§ 436.35 21 CFR Ch. I (4–1–98 Edition)
(f) Method 6. Proceed as directed in Con- Volume paragraph (a) of this section, except di- Dilu- centra- of test Antibiotic 1 tion of solution lute sample with 0.05N sodium hydrox- ent test so- to be in- ide (diluent 9). lution 2 jected 3 (g) Method 7. Proceed as directed in Tetracycline hydrochloride ..... 4 5.0 .6 paragraph (a) of this section, except di- Tetracycline phosphate 5 ...... 3 5.0 .6 lute sample with sodium carbonate so- Vidarabine monohydrate 5 ...... 4 1.0 1.0 lution (diluent 13). 1 Diluent number as listed in sec. 436.31(b). (h) Method 8. Proceed as directed in 2 Milligrams of activity per milliliter. paragraph (a) of this section, except in- 3 Milliliters per kilogram of body weight. 4 The concentration of the test solution is expressed in units ject a test dose of 2.0 milliliters of the per milliliter in lieu of milligrams of activity per milliliter. diluted sample per kilogram of rabbit 5 To prepare the test solution, proceed as directed in the in- dividual section of the antibiotic drug regulation in this chapter weight. for the antibiotic to be tested. (i) Method 9. Proceed as directed in paragraph (a) of this section, except di- [46 FR 60568, Dec. 11, 1981, as amended at 46 lute sample with pyrogen-free sodium FR 61071, Dec. 15, 1981; 49 FR 5096, Feb. 10, carbonate solution (diluent 15). 1984] (j) Method 10. Proceed as directed in paragraph (a) of this section, except di- Subpart D—Microbiological Assay lute the sample with sodium carbonate Methods solution (diluent 16). [39 FR 18944, May 30, 1974, as amended at 40 § 436.100 Laboratory equipment. FR 51625, Nov. 6, 1975; 45 FR 22921, Apr. 4, Equipment should be selected which 1980; 50 FR 48397, Nov. 25, 1985; 53 FR 13401, Apr. 25, 1988] is adequate for its intended use and should be thoroughly cleansed after § 436.35 Depressor substances test. each use to remove any antibiotic resi- dues. The equipment should be kept Proceed as directed in the USP XX covered when not in use. Clean glass- depressor substances test. Prepare the ware intended for holding and transfer- sample test solution as follows: For ring the test organisms should be steri- each antibiotic listed in the table lized in a hot air oven at 200–220° C. for below, select the appropriate diluent 2 hours. Volumetric flasks, pipettes, or and test dose (concentration and vol- accurately calibrated diluting devices ume). If the product is packaged for should be used when diluting standard dispensing and is in a combination and sample solutions. package with a container of diluent, di- lute the product as directed in the la- (a) Microbiological agar diffusion beling. assay—(1) Cylinders. Use stainless steel cylinders with an outside diameter of 8 Con- Volume millimeters (±0.1 millimeter), an inside Dilu- centra- of test diameter of 6 millimeters (±0.1 millime- Antibiotic 1 tion of solution ent test so- to be in- ter), and a length of 10 millimeters lution 2 jected 3 (±0.1 millimeter). Bleomycin sulfate ...... 4 4 0.5 1.0 (2) Plates. Plastic or glass Petri Capreomycin sulfate ...... 4 3.0 1.0 dishes may be used, having dimensions Chlortetracycline hydro- chloride ...... 3 5.0 .6 of 20 by 100 millimeters. Covers should Clindamycin phosphate ...... 4 5.0 1.0 be of suitable material. Daunorubicin hydrochloride ... 4 1.5 1.0 (b) Microbiological turbidimetric Dihydrostreptomycin sulfate ... 4 3.0 1.0 Doxorubicin hydrochloride ...... 4 1.5 1.0 assay—(1) Tubes. Tubes which give sat- Doxycycline hyclate ...... 4 5.0 1.0 isfactory results and have uniform Lincomycin hydrochloride length and diameter should be used. If monohydrate ...... 4 3.0 1.0 Minocycline hydrochloride ...... 4 5.0 .6 reusable tubes are employed, care must Plicamycin ...... 3 0.050 1.0 be taken to remove not only all anti- Mitomycin ...... 4 0.050 1.0 biotic residues from the previous test Oxytetracycline 5 ...... 3 5.0 .6 Oxytetracycline hydrochloride 4 5.0 .6 but also all traces of cleaning solution. Rolitetracycline ...... 4 5.0 .6 (2) Colorimeter. Use a suitable photo- Rolitetracycline nitrate ...... 4 5.0 .6 electric colorimeter at a wavelength of Sodium colistimethate ...... 4 3.0 1.6 Spectinomycin hydrochloride 4 15.0 1.0 530 millimicrons. Set the instrument at Streptomycin sulfate ...... 4 3.0 1.0 zero absorbance with clear,
