The activity (potency) of antibiotics may be demonstrated under suitable conditions by their inhibitory effect on microorganisms. A reduction in antimicrobial activity also will reveal subtle changes not demonstrable by chemical methods. Accordingly, microbial or biological assays remain generally the standard for resolving doubt with respect to possible loss of activity. This chapter summarizes these procedures for the antibiotics recognized in this Pharmacopeia for which microbiological assay remains the definitive method.
Two general methods are employed, the cylinder-plate or “plate” assay and the turbidimetric or “tube” assay. The first depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a petri dish or plate to an extent such that growth of the added microorganism is prevented entirely in a circular area or “zone” around the cylinder containing a solution of the antibiotic. The turbidimetric method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favorable to its rapid growth in the absence of the antibiotic.
APPARATUS
All equipment is to be thoroughly cleaned before and after each use. Glassware for holding and transferring test organisms is sterilized by dry heat or by steam.
Temperature Control
Thermostatic control is required in several stages of a microbial assay, when culturing a microorganism and preparing its inoculum, and during incubation in plate and tube assays. Maintain the temperature of assay plates at ±0.5 of the temperature selected. Closer control of the temperature (±0.1 of the selected temperature) is imperative during incubation in a tube assay, and may be achieved in either circulated air or water, the greater heat capacity of water lending it some advantage over circulating air.
Spectrophotometer
Measuring transmittance within a fairly narrow frequency band requires a suitable spectrophotometer in which the wavelength of the light source can be varied or restricted by the use of a 580-nm filter or a 530-nm filter for reading the absorbance in a tube assay. For the latter purpose, the instrument may be arranged to accept the tube in which incubation takes place (see Turbidimetric Assay Receptacles), to accept a modified cell fitted with a drain that facilitates rapid change of content, or preferably, fixed with a flow-through cell for a continuous flow-through analysis; set the instrument at zero absorbance with clear, uninoculated broth prepared as specified for the particular antibiotic, including the same amount of test solution and formaldehyde as found in each sample.
note—Either absorbance or transmittance measurement may be used for preparing inocula.
Cylinder-Plate Assay Receptacles
For assay plates, use glass or plastic petri dishes (approximately 20 × 100 mm) having covers of suitable material. For assay cylinders, use stainless steel or porcelain cylinders with the following dimensions, each dimension having a tolerance of ±0.1 mm: outside diameter 8 mm; inside diameter 6 mm; and length 10 mm. Carefully clean cylinders to remove all residues. An occasional acid bath, e.g., with about 2 N nitric acid or with chromic acid (see Cleaning Glass Apparatus 1051 ) is needed.
Turbidimetric Assay Receptacles
For assay tubes, use glass or plastic test tubes, e.g., 16 × 125 mm or 18 × 150 mm that are relatively uniform in length, diameter, and thickness and substantially free from surface blemishes and scratches. Tubes that are to be placed in the spectrophotometer are matched and are without scratches or blemishes. Cleanse thoroughly to remove all antibiotic residues and traces of cleaning solution, and sterilize tubes that have been used previously, before subsequent use.
MEDIA AND DILUENTS
Media
The media required for the preparation of test organism inocula are made from the ingredients listed herein. Minor modifications of the individual ingredients, or reconstituted dehydrated media, may be substituted, provided the resulting media possess equal or better growth-promoting properties and give a similar standard curve response.
Dissolve the ingredients in water to make 1 L, and adjust the solutions with either 1 N sodium hydroxide or 1 N hydrochloric acid as required, so that after steam sterilization the pH is as specified.
medium 1
Peptone
6.0 g
Pancreatic Digest of Casein
4.0 g
Yeast Extract
3.0 g
Beef Extract
1.5 g
Dextrose
1.0 g
Agar
15.0 g
Water
1000 mL
pH after sterilization: 6.6 ± 0.1.
medium 2
Peptone
6.0 g
Yeast Extract
3.0 g
Beef Extract
1.5 g
Agar
15.0 g
Water
1000 mL
pH after sterilization: 6.6 ± 0.1.
