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〈61〉 MICROBIAL LIMIT TESTS
This chapter provides tests for the estimation of the number of viable aerobic microorganisms prent and for freedom from designated microbial species in pharmaceutical articles of all kinds, from raw materials to the finished forms. An automated method may be substituted for the tests prented here, provided it has been properly validated as giving equivalent or better results. In preparing for and in applying the tests, obrve aptic precautions in handling the specimens. Unless otherwi directed, where the procedure specifies simply ―incubate,‖ hold the container in air that is thermostatically controlled at a temperature between 30 and 35, for a period of 24 to 48 hours. The term ―growth‖ is ud in a special n herein, i.e., to designate the prence and presumed proliferation of viable microorganisms.
Preparatory Testing
The validity of the results of the tests t forth in this chapter rests largely upon the adequacy of a demonstration that the test specimens to which they are applied do not, of themlves, inhibit the multiplication, under the test conditions, of microorganisms that may be prent. Therefore, preparator
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to conducting the tests on a regular basis and as circumstances require subquently, inoculate diluted specimens of the material to be tested with parate viable cultures of Staphylococcus aureus, Escherichia coli, Pudomonas aeruginosa, and Salmonella. This can be done by adding 1 mL of not less than 10-3 dilution of a 24-hour broth culture of the microorganism to the first dilution (in pH 7.2 Phosphate Buffer, Fluid Soybean–Cain Digest Medium, or Fluid Lacto Medium) of the test material and following the test procedure. Failure of the organism(s) to grow in the relevant medium invalidates that portion of the examination and necessitates a modification of the procedure by (1) an increa in the volume of diluent, the quantity of test material remaining the same, or by (2) the incorporation of a sufficient quantity of suitable inactivating agent(s) in the diluents, or by (3) an appropriate combination of modifications (1) and (2) so as to permit growth of the inocula.
The following are examples of ingredients and their concentrations that may be added to the culture medium to neutralize inhibitory substances prent in the sample: soy lecithin, 0.5%; and polysorbate 20, 4.0%. Alternatively, repeat the test as described in the preceding paragraph, usingFluid Cain Digest–Soy Lecithin–Polysorbate 20 Medium to demonstrate neutralization of prervatives or other antimicrobial agents in the test material. Where inhibitory substances are cont
ained in the product and the latter is soluble, a
suitable, validated adaptation of a procedure t forth in the
ction Membrane Filtration under Test for Sterility of the Product to be Examined under Sterility Tests 〈71〉, may be ud.
If in spite of the incorporation of suitable inactivating agents and a substantial increa in the volume of diluent, it is still not possible to recover the viable cultures described above and where the article is not suitable for employment of membrane filtration, it can be assumed that the failure to isolate the inoculated organism is attributable to the bactericidal activity of the product. This information rves to indicate that the article is not likely to be contaminated with the given species of microorganism. Monitoring should be continued in order to establish the spectrum of inhibition and bactericidal activity of the article.
Buffer Solution and Media
Culture media may be prepared as follows, or dehydrated culture media may be ud provided that, when reconstituted as directed by the manufacturer or distributor, they have similar ingredients and/or yield media comparable to tho obtained from the formulas given herein.
In preparing media by the formulas t forth herein, dissolve the soluble solids in the water, using heat, if necessary, to effect complete solution, and add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the desired pH in the medium when it is ready for u. Determine the pH at 25 ± 2.
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Where agar is called for in a formula, u agar that has a moisture content of not more than 15%. Where water is called for in a formula, u Purified Water.
山水诗PH 7.2 Phosphate Buffer
Stock Solution— Dissolve 34 g of monobasic potassium phosphate in about 500 mL of water contained in a 1000-mL volumetric flask. Adjust to pH 7.2 ± 0.1 by the addition of sodium hydroxide TS (about 175 mL), add water to volume, and mix. Dispen and sterilize. Store under refrigeration.
For u, dilute the Stock Solution with water in the ratio of 1 to 800, and sterilize.
Media
Unless otherwi indicated, the media should be sterilized by heating in an autoclave (e Steam Sterilization under Sterilization 〈1211〉), the exposure time depending on the volume to be sterilized.
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heating in a water bath at 48 to 50 for about 30 minutes to effect solution. Add 40 mL of polysorbate 20. Mix, and dispen as desired.
solution.
pH after sterilization: 7.4 ± 0.2.
between 45 and 50, and add 10 mL of sterile potassium tellurite solution (1 in 100) and 50 mL of egg-yolk emulsion. Mix intimately but gently, and pour into plates. (Prepare the egg-yolk emulsion by disinfecting the surface of whole shell eggs, aptically cracking the eggs, and parating out intact yolks into a sterile graduated cylinder. Add sterile saline TS to obtain a 3 to 7 ratio of egg yolk to saline. Add to a sterile blender cup, and mix at high speed for 5 conds.)
口技pH after sterilization: 6.8 ± 0.2.
Boil the solution of solids for 1 minute. Sterilize, cool to between 45 and
50, and add 20 mL of sterile potassium tellurite solution (1 in 100).
pH after sterilization: 7.2 ± 0.2.
等式和方程的关系frequent agitation, and boil for 1 minute to effect solution.
尚于博pH after sterilization: 7.2 ± 0.2.
VIII. Pudomonas Agar Medium for Detection of Fluorescin
with frequent agitation, and boil for 1 minute to effect solution. pH after sterilization: 7.2 ± 0.2.
动物王国with frequent agitation, and boil for 1 minute to effect solution. pH after sterilization: 7.2 ± 0.2.立体风筝
pH after sterilization: 6.9 ± 0.2.
Final pH: 7.0 ±