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uninoculated broth prepared as de- Monobasic potassium phosphate: 80.0 gm. scribed in the applicable method for Distilled water, q.s: 1,000.0 ml. the antibiotic being assayed. Adjust with 18N phosphoric acid or [39 FR 18944, May 30, 1974, as amended at 41 10N potassium hydroxide to yield a pH FR 34743, Aug. 17, 1976] 5.95 to 6.05 after sterilization. (7)–(9) [Reserved] § 436.101 Solutions. (10) Solution 10 (0.2M potassium phos- (a) Antibiotic assay solutions are phate buffer, pH 10.5). prepared as follows (solution numbers Dibasic potassium phosphate: 35.0 gm. 1, 2, 3, 4, and 6 correspond to those used 10 N potassium hydroxide: 2.0 ml. in ‘‘Assay Methods of Antibiotics,’’ D. Distilled water, q.s: 1,000.0 ml. C. Grove and W. A. Randall, Medical Adjust with 18N phosphoric acid or Encyclopedia, Inc., New York, N.Y. 10N potassium hydroxide to yield a pH (1955), p. 222), which is incorporated by 10.4 to 10.6 after sterilization. reference. Copies are available from (11) Solution 11 (10 percent potassium the Medical Encyclopedia Inc., 30 East phosphate buffer, pH 2.5). 60th St., New York, NY 11220, or avail- able for inspection at the Office of the Monobasic potassium phosphate: 100.0 gm. Federal Register, 800 North Capitol Concentrated hydrochloric acid: 0.2 ml. (ap- Street, NW., suite 700, Washington, DC. proximately). Distilled water, q.s: 1,000.0 ml. (1) Solution 1 (1 percent potassium phosphate buffer, pH 6.0). Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH Dibasic potassium phosphate: 2.0 gm. Monobasic potassium phosphate: 8.0 gm. 2.0 to 2.8 after sterilization. Distilled water, q.s: 1,000.0 ml. (12) Solution 12 (10 percent potassium phosphate buffer, pH 7.0). Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH Monobasic potassium phosphate: 100.0 gm. 5.95 to 6.05 after sterilization. Distilled water, q.s: 1,000.0 ml. (2) Solution 2 (citrate buffer solution pH Adjust with 18N phosphoric acid or 6.3). 10N potassium hydroxide to yield a pH Citric acid: 13.2 gm. 6.95 to 7.05 after sterilization. Sodium hydroxide: 7.06 gm. (13) Solution 13 (0.01N methanolic hy- Sodium citrate: 97.0 gm. drochloric acid). Distilled water, q.s: 1,000.0 ml. 1.0N hydrochloric acid: 10.0 ml. Adjust with 10 percent citric acid so- Methyl alcohol, q.s: 1,000.0 ml. lution or 10N sodium hydroxide to yield (14) Solution 14 (2 percent sodium bicar- pH 6.2 to 6.4 after sterilization. bonate solution). (3) Solution 3 (0.1M potassium phos- phate buffer, pH 8.0). Sodium bicarbonate: 20.0 gm. Distilled water, q.s: 1,000.0 ml. Dibasic potassium phosphate: 16.73 gm. Monobasic potassium phosphate: 0.523 gm. Prepare daily. Distilled water, q.s: 1,000.0 ml. (15) Solution 15 (80 percent isopropyl al- Adjust with 18N phosphoric acid or cohol solution). 10N potassium hydroxide to yield a pH Isopropyl alcohol: 800.0 ml. 7.9 to 8.1 after sterilization. Distilled water, q.s: 1,000.0 ml. (4) ( Solution 4 0.1M potassium phosphate (16) Solution 16 (0.1 M potassium phos- ). buffer, pH 4.5 phate buffer, pH 7.0). Monobasic potassium phosphate: 13.6 gm. Dibasic potassium phosphate: 13.6 gm. Distilled water, q.s: 1,000.0 ml. Monobasic potassium phosphate: 4.0 gm. Adjust with 18N phosphoric acid or Distilled water, q.s.: 1,000.0 ml. 10N potassium hydroxide to yield a pH Adjust with 18 N phosphoric acid or 4.45 to 4.55 after sterilization. 10 N potassium hydroxide to yield a pH (5) [Reserved] 6.8 to 7.2 after sterilization. (6) Solution 6 (10 percent potassium (17) Solution 17 (5 percent methyl alco- phosphate buffer, pH 6.0). hol in 1 percent potassium phosphate Dibasic potassium phosphate: 20.0 gm. buffer, pH 6.0).