medium 3
Peptone
5.0 g
Yeast Extract
1.5 g
Beef Extract
1.5 g
Sodium Chloride
3.5 g
Dextrose
1.0 g
Dibasic Potassium Phosphate
3.68 g
Monobasic Potassium Phosphate
1.32 g
Water
1000 mL
pH after sterilization: 7.0 ± 0.05.
medium 4
Same as Medium 2, except for the additional ingredient 1.0 g of Dextrose.
medium 5
Same as Medium 2, except that the final pH after sterilization is 7.9 ± 0.1.
medium 8
Same as Medium 2, except that the final pH after sterilization is 5.9 ± 0.1.
medium 9
Pancreatic Digest of Casein
17.0 g
Papaic Digest of Soybean
3.0 g
Sodium Chloride
5.0 g
Dibasic Potassium Phosphate
2.5 g
Dextrose
2.5 g
Agar
20.0 g
Water
1000 mL
pH after sterilization: 7.2 ± 0.1.
medium 10
Same as Medium 9, except to use 12.0 g of Agar instead of 20.0 g, and to add 10 mL of Polysorbate 80 after boiling the medium to dissolve the agar.
pH after sterilization: 7.2 ± 0.1.
medium 11
Same as Medium 1, except that the final pH after sterilization is 8.3 ± 0.1.
medium 13
Dextrose
20.0 g
Peptone
10.0 g
Water
1000 mL
pH after sterilization: 5.6 ± 0.1.
medium 19
Peptone
9.4 g
Yeast Extract
4.7 g
Beef Extract
2.4 g
Sodium Chloride
10.0 g
Dextrose
10.0 g
Agar
23.5 g
Water
1000 mL
pH after sterilization: 6.1 ± 0.1.
medium 32
Same as Medium 1, except for the additional ingredient 0.3 g of Manganese Sulfate.
medium 34
Glycerol
10.0 g
Peptone
10.0 g
Beef Extract
10.0 g
Sodium Chloride
3.0 g
Water
1000 mL
pH after sterilization: 7.0 ± 0.1.
medium 35
Same as Medium 34, except for the additional ingredient 17.0 g of Agar.
medium 36
Pancreatic Digest of Casein
15.0 g
Papaic Digest of Soybean
5.0 g
Sodium Chloride
5.0 g
Agar
15.0 g
Water
1000 mL
pH after sterilization: 7.3 ± 0.1.
medium 39
Same as Medium 3, except that the final pH after sterilization is 7.9 ± 0.1.
medium 40
Yeast Extract
20.0 g
Polypeptone
5.0 g
Dextrose
10.0 g
Monobasic Potassium Phosphate
2.0 g
Polysorbate 80
0.1 g
Agar
10.0 g
Water
1000 mL
pH after sterilization: 6.7 ± 0.2.
medium 41
Pancreatic Digest of Casein
9.0 g
Dextrose
20.0 g
Yeast Extract
5.0 g
Sodium Citrate
10.0 g
Monobasic Potassium Phosphate
1.0 g
Dibasic Potassium Phosphate
1.0 g
Water
1000 mL
pH after sterilization: 6.8 ± 0.1.
Phosphate Buffers and Other Solutions
Prepare as follows, or by other suitable means, the potassium phosphate buffers required for the antibiotic under assay. The buffers are sterilized after preparation, and the pH specified in each case is the pH after sterilization.
buffer no. 1, 1 percent, ph 6.0— Dissolve 2.0 g of dibasic potassium phosphate and 8.0 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 6.0 ± 0.05.
buffer no. 3, 0.1 m, ph 8.0— Dissolve 16.73 g of dibasic potassium phosphate and 0.523 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 8.0 ± 0.1.
buffer no. 4, 0.1 m, ph 4.5— Dissolve 13.61 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 4.5 ± 0.05.
buffer no. 6, 10 percent, ph 6.0— Dissolve 20.0 g of dibasic potassium phosphate and 80.0 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 6.0 ± 0.05.
buffer no. 10, 0.2 m, ph 10.5— Dissolve 35.0 g of dibasic potassium phosphate in 1000 mL of water, and add 2 mL of 10 N potassium hydroxide. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 10.5 ± 0.1.
buffer no. 16, 0.1 m, ph 7.0— Dissolve 13.6 g of dibasic potassium phosphate and 4.0 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 7.0 ± 0.2.
other solutions— Use the substances specified under Reagents, Indicators, and Solutions. For water, use Purified Water. For saline, use Sodium Chloride Injection. Dilute formaldehyde is Formaldehyde Solution diluted with water
Two general methods are employed, the cylinder-plate or “plate” assay and the turbidimetric or “tube” assay. The first depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a petri dish or plate to an extent such that growth of the added microorganism is prevented entirely in a circular area or “zone” around the cylinder containing a solution of the antibiotic. The turbidimetric method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favorable to its rapid growth in the absence of the antibiotic.
