take 1 ml solution in
micropipette. Unplug 10-3 saline tube and pour the solution of
micropipette inside it.

16. • Follow the previous step to attain the 10-6 dilution.
• Follow the pour plate method to pour saline tube in petri plate with 3 replica
of each.
• Incubate all the petri plate in inverted position for 72 hrs. at ºC and later 24
hrs. at 30-35ºC.
• Observe the petri plate at regular interval of time.
• After incubation complete then count the colonies with digital colony
counter under SOP.
• Record the colony count and documented in report of product.
1ml sample : 9 ml saline = 10 ml sol. (1/10) 10-11ml of sol. : 9 ml of saline =
10 ml Sol. (1/100) 10-21ml of sol. : 9 ml of saline = 10 ml Sol. (1/1000) 10-
31ml of sol. : 9 ml of saline = 10 ml Sol. (1/10000) 10-41ml of sol. : 9 ml of
saline = 10 ml Sol. (1/100000) 10-51ml of sol. : 9 ml of saline = 10 ml Sol. (1/
) 10- 6

17. • Enrichment for qualitative estimation
• Take the pre-treated sample.Follow the membrane filtration method.Dip the
filter paper inside the enrichment broth (SCDM) and incubate for 24 hrs. at ºC.
• Qualitative estimation (Pathogen detection)
• In qualitative estimation the four more pathogenic bacteria are detecting under
this test, which are following:Escherichia coli Pseudomonas
aeruginosaStaphylococcus aureusSalmonella
• In qualitative estimation the two more pathogenic fungi are detecting under this
test, which are following:Candida albicans Aspergillus niger
• Escherichia coli
• These include identification of E. coli, bacteria pathogenic to human body and
causes infection of stomach. It is detected by using specific, differential media
which support growth of only E. coli.Primary test:Pipette 1 ml of enrichment
culture into tubes containing 5 ml Mac Conkey’s broth and incubate at ˚C for 48
hrs.If the content shows acid and gas carry out the secondary test.

18. • Escherichia coli Secondary test:
• After incubation, if the tube shows presence of acid and gas, transfer 0.1 ml
from tube to each of two tubes containing, 5 ml Mac Conkey’s broth and other
containing 5 ml peptone water and Incubate broth tubes in a water bath at
43.5˚C to 44.5˚C for 24 hrs.
• After incubation examine tube (a) for acid and gas (b) for indole.If the tubes
shows turbidity then one loop full culture is streaked over EMB and MCA and
incubate them for 24 hrs. at 43.5˚C to 44.5˚C.
• Escherichia coli tertiary test:
• To test for indole production add 0.5 ml of kovac’s reagent, shake well and
allow to stand for 1 min., if a red color is observed in the reagent layer, indole
is present,
• The presence of acid and gas and of indole indicates presence of Escherichia
coli.

19. Staphylococcus aureus
• Identification of Staphylococcus aureus is the detection of pathogenic
bacteria which causes infection in human body. It can be identified by
using specific, differentiation media which supports growth of only
Staphylococcus aureus.
• Primary test:Place the prescribed quantity in a sterile screw-capped jar
containing 100 ml of soybean casein digest medium and incubate at 32-
37˚C for hrs.
• Subculture on a plate containing a layer of mannitol salt agar medium or
Vogel Johnson agar medium and incubate at 32-37˚C for hrs.Examine
the resulting growth by Gram’s stain and apply the coagulase test. Gram
positive cocci (in cluster) in yellow colonies (on mannitol salt agar
medium) and in colonies, black surrounded by yellow zones (on Vogel
Johnson agar medium) and giving a positive coagulase test indicate the
presence of Staphylococcus aureus.

20. • Confirmatory test: (coagulase test)Transfer representative suspect
colonies from the agar surface of mannitol salt agar medium or Vogel
Johnson agar medium to individual tubes, each containing 0.5 ml of
mammalian, preferably rabbit or horse plasma with or without
additives. Incubate in water bath at 37˚C examining the tubes after 24
hrs.If coagulation in any degree is observed, the test is positive.

21. Pseudomonas aeruginosa
The identification or detection of pathogenic bacteria which causes infections
to the human body can be identified by using specific differential media
which support only the growth of Pseudomonas aeruginosa.Primary
test:Pretreat the preparation as described above.Place the prescribed quantity
in a sterile screw caped jar containing 100 ml of soybean casein digest
medium and incubate at 35-37˚C for 24 to 48 hrs.Observe the medium for
growth.If any growth is observed, subculture a portion of medium on a plate
containing a layer of Cetrimide Agar and incubate ˚C for 18 to 24 hrs.If none
of the plate contains colonies having characteristics given in the table, carry
out the confirmatory test.
Confirmatory test: (oxidase and pigment test)Streak representative suspect
colonies from the agar surface of cetrimide agar medium on the agar surface
of pseudomonas agar medium for detection of fluorescein and pseudomonas
agar medium for detection of pyocyanin contained in petri dishes.

22. • Cover and invert the inoculated media, and incubate at 35±2˚C for not less
than three days.Examine the streaked surface under UV light.Examine the
plate to determine the whether colonies having the characteristics, given in
table, are present.Confirm any suspect colonial growth on one or more of the
media as Pseudomonas aeruginosa by means of oxidase test.Upon the
colonial growth place or transfer colonies to strip or disks of filter papers
that previously has been impregnated with N, N, N, N,-tetra-methyl, 4
phenyl adenine; if there is no development of pink color, changing to purple,
the specimen meets the requirement of the test for the absence of
Pseudomonas aeruginosa.The presence of Pseudomonas aeruginosa may be
confirmed by other suitable cultural and bio-chemical test, if necessary.
• Add 1 ml of enrichment culture to 10 ml of Rappaport vassiliadis salmonella
enrichment broth and incubate at 30-35˚C for 18 to 24 hrs. Subculture
Salmonella on xylose lysine Deoxychollate agar media with inoculating
loop.

23. • Result of Pathogen Detection
• Escherichia coliMediumDescription of colonyMCABrick red colonies with a
surrounding zone of ppt. bileEMBMetallic sheen under reflected light and blue
black appearance under transmitted light.Sample plate have no
colonyStaphylococcus aureusMediumDescription of colonyMSAYellow color with
yellow zoneVJABlack, surrounded by yellow zoneSample plate have no colony
• Pseudomonas aeruginosa MediumCetrimide Agar MediumPseudomonas agar
medium for detection of fluoresceinPseudomonas agar medium for detection of
pyocyaninCharacteristic colonial morphologyGenerally greenishGenerally colorless
to yellowishFlorescence in UV lightGreenishYellowishBlueSample plate have no
colonySalmonellaMediumDescription of colonyRVSEBbrinjal color fiber seen and
medium color changeXLDARed colonies with or without black center.Sample plate
have no colon

24. REFERENCES :
1. Vogel's Textbook of Quantitative Chemical Analysis-Jeffery J Bassett, J.
Mendham, I C. Denney, 5 ed. ELBS, 1991.
2 Practical Pharmaceutical Chemistry Beckett and Stenlake. Vol 2, 4 ed. CBS
Publishers, New Delhi
3. Textbook of Pharmaceutical Analysis-K.A. Connors. 3 ed. John Wiley & Sons,
1982
4. Pharmaceutical Analysis-Higuchi, 2d ed. Wiley-Inter Science Publication,
1961.
5. Quantitative Analysis of Drugs in Pharmaceutical Formulation-P.D. Sethi, 3
ed. CBS Publishers, New Delhi
6. Pharmaceutical Analysis Modern Methods-1.W. Mumson-Part B. Volume 11,
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