Microbial

Limit Test
Shrivardhan Dheeman
shrivans@gmail.com
svdheeman@gmail.com

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).

Quantitative estimation
There

is two test carried for quantitative

estimation:

TBC (Total Bacterial Count)

TFC (Total Fungal count)

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****

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

TBC (Total Bacterial

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

Filtration method
Pour pate method
Spread plate method
Serial Dilution Method

Membrane Filtration
Method

Pretreatment of sample:
Water soluble product:

Water insoluble product:

10 gm.*/10 ml** sample +70 ml buffered normal

saline solution+ make up volume up to 100
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
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
Remove
cotton plug from
filtration the
assembly.
flask with your left hands last
two finger and let remain plug
in your hand. Pour all treated
sample in the filtration cup.
Wait until all sample is filter.

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
Adequately
open the led of
assembly again.
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.

Membrane Filtration
Method
Procedure:
Incubate

plate in inverted
position for 72 hrs. at 2225C and later 24 hrs. at 3035C.

Observe

the plate and count

total colony appear by digital
colony counter.

Pour Plate Method

Procedure:

Take the treated sample.

Remove cotton plug with your right hands 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 hands 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 Method

Let the plate solidify. After solidification invert

all the petri plate.
Incubate all the petri plate in inverted position
for 72 hrs. at 22-25C and later 24 hrs. at 3035C.
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 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 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 22-25C and later 24 hrs. at 3035C.
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.

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.

Serial Dilution Method

Vortex the tube 10-1 for 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 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 Method

Incubate all the petri plate in inverted

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

Serial Dilution Method

1ml
1ml
1ml
1ml
10-4
1ml
10-5
1ml
10-6

sample : 9 ml saline
of sol. : 9 ml of saline
of sol. : 9 ml of saline
of sol. : 9 ml of saline

=
=
=
=

10
10
10
10

ml
ml
ml
ml

sol. (1/10) 10-1

Sol. (1/100) 10-2
Sol. (1/1000) 10-3
Sol. (1/10000)

of sol. : 9 ml of saline = 10 ml Sol. (1/100000)

of sol. : 9 ml of saline = 10 ml Sol. (1/1000000)

Serial Dilution Method

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 30-35C.

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 abony

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 Conkeys broth and incubate
at 36-38 C for 48 hrs.
If the content shows acid and gas carry out the
secondary test.

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 Conkeys
broth and other containing 5 ml peptone water
and Incubate broth tubes in a water bath at
43.5C to 44.5C 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 43.5C to 44.5C.

Escherichia coli
tertiary

test:

To test for indole production add 0.5 ml of

kovacs 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 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 32-37C
for 24-48 hrs.
Subculture on a plate containing a layer of mannitol salt agar
medium or Vogel Johnson agar medium and incubate at 32-37C for
18-24 hrs.
Examine the resulting growth by Grams 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 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
37C examining the tubes after 24 hrs.
If coagulation in any degree is observed, the
test is positive.

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 35-37C 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 35-37C for 18 to 24 hrs.
If none of the plate contains colonies having characteristics
given in the table, carry out the confirmatory test.

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 352C 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 abony
Add

1 ml of enrichment culture to 10 ml
of Rappaport vassiliadis salmonella
enrichment broth and incubate at 3035C for 18 to 24 hrs. Subculture
Salmonella
on
xylose
lysine
Deoxychollate
agar
media
with
inoculating loop.

Result of Pathogen
Detection
Escherichia coli
Medium

Description of colony

MCA
EMB

Brick red colonies with a surrounding zone of ppt. bile

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

Result of Pathogen
Detection
Pseudomonas aeruginosa
Medium

Cetrimide
Agar
Medium

Pseudomon
as agar
medium for
detection of
fluorescein

Pseudomon
as agar
medium for
detection of
pyocyanin

Characteristic
colonial
morphology
Florescence in
UV light

Generally
greenish

Generally
colorless to
yellowish
Yellowish

Generally
greenish

Greenish

Blue

Sample plate have no colony

Salmonella abony
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

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

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.

Thank
you