1 of 8

Form No. FZ-164

Revision No. 01
Effective Date: 25 February 2014
Next Review on: 24 February 2017

MICROBIAL LIMIT TEST

(MLT)
REPORT No:
PRODUCT: Acylex Tablets
SAMPLE SOURCE:

TESTING DATE:

Production.

31/12/2014.

FP-658
400 mg. BATCH/GRN No.:

9Z124.

Q.C. No.:

-----------

SAMPLE QUANTITY:

10 Tablets.

REPORTING DATE:

05/01/2015.

SUMMARY OF RESULTS
TEST

LIMITS

RESULTS

Total Aerobic Microbial Count

(TAMC)

Not more than 1,000 CFU per Tab.

185 CFU/Tab

Total Combined Yeast/Mold Count

(TYMC)

Not more than 100 per Tab.

35 CFU/Tab

Bile-tolerant
Gram-negative Bacteria

Should be absent.

Absent

Pseudomonas aeruginosa

Must be absent

Absent

Staphylococcus aureus

Must be absent

Absent

Escherichia coli

Must be absent

Absent

Salmonella Species

Must be absent

Absent

Clostridia Species

Must be absent

Absent

Candida albicans

Must be absent

Absent

CFU = Colony Forming Unit.

REMARKS: Sample complies the acceptance criteria, (Colored box shows the product's status).

Yes

No
2 of 8

Form No. FZ-164

Revision No. 01
Effective Date: 25 February 2014
Next Review on: 24 February 2017

MICROBIAL LIMIT TEST

(MLT)
REPORT No:

FP-658

DAY - 1:
1.

Growth Promotion Test and Negative Controls of First Day Media Used:
Media

Lot No.

Growth Promotion
Test No.

Incubation
Temperature & Time

Tryptic Soy Agar

5390

61

30 - 35 C for 72 Hours

Tryptic Soy Broth

5391

62

30 - 35 C for 24 Hours

Sabouraud Dextrose Agar

5392

58

20 - 25 C for 120 Hours

Sabouraud Dextrose Broth

5393

53

20 - 25 C for 72 Hours

Reinforced Clostridial Medium

5394

56

30 - 35 C for 48 Hours

Enterobacteria Enrichment BrothMossel

5395

59

30 - 35 C for 24 Hours

Test Performed By/On:

Microbiologist
2.

31/12/2014.

PREPARATION OF THE SAMPLE:

2.1 Water-Soluble Products: Dissolve or dilute (usually a 1:10 dilution is prepared) 10 g / 10 mL
according to the nature of the sample to be tested in Sterile Peptone Buffered Solution / Sterile
Tryptic Soy Broth. If necessary, adjust the pH to 6 - 8. Further dilutions, where necessary, are
prepared with the same diluent.
2.2 Nonfatty and Fatty Products Insoluble in Water: Suspend the product to be tested
(usually a 1:10 dilution is prepared) 10 g / 10 mL in Sterile Peptone Solution / Sterile Tryptic Soy
Broth, mix for wetting the sample then add surface-active agent such as 1 g / liter of sterile Polysorbate
80 (use sterile polysorbate 80 contaning diluent) or use sterile isopropyl myristate. If necessary, adjust
the pH to 6 - 8. Further dilutions, where necessary, are prepared with the same diluent.

3.

Neutralization/Removal of Antimicrobial Activity: If product contains any antimicrobial agent / product

itself is antibiotic, then add polysorbate 80 to diluent and sterile it before using / increase the volume of
diluent for inactivation of antimicrobial activity of the product.

4.

Total Aerobic Microbial Count (TAMC): Pipette 1 mL of the prepared sample into each of two
sterile petri-dishes. Add 15 - 20 mL cooled Sterile liquefied Tryptic Soya Agar to each plates. Cover
the dishes, mix the sample with agar by tilting or rotating the dishes, and the contents to solidify at room
temperature. Invert the petri-dishes and incubate these two plates at 30 - 35 C initially for 3 days.
Limit: As per COA

5.

