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SOP for Antimicrobial Effectiveness Testing


Standard operating procedure to test the effectiveness of antimicrobial substances added in pharmaceutical products by using standard cultures.

1.0  PURPOSE

       To lay down the procedure to perform Antimicrobial effectiveness testing to be performed using standard culture.

2.0  SCOPE

       It is applicable to the microbiology lab for Antimicrobial effectiveness testing.

3.0  RESPONSIBILITY

       Microbiology personnel

4.0  ACCOUNTABILITY

       Head of Department

5.0  PROCEDURE

5.1  Precaution taken during antimicrobial effectiveness testing.

5.1.1  Glassware to be used shall be sterilized.
5.1.2  Media to be used shall be pre incubated
5.1.3  Standard culture to be used shall not be more than 4 passage

5.2  Test organisms used

Use cultures of the following microorganisms Candida albicans ATCC No. 10231, Aspergillus niger ATCC No. 16404, Escherichia coli ATCC No. 8739, Pseudomonas aeruginosa ATCC No. 9027, and Staphylococcus aureus ATCC No. 6538. The viable microorganisms used in the test must not be more than five passages removed from the original ATCC culture or any other equivalent cultures.

5.3  Preparation of inoculums

5.3.1  Prepare the inoculums as per the SOP for preparation of culture suspension.
5.3.2  To harvest the bacterial and Candida albicans cultures, use sterile peptone saline, wash the surface growth, collecting it in suitable glassware, and adding sufficient sterile peptone saline to obtain a microbial count of about 1 × 108 colony-forming units (CFU) per ml. To harvest the cells of Aspergillus niger, use sterile peptone saline containing 0.05% of polysorbate 80, and add sufficient sterile peptone saline to obtain a count of about 1 × 108 CFU per ml.
5.3.3  Determine the number of CFU per ml in each suspension, using the conditions of media and microbial recovery incubation times listed in Table below to confirm the initial CFU per ml. This value serves to calibrate the size of inoculum used in the test. The bacterial and yeast suspensions are to be used within 24 hours of harvest, but the fungal preparation may be stored under refrigeration for up to 7 days.
5.3.4  Culture Conditions for Inoculum Preparation as below table-
Organism
Suitable Medium
Incubation
Temperature
Incubation Time
Microbial Recovery
Incubation Time
Escherichia coli
SCM /SCA
32.5 ± 2.5 C
18 to 24 hours
3 to 5 days
Pseudomonas aeruginosa
SCM /SCA
32.5 ± 2.5 C
18 to 24 hours
3 to 5 days
Staphylococcus aureus
SCM /SCA
32.5 ± 2.5 C
18 to 24 hours
3 to 5 days
Candida albicans
SCM /SDA
22.5 ± 2.5 C
44 to 52 hours
3 to 5 days
Aspergillus niger
SCM /SDA
22.5 ± 2.5 C
6 to 10 days
3 to 7 days

5.4  Test Procedure

5.4.1  The test can be conducted either in five original containers if sufficient volume of product is available in each container or in five sterile capped bacteriological containers of suitable size into which a sufficient volume (Not less than) of product has been transferred.
5.4.2  Inoculate each container with one of the prepared and standardized inoculum, and mix. The volume of the suspension inoculum used is between 0.5% and 1.0% of the volume of the product.
5.4.3  The concentration of test microorganisms that is added to the product are such that the final concentration of the test preparation after inoculation is between 1 × 105 and 1 × 106 CFU per ml of the product.
5.4.4  The initial concentration of viable microorganisms in each test preparation is estimated based on the concentration of microorganisms in each of the standardized inoculum as determined by the plate-count method.
5.4.5  Incubate the inoculated containers at 22.5 ± 2.5° C. Sample each container at the appropriate intervals specified in point number 5.5. Record any changes observed in appearance at these intervals.
5.4.6  Determine by the plate-count procedure the number of CFU present in each test preparation for the applicable intervals
5.4.7  Incorporate an inactivator (neutralizer) of the specific antimicrobial in the plate count or in the appropriate dilution prepared for plating (if required).
5.4.8  Using the calculated concentrations of CFU per ml present at the start of the test, calculate the change in log10 values of the concentration of CFU per ml for each microorganism at the applicable test intervals, and express the changes in terms of log reductions.

Related: Incubation Conditions for Common Media used for Fungus and Bacteria

5.5  Frequency and Acceptance criteria

As per EP:
Category of products
Case
Bacterial log reduction
Fungal log reduction
6 hrs
24 hrs
2 days
7 th day
14 th day
28 th day
7 th day
14 th day
28 th day
Parenteral and ophthalmic preparations
A
2
3
-
-
-
NR
2
-
NI
B
-
1
-
3
-
NI
-
1
NI
Topical preparations
A
-
-
2
3
-
NI
-
2
NI
B
-
-
-
-
3
NI
-
1
NI
Oral preparations
A
-
-
-
-
3
NI
-
1
NI










As per USP:
Category
Bacteria
Yeast and Molds
Injections, other parenterals including emulsions, otic products, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles
Not less than 1.0 log reduction from the initial calculated count at 7 days, not less than 3.0 log reduction from the initial count at 14 days, and no increase from the 14 days' count at 28 days.
No increase from the initial calculated count at 7, 14, and 28 days.
Topically used products made with aqueous bases or vehicles, nonsterile nasal products, and emulsions, including those applied to mucous membranes.
Not less than 2.0 log reduction from the initial count at 14 days, and no increase from the 14 days' count at 28 days.
No increase from the initial calculated count at 14 and 28 days.
Oral products other than antacids, made with aqueous bases or vehicles.
Not less than 1.0 log reduction from the initial count at 14 days, and no increase from the 14 days' count at 28 days.
No increase from the initial calculated count at 14 and 28 days.
Antacids made with an aqueous base
No increase from the initial calculated count at 14 and 28 days.

















As per Harmonized criteria:
Category of products
Case
Bacterial log reduction
Fungal log reduction
6 hrs
24 hrs
2 days
7 th day
14 th day
28 th day
7 th day
14 th day
28 th day
Parenteral and ophthalmic preparations
A
2
3
-
NI
NI
NR
2
NI
NI
B
-
1
-
3
NI
NI
NI
1
NI
Topical preparations
A
-
-
2
3
NI
NI
-
2
NI
B
-
-
-
-
3
NI
-
1
NI
Oral preparations
A
-
-
-
-
3
NI
-
1
NI
Antacids
-
-
-
-
-
NI
NI
-
NI
NI












6.0  ABBREVIATIONS

6.1  SOP - Standard Operating Procedure
6.2  ATCC - American Type of Culture Collection
6.3  CFU - Colony Forming Unit
6.4  SCM - Soyabean Casein Digest Medium
6.5  SCA - Soyabean Casein Digest Agar
6.6  SDA - Sabouraud Dextrose Agar
6.7  °C - Degree Centigrade
6.8  % - Percentage
6.9  EP - European Pharmacopoeia
6.10  USP - United State Pharmacopoeia
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


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