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Methyl alcohol: 50.0 ml. Dextrose: 1.0 gm. 1 percent potassium phosphate buffer, pH 6.0, Agar: 15.0 gm. q.s.: 1,000.0 ml. Distilled water, q.s: 1,000.0 ml. pH 6.5 to 6.6 after sterilization. (18) Solution 18 (0.054M sodium phos- phate buffer, pH 6.9). (2) Medium 2. Sodium dihydrogen phosphate Peptone: 6.0 gm. monohydrate: 3.97 gm. Yeast extract: 3.0 gm. Disodium hydrogen phosphate anhydrous: Beef extract: 1.5 gm. 3.55 gm. Agar: 15.0 gm. Distilled water, q.s.: 1,000.0 mL. Distilled water, q.s: 1,000.0 ml. pH 6.5 to 6.6 after sterilization. [39 FR 18944, May 30, 1974, as amended at 40 FR 52004, Nov. 7, 1975; 45 FR 75194, Nov. 14, (3) Medium 3. 1980; 47 FR 9396, Mar. 5, 1982] Peptone: 5.0 gm. § 436.102 Culture media. Yeast extract: 1.5 gm. Beef extract: 1.5 gm. (a) Ingredients. Use ingredients that Sodium chloride: 3.5 gm. conform to the standards, if any, pre- Dextrose: 1.0 gm. scribed by the U.S.P. or N.F. In lieu of Dipotassium phosphate: 3.68 gm. preparing the media from the individ- Potassium dihydrogen phosphate: 1.32 gm. ual ingredients specified, they may be Distilled water, q.s: 1,000.0 ml. made from dehydrated mixtures that, pH 6.95 to 7.05 after sterilization. when reconstituted with distilled (4) Medium 4. water, have the same composition as Peptone: 6.0 gm. such media. Minor modifications of the Yeast extract: 3.0 gm. individual ingredients specified in this Beef extract: 1.5 gm. section are permissible if the resulting Dextrose: 1.0 gm. media possess growth-promoting prop- Agar: 15.0 gm. erties at least equal to the media de- Distilled water, q.s: 1,000.0 ml. scribed. pH 6.5 to 6.6 after sterilization. (b) Description of media. Medium num- (5) Medium 5. Medium 5 is the same as bers 1, 2, 3, 4, 5, 8, 9, 10, 11, and 13 cor- medium 2, except adjust the final pH to respond to those used in ‘‘Assay Meth- 7.8 to 8.0 after sterilization. ods of Antibiotics,’’ D. C. Grove and W. (6)–(7) [Reserved] A. Randall, Medical Encyclopedia, Inc., (8) Medium 8. Medium 8 is the same as New York, N.Y. (1955) p. 220, which is medium 2, except adjust the final pH to incorporated by reference. Copies are 5.8 to 6.0 after sterilization. available from Medical Encyclopedia (9) Medium 9. Inc., 30 East 60th St., New York, NY, or available for inspection at the Office of Pancreatic digest of casein: 17.0 gm. Papaic digest of soybean: 3.0 gm. the Federal Register, 800 North Capitol Sodium chloride: 5.0 gm. Street, NW., suite 700, Washington, DC. Dipotassium phosphate: 2.5 gm. Medium numbers 18 through 21 cor- Dextrose: 2.5 gm. respond to those used in ‘‘Outline of Agar: 20.0 gm. Details for Official Microbiological As- Distilled water, q.s: 1,000.0 ml. says of Antibiotics,’’ A. Kirshbaum and pH 7.2 to 7.3 after sterilization. B. Arret, ‘‘Journal of Pharmaceutical (10) Medium 10. Medium 10 is the same Sciences,’’ vol. 56, No. 4, April 1967, p. as medium 9, except: 512, which is incorporated by reference. Copies are available from the American Agar: 12.0 gm. Polysorbate 80 (add polysorbate 80 after boil- Pharmaceutical Association, 2215 Con- ing the medium to dissolve the agar): 10.0 stitution Ave. NW., Washington, DC ml. 20037, or available for inspection at the pH 7.2 to 7.3 after sterilization. Office of the Federal Register (see ad- dress in this paragraph). (11) Medium 11. Medium 11 is the same (1) Medium 1. as medium 1, except adjust the final pH to 7.8 to 8.0 after sterilization. Peptone: 6.0 gm. (12) [Reserved] Pancreatic digest of casein: 4.0 gm. (13) Medium 13. Yeast extract: 3.0 gm. Beef extract: 1.5 gm. Peptone: 10.0 gm.
