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.
Preparatory Testing
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 á71ñ, 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
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 á1211ñ), 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 |
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 |
III. Fluid Soybean–Casein Digest Medium
| Prepare as directed for Soybean–Casein Digest Medium under Sterility Tests á71ñ. |
| 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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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.
Sampling
Provide separate 10-mL or 10-g specimens for each of the tests called for in the individual monograph.
Procedure
Prepare the specimen to be tested by treatment that is appropriate to its physical characteristics and that does not alter the number and kind of microorganisms originally present, in order to obtain a solution or suspension of all or part of it in a form suitable for the test procedure(s) to be carried out.
For a solid that dissolves to an appreciable extent but not completely, reduce the substance to a moderately fine powder, suspend it in the vehicle specified, and proceed as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.
For a fluid specimen that consists of a true solution, or a suspension in water or a hydroalcoholic vehicle containing less than 30 percent of alcohol, and for a solid that dissolves readily and practically completely in 90 mL of pH 7.2 Phosphate Buffer or the media specified, proceed as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.
For water-immiscible fluids, ointments, creams, and waxes, prepare a suspension with the aid of a minimal quantity of a suitable, sterile emulsifying agent (such as one of the polysorbates), using a mechanical blender and warming to a temperature not exceeding 45, if necessary, and proceed with the suspension as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.
For a fluid specimen in aerosol form, chill the container in an alcohol-dry ice mixture for approximately 1 hour, cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible, and transfer the quantity of test material required for the procedures specified in one of the two preceding paragraphs, as appropriate. Where 10.0 g or 10.0 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residues. If the results of the test are inconclusive or doubtful, repeat the test with a specimen from 20 more containers.
Total Aerobic Microbial Count
For specimens that are sufficiently soluble or translucent to permit use of the Plate Method, use that method; otherwise, use the Multiple-Tube Method. With either method, first dissolve or suspend 10.0 g of the specimen if it is a solid, or 10 mL, accurately measured, if the specimen is a liquid, in pH 7.2 Phosphate Buffer, Fluid Soybean–Casein Digest Medium, or Fluid Casein Digest–Soy Lecithin-Polysorbate 20 Medium to make 100 mL. For viscous specimens that cannot be pipeted at this initial 1:10 dilution, dilute the specimen until a suspension is obtained, i.e., 1:50 or 1:100, etc., that can be pipeted. Perform the test for absence of inhibitory (antimicrobial) properties as described under Preparatory Testing before the determination of Total Aerobic Microbial Count. Add the specimen to the medium not more than 1 hour after preparing the appropriate dilutions for inoculation.
PLATE METHOD
Dilute further, if necessary, the fluid so that 1 mL will be expected to yield between 30 and 300 colonies. Pipet 1 mL of the final dilution onto each of two sterile petri dishes. Promptly add to each dish 15 to 20 mL of Soybean–Casein Digest Agar Medium that previously has been melted and cooled to approximately 45. Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature. Invert the petri dishes, and incubate for 48 to 72 hours. Following incubation, examine the plates for growth, count the number of colonies, and express the average for the two plates in terms of the number of microorganisms per g or per mL of specimen. If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as “less than 10 microorganisms per g or per mL of specimen.”
MULTIPLE-TUBE METHOD
Into each of fourteen test tubes of similar size place 9.0 mL of sterile Fluid Soybean–Casein Digest Medium. Arrange twelve of the tubes in four sets of three tubes each. Put aside one set of three tubes to serve as the controls. Into each of three tubes of one set (“100”) and into a fourth tube (A) pipet 1 mL of the solution or suspension of the specimen, and mix. From tube A, pipet 1 mL of its contents into the one remaining tube (B) not included in a set, and mix. These two tubes contain 100 mg (or 100 µL) and 10 mg (or 10 µL) of the specimen, respectively. Into each of the second set (“10”) of three tubes pipet 1 mL from tube A, and into each tube of the third set (“1”) pipet 1 mL from tube B. Discard the unused contents of tubes A and B. Close well, and incubate all of the tubes. Following the incubation period, examine the tubes for growth: the three control tubes remain clear and the observations in the tubes containing the specimen, when interpreted by reference to Table 1, indicate the most probable number of microorganisms per g or per mL of specimen.
