(c) Procedure for assay. For the standard response line, use a total of 12 plates-three plates for each response line solution, except the reference concentration solution which is included on each plate. On each set of three plates, fill three alternate cylinders with the reference concentration solution and the other three cylinders with the concentration of the response line under test. Thus, there will be 36 reference concentration zones of inhibition and nine zones of inhibition for each of the four other concentrations of the response line. For each sample tested use three plates. Fill three alternate cylinders on each plate with the standard reference concentration solution and the other three cylinders with the sample reference concentration solution. After all the plates have incubated for 16 to 18 hours at the appropriate incubation temperature for each antibiotic listed in the table in paragraph (b) of this section, measure the diameters of the zones of inhibition using an appropriate measuring device such as a millimeter rule, calipers, or an optical projector.

(d) Estimation of potency. To prepare the standard response line, average the diameters of the standard reference concentration and average the diameters of the standard response line concentration tested for each set of three plates. Average also all 36 diameters of the reference concentration for all four sets of plates. The average of the 36 diameters of the reference concentration is the correction point of the response line. Correct the average diameter obtained for each concentration to the figure it would be if the average reference concentration diameter for that set of three plates were the same as the correction point. Thus, if in correcting the highest concentration of the response line, the average of the 36 diameters of the reference concentration is 16.5 millimeters and the average of the reference concentration of the set of three plates (the set containing the highest concentration of the response line) is 16.3 millimeters, the correction is +0.2 millimeter. If the average reading of the highest concentration of the response line of these same three plates is 16.9 millimeters, the corrected diameter is

L Calculated zone diameter for the lowest concentration of the standard response line;

H=Calculated zone diameter for the highest concentration of the standard response line;

c=Average zone diameter of 36 readings of the reference point standard solution;

a, b, d, e=Corrected average values for the other standard solutions, lowest to highest concentration, respectively.

To estimate the potency of the sample, average the zone diameters of the standard and the zone diameters of the sample on the three plates used. If the average zone diameter of the sample is larger than that of the standard, add the difference between them to the reference concentration diameter of the standard response line. If the average zone diameter of the sample is lower than that of the standard, subtract the difference between them from the reference concentration diameter of the standard response line. From the response line, read the concentrations corresponding to these corrected values of zone diameters. Multiply the concentration by the appropriate dilution factor to obtain the antibiotic content of the sample.

[39 FR 18944, May 30, 1974]

and storage time for the standard solutions and proceed as follows: If necessary, dry the working standard as described in § 436.200; dissolve and dilute an accurately weighed portion to the proper concentration for the working standard stock solution. Store the working standard stock solution under refrigeration and do not use longer than the recommended storage time. Prepare the proper concentrations for the standard response line solutions by further diluting an aliquot of the working standard stock solution. The reference concentration of the assay is the mid concentration of the standard response line.

(b) Procedure for assay. For each antibiotic listed in the table in this paragraph, select the test organism (as listed in § 436.103(a)), nutrient broth (as listed by medium number in § 436.102(b)), and suggested inoculum and proceed as follows: Place 1.0 milliliter (or 0.1 milliliter in the case of gramicidin and tyrothricin) of each concentration of the standard response line (prepare as described in paragraph (a) of this section) and of the sample solution in each set of three replicate tubes (as described in § 436.100(b)(1)). Fifteen tubes are used for the five-point standard response line and three for each sample. To each tube add 9 milliliters of the inoculated broth and place immediately in a water bath at the appropriate

temperature for 2 to 4 hours. The exact length of the incubation period should be determined by observation of growth in the reference concentration tube of the standard. Remove the tubes from the water bath and add 0.5 milliliter of a 12-percent formaldehyde solution to each tube. Determine the absorbance value of each tube in a suitable photoelectric colorimeter, at a wavelength of 530 millimicrons. Set the instrument at zero absorbance with an uninoculated blank composed of the same amounts of nutrient broth and formaldehyde used in the assay.

NOTE: The amount of working standard and sample solutions may be reduced as long as all other solutions used are reduced proportionately.

L=Calculated absorbance value for the lowest concentration of the standard response line. H=Calculated absorbance value for the highest concentration of the standard response line.

a, b, c, d, e=Average absorbance values for each concentration of the standard response line, lowest to the highest, respectively.

(c) Estimation of potency. To prepare the standard response line, plot the average absorbance values for each concentration of the standard response line on one-cycle semilogarithmic graph paper with the absorbance values on the arithmetric scale and concentrations on the logarithmic scale. The response line is drawn either through these points by inspection or through points plotted for highest and lowest absorbance values obtained by means of the following equations.

To estimate the potency of the sample, average the absorbance values for the sample and determine the antibiotic concentration from the standard response line. Multiply the concentration by the appropriate dilution factor to obtain the antibiotic content of the sample.

[39 FR 18944, May 30, 1974, as amended at 40 FR 57797, Dec. 12, 1975; 41 FR 49483, Nov. 9, 1976; 44 FR 22057, Apr. 13, 1979; 46 FR 3835, 3839, Jan. 16, 1981; 46 FR 16682, Mar. 13, 1981; 46 FR 33512, June 30, 1981; 46 FR 61072, Dec. 15, 1981; 47 FR 22515, May 25, 1982; 47 FR 23708, June 1, 1982; 48 FR 3960, Jan. 28, 1983; 53 FR 32607, Aug. 26, 1988]

Subpart E-General Chemical Tests for Antibiotics

§ 436.200 Loss on drying.

Use the method specified in the individual section for each antibiotic.

(a) Method 1. In an atmosphere of about 10 percent relative humidity,

grind the sample, if necessary, to obtain a fine powder. When tablets, troches, or capsules are to be tested, use four tablets, troches, or capsules in preparing the sample. Transfer about 100 milligrams of the sample to a tared weighing bottle equipped with a ground-glass stopper. Weigh the bottle and place it in a vacuum oven, tilting the stopper on its side so that there is no closure during the drying period. Dry at a temperature of 60° C. and a pressure of 5 millimeters of mercury or less for 3 hours. At the end of the drying period, fill the vacuum oven with air dried by passing it through a drying agent such as sulfuric acid or silica gel. Replace the stopper and place the weighing bottle in a desiccator over a desiccating agent, such as phosphorous pentoxide or silica gel, allow to cool to room temperature, and reweigh. Calculate the percent of loss.

(b) Method 2. Proceed as directed in paragraph (a) of this section, except use a tared weighing bottle or weighing tube equipped with a capillarytube stopper, the capillary having an inside diameter of 0.20-0.25 millimeter, and place it in a vacuum oven without removing the stopper.

(c) Method 3. Proceed as directed in paragraph (a) of this section, except dry the sample at a temperature of 110° C. and a pressure of 5 millimeters of mercury or less for 3 hours.

(d) Method 4. Proceed as directed in paragraph (a) of this section, except dry the sample at a temperature of 40° C. and a pressure of 5 millimeters of mercury or less for 2 hours.

(e) Method 5. Proceed as directed in paragraph (a) of this section, except dry the sample at a temperature of 100° C. and a pressure of 5 millimeters of mercury or less for 4 hours.

(f) Method 6. Proceed as directed in paragraph (a) of this section, except dry the sample at a temperature of 40° C. and a pressure of 5 millimeters of mercury or less for 3 hours.

(g) Method 7. Proceed as directed in paragraph (a) of this section, except dry the sample at a temperature of 25° C. and a pressure of 5 millimeters of mercury or less for 4 hours.

(h) Method 8. Proceed as directed in paragraph (a) of this section, except transfer approximately 300 milligrams