Pages

Tuesday, 9 October 2012

81 Antibiotics - Microbial Assays

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

BET - Gel-Clot Technique

The Gel-Clot Technique is for detecting or quantifying endotoxins based on clotting of the lysate reagent in the presence of endotoxin. The minimum concentration of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of the lysate reagent. To ensure both the precision and validity of the test, perform the tests for confirming the labeled lysate sensitivity and for interfering factors as described under Preparatory Testing.
Preparatory Testing
Test for Confirmation of Labeled Lysate Sensitivity— Confirm in four replicates the labeled sensitivity, , expressed in EU/mL of the lysate prior to use in the test. The test for confirmation of lysate sensitivity is to be carried out when a new batch of lysate is used or when there is any change in the test conditions that may affect the outcome of the test. Prepare standard solutions having at least four concentrations equivalent to 2 , , 0.5 , and 0.25 by diluting the USP Endotoxin RS with Water for BET.
Mix a volume of the Lysate TS with an equal volume (such as 0.1-mL aliquots) of one of the Standard Endotoxin Solutions in each test tube. When single test vials or ampuls containing lyophilized lysate are used, add solutions directly to the vial or ampul. Incubate the reaction mixture for a constant period according to the directions of the lysate manufacturer (usually at 37 ± 1 for 60 ± 2 minutes), avoiding vibration. To test the integrity of the gel, take each tube in turn directly from the incubator and invert it through about 180 in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed. The test is considered valid when the lowest concentration of the standard solutions shows a negative result in all replicate tests.
The endpoint is the smallest concentration in the series of decreasing concentrations of standard endotoxin that clots the lysate. Determine the geometric mean endpoint by calculating the mean of the logarithms of the endpoint concentrations of the four replicate series and then taking the antilogarithm of the mean value, as indicated in the following formula:
Geometric Mean Endpoint Concentration = antilog (Se/f)
where Se is the sum of the log endpoint concentrations of the dilution series used, and f is the number of replicate test tubes. The geometric mean endpoint concentration is the measured sensitivity of the lysate (in EU/mL). If this is not less than 0.5 and not more than 2 , the labeled sensitivity is confirmed and is used in tests performed with this lysate.
Test for Interfering Factors— Usually prepare solutions (A–D) as shown in Table 1, and perform the inhibition/enhancement test on the Sample Solutions at a dilution less than the MVD, not containing any detectable endotoxins, operating as described for Test for Confirmation of Labeled Lysate Sensitivity. The geometric mean endpoint concentrations of Solutions B and C are determined using the formula described in the Test for Confirmation of Labeled Lysate Sensitivity.
Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques
Solution
Endotoxin Concentration/
Solution to which Endotoxin
is Added
Diluent
Dilution
Factor
Endotoxin
Concentration
Number of
Replicates
Aa
None/Sample Solution
4
Bb
2 /Sample Solution
Sample Solution
1
2
4
2
1
4
4
0.5
4
8
0.25
4
Cc
2 /Water for BET
Water for BET
1
2
2
2
1
2
4
0.5
2
8
0.25
2
Dd
None/Water for BET
2
a  Solution A: A Sample Solution of the preparation under test that is free of detectable endotoxins.
b  Solution B: Test for interference.
c  Solution C: Control for labeled lysate sensitivity.
d  Solution D: Negative control of Water for BET
The test is considered valid when all replicates of Solutions A and D show no reaction and the result of Solution C confirms the labeled sensitivity.
If the sensitivity of the lysate determined in the presence of Solution B is not less than 0.5 and not greater than 2 , the Sample Solution does not contain factors that interfere under the experimental conditions used. Otherwise, the Sample Solution to be examined interferes with the test.
If the sample under test does not comply with the test at a dilution less than the MVD, repeat the test using a greater dilution, not exceeding the MVD. The use of a more sensitive lysate permits a greater dilution of the sample to be examined and this may contribute to the elimination of interference.
Interference may be overcome by suitable treatment, such as filtration, neutralization, dialysis, or heating. To establish that the chosen treatment effectively eliminates interference without loss of endotoxins, perform the assay described above using the preparation to be examined to which USP Endotoxin RS has been added and which has then been submitted to the chosen treatment.
Limit Test
Procedure— Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on these solutions following the procedure for Test for Confirmation of Labeled Lysate Sensitivity under Preparatory Testing.
Table 2. Preparation of Solutions for the Gel-Clot Limit Test
Solution*
Endotoxin Concentration/Solution to which Endotoxin is Added
Number of Replicates
A
None/Diluted Sample Solution
2
B
2 /Diluted Sample Solution
2
C
2 /Water for BET
2
D
None/Water for BET
2
*  Prepare Solution A and the positive product control Solution B using a dilution not greater than the MVD and treatments as for the Test for Interfering Factors under Preparatory Testing. The positive control Solutions B and C contain the Standard Endotoxin Solution at a concentration corresponding to twice the labeled lysate sensitivity. The negative control Solution D consists of Water for BET.
Interpretation— The test is considered valid when both replicates of Solution B and C are positive and those of Solution D are negative. When a negative result is found for both replicates of Solution A, the preparation under test complies with the test. When a positive result is found for both replicates of Solution A, the preparation under test does not comply with the test.