Table 1. Most Probable Total Count by Multiple-Tube Method
| Observed Combinations of Numbers of Tubes Showing Growth in Each Set | Most Probable Number of Microorganisms per g or per mL | ||
| No. of mg (or mL) of Specimen per Tube | |||
| 100 (100 µL) | 10 (10 µL) | 1 (1 µL) | |
| 3 | 3 | 3 | >1100 |
| 3 | 3 | 2 | 1100 |
| 3 | 3 | 1 | 500 |
| 3 | 3 | 0 | 200 |
| 3 | 2 | 3 | 290 |
| 3 | 2 | 2 | 210 |
| 3 | 2 | 1 | 150 |
| 3 | 2 | 0 | 90 |
| 3 | 1 | 3 | 160 |
| 3 | 1 | 2 | 120 |
| 3 | 1 | 1 | 70 |
| 3 | 1 | 0 | 40 |
| 3 | 0 | 3 | 95 |
| 3 | 0 | 2 | 60 |
| 3 | 0 | 1 | 40 |
| 3 | 0 | 0 | 23 |
To the specimen add Fluid Soybean–Casein Digest Medium to make 100 mL, mix, and incubate. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of Vogel–Johnson Agar Medium (or Baird–Parker Agar Medium, or Mannitol–Salt Agar Medium) and of Cetrimide Agar Medium, each plated on petri dishes. Cover and invert the dishes, and incubate. If, upon examination, none of the plates contains colonies having the characteristics listed in Tables 2 and 3 for the media used, the test specimen meets the requirements for freedom from Staphylococcus aureus and Pseudomonas aeruginosa.
Table 2. Morphologic Characteristics of Staphylococcus aureus on Selective Agar Media
| Selective Medium | Characteristic Colonial Morphology | Gram Stain |
| Vogel-Johnson Agar Medium | Black Surrounded by yellow zone | Positive cocci (in clusters) |
| Mannital-Salt Agar Medium | Yellow colonies with yellow zones | Positive cocci (in clusters) |
| Baird-Parker Agar Medium | Black, shiny, surrounded by clear zones 2 to 5 mm | Positive cocci (in clusters) |
| Selective Medium | Characteristic Colonial Morphology | Fluorescence in UV Light | Oxidase Test | Gram Stain |
| Centrimide Agar Medium | Generally greenish | Greenish | Positive | Negative rods |
| Pseudomonas Agar Medium for Detection of Fluorescin | Generally colorless to yellowish | Yellowish | Positive | Negative rods |
| Pseudomonas Agar Medium for Detection of Pyocyanin | Generally greenish | Blue | Positive | Negative rods |
Oxidase and Pigment Tests (for Pseudomonas aeruginosa)— With the aid of an inoculating loop, streak representative suspect colonies from the agar surface of Cetrimide Agar Medium on the agar surfaces of Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin contained in petri dishes. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be inoculated from a separate colony. Cover and invert the inoculated media, and incubate at 35 ± 2 for not less than three days. Examine the streaked surfaces under UV light. Examine the plates to determine whether colonies having the characteristics listed in Table 3 are present.
Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,N-dimethyl-p-phenylenediamine dihydrochloride: if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa. The presence of Pseudomonas aeruginosa may be confirmed by other suitable cultural and biochemical tests, if necessary.
Test for Salmonella species and Escherichia coli
To the specimen, contained in a suitable vessel, add a volume of Fluid Lactose Medium to make 100 mL, and incubate. Examine the medium for growth, and if growth is present, mix by gently shaking. Pipet 1-mL portions into vessels containing, respectively, 10 mL of Fluid Selenite–Cystine Medium and Fluid Tetrathionate Medium, mix, and incubate for 12 to 24 hours. (Retain the remainder of the Fluid Lactose Medium.)
Test for Salmonella Species— By means of an inoculating loop, streak portions from both the selenite-cystine and tetrathionate media on the surface of Brilliant Green Agar Medium, Xylose–Lysine–Desoxycholate Agar Medium, and Bismuth Sulfite Agar Medium contained in petri dishes. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 4, the specimen meets the requirements of the test for absence of the genus Salmonella.
Table 4. Morphologic Characteristics of Salmonella Species on Selective Agar Media
| Selective Medium | Characteristic Colonial Morphology |
| Brilliant Green Agar Medium | Small, transparent, colorless or pink to white opaque (frequently surrounded by pink to red zone) |
| Xylose-Lysine- Desoxycholate Agar Medium | Red, with or without black centers |
| Bismuth Sulfite Agar Medium | Black or green |
Test for Escherichia coli— By means of an inoculating loop, streak a portion from the remaining Fluid Lactose Medium on the surface of MacConkey Agar Medium. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 5 for this medium, the specimen meets the requirements of the test for absence of Escherichia coli.
Table 5. Morphologic Characteristics of Escherichia coli on MacConkey Agar Medium
| Gram Stain | Characteristic Colonial Morphology |
| Negative rods (cocco-bacilli) | Brick-red; may have surrounding zone of precipitated bile |
Total Combined Molds and Yeasts Count
Proceed as for the Plate Method under Total Aerobic Microbial Count, except for using the same amount of Sabouraud Dextrose Agar Medium or Potato Dextrose Agar Medium, instead of Soybean Casein Digest Medium, and except for incubating the inverted petri dishes for 5 to 7 days at 20 to 25.
Retest
For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following their application to a 10.0-g specimen, a retest on a 25-g specimen of the product may be conducted. Proceed as directed for Procedure, but make allowance for the larger specimen size.
* Additional, confirmatory evidence may be obtained by use of procedures set forth in Official Methods of Analysis of the AOAC, 12th ed. (1975), sections 46.013-46.026.
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