1. Microbial Limit Test

2. Microbial Limit Test This test is designed to perform:
Total Viable Count (TVC) of bacteria and fungi (Quantitative estimation).
Enrichment for qualitative estimation:
Identification of microorganism by cultivating on selective media comparing ATCC/MTCC culture of pathogen (Qualitative estimation).

3. INTRODUCTION TO MICROBIAL LIMIT TEST
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).
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 AGAR
EOSINE METHYLENE BLUE AGAR
MACCONKEY AGAR
MANNITOL SALT AGAR
PSEUDOMONAS AGAR
SABOURAUD CHLORAMPHENICOL AGAR
SOYABEAN CASEIN DIGEST AGAR
TRIPLE SUGAR IRON AGAR

6. BUFFERED PEPTONE WATER
FLUID SELENITE CYSTEINE BROTH
MACCONKEY BROTH
PEPTONE WATER
SOYABEAN CASEIN DIGEST MEDIUM
TETRATHIONATE BRILLINT GREEN BILE BROTH

7. Quantitative estimation
There is two test carried for quantitative estimation:
TBC (Total Bacterial Count)
TFC (Total Fungal count)

8. TBC (Total Bacterial Count) or TFC (Total Fungal Count)
Objective: to determine the population of bacteria/fungi in sample of pharmaceutical product.
Reqiurement:
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

9. TBC (Total Bacterial Count) or TFC (Total Fungal Count)
Four Method are employed for this test:
Filtration method
Pour pate method
Spread plate method
Serial Dilution Method
* Filtration method, **Pour plate method, ***Serial dilution method, ****Spread plate method.

10. 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
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
* Solid Like tablets, **liquid like water.

11. 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 filter.

12. Membrane Filtration Method
Procedure:
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 forcep. Press gentle and close the lid and invert the plate.

13. Membrane Filtration Method
Procedure:
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.

14. Pour Plate Method Take the treated sample.
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 mention 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. Be precautious that media should not touch the lid of petri plate.

15. Pour Plate Method
Let the plate solidify. After solidification invert all the petri plate.
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 document in report of product.

16. Spread Plate Method Take the pre-treated sample.
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 led 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 it touch with surface until all sample spread well.

17. Spread Plate Method 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 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.

18. 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.

19. Serial Dilution Method
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.
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.

20. Serial Dilution Method
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.

21. Serial Dilution Method
1ml sample : 9 ml saline = 10 ml sol. (1/10) 10-1
1ml of sol. : 9 ml of saline = 10 ml Sol. (1/100) 10-2
1ml of sol. : 9 ml of saline = 10 ml Sol. (1/1000) 10-3
1ml of sol. : 9 ml of saline = 10 ml Sol. (1/10000) 10-4
1ml of sol. : 9 ml of saline = 10 ml Sol. (1/100000) 10-5
1ml of sol. : 9 ml of saline = 10 ml Sol. (1/ ) 10- 6

22. Serial Dilution Method

23. 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.

24. Qualitative estimation (Pathogen detection)
In qualitative estimation the four more pathogenic bacteria are detecting under this test, which are following:
Escherichia coli
Pseudomonas aeruginosa
Staphylococcus aureus
Salmonella
In qualitative estimation the two more pathogenic fungi are detecting under this test, which are following:
Candida albicans
Aspergillus niger

25. 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.

26. 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.

27. 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.

29. 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.

30. Staphylococcus aureus
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.

32. 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.

33. Pseudomonas aeruginosa
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.
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.

35. Salmonella
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.

37. Result of Pathogen Detection
Escherichia coli
Medium
Description of colony
MCA
Brick red colonies with a surrounding zone of ppt. bile
EMB
Metallic sheen under reflected light and blue black appearance under transmitted light.
Sample plate have no colony
Staphylococcus aureus
Medium
Description of colony
MSA
Yellow color with yellow zone
VJA
Black, surrounded by yellow zone
Sample plate have no colony

38. Result of Pathogen Detection
Pseudomonas aeruginosa
Medium
Cetrimide Agar Medium
Pseudomonas agar medium for detection of fluorescein
Pseudomonas agar medium for detection of pyocyanin
Characteristic colonial morphology
Generally greenish
Generally colorless to yellowish
Florescence in UV light
Greenish
Yellowish
Blue
Sample plate have no colony
Salmonella
Medium
Description of colony
RVSEB
brinjal color fiber seen and medium color change
XLDA
Red colonies with or without black center.
Sample plate have no colony

39. Conclusion
In Microbial limit test, microorganisms are count and microorganisms are compared with the ATCC/MTCC culture for detection of pathogenic bacteria. The bacterial culture of ATCC/NCTC/MTCC used, are given below:
Escherichia coli ATCC No. 8739
Pseudomonas aeruginosa ATCC No. 9027
Staphylococcus aureus ATCC No. 6539
Salmonella NCTC No. 6017
Candida albicans ATCC No
Aspergillus niger ATCC No

40. Conclusion
No colony on selective media shows the absence of pathogenic microorganisms in the sample. Hence it is compliance with the IP, BP, USP specification.

41. THANK YOU -PHARMA STREET