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1 Determine the amount of the inoculum by the use of test plates. 2 Use dilution of the suspension that gives 25 percent light transmission in lieu of the stock suspension.

(b) Preparation of working standard stock solutions and standard response line solutions. For each antibiotic listed in the table in this paragraph, select the working standard drying conditions, solvent(s), concentrations, and storage time for the standard solutions and proceed as follows: If necessary, dry the working standard as described in 8 436.200; dissolve and dilute an accurately weighed portion to the proper concentration to prepare the working

standard stock solution. Store the working standard stock solution under refrigeration and do not use longer than the recommended storage time. Further dilute an aliquot of the working standard stock solution to the proper concentrations to prepare the standard response line solutions. The reference concentration of the assay is the mid concentration of the response line.

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'Further dilute aliquots of the working standard stock solution with dimethylsulfoxide to give concentrations of 12.8, 16, 20.0, 25, and 31.2 micrograms per milliliter.
2 Further dilute aliquots of the working standard stock solution with dimethylformamide to give concentrations of 256, 320, 400, 500, and 624 units per milliliter.
3 Add 2 milliliters of distilled water for each 5 milligrams of weighed working standard material.
* Further dilute aliquots of the working standard stock solution with dimethylformamide to give concentrations of 84, 80, 100, 125, and 156 micrograms per milliliter.
6 The final concentration of the working standard stock solution is allowed to hydrolyze in a 37° C. constant temperature water bath for 60 minutes.
6 Working standard should be stored below minus 20° C under an atmosphere of nitrogen. Sisomicin is hygroscopic and care should be exercised during weighing.
Further dilute aliquots of the working standard stock solution with dimethylsulfoxide to give concentrations 64.0, 80.0, 100, 125, and 156 micrograms per milliliter.

& Weigh a separate portion of the working standard and determine the loss on drying by the method described in $436.200(c) of this chapter. Use this value to determine the anhydrous
content of the working standard.

Working standard should be stored below minus 10o C under an atmosphere of nitrogen. Netilmicin sulfate is hygroscopic and care should be exercised during weighing.
10 For assay of nystatin pastilles, use 80 percent aqueous dimethylformamide as the initial solvent and as diluent for all dilutions where dimethylformamide is required.

corrected diameters, including the average of the 36 diameters of the reference concentration on 2-cycle semilog paper, using the concentration of the antibotic in micrograms or units per milliliter as the ordinate (the logarithmic scale), and the diameter of the zone of inhibition as the abscissa. The response line is drawn either through these points by inspection or through points plotted for highest and lowest zone diameters obtained by means of the following equation:

3a + 2b +c-e L=

5

3e +20 +c-a H =

5

where: L=Calculated zone diameter for the lowest

concentration of the standard response

(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 then 17.1 millimeters. Plot these

H=Calculated zone diameter for the high

est concentration of the standard re

sponse 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 dilutior factor to obtain the antibiotic content of the sample.

(39 FR 18944, May 30, 1974)

EDITORIAL NOTE: For FEDERAL REGISTER CItations affecting $ 436.105, see the List of CFR Sections Affected appearing in the Finding Aids section of this volume.

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