APPARATUS
All equipment is to be thoroughly cleaned before and after each use. Glassware for holding and transferring test organisms is sterilized by dry heat or by steam.
Temperature Control
Thermostatic control is required in several stages of a microbial assay, when culturing a microorganism and preparing its inoculum, and during incubation in plate and tube assays. Maintain the temperature of assay plates at ±0.5 of the temperature selected. Closer control of the temperature (±0.1 of the selected temperature) is imperative during incubation in a tube assay, and may be achieved in either circulated air or water, the greater heat capacity of water lending it some advantage over circulating air.
Spectrophotometer
Measuring transmittance within a fairly narrow frequency band requires a suitable spectrophotometer in which the wavelength of the light source can be varied or restricted by the use of a 580-nm filter or a 530-nm filter for reading the absorbance in a tube assay. For the latter purpose, the instrument may be arranged to accept the tube in which incubation takes place (see Turbidimetric Assay Receptacles), to accept a modified cell fitted with a drain that facilitates rapid change of content, or preferably, fixed with a flow-through cell for a continuous flow-through analysis; set the instrument at zero absorbance with clear, uninoculated broth prepared as specified for the particular antibiotic, including the same amount of test solution and formaldehyde as found in each sample.
note—Either absorbance or transmittance measurement may be used for preparing inocula.
Cylinder-Plate Assay Receptacles
For assay plates, use glass or plastic petri dishes (approximately 20 × 100 mm) having covers of suitable material. For assay cylinders, use stainless steel or porcelain cylinders with the following dimensions, each dimension having a tolerance of ±0.1 mm: outside diameter 8 mm; inside diameter 6 mm; and length 10 mm. Carefully clean cylinders to remove all residues. An occasional acid bath, e.g., with about 2 N nitric acid or with chromic acid (see Cleaning Glass Apparatus 1051 ) is needed.
Turbidimetric Assay Receptacles
For assay tubes, use glass or plastic test tubes, e.g., 16 × 125 mm or 18 × 150 mm that are relatively uniform in length, diameter, and thickness and substantially free from surface blemishes and scratches. Tubes that are to be placed in the spectrophotometer are matched and are without scratches or blemishes. Cleanse thoroughly to remove all antibiotic residues and traces of cleaning solution, and sterilize tubes that have been used previously, before subsequent use.
MEDIA AND DILUENTS
Media
The media required for the preparation of test organism inocula are made from the ingredients listed herein. Minor modifications of the individual ingredients, or reconstituted dehydrated media, may be substituted, provided the resulting media possess equal or better growth-promoting properties and give a similar standard curve response.
Dissolve the ingredients in water to make 1 L, and adjust the solutions with either 1 N sodium hydroxide or 1 N hydrochloric acid as required, so that after steam sterilization the pH is as specified.
medium 1
Peptone
6.0 g
Pancreatic Digest of Casein
4.0 g
Yeast Extract
3.0 g
Beef Extract
1.5 g
Dextrose
1.0 g
Agar
15.0 g
Water
1000 mL
pH after sterilization: 6.6 ± 0.1.
medium 2
Peptone
6.0 g
Yeast Extract
3.0 g
Beef Extract
1.5 g
Agar
15.0 g
Water
1000 mL
pH after sterilization: 6.6 ± 0.1.
medium 3
Peptone
5.0 g
Yeast Extract
1.5 g
Beef Extract
1.5 g
Sodium Chloride
3.5 g
Dextrose
1.0 g
Dibasic Potassium Phosphate
3.68 g
Monobasic Potassium Phosphate
1.32 g
Water
1000 mL
pH after sterilization: 7.0 ± 0.05.
medium 4
Same as Medium 2, except for the additional ingredient 1.0 g of Dextrose.
