中国药典微生物检测翻译稿Appendix XI J Microbial Limit Test Metho...

Appendix XI J Microbial Limit Test Method

The tests for microbial limit are designed for non-sterile products and their APIs, excipients to determine the microbial contamination, including tests for bacteria count, mold count and the test for specified micro-organisms.

The microbial limit test should be performed in the 100-Class clean area with one-direction air flow introduced in, which settled in the 10000-Class clean environment. And all the tests should strictly comply with sterile technique to protect recontamination. The cleanliness of the one-direction air flow area, working bench and the environment should be validated based upon current national criteria on Test Method for Airborne Particles, Airborne Microbe and Settling Microbe in Clean Room (area) of the Pharmaceutical Industry regularly.

If the substances to be examined have surface-active agents, neutralizing agents or inactivators, their efficacy and absence of influence for micro-organisms must be demonstrated.

The culturing temperature for bacteria is 30~35˚C; for molds and yeasts 23~28˚C; and for specified micro-organisms 35~37˚C,unless otherwise prescribed.

The results should be reported in 1g, 1ml, 10g, 10ml or 10cm2.

Test Amount

Test amount is the amount of the sample to be examined once (in g, ml or cm2).

The test amount is normally 10g or 10ml, unless otherwise prescribed for chemical films 100 cm2; for expensive or micro-packaged drugs, reduced test amount is acceptable. The sample to be examined for Salmonella another 10g or 10ml is required.

While testing, sample from more than 2 minimum packages, for films at least 4 sheets.

Generally speaking, randomly sample 3 times of the test amount (more than two minimum packages).

Preparation of Test Solution

The suitable method for test solution preparation depends upon the physical and biological characteristics of the substance. If the preparation introduces water bath for heating, the temperature shouldn’t exceed 45˚C. As soon as the test solution is prepared, the culture medium should be added within 1 hour.

Unless otherwise prescribed, the common preparation methods are presented as follows.

1.Liquid products

Take 10ml of the sample, add in sterile buffered sodium chloride-peptone solution pH 7.0 to 100ml, and mix well to obtain 1 in 10 dilution. For oils, add in suitable quantity of sterile polysorbate 80 to evenly-disperse the test solution; and for water-soluble liquid products, mixed sample concentrate can be used as test solution.

2.Solid, semisolid or viscous products

Take 10g of the sample, add in sterile buffered sodium chloride-peptone solution pH 7.0 to 100ml, and mix well with homogenizer or other suitable methods, to obtain 1 in 10 dilution. Add in suitable quantity of sterile polysorbate 80 and properly heat with water bath to disperse the sample evenly.

3.Products prepared by special procedures

(1) Water-insoluble products

Method 1 Take 5g (or 5ml) of the sample, and add to the beaker in which there is dissolved sterile mixture(temperature not more than 45˚C) containing 5g of Tween 80, 3g of glyceryl monostearate and 10g of polysorbate 80. Stir to be mass with sterile glass rod. Then slowly add in 45˚C sterile buffered sodium chloride-peptone solution pH 7.0 to 100ml, while stirring. Emulsify completely to obtain 1 in 20 dilution.

Method 2 Take 10g of the sample, and add to the suitable container in which there are 20ml of sterile isopropyl myristate (refer to the Sterility Test in Appendix XI H Sterility Test for preparation methods) and sterile glass balls. If necessary, increase the amount of isopropyl myristate used. Shake and vibrate to dissolve completely. Then add in 45˚C sterile buffered sodium chloride-peptone solution pH 7.0 to 100ml, vibrate for 5~10 minutes to extract, and stand still to laminate the water and oil. Take the water layer as 1 in 10 dilution.

(2) Films products

Take 100 cm2 of the sample, cut into pieces, add in 100ml of sterile buffered sodium chloride-peptone solution pH 7.0 (If necessary increase the amount added), soak, and vibrate to obtain 1 in 10 dilution.

(3) Enteric or colonic targeting products