USP微生物限度法

MICROBIAL LIMIT TESTS

（微生物限度检查

USP

）

This chapter provides tests for the estimation of the number of viable

aerobic microorganisms present 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

presented here, provided it has been properly validated as giving equivalent

or better results. In preparing for and in applying the tests, observe aseptic

precautions in handling the specimens. Unless otherwise 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 used in a spec

ial sense herein,

i.e., to designate the presence and presumed proliferation of viable

microorganisms.

这一章规定，测试估计的数目可行的好氧微生物，从指定的微生物物种在制药文章的所有

品种，从原料到成品的形式。一个自动化的方法，可取代的考 验就在这里，只要它已妥善

验证给予同等或更好的结果。在准 备和在应用试验，观察无菌的预防措施，再处理标本。

除非另有指示，凡指定的程序是简单的“孵化”

，举行容器中的空气是恒温的控制在温度

30

至

35

，时间在

24

至

48

< br>小时。相对而言，

“增长”是用来在一个特殊的意义在 这里，

即

指定的存在和扩散的微生物。

........

Preparatory Testing

（筹备测试）

The validity of the results of the tests set forth in this chapter rests

largely upon the adequacy of a demonstration that the test specimens to which

they are applied do not, of themselves, inhibit the multiplication, under the

test conditions, of microorganisms that may be present. Therefore, preparatory

to conducting the tests on a regular basis and as circumstances require

subsequently, inoculate diluted specimens of the material to be tested with

separate viable cultures of Staphylococcus aureus, Escherichia coli,

Pseudomonas 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

–

Casein Digest

Medium, or Fluid Lactose 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 increase 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 present in

the sample: soy lecithin, 0.5%; and polysorbate 20, 4.0%. Alternatively,

repeat the test as described in the preceding paragraph, using Fluid Casein

Digest

–

Soy Lecithin

–

Polysorbate 20 Medium to demonstrate neutralization of

preservatives or other antimicrobial agents in the test material. Where

inhibitory substances are contained in the product and the latter is soluble,

a suitable, validated adaptation of a procedure set forth in the section

Membrane Filtration under Test for Sterility of the Product to be Examined

under Sterility Tests 71, may be used.

If in spite of the incorporation of suitable inactivating agents and a

substantial increase 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 serves 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

（缓冲溶液和培养基）

Culture media may be prepared as follows, or dehydrated culture media may be

used provided that, when reconstituted as directed by the manufacturer or

distributor, they have similar ingredients and/or yield media comparable to

those obtained from the formulas given herein.

In preparing media by the formulas set 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 use.

Determine the pH at 25 ±

2.

Where agar is called for in a formula, use agar that has a moisture content of

not more than 15%. Where water is called for in a formula, use Purified Water.

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. Dispense and sterilize. Store under refrigeration.

For use, dilute the Stock Solution with water in the ratio of 1 to 800, and

sterilize.

Media

Unless otherwise indicated, the media should be sterilized by heating in an

autoclave (see Steam Sterilization under Sterilization 1211), the exposure

time depending on the volume to be sterilized.

I. Fluid Casein Digest

–

Soy Lecithin

–

Polysorbate 20 Medium

Pancreatic Digest of Casein 20 g

Soy Lecithin 5 g

Polysorbate 20 40 mL

Water 960 mL

Dissolve the pancreatic digest of casein and soy lecithin in 960 mL of water,

heating in a water bath at 48 to 50 for about 30 minutes to effect solution.

Add 40 mL of polysorbate 20. Mix, and dispense as desired.

II. Soybean

–

Casein Digest Agar Medium

Pancreatic Digest of Casein 15.0 g

Papaic Digest of Soybean Meal 5.0 g

Sodium Chloride 5.0 g

Agar 15.0 g

Water 1000 mL

pH after sterilization: 7.3 ±

0.2.

III. Fluid Soybean

–

Casein Digest Medium

Prepare as directed for Soybean

–

Casein Digest Medium under Sterility Tests

IV

. Mannitol

–

Salt Agar Medium

Pancreatic Digest of Casein 5.0 g

Peptic Digest of Animal Tissue 5.0 g

Beef Extract 1.0 g

D-Mannitol 10.0 g

Sodium Chloride 75.0 g

Agar 15.0 g

Phenol Red 0.025 g

Water 1000 mL

Mix, then heat with frequent agitation, and boil for 1 minute to effect

solution.

pH after sterilization: 7.4 ±

0.2.

V

. Baird

–

Parker Agar Medium

Pancreatic Digest of Casein 10.0 g

Beef Extract 5.0 g

Yeast Extract 1.0 g

Lithium Chloride 5.0 g

Agar 20.0 g

Glycine 12.0 g

Sodium Pyruvate 10.0 g

Water 950 mL

Heat with frequent agitation, and boil for 1 minute. Sterilize, cool to

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, aseptically cracking the eggs, and separating 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 seconds.)

pH after sterilization: 6.8 ±

0.2.

VI. V

ogel

–

Johnson Agar Medium

Pancreatic Digest of Casein 10.0 g

Yeast Extract 5.0 g

Mannitol 10.0 g

Dibasic Potassium Phosphate 5.0 g

Lithium Chloride 5.0 g

Glycine 10.0 g

Agar 16.0 g

Phenol Red 25.0 mg

Water 1000 mL

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.

VII. Cetrimide Agar Medium

Pancreatic Digest of Gelatin 20.0 g

Magnesium Chloride 1.4 g

Potassium Sulfate 10.0 g

Agar 13.6 g

Cetyl Trimethylammonium Bromide (Cetrimide) 0.3 g

Glycerin 10.0 mL

Water 1000 mL

Dissolve all solid components in the water, and add the glycerin. Heat, with

frequent agitation, and boil for 1 minute to effect solution.

pH after sterilization: 7.2 ±

0.2.

VIII. Pseudomonas Agar Medium for Detection of Fluorescin

Pancreatic Digest of Casein 10.0 g