Total Combined Yeast/ Mould Count (TYMC): Pipette 1 mL of the prepared sample into each
of two sterile petri-dishes. Add 15 - 20 mL cooled Sterile liquefied Sabouraud Dextrose Agar to
each plates. Cover the dishes, mix the sample with agar by tilting or rotating the dishes, and the

contents to solidify at room temperature. Invert petri-dishes and incubate these two plates at 20 - 25 C
for 5 days.
Limit: As per COA
3 of 8

Form No. FZ-164

Revision No. 01
Effective Date: 25 February 2014
Next Review on: 24 February 2017

MICROBIAL LIMIT TEST

(MLT)
REPORT No:

FP-658

6.

Calculation: Record the number of colonies per plate and calculate the result by multiplying by a dilution
factor of 10 to give results in CFU/g or mL or tablet / capsule.

7.

TEST FOR SPECIFIED MICRO-ORGANISM:

7.1 Bile-Tolerant Gram-Negative Bacteria: Dissolve 10 g of sample or powder of 10 tablets/cap,
10 mL of sample in Tryptic Soy Broth and make up the volume to 100 mL with the same, shake well
and incubate at 20 - 25 C for a time sufficient to resuscitate the bacteria but not sufficient to
encourage multiplication of the organism (usually 2 hour but not more than 5 hour).
7.1.1 Test for Absence: Unless otherwise prescribed, use 10 mL or the volume corresponding to 1 g
of the product, to inoculate 100 mL Enterobacteria Enrichment Broth-Mossel. Incubate at
30 - 35 C for 24 hours.
7.2 Sample preparation and pre-incubation for Escherichia coli / Pseudomonas aeruginosa /
Staphylococcus aureus /Salmonellae:
Prepare a sample using a 1 in 10 dilution of not less than 10 g or 10 mL or powder of 10 tablets
or capsules in 100 mL Tryptic Soy Broth, mix and incubate at 30 - 35 C for 24 hrs.
7.3 Sample Preparation and Heat Treatment for Clostridia: Prepare a sample using a 1 in 10 dilution
of not less than 2 g /2 mL of the product to be examined in Tryptic Soy Broth. Take two equal portions
corresponding to not less than 1 g / mL of the product to be examined. Heat 1 portion at 80 C for
10 minutes & cool rapidly. Don't heat the other portion. Transfer 10 mL or the quantity corresponding
to 1 g /1 mL of each portion to 2 containers containing 100 mL / suitable amounts of Reinforced
Clostridial Medium. Incubate under anaerobic conditions at 30 - 35 C for 48 hours.
7.3 Sample Preparation for Candida albicans : Prepare a sample using a 1 : 10 dilution of not less than
10 g/10 mL/tablets or capsules Tryptic Soy Broth and mix well. Transfer 10 mL or the quantity
corresponding to not less than 1 g/1 mL to Sabouraud Dextrose Broth, mix well and incubate at
incubate at 30 -35 C for 3 days.

DAY - 2:
8.

Physical Check of Media: (Colored box shows test and media status)
Observation of
Media
Positive Control
Growth
Tryptic Soy Broth
Yes
No
Yes
No

Enterobacteria Enrichment BrothMossel

Results Observed By/On:
Microbiologist
9.

Yes

No

Yes

No

Negative Control
No

Yes

No

Yes

01/01/2015.

Growth Promotion Test and Negative Controls of Second Day Media Used:
Growth Promotion
Media
Lot No.
Test No.
Cetrimide Agar
5402
66

Incubation Temp. &

Time
30 - 35 C for 72 Hours

MacKonkey Broth

5403

63

42 - 44 C for 24-48 Hrs

Mannitol Salt Agar

5404

57

30 - 35 C for 72 Hours

Violet Red Bile Agar

Rappaport Vassiliadis Salmonella
Broth

5405

55

30 - 35 C for 24 Hours

5406

30

30 - 35 C for 24 Hours
4 of 8

Form No. FZ-164

Revision No. 01
Effective Date: 25 February 2014
Next Review on: 24 February 2017

MICROBIAL LIMIT TEST

(MLT)
REPORT No:
10.

