MICROBIAL LIMIT TESTS （微生物限度檢查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 special sense herein,
i.e., to designate the presence and presumed proliferation of viable
microorganisms.
這一章規定，測試估計的數目可行的好氧微生物，從指定的微生物物種在製藥文章的所有
品種，從原料到成品的形式。一個自動化的方法，可取代的考驗就在這裏，隻要它已妥善
驗證給予同等或更好的結果。在準備和在應用試驗，觀察無菌的預防措施，再處理標本。
除非另有指示，凡指定的程序是簡單的“孵化” ，舉行容器中的空氣是恒溫的控製在溫度
30至35 ，時間在24至48小時。相對而言， “增長”是用來在一個特殊的意義在這裏，即
指定的存在和擴散的微生物。........
Preparatory Testing （籌備測試）
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 （緩衝溶液和培養基）
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. Vogel–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
Peptic Digest of Animal Tissue 10.0 g
Anhydrous Dibasic Potassium Phosphate 1.5 g
Magnesium Sulfate (MgSO4·7H2O) 1.5 g
Glycerin 10.0 mL
Agar 15.0 g
Water 1000 mL
Dissolve the solid components in the water before adding the glycerin. Heat,
with frequent agitation, and boil for 1 minute to effect solution.
pH after sterilization: 7.2 ± 0.2.
IX. Pseudomonas Agar Medium for Detection of Pyocyanin
Pancreatic Digest of Gelatin 20.0 g
Anhydrous Magnesium Chloride 1.4 g
Anhydrous Potassium Sulfate 10.0 g
Agar 15.0 g
Glycerin 10.0 mL
Water 1000 mL
Dissolve the solid components in the water before adding the glycerin. Heat,
with frequent agitation, and boil for 1 minute to effect solution.
pH after sterilization: 7.2 ± 0.2.
X. Fluid Lactose Medium
Beef Extract 3.0 g
Pancreatic Digest of Gelatin 5.0 g
Lactose 5.0 g
Water 1000 mL
Cool as quickly as possible after sterilization.
pH after sterilization: 6.9 ± 0.2.
XI. Fluid Selenite–Cystine Medium
Pancreatic Digest of Casein 5.0 g
Lactose 4.0 g
Sodium Phosphate 10.0 g
Sodium Acid Selenite 4.0 g
L-Cystine 10.0 mg
Water 1000 mL
Final pH: 7.0 ± 0.2.
Mix, and heat to effect solution. Heat in flowing steam for 15 minutes. Do not
sterilize.
XII. Fluid Tetrathionate Medium
Pancreatic Digest of Casein 2.5 g
Peptic Digest of Animal Tissue 2.5 g
Bile Salts 1.0 g
Calcium Carbonate 10.0 g
Sodium Thiosulfate 30.0 g
Water 1000 mL
Heat the solution of solids to boiling. On the day of use, add a solution
prepared by dissolving 5 g of potassium iodide and 6 g of iodine in 20 mL of
water. Then add 10 mL of a solution of brilliant green (1 in 1000), and mix.
Do not heat the medium after adding the brilliant green solution.
XIII. Brilliant Green Agar Medium
Yeast Extract 3.0 g
Peptic Digest of Animal Tissue 5.0 g
Pancreatic Digest of Casein 5.0 g
Lactose 10.0 g
Sodium Chloride 5.0 g
Sucrose 10.0 g
Phenol Red 80 mg
Agar 20.0 g
Brilliant Green 12.5 mg
Water 1000 mL
Boil the solution of solids for 1 minute. Sterilize just prior to use, melt
the medium, pour into petri dishes, and allow to cool.
pH after sterilization: 6.9 ± 0.2.
XIV. Xylose–Lysine–Desoxycholate Agar Medium
Xylose 3.5 g
L-Lysine 5.0 g
Lactose 7.5 g
Sucrose 7.5 g
Sodium Chloride 5.0 g
Yeast Extract 3.0 g
Phenol Red 80 mg
Agar 13.5 g
Sodium Desoxycholate 2.5 g
Sodium Thiosulfate 6.8 g
Ferric Ammonium Citrate 800 mg
Water 1000 mL
Final pH: 7.4 ± 0.2.