medium 5
Same as Medium 2, except that the final pH after sterilization is 7.9 ± 0.1.
medium 8
Same as Medium 2, except that the final pH after sterilization is 5.9 ± 0.1.
medium 9
Pancreatic Digest of Casein
17.0 g
Papaic Digest of Soybean
3.0 g
Sodium Chloride
5.0 g
Dibasic Potassium Phosphate
2.5 g
Dextrose
2.5 g
Agar
20.0 g
Water
1000 mL
pH after sterilization: 7.2 ± 0.1.
medium 10
Same as Medium 9, except to use 12.0 g of Agar instead of 20.0 g, and to add 10 mL of Polysorbate 80 after boiling the medium to dissolve the agar.
pH after sterilization: 7.2 ± 0.1.
medium 11
Same as Medium 1, except that the final pH after sterilization is 8.3 ± 0.1.
medium 13
Dextrose
20.0 g
Peptone
10.0 g
Water
1000 mL
pH after sterilization: 5.6 ± 0.1.
medium 19
Peptone
9.4 g
Yeast Extract
4.7 g
Beef Extract
2.4 g
Sodium Chloride
10.0 g
Dextrose
10.0 g
Agar
23.5 g
Water
1000 mL
pH after sterilization: 6.1 ± 0.1.
medium 32
Same as Medium 1, except for the additional ingredient 0.3 g of Manganese Sulfate.
medium 34
Glycerol
10.0 g
Peptone
10.0 g
Beef Extract
10.0 g
Sodium Chloride
3.0 g
Water
1000 mL
pH after sterilization: 7.0 ± 0.1.
medium 35
Same as Medium 34, except for the additional ingredient 17.0 g of Agar.
medium 36
Pancreatic Digest of Casein
15.0 g
Papaic Digest of Soybean
5.0 g
Sodium Chloride
5.0 g
Agar
15.0 g
Water
1000 mL
pH after sterilization: 7.3 ± 0.1.
medium 39
Same as Medium 3, except that the final pH after sterilization is 7.9 ± 0.1.
medium 40
Yeast Extract
20.0 g
Polypeptone
5.0 g
Dextrose
10.0 g
Monobasic Potassium Phosphate
2.0 g
Polysorbate 80
0.1 g
Agar
10.0 g
Water
1000 mL
pH after sterilization: 6.7 ± 0.2.
medium 41
Pancreatic Digest of Casein
9.0 g
Dextrose
20.0 g
Yeast Extract
5.0 g
Sodium Citrate
10.0 g
Monobasic Potassium Phosphate
1.0 g
Dibasic Potassium Phosphate
1.0 g
Water
1000 mL
pH after sterilization: 6.8 ± 0.1.
Phosphate Buffers and Other Solutions
Prepare as follows, or by other suitable means, the potassium phosphate buffers required for the antibiotic under assay. The buffers are sterilized after preparation, and the pH specified in each case is the pH after sterilization.
buffer no. 1, 1 percent, ph 6.0— Dissolve 2.0 g of dibasic potassium phosphate and 8.0 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 6.0 ± 0.05.
buffer no. 3, 0.1 m, ph 8.0— Dissolve 16.73 g of dibasic potassium phosphate and 0.523 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 8.0 ± 0.1.
buffer no. 4, 0.1 m, ph 4.5— Dissolve 13.61 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 4.5 ± 0.05.
buffer no. 6, 10 percent, ph 6.0— Dissolve 20.0 g of dibasic potassium phosphate and 80.0 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 6.0 ± 0.05.
buffer no. 10, 0.2 m, ph 10.5— Dissolve 35.0 g of dibasic potassium phosphate in 1000 mL of water, and add 2 mL of 10 N potassium hydroxide. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 10.5 ± 0.1.
buffer no. 16, 0.1 m, ph 7.0— Dissolve 13.6 g of dibasic potassium phosphate and 4.0 g of monobasic potassium phosphate in 1000 mL of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 7.0 ± 0.2.
other solutions— Use the substances specified under Reagents, Indicators, and Solutions. For water, use Purified Water. For saline, use Sodium Chloride Injection. Dilute formaldehyde is Formaldehyde Solution diluted with water
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