Microbial Limit Test

Microbial Microbial Limit TestLimit TestShrivardhan [email protected]@gmail.com

Microbial Limit TestMicrobial 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).

Quantitative estimationQuantitative estimation There is two test carried for quantitative

estimation: TBC (Total Bacterial Count) TFC (Total Fungal count)

TBC (Total Bacterial TBC (Total Bacterial Count) or TFC (Total Count) or TFC (Total Fungal Count)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****

* Filtration method, **Pour plate method, ***Serial dilution method, ****Spread plate method.

TBC (Total Bacterial TBC (Total Bacterial Count) or TFC (Total Count) or TFC (Total Fungal Count)Fungal Count) Four Method are employed for this test:

Filtration method Pour pate method Spread plate method Serial Dilution Method

Membrane Filtration Membrane Filtration MethodMethod 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 100 + 0.1% 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 20 + gentle heat + make up volume up to 100

* Solid Like tablets, **liquid like water.

Membrane Filtration Membrane Filtration MethodMethod 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.


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 RODAC 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.


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.

Pour Plate MethodPour 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 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.

Pour Plate MethodPour 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 22-25ºC 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.

Spread Plate MethodSpread 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 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.

Spread Plate MethodSpread 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 22-25ºC and later 24 hrs. at 30-35ºC.


Record the colony count and documented in report of product.

Serial Dilution MethodSerial 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.

Serial Dilution MethodSerial 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 10-2 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 10-3 -10-6

saline tube in petri plate with 3 replica of each.

Serial Dilution MethodSerial Dilution Method Incubate all the petri plate in inverted

position for 72 hrs. at 22-25ºC and later 24 hrs. at 30-35ºC.


Record the colony count and documented in report of product.

Serial Dilution MethodSerial 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. (1/1000000) 10-6

Serial Dilution MethodSerial Dilution Method

Enrichment for qualitative Enrichment for qualitative estimationestimation 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 30-35ºC.

Qualitative estimation Qualitative estimation (Pathogen detection) (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 abony

In qualitative estimation the two more pathogenic fungi are detecting under this test, which are following: Candida albicans Aspergillus niger

Escherichia coliEscherichia 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 36-38 ˚C for 48 hrs.

If the content shows acid and gas carry out the secondary test.

Escherichia coliEscherichia 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 coliEscherichia 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.

Staphylococcus aureusStaphylococcus 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 24-48 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 18-24 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.

Staphylococcus aureusStaphylococcus 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.

Pseudomonas Pseudomonas aeruginosaaeruginosa 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 35-37˚C for 18 to 24 hrs.

If none of the plate contains colonies having characteristics given in the table, carry out the confirmatory test.

Pseudomonas Pseudomonas aeruginosaaeruginosa

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.

Salmonella abonySalmonella abony 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.

Result of Pathogen Result of Pathogen DetectionDetection

Medium Description of colonyMCA Brick red colonies with a surrounding zone of ppt. bileEMB Metallic sheen under reflected light and blue black

appearance under transmitted light.

Medium Description of colony MSA Yellow color with yellow zone

VJA Black, surrounded by yellow zone

Sample plate have no colony

Escherichia coli

Staphylococcus aureus


Result of Pathogen Result of Pathogen DetectionDetection

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

Generally greenish

Florescence in UV light

Greenish Yellowish Blue

Medium Description of colony RVSEB brinjal color fiber seen and medium color change

XLDA Red colonies with or without black center.

Pseudomonas aeruginosa



Salmonella abony

ConclusionConclusion 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 abony NCTC No. 6017 Candida albicans ATCC No. 10231 Aspergillus niger ATCC No. 16404

ConclusionConclusion No colony on selective media shows the

absence of pathogenic microorganisms in the sample. Hence it is compliance with the IP, BP, USP specification.

Thankyou

Microbial Limit Test

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