Heat the mixture of solids and water, with swirling, just to the boiling
point. Do not overheat or sterilize. Transfer at once to a water bath
maintained at about 50, and pour into plates as soon as the medium has cooled.
XV. Bismuth Sulfite Agar Medium
Beef Extract 5.0 g
Pancreatic Digest of Casein 5.0 g
Peptic Digest of Animal Tissue 5.0 g
Dextrose 5.0 g
Sodium Phosphate 4.0 g
Ferrous Sulfate 300 mg
Bismuth Sulfite Indicator 8.0 g
Agar 20.0 g
Brilliant Green 25 mg
Water 1000 mL
Final pH: 7.6 ± 0.2.
Heat the mixture of solids and water, with swirling, just to the boiling
point. Do not overheat or sterilize. Transfer at once to a water bath
maintained at about 50, and pour into plates as soon as the medium has cooled.
XVI. Triple Sugar–Iron–Agar Medium
Pancreatic Digest of Casein 10.0 g
Pancreatic Digest of Animal Tissue 10.0 g
Lactose 10.0 g
Sucrose 10.0 g
Dextrose 1.0 g
Ferrous Ammonium Sulfate 200 mg
Sodium Chloride 5.0 g
Sodium Thiosulfate 200 mg
Agar 13.0 g
Phenol Red 25 mg
Water 1000 mL
pH after sterilization: 7.3 ± 0.2.
XVII. MacConkey Agar Medium
Pancreatic Digest of Gelatin 17.0 g
Pancreatic Digest of Casein 1.5 g
Peptic Digest of Animal Tissue 1.5 g
Lactose 10.0 g
Bile Salts Mixture 1.5 g
Sodium Chloride 5.0 g
Agar 13.5 g
Neutral Red 30 mg
Crystal Violet 1.0 mg
Water 1000 mL
Boil the mixture of solids and water for 1 minute to effect solution.
pH after sterilization: 7.1 ± 0.2.
XVIII. Levine Eosin–Methylene Blue Agar Medium
Pancreatic Digest of Gelatin 10.0 g
Dibasic Potassium Phosphate 2.0 g
Agar 15.0 g
Lactose 10.0 g
Eosin Y 400 mg
Methylene Blue 65 mg
Water 1000 mL
Dissolve the pancreatic digest of gelatin, the dibasic potassium phosphate,
and the agar in the water, with warming, and allow to cool. Just prior to
use, liquefy the gelled agar solution, add the remaining ingredients, as
solutions, in the following amounts, and mix: for each 100 mL of the liquefied
agar solution—5 mL of lactose solution (1 in 5), 2 mL of the eosin Y solution
(1 in 50), and 2 mL of methylene blue solution (1 in 300). The finished medium
may not be clear.
pH after sterilization: 7.1 ± 0.2.
XIX. Sabouraud Dextrose Agar Medium
Dextrose 40 g
Mixture of equal parts of Peptic Digest of Animal Tissue and Pancreatic Digest
of Casein 10 g
Agar 15 g
Water 1000 mL
Mix, and boil to effect solution.
pH after sterilization: 5.6 ± 0.2.
XX. Potato Dextrose Agar Medium
Cook 300 g of peeled and diced potatoes in 500 mL of water prepared by
distillation, filter through cheesecloth, add water prepared by distillation
to make 1000 mL, and add the following:
Agar 15 g
Glucose 20 g
Dissolve by heating, and sterilize.
pH after sterilization: 5.6 ± 0.2.
For use, just prior to pouring the plates, adjust the melted and cooled to 45
medium with sterile tartaric acid solution (1 in 10) to a pH of 3.5 ± 0.1.
Do not reheat the pH 3.5 medium.
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