Preservative Efficacy Test : Pharmaguideline

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Preservative Efficacy Test

Learn how to test the preservative efficacy of Pharmaceutical products using bacterial and fungal cultures.

1.0 Required Equipment

Micropipette tips 200 ml

2.0 Material Required

Sterile saline solution with 0.1% peptone
Sterile saline solution with 0.05% polysorbate -80
70% IPA solution
Sample for testing
Test culture suspension 1 X 108 cells of each organisms: - Candida albicans NCIM 3471 (ATCC 10231), Aspergillus niger   NCIM 1196 (ATCC 16404), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus NCIM 2079 (ATCC 6538), Escherichia coli NCIM 2065 (ATCC 8739) NCIM 2200

3.0 Procedure

3.1 From a recently grown stock culture of each of the test organisms; subculture on the surface of respective media slants as given below.
Incubation temperature and duration
Candida albicans
ATCC 10231
Dextrose Agar
20-25°C for 3 days
Aspergillus niger
ATCC 16404
Dextrose Agar
20-25°C for 7 days
ATCC 8739
Soyabean Casein
Digest Agar
30-35°C for 24 to 48 hours
Ps. aeruginosa
ATCC 9027
Soyabean Casein
Digest Agar
30-35°C for 24 to 48 hours
S. aureus
ATCC 6538
Soya bean Casein
Digest Agar
30-35°C for 24 to 48 hours

3.2 Using sterile solution about 10 ml of sterile normal saline with peptone (0.1 % w/v), harvest the bacteria and C. albicans culture and diluted suitably with the sterile saline solution to bring the count to about 1 X 108 per ml.
3.3 Similarly harvest A. niger culture with the sterile saline solution containing 0.05% w/v of Tween 80 & adjust the spore count to about 1 X 108 per ml with the sterile saline solution.
3.4 Determine the number of cfu per ml in each suspension by serial dilution method. Prepare ten-fold serial dilutions of each of the bacterial cultures and Candida albicans using sterile normal saline (0.9 % w/v) with peptone (0.1 % w/v); and Aspergillus niger using sterile normal saline, with polysorbate 80.
3.5 Using the pour plate technique, plate out, in duplicate ­the last 7 dilutions using the appropriate media. Allow solidifying. Invert the Petri dishes and incubate the plates for the bacterial count at 30­ -35°C for 2 days and the plates for the fungal count at 20°C - 25°C for 2 - 7days. Count the cfu per ml. Tabulate the counts obtained in the Test Data Sheet.
3.6 Inoculate each original product container or product tube (when original container is not suitable for inoculation with sterile syringe fitted with needle, transfer 10 -20 ml per capped bacteriological tube) with one of the standardized microbial suspensions using a ratio equivalent to 0.1 ml of inoculums suspension to 20 ml of product and mix.
3.7 The final concentration should be between 1x105 and 1x106 microorganisms per ml of product.
3.8 Determine the number of viable microorganisms by plate count method in each inoculums suspension. Prepare ten-fold serial dilutions using sterile normal saline (0.9 % w/v) with peptone (0.1 % w/v) and for Aspergillus niger uses 9 ml sterile saline with polysorbate 80 (0.05%) up to 10 fold dilution and plating all the dilutions. Calculate the initial concentration of inoculums in the product (at zero hours)
3.9 Incubate the inoculated containers or tubes at 20 to 25°C.
3.10 Determine the viable count (serial dilution, plate count method) at 7, 14, and 28 days subsequent to inoculation. Record any changes observed in appearance.

4.0 Precaution

4.1 Keep the hands clean and used strictly 70 % IPA to rinsed hand gloves throughout the operations.
4.2 Dilution of each microbial inoculums should be made properly
4.3 Plating of final dilution and counting of cfu for each microbial culture should be recorded properly which forms the baseline for comparison with the test.
4.4 Use separate tube with the product or separate container for each culture.
4.5 All the procedures of Preservative Efficacy test should be Carried out under the laminar air flow unit chamber

5.0 Limits/ Acceptance Criteria

5.1 The preservative is effective in the product examined if (a) the concentration of viable bacteria are not more than 0.1% of the initial concentrations by 14th day, (b) the concentrations of viable yeast & moulds remain at or below the initial concentration during the first 14 days and, (c) the concentration of each test microorganism remains at or below these designated levels during the remainder of the 28-day period.

6.0 Abbreviation

SOP: Standard Operating Procedure
QA: Quality Assurance
QC: Quality Control
NA: Not Applicable
IPA: Isopropyl Alcohol
LAF: Laminar Air Flow
cfu: Colony forming unit
SCDA: Soybean casein digest Agar
SDA: Sabouraud Dextrose Agar

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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