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Dextrose: 20.0 gm. Pancreatic digest of casein: 17.0 gm. Distilled water, q.s: 1,000.0 ml. Soybean peptone: 3.0 gm. pH 5.6 to 5.7 after sterilization. Dextrose: 2.5 gm. (14)–(18) [Reserved] Sodium chloride: 5.0 gm. (19) Medium 19. Dipotassium phosphate: 2.5 gm. Distilled water, q.s.: 1,000.0 ml. Peptone: 9.4 gm. pH 7.3 after sterilization. Yeast extract: 4.7 gm. Beef extract: 2.4 gm. (38) Medium 38. Sodium chloride: 10.0 gm. Dextrose: 10.0 gm. Peptone: 15.0 gm. Agar: 23.5 gm. Papaic digest of soybean meal: 5.0 gm. Distilled water, q.s: 1,000.0 ml. Sodium chloride: 4.0 gm. pH 6.0 to 6.2 after sterilization. Sodium sulfite: 0.2 gm. (20)–(31) [Reserved] L-cystine: 0.7 gm. (32) Medium 32. Prepare as medium 1, Dextrose: 5.5 gm. except add 300 milligrams of hydrated Agar: 15.0 gm. Distilled water, q.s.: 1,000.0 ml. manganese sulfate (MnSO4·H2O) to each liter of medium. pH 7.0 after sterilization. (33) Medium 33. Use medium 1, steri- [39 FR 18944, May 30, 1974, as amended at 40 lized and cooled to 50° C. Aseptically FR 52004, Nov. 7, 1975; 42 FR 14092, Mar. 15, add sufficient sterile sodium 1977; 47 FR 9396, Mar. 5, 1982; 47 FR 22514, May novobiocin solution to give a final con- 25, 1982] centration of 10 micrograms of novobiocin activity per milliliter of § 436.103 Test organisms. medium. Sterile sodium novobiocin so- (a) Preparation of test organism suspen- lution is prepared by filtering a solu- sions. For each test organism listed in tion containing 2.5 milligrams of the following table, select the media novobiocin per milliliter of distilled (as listed by medium number in water through a membrane filter of § 436.102(b)), incubation period of the 0.22–micron porosity. Roux bottle, suggested dilution factor, (34) Medium 34. and suggested storage period for the particular test organism and proceed Glycerol: 10.0 gm. Peptone: 10.0 gm. by the appropriate method described in Beef extract: 10.0 gm. paragraph (b) of this section. Test or- Sodium chloride: 3.0 gm. ganism letters A through K, M, and N Distilled water, q.s.: 1,000.0 ml. correspond to those used in ‘‘Outline of pH 7.0 after sterilization. Details for Official Microbiological As- (35) Medium 35. Same as medium 34, says of Antibiotics,’’ A. Kirshbaum and except add 17.0 grams of agar to each B. Arret, ‘‘Journal of Pharmaceutical liter of medium. Sciences,’’ Vol. 56, No. 4, p. 512 (April (36) Medium 36. 1967), which is incorporated by ref- Pancreatic digest of casein ...... 15.0 gm. erence. Copies are available from the Papaic digest of soybean ...... 5.0 gm. American Pharmaceutical Association, Sodium chloride ...... 5.0 gm. 2215 Constitution Ave. NW., Washing- Agar ...... 15.0 gm. Distilled water, q.s ...... 1,000.0 ml. ton, DC 20037, or available for inspec- pH 7.3 after sterilization ...... tion at the Office of the Federal Reg- (37) Medium 37. ister, 800 North Capitol Street, NW., suite 700, Washington, DC.
