Preservative Efficacy Test – The Complete Procedure

Standard Operating Procedure (SOP) and Guideline for Preservative Efficacy Test in Microbiology Laboratory for checking of effectiveness of preservatives added in the drug formulations.

Procedure for Preservative Efficacy Test

1.0   Objective :

• • To lay down a procedure for preservative efficacy test.

2.0   Scope :

• • This guideline is applicable  in Microbiology Laboratory for checking of effectiveness of preservatives added in the drug formulations.

3.0   Procedure for Preservative Efficacy Test:

• • The test consists of challenging to preparation, wherever possible in its final container, with a prescribed inoculum of suitable microorganism, storing the inoculated preparation at a prescribed temperature, withdrawing samples from the container at specified intervals of time and counting the organisms in the samples so removed.

• • Preservative properties of the preparation are adequate if in the conditions of the test there is a significant fall or no increase, as appropriate, in the number of microorganisms in the inoculated preparation after the times and at the temperature prescribed.

• • Product Categories: (Preservative Efficacy Test)

• • For the purpose of testing, compendial articles have been divided into four categories (Table 01).

• • The criteria of antimicrobial effectiveness for these products are a function of the route of administration:

Table 01-Compendial Product Categories

• • Test Organisms: Preservative Efficacy Test

• • Use cultures of the following organisms:

• • Candida albicans ATCC10231 / MTCC No.227

• • Aspergillus brasielliensis ATCC No. 16404/ MTCC No.1344

• • coli ATCC No.8739 / MTCC 1687

• • Pseudomonas aeruginosa ATCC9027 / MTCC 1688

• • Staphylococcus aureus ATCC6538 / MTCC 737

• for oral preparation containing a high concentration of sugar.

• • The viable microorganism used in the test must not be more than five passage removed from the original ATCC culture and not more than four passage of MTCC culture.

• • Media:

• • All media used in the test must be tested for growth promotion.

• • Suitable media used for the test are : Soyabean Casein Digest Agar and Sabouraud Dextrose Agar.

• • Preparation of Inoculum for Preservative Efficacy Test:

• • Preparatory to the test, inoculate the surface of a suitable volume o solid agar media from a recently revived stock culture of each of the specified microorganisms.

• • The culture conditions of the inoculum culture are described in Table 2.

• • To harvest the bacterial and Candida albicans cultures, use sterile saline TS, washing the surface growth, collecting it in a suitable vessel and adding sufficient sterile saline TS to obtain a microbial count of about 1×10⁸ cfu per ml.

• • To harvest the cells of Aspergillus brasielliensis, use sterile saline TS containing 0.05% of Polysorbate 80 and add sufficient sterile saline TS to obtain a count of about 1×10⁸ cfu per ml.

• • Alternatively the stock culture organisms may be grown in a suitable liquid medium (i.e. Soyabean Casein Digest Broth or Sabouraud Dextrose Broth) and the cells harvested by centrifugation, then washed and re-suspended in sterile saline TS to obtain a microbial count of about 1×10⁸ cfu per ml.

• • Determine the number of cfu per ml in each suspension using the conditions listed in Table 02 to confirm the initial cfu per ml estimate.

Table 02:Culture conditions for Inoculum Preparations

• • Method – Preservative Efficacy Test :

• • The test can be conducted either in five original containers and the product container can be entered aseptically or in five sterile, capped bacteriological containers of suitable size into which a sufficient volume of product has been transferred.

Related : Disinfectant Efficacy Test

• • Inoculate each container with one of the prepared and standardized inoculums and mix.

• • The volume of the suspension inoculums used is between 0.5% and 1.0% of the volume of product.

• • The concentration of test microorganism that is added to the product are such that the final concentration of the test preparation after inoculation is between 1×10⁷ and 1×10⁶ cfu per ml of the product.

• • The initial concentration of viable microorganisms in each preparation is estimated based on the concentration of microorganism in each of the standardized inoculums as determined by plate count method.

• • Incubate the inoculated containers at 22.5±2.5°C.

• • Remove a suitable amount of sample from each container typically 1ml or 1g at 0 hr and at appropriate intervals according to the type of product specified in table 03.

• • Determine by the plate count procedure the number of cfu present in each test preparation for the applicable intervals.

• • Record any changes observed in appearance at these intervals.

• • 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 reduction.

• • Plates are then incubated for 2-3 days at 30-35°C for bacterial and yeast growth and for 5-10 days at 20-25° C for fungal growth.

• • After incubation the number of colonies is counted and the resulting figure is multiplied by dilution factor.

• • Criteria For Antimicrobial Effectiveness – Preservative Efficacy Test:

• • The requirements for antimicrobial effectiveness are met if the criteria specified under table 3 are met.

• • No increase is defined as not more than 0.5 log unit higher than the previous value measured.

Table 03 Criteria for tested microorganism

4.0  Reference (S) – Preservative Efficacy Test :

• • Chapter No. 2.2.2 of IP 2014

• • Chapter No. 51 of USP 38

• • Appendix XVI C, Volume –V of BP 2015

5.0   Glossary : Preservative Efficacy Test :

• • SOP                    :  Standard Operating Procedure

• • QC                      :  Quality Control

• • ºC                        :  Degree Celsius

• • IP                        :  Indian Pharmacopoeia

• • BP                       :  British Pharmacopoeia

• • USP                    :  United State Pharmacopoeia

• • ATCC                  :  American type culture collection

• • MTCC                 :  Microbial type culture collection

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