FP-658

Selection and Subculture:

10.1 Test for Bile-Tolerant Gram-Negative Bacteria: After completion of 24 hours incubation,
subculture a small aliquot from Enterobacteria Enrichment Broth-Mossel to Violet Red Bile Glucose
Agar plates, incubate at 30 - 35 C for 24 hours.
10.2 Test for Escherichia coli : Shake the bottle, transfer 1 mL of Tryptic Soy Broth to 100 mL of
MacKonkey Broth, and incubate at 42 - 44 C for 24 - 48 hours.
10.3 Test for Pseudomonas aeruginosa : After completion of 24 hours incubation, subculture a small
aliquot from Tryptic Soy Broth to Cetrimide Agar plates, and incubate at 30 - 35 C for 72 hours.
10.4 Test for Staphylococcus aureus : After completion of 24 hours incubation, subculture a small
aliquot from Tryptic Soy Broth to Mannitol Salt Agar plates, and incubate at 30 - 35 C for 72 hours.
10.5 Test for Salmonella : After completion of 24 hours incubation, transfer 0.1 mL Tryptic Soy Broth
to 10 mL of Rappaport Vassiliadis Salmonella broth, and incubate at 30 - 35 C for 24 hours.

Test Performed By/On:

Microbiologist

01/01/2015.

DAY - 3:
11.

Physical Check of Media: (Colored box shows test and media stauts)
Observation of
Media
Positive Control
Growth

Negative Control

MacKonkey Broth

Yes

No

Yes

No

No

Yes

Violet Red Bile Agar

Yes

No

Yes

No

No

Yes

Reinforced Clostridial Medium

Yes

No

Yes

No

No

Yes

Rappaport Vassiliadis Salmonella

Enrichment Broth

Yes

No

Yes

No

No

Yes

12.

Physical Check of Media for growth and Record: Examine the plates for growth and record if present
on specified media plates for specified microorganisms.
Specimen Plates
Absent / Present
Media
Dilution
Negative Control
#2
#1
Clostridia Species :
Reinforced Clostridial
No
Yes
NIL
Absent
1 : 10
NIL
Medium
Bile-Tolerant Gram-Negative Bacteria:
Violet Red Bile Agar
Escherichia coli :

1 : 10

NIL

NIL

Absent

No

Yes

MacKonkey Broth
Salmonella Speices :
Rappaport Vassiliadis
Salmonella Broth

1 : 10

NIL

NIL

Absent

No

Yes

1 : 10

NIL

NIL

Absent

No

Yes
5 of 8

Form No. FZ-164

Revision No. 01
Effective Date: 25 February 2014
Next Review on: 24 February 2017

MICROBIAL LIMIT TEST

(MLT)
REPORT No:
13.

FP-658

Interpretation of Results:
13.1 For Clostridia Speices : Product complies if there is no growth / turbidity observed in the medium,
if so, the identification tests are negative in subculturing step on Columbia Agar.
13.2 For Bile-tolerant Gram-negative Bacteria: Product complies if there is no growth.
12.3 For Escherichia coli : Product complies if there is no growth observed or confirmatory
identification tests are negative in subculturing step on MacKonkey Agar.
13.4 For Salmonella Species : No color change or turbidity observed in medium,
complies if colonies of the types described are not present or identification shows negative result.

Results Observed By/On:

Microbiologist
14.

02/01/2015.

Growth Promotion Test and Negative Controls of Three Day Media Used:
Growth Promotion
Media
Lot No.
Test No.

Incubation Temp. &

Time

Columbia Agar

5407

49

30 - 35 C for 48 Hours

MacKonkey Agar

5408

65

30 - 35 C for 48 Hours

Sabouraud Dextrose Agar

5409

58

30 - 35 C for 48 Hours

Xylose, Lysine, Deoxycholate Agar

5410

64

30 - 35 C for 48 Hours

15.

