SOP for Suitability Testing

PURPOSE

The purpose of this document is to lay down the procedure for establishing the ability of the test method used to detect microorganisms in the presence of a product to be tested.

SCOPE

This procedure should be done in all pharmaceutical samples including that of cosmetic formulations, prior to the actual examination of the product. If historical information has been gathered already, it might be not necessary to perform such a test. 

The method describes in this article take into account from inoculum preparation, sample preparation, inoculation and dilution, removal of antimicrobial activity, recovery of microorganisms in the presence of the product, to results interpretation. The method applies to both quantitative and qualitative tests.

PROCEDURE

Preparation of Test Strains

• Use standardized stable suspensions of test strains so that the final plate count will be less than 100 cfu/g or mL.
• Seed-lot culture maintenance techniques should be used so that the viable microorganisms used for inoculation are not more than five passages removed from the original master seed-lot.
• Grow each of the bacterial and fungal test strains separately as described in Annex B.
• Select the test organism based on the technical perspective of the analysts depending on the nature of the method and target organisms.

Preparation of sample

• The method for sample preparation depends on the physical characteristics of the product to be tested.
• If none of the procedures described in WI. Preliminary Sample Preparation of Cosmetic Products can be demonstrated to be satisfactory; a suitable alternative procedure must be developed.

Inoculation and Dilution

1. Quantitative Tests

• Add to the sample prepared as directed above and to control (with no test material included) a sufficient volume of the microbial suspension to obtain an inoculum of not more than 100 cfu.
• To demonstrate acceptable microbial recovery from the product, the lowest possible dilution factor of the prepared sample must be used for the test. Where this is not possible due to antimicrobial activity or poor solubility, further appropriate protocols must be developed.
• If inhibition of growth by the sample cannot otherwise be avoided, the aliquot of the microbial suspension may be added after neutralization, dilution, or filtration.

2. Qualitative Tests

• For each new product to be tested perform sample preparation as described above.
• At the time of mixing, add each test strain in the prescribed growth medium.
• Inoculate the test strains individually.
• Use a number of microorganisms equivalent to not more than 100 cfu in the inoculated test preparation.
• Prepare a control, wherein test organisms are inoculated in the broth/diluent without the test product.
• Perform the test as described in the relevant test method procedure for the detection of specific microorganisms using the shortest incubation period prescribed.
• The specified microorganism must be detected with the indication reactions as described under the relevant test methods.
• Any antimicrobial activity of the product necessitates a modification of the test procedure. Refer WI. Validation of Neutralization Method.
• For a given product, if the antimicrobial activity with respect to a microorganism for which testing is prescribed cannot be neutralized, then it is to be assumed that the inhibited microorganism will not be present in the product.

Recovery of Microorganisms in the Presence of Product

1. Membrane Filtration

• Use membrane filters having a nominal pore size not greater than 0.45 µm.
• The type of filter material is chosen in such a way that the bacteria-retaining efficiency is not affected by the components of the sample to be investigated. For each microorganism selected one membrane filter is used.
• Transfer a suitable quantity of sample prepared as described above to the membrane filter, filter immediately, and rinse the membrane filter with an appropriate volume of diluent.

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2. Plate-Count Method

A. Pour-Plate Method

• Perform plate-count methods at least in duplicate for each medium, and use the mean count of the result.
• For petri-dishes, 9 cm in diameter, add to the dish 1 mL of the sample prepared as described as above and 15-20 mL of prescribed agar maintained at not more than 45°C, volume of the medium is increased when a larger petri dish is used.

• Incubate the plates as indicated in the specific test method procedure.
• Take the arithmetic mean of the counts per medium, and calculate the number of cfu in the original inoculum

B. Spread-Plate Method

• For petri-dishes, 9 cm in diameter, add 15-20 mL of the recommended agar medium at about 45°C to each dish, and allow solidifying. If larger plates are used, the volume of the agar medium is increased accordingly.
• Dry the plates, e.g. in a laminar-flow cabinet or in an incubator.
• Spread a measured volume of the sample preparations of not less than 0.1 mL.
• Incubate and count as directed above.

3. Qualitative Test

• Perform the test as described in the relevant specific test method procedure using the sample preparation with inoculum.
• The growth of microorganisms must be confirmed so that only targeted ones are recovered.

INTERPRETATION

• When verifying the suitability of the Membrane Filtration or the Plate-Count Method, a mean count of any of the test organisms not differing by a factor greater than 2 from the value of the control defined in the Qualitative Test in the absence of the product must be obtained.
• In verifying a qualitative test, the specified microorganisms must be detected with the indication reactions as described in the relevant test method procedure.
• If these criteria cannot be met for one of more of the organisms tested with any of the described methods, the method and test conditions that come closest to the criteria are used to test the product.

REVISION HISTORY

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