Medium used for theÐ Suggested Incubation Suggested storage period Test organisms Method period of dilution of suspensions used Slants Roux bot- Roux bot- factor under refrigera- tles tle tion
Test organism AÐStaphylococcus aureus 1 1 1 24 hours 1:20 1 week. (ATCC 6538P) 2. Test organism BÐMicrococcus luteus 1 1 1 24 hours 1:30 2 weeks. (ATCC 7468) 2. Test organism CÐMicrococcus luteus 1 1 1 24 hours 1:40 2 weeks. (ATCC 9341) 2. Test organism DÐStaphyloccocus 1 1 1 24 hours 1 1:14 1 week. epidermidis (ATCC 12228) 2.
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Medium used for theÐ Suggested Incubation Suggested storage period Test organisms Method period of dilution of suspensions used Slants Roux bot- Roux bot- factor under refrigera- tles tle tion
Test organism EÐSaccharomyces 6 19 ...... 1:30 4 weeks. cerevisiae (ATCC 9763) 2. or 7 19 19 48 hours 1:30 4 weeks. Test organism FÐBordetella 1 1 1 24 hours 1:20 2 weeks. bronchiseptica (ATCC 4617) 2. Test organism GÐBacillus cereus var. 3 1 1 1 week ...... 6 months. mycoides (ATCC 11778) 2. Test organism HÐBacillus subtilis (ATCC 1 1 1 24 hours ...... 6 months. 6633) 2. or 2 1 32 5 days ...... 6 months. Test organism IÐKlebsiella pneumoniae 1 1 1 24 hours 1:25 1 week. (ATCC 10031) 2. Test organism JÐEscherichia coli (ATCC 1 1 1 24 hours 1:20 2 weeks. 10536). Test organism KÐStreptococcus faecium 5 ...... 24 hours. (ATCC 10541) 2. Test organism LÐMicrococcus luteus 1 1 1 24 hours 1:35 4 weeks. (ATCC 10240) 2. Test organism OÐStaphylococcus aureus, 1 33 33 24 hours 1:10 4 weeks. resistant to novobiocin (ATCC 12692) 2. Test organism TÐSaccharomyces 7 19 19 48 hours 1:30 4 weeks. cerevisiae (ATCC 2601) 2. Test organism VÐMicrococcusluteus, re- 1 1 1 48 hours 1:35 4 weeks. sistant to dihydrostreptomycin (ATCC 10240A) 2. Test organism WÐPseudomonas 1 1 1 24 hours 1:25 2 weeks. aeruginosa (ATCC 25619) 2. Test organism XÐMycobacterium 8 36 ...... 2 weeks. smegmatis (ATCC 607).. Test organism YÐPseudomonas 9 36 36 24 hours 1:50 1 week. aeruginosa (ATCC 29336) 2. 1 If the antibiotic to be tested is paromomycin, the dilution factor is 1:25. 2 Available from American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD. 20852.
(b) Methods for preparation of test or- mine the amount of suspension to be ganism suspensions—(1) Method 1—(i) added to each 100 milliliters of agar or Preparation of suspension. Maintain or- nutrient broth by the use of test plates ganisms on agar slants containing 10 or test broth. Store the test organism milliliters of the appropriate medium. suspension under refrigeration. Incubate the slants at 32° C.–35° C. for (2) Method 2. Proceed as directed in 24 hours. Using 3 milliliters of sterile paragraph (b)(1) of this section, except U.S.P. saline T.S., wash the growth in lieu of paragraph (b)(1)(ii) thereof, from the agar slant onto a large agar heat-shock and standardize the suspen- surface, such as a Roux bottle, contain- sion as follows: Centrifuge and decant ing 250 milliliters of the appropriate the supernatant liquid. Resuspend the medium. Spread the suspension of or- sediment with 50 to 70 milliliters of ganisms over the entire surface of the sterile U.S.P. saline T.S. and heat the Roux bottle with the aid of sterile suspension for 30 minutes at 70° C. Use glass beads. Incubate the Roux bottle test plates to assure the viability of at 32° C.–35° C. Wash the resulting the spores and to determine the growth from the agar surface with 50 amount of spore suspension to be added milliliters of sterile U.S.P. saline T.S. to each 100 milliliters of agar. Main- (ii) Standardization of suspension. De- tain the spore suspension under refrig- termine the dilution factor that will eration. give a 25–percent light transmission at (3) Method 3. Proceed as directed in a wavelength of 580 millimicrons using paragraph (b)(1) of this section, except a suitable photoelectric colorimeter in lieu of paragraph (b)(1)(ii) thereof, and a 13–millimeter diameter test tube heat-shock and standardize the suspen- as an absorption cell. It may be nec- sion as follows: Heat the suspension for essary to adjust the suspension. Deter-