Selection and Subculture:

15.1

Test for Escherichia coli : Shake the bottle, subculture 1 mL of MacKonkey Broth to
MacKonkey Agar plates and incubate at 30 - 35 C for 48 hours.

15.2

Test for Salmonella : Shake the bottle, subculture 1 mL of Rappaport Vassiliadis Salmonella
Enrichment Broth to Xylose, Lysine, Deoxycholate Agar plates, incubate at 30 - 35 C 48 hours.

15.3

Test for Clostridia : Shake the bottle, subculture 1 mL of Reinforced Clostridial Medium from
each two bottle / tubes on Columbia Agar, incubate anaerobically condition at 30 - 35 C 48 hours.

15.4

Test for Candida albicans : Shake the bottle, subculture 1 mL of Sabouraud Dextrose Broth
on Sabouraud Dextrose Agar plates and incubate at 30 - 35 C 48 hours.

Test Performed By/On:

Microbiologist

02/01/2015.
6 of 8

Form No. FZ-164

Revision No. 01
Effective Date: 25 February 2014
Next Review on: 24 February 2017

MICROBIAL LIMIT TEST

(MLT)
REPORT No:

FP-658

DAY - 4:
16.

Physical Check of Media: (Colored box shows test and media status)
Observation of
Media
Positive Control
Growth
Tryptic Soy Agar

Yes

No

Yes

No

No

Yes

Sabouraud Dextrose Broth

Yes

No

Yes

No

No

Yes

Results Observed By/On:

Microbiologist
17.

Negative Control

03/01/2015.

Physical Check of Media for Growth and Record: Examine the plates for growth, count the number
of colonies, and express the average for the two plates in terms of Colony Forming Unit per g (CFU/g)
or per mL or per 100 cm or bottle/tube of specimen.
If no growth or microbial colonies recover from the specimen's duplicate plates, express the result(s) as,

"Less Than 10 CFU/g or mL or 100 cm or bottle/tube of Specimen".

Specimen Plates
Average
CFU/g or
Media
Dilution
Count
mL
#2
#1
Total Aerobic Microbial Count (TAMC):
Tryptic Soy Agar

1 : 10

Enrichment for Candida albicans :

Sabouraud Dextrose
1 : 10
Broth
18.

21

16

18.5

Negative Control

185 CFU No

Yes

Clear / Turbid
Clear

Clear

No

Yes

Interpretation of Results:
18.1 For Candida albicans : If the turbidity is detected / observed in Sabouraud Dextrose Broth,
then do identification tests and also subculture on Sabouraud Dextrose Agar plates.

Result Observed By/On:

Microbiologist

03/01/2015.

DAY - 5:
19.

Physical Check of Media: (Colored box shows test and media status)
Observation of
Media
Positive Control
Growth

Negative Control

Cetrimide Agar

Yes

No

Yes

No

No

Yes

Mannitol Salt Agar

Yes

No

Yes

No

No

Yes

Xylose, Lysine, Deoxycholate Agar Yes

Sabouraud Dextrose Agar

Yes

Result Observed By/On:

Microbiologist

No

Yes

No

No

Yes

No

Yes

No

No

Yes

04/01/2015.
7 of 8

Form No. FZ-164

Revision No. 01
Effective Date: 25 February 2014
Next Review on: 24 February 2017

MICROBIAL LIMIT TEST

(MLT)
REPORT No:

FP-658

20.

Physical Check of Media for growth and Record: Examine the plates for growth and record if present
on specified media plates for specified microorganisms.
Specimen Plates
Absent / Present
Media
Dilution
Negative Control
#2
#1
Pseudomonoas aeruginosa :
Cetrimide Agar
No
Yes
NIL
Absent
1 : 10
NIL
Staphylococcus aureus :
Mannitol Salt Agar

1 : 10

NIL

NIL

Absent

No

Yes

Escherichia coli :
MacKonkey Agar

1 : 10

NIL

NIL

Absent

No

Yes

1 : 10

NIL

NIL

Absent

No

Yes

Salmonella :
Xylose, Lysine,
Deoxycholate Agar
Clostridia :
Columbia Agar
21.