When a positive result is found for one replicate of Solution A and a negative result is found for the other, repeat the test. In the repeat test, the preparation under test complies with the test if a negative result is found for both replicates of Solution A. The preparation does not comply with the test if a positive result is found for one or both replicates of Solution A. However, if the preparation does not comply with the test at a dilution less than the MVD, the test may be repeated using a greater dilution, not exceeding the MVD.
Quantitative Test
Procedure— The test quantifies bacterial endotoxins in Sample Solutions by titration to an endpoint. Prepare Solutions A, B, C, and D as shown in Table 3, and test these solutions by following the procedure in the Test for Confirmation of Labeled Lysate Sensitivity under Preparatory Testing.
Table 3. Preparation of Solutions for the Gel-Clot Assay
Solution
Endotoxin Concentration/
Solution to which Endotoxin
is Added
Diluent
Dilution Factor
Endotoxin
Concentration
Number of
Replicates
Aa
None/Sample Solution
Water for BET
1
2
2
2
4
2
8
2
Bb
2 /Sample Solution
1
2
2
Cc
2 /Water for BET
Water for BET
1
2
2
2
1
2
4
0.5
2
8
0.25
2
Dd
None/Water for BET
2
a  Solution A: Sample Solution under test at the dilution, not to exceed the MVD, with which the Test for Interfering Factors was completed. Subsequent dilution of the Sample Solution must not exceed the MVD. Use Water for BET to make a dilution series of four tubes containing the Sample Solution under test at concentrations of 1, ½, ¼, and 1/8 relative to the concentration used in the Test for Interfering Factors. Other dilutions up to the MVD may be used as appropriate.
b  Solution B: Solution A containing standard endotoxin at a concentration of 2 (positive product control).
c  Solution C: Two replicates of four tubes of Water for BET containing the standard endotoxin at a concentration of 2 , , 0.5 , and 0.25 , respectively.
d  Solution D: Water for BET (negative control).
Calculation and Interpretation— The test is considered valid when the following three conditions are met: (1) Both replicates of negative control Solution D are negative; (2) Both replicates of positive product control Solution B are positive; and (3) The geometric mean endpoint concentration of Solution C is in the range of 0.5 to 2 .
To determine the endotoxin concentration of Solution A, calculate the endpoint concentration for each replicate by multiplying each endpoint dilution factor by . The endotoxin concentration in the Sample Solution is the endpoint concentration of the replicates. If the test is conducted with a diluted Sample Solution, calculate the concentration of endotoxin in the original Sample Solution by multiplying by the dilution factor. If none of the dilutions of the Sample Solution is positive in a valid assay, report the endotoxin concentration as less than (if the diluted sample was tested, report as less than times the lowest dilution factor of the sample.) If all dilutions are positive, the endotoxin concentration is reported as equal to or greater than the greatest dilution factor multiplied by (e.g., initial dilution factor times 8 times in Table 3).
The preparation under test meets the requirements of the test if the concentration of endotoxin in both replicates is less than that specified in the individual monograph.
PREPARATION OF SOLUTIONS
Standard Endotoxin Stock Solution— A Standard Endotoxin Stock Solution is prepared from a USP Endotoxin Reference Standard that has been calibrated to the current WHO International Standard for Endotoxin. Follow the specifications in the package leaflet and on the label for preparation and storage of the Standard Endotoxin Stock Solution. Endotoxin is expressed in Endotoxin Units (EU). [Note—One USP Endotoxin Unit (EU) is equal to one International Unit (IU) of endotoxin. ]
Standard Endotoxin Solutions—After mixing the Standard Endotoxin Stock Solution vigorously, prepare appropriate serial dilutions of Standard Endotoxin Solution, using Water for BET. Use dilutions as soon as possible to avoid loss of activity by adsorption.
Sample Solutions—Prepare the Sample Solutions by dissolving or diluting drugs, or taking washes from medical devices using Water for BET. Some substances or preparations may be more appropriately dissolved, diluted, or extracted in other aqueous solutions. If necessary, adjust the pH of the solution to be examined (or dilution thereof) so that the pH of the mixture of the lysate and Sample Solution falls within the pH range specified by the lysate manufacturer, usually 6.0 to 8.0. The pH may be adjusted by use of an acid, base, or suitable buffer as recommended by the lysate manufacturer. Acids and bases may be prepared from concentrates or solids with Water for BET in containers free of detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and interfering factors.

REAGENTS AND TEST SOLUTIONS
Amoebocyte Lysate— A lyophilized product obtained from the lysate of amoebocytes (white blood cells) from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). This reagent refers only to a product manufactured in accordance with the regulations of the competent authority. [Note—Amoebocyte Lysate reacts to some -glucans in addition to endotoxins. Amoebocyte Lysate preparations that do not react to glucans are available: they are prepared by removing the G factor reacting to glucans from Amoebocyte Lysate or by inhibiting the G factor reacting system of Amoebocyte Lysate and may be used for endotoxin testing in the presence of glucans. ]
Water for Bacterial Endotoxins Test (BET)—Use Water for Injection or water produced by other procedures that shows no reaction with the lysate employed, at the detection limit of the reagent.
Lysate TS—Dissolve Amoebocyte Lysate in Water for BET, or in a buffer recommended by the lysate manufacturer, by gentle stirring. Store the reconstituted lysate, refrigerated or frozen, according to the specifications of the manufacturer.