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30 minutes at 70° C. Wash the spore sus- incubate the slant and Roux bottle at pension three times with 25 to 50 milli- 37° C and wash the resulting growth liters of sterile distilled water. Resus- from the agar surface with 50 milli- pend the organisms in 50 to 70 milli- liters of Medium 37 as described in liters of sterile distilled water and § 436.102(b)(37). heat-shock again for 30 minutes at 70° [39 FR 18944, May 30, 1974, as amended at 40 C. Use test plates to assure the viabil- FR 52004, Nov. 7, 1975; 42 FR 14092, Mar. 15, ity of the spores and to determine the 1977; 42 FR 18058, Apr. 5, 1977; 44 FR 10378, amount of spore suspension to be added Feb. 20, 1979; 47 FR 22514, May 25, 1982; 47 FR to each 100 milliliters of agar. Main- 27552, June 25, 1982] tain the spore suspension under refrig- eration. § 436.104 Penicillin activity. (4) [Reserved] Use penicillin-free equipment and (5) Method 5. Maintain the test orga- glassware. nisms in 100–milliliter quantities of nu- (a) Preparation of inoculated plates. trient broth—Medium 3 as described in Proceed as directed in § 436.105(a), using § 436.102(b)(3). For the test prepare a 10 milliliters of medium 1 for the base fresh subculture by transferring a layer. For the seed layer, use 4 milli- loopful of the stock culture to 100 mil- liters of medium 4, inoculated with the liliters of the same nutrient broth and amount of test organism C which gave incubate for 16 to 18 hours at 37° C. the clearest, sharpest zones of inhibi- Store this broth culture under refrig- tion measuring 17 to 21 millimeters in eration. diameter when standardized as de- (6) Method 6. Maintain the test orga- scribed in § 436.103(b)(1)(ii). Use the nisms on agar slants containing 10 mil- plates the same day they are prepared. liliters of the medium specified in (b) Preparation of working standard paragraph (a) of this section. Incubate stock solutions and standard response the slants at 32° C.–35° C. for 24 hours. lines solutions. Proceed as directed for Inoculate 100 milliliters of nutrient penicillin G in § 436.105(b), except dilute broth—Medium 13 as described in the working standard stock solution to § 436.102(b)(13). Incubate for 16 to 18 a final concentration of 100 units of hours at 37° C. Proceed as directed in penicillin G per milliliter and use the paragraph (b)(1)(ii) of this section. following final concentrations for the (7) Method 7. Proceed as directed in standard response line: 0.005, 0.0125, paragraph (b)(1) of this section, except 0.025, 0.050, 0.100, and 0.200 unit of peni- incubate the slants at 30° C. for 24 cillin G per milliliter. The 0.050 unit of hours and incubate the Roux bottle at penicillin G–per–milliliter solution is 30° C. for 48 hours. the reference concentration of the (8) Method 8. Maintain organisms on assay. agar slants containing 10 milliliters of (c) Sample preparation. Dissolve 1.0 the appropriate medium and transfer gram of the sample in sufficient dis- to a fresh slant about once a week. In- tilled water to make 18 milliliters. Fil- cubate the slants at 37° C for 48 hours. ter if not clear. Transfer 9.0 milliliters Using 3 milliliters of sterile U.S.P. sa- to a separatory funnel, and add 20 mil- line T.S., wash the growth from the liliters of amyl acetate. Add 1 milli- agar slant into a 500–milliliter Erlen- liter of 10 percent potassium phosphate meyer flask containing 100 milliliters buffer, pH 2.5 (solution 11 as described of medium 34, as described in in § 436.101), shake, allow to separate, § 436.102(b) (34), and 50 grams of glass and draw off the aqueous layer into a beads. Agitate the culture by rotation second separatory funnel. Check the pH at a speed of 130 cycles per minute and of the aqueous solution with pH paper, a radius of 3.5 centimeters at 27° C for and readjust with concentrated hydro- 5 days. Determine the amount of sus- chloric acid if the pH is three or above. pension to be added to each 100 milli- Extract again with 20 milliliters or liters of agar by the use of test plates. amyl acetate, discard the aqueous Store the test organism suspension phase, and combine the amyl acetate under refrigeration. extracts. Wash the extracts with 10 (9) Method 9. Proceed as directed in milliliters of 1 percent potassium phos- paragraph (b)(1) of this section, except phate buffer, pH 2.5, and discard the