No
Yes
NIL
Absent
1 : 10
NIL
Interpretation of Results:
21.1 For Pseudomonas aeruginosa : Product complies if there is no growth observed or confirmatory
identification tests are negative.
21.2 For Staphylococcus aureus : Yellow or white colonies surrounded by a yellow zone, product
product complies if colonies of the types described are not present or identification tests are negative.
21.3 For Escherichia coli : Product complies if no growth observed or confirmatory identification
tests show negative result.
21.4 For Salmonella : Well developed, red-colonies, with or without black centers, product complies
if colonies of the types described are not present or identification shows negative result.
21.5 For Clostridia : The occurrence of anaerobic growth of rods (with or without endospores) giving
a negative catalase reaction indicates the presence of Clostridia .
This is confirmed by identification tests. The product complies with the test if colonies of the types
described are not present or if the confirmatory identification tests are negative.

Result Observed By/On:

Microbiologist

04/01/2015.

DAY - 6:
22.

Physical Check of Media for growth and Record:

Specimen Plates
Media
Dilution
#2
#1
Candida albicans :

Average
Count

CFU/g or
mL

Negative Control

Sabouraud Dextrose
No
Yes
NIL
Absent
1 : 10
NIL
Agar
For Candida albicans : Growth of white colonies may indicate the presence of Candida albicans .
This if confirmed by identification tests.
The product complies if such colonies are not present or identification shows negative result.
Result Observed By/On:
Microbiologist

05/01/2015.
8 of 8

Form No. FZ-164

Revision No. 01
Effective Date: 25 February 2014
Next Review on: 24 February 2017

MICROBIAL LIMIT TEST

(MLT)
REPORT No:
22.

FP-658

Physical Check of Media for growth and Record: (Colored box shows test and medium status)
Observation of
Media
Positive Control
Negative Control
Growth
Sabouraud Dextrose Agar

Yes

No

Yes

Specimen Plates
#2
#1
Total Combined Yeast / Mold Count (TYMC):
Sabouraud Dextrose
02
1 : 10
05
Agar
Media

23.

Dilution

No

Average
Count

CFU/g or
mL

3.5

35 CFU

No

Yes
Negative Control

No

Yes

Interpretation of Results:
23.1 For Total Aerobic Microbial Count (TAMC) & Total Combined Yeast / Mold Count (TYMC):
If Fungi detected on TSA plates OR Bacteria dectected on SDA plates, they are counted as a part of
TAMC as well as TYMC.
When an acceptance criterion for microbiological quality of product is prescribed, it is interpreted
as follows:

10

CFU: Maximum Acceptable Count

= 20;

10
10

CFU: Maximum Acceptable Count

= 200;

CFU: Maximum Acceptable Count

= 2000.

Result Observed By/On:

Microbiologist
24.

05/01/2015.

Acceptance Criteria:
Product complies with the test if no growth observed / detected on the surface of specified culture media.
If growth observed / detected on any specified medium, the product test must be repeated. After retesting,
if again observed / detected any sign of growth then reject the batch at non-compliance of the test.

25.

Negative Controls:
To verify testing conditions, a negative control is performed using the chosen diluent in place of the test
preparation. There must be no growth of microorganisms observed / detected. A negative control is also
performed when testing the products as described under the Testing of Products .
If product sample shows no growth but negative control shows any growth then retest the product sample.
If product sample shows growth but negative control also shows any growth then again retest the sample.
A failed negative control requires an investigation.

26.

Reference:
United States Pharmacopeia (USP - 35), Chapters: <61> & <62>.
British Pharmacopeia (BP - 2012), Chapters <2.6.12> & <2.6.13>.
Center for Drug Evaluation and Research (CDER) May, 1999.

Performed by:

Checked by:

Reviewed by:

Microbiologist (Name/Sign./Date)

AM Microbiology (Name/Sign./Date)

MQC Microbiology (Name/Sign./Date)