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buffer wash. Extract the penicillin 45 readings of the 0.050 unit of penicil- from the amyl acetate with a 10–milli- lin G–per–milliliter concentration is liter aliquot of 1 percent potassium 18.5 millimeters and the average of the phosphate buffer, pH 6.0 (solution 1 as 0.050 unit of pencillin G–per milliliter described in § 436.101). This is the assay concentration of this set of three solution. plates is 18.3 millimeters, the correc- (d) Procedure for assay. For the stand- tion is +0.2 millimeters. If the average ard response line, use a total of 15 reading of the 0.025 unit of pencillin G– plates (three plates for each response per–milliliter concentration of these line solution, except the reference con- same three plates is 15.5 millimeters, centration solution, which is included the corrected value is 15.7 millimeters. on each plate). On each set of three Plot these corrected values, including plates, fill three alternate cylinders the average of the 0.050 unit of penicil- with the reference concentration solu- lin G-per-milliliter concentration, on tion and the other three cylinders with semilogarithmic graph paper using the the concentration of the response line pencillin concentration in units per under test. Thus, there will be 45 ref- milliliter on the logarithmic scale and erence concentration zones of inhibi- the diameter of the zone of inhibition tion and nine zones of inhibition for on the arithmetic scale. Draw the line each of the other concentrations of the of best fit through these points. To es- response line. Treat a portion of the timate the sample potency, average the sample solution (2 to 5 milliliters) with zone diameters of the standard and the 0.1 milliliter of penicillinase solution zone diameters of the sample on the and incubate at 37° C. for 1 hour. For three plates used. If the average zone each sample tested, use three plates. diameter of the sample is lower than On each plate fill two cylinders with that of the standard, subtract the dif- the 0.050 unit of penicillin G per milli- ference between them from the ref- liter standard, two cylinders with the erence concentration diameter of the untreated sample, and two cylinders standard response line. From the re- with the penicillinase-treated sample. sponse line, read the concentrations Incubate all plates, including those of corresponding to these corrected values the standard response line, overnight of zone diameters. Multiply the con- ° at 30 C. A zone of inhibition with the centration by the dilution factor to ob- untreated sample and no zone with the tain the units of penicillin G per sam- penicillinase-treated sample are a posi- ple size tested. tive test for penicillin. If a positive test is obtained, measure the diameters [39 FR 18944, May 30, 1974, as amended at 41 of the zones of inhibition using an ap- FR 34743, Apr. 17, 1976] propriate measuring device such as a millimeter rule, calipers, or an optical § 436.105 Microbiological agar diffu- sion assay. projector. (e) Estimation of penicillin G activity. Using the sample solution prepared To prepare the standard response line, as described in the section for the par- average the diameters of the standard ticular antibiotic to be tested, proceed reference concentration and average as described in paragraphs (a), (b), (c), the diameters of the standard response and (d) of this section. line concentration tested for each set (a) Preparation of inoculated plates. of three plates. Average also all 45 di- For each antibiotic listed in the table ameters of the reference concentration. in this paragraph, select the media (as The average of the 45 diameters of the listed by medium number in reference concentration is the correc- § 436.102(b)), the amount of media to be tion point of the response line. Correct used in the base and seed layers, the the average diameter obtained for each test organism (as listed in § 436.103(a)), concentration to the figure it would be and the suggested inoculum and pre- if the average reference concentration pare the inoculated plates as follows: diameter for that set of three plates Prepare the base layer by adding the were the same as the correction point. appropriate amount of melted agar to Thus, if in correcting the 0.025 penicil- each Petri dish (nominal dimensions 20 lin G concentration, the average of the by 100 millimeters). Distribute the agar
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evenly in each dish on a flat, level sur- flask to obtain a homogeneous suspen- face, placing a cover on each plate in sion, and add the appropriate amount turn; if a nonporous cover is used, of the inoculated media to each of the leave it slightly ajar to prevent accu- plates containing the uninoculated mulation of condensed moisture from base agar. Spread evenly over the agar the hot agar base layer. After the agar surface, cover, and allow to harden on hardens, seat the nonporous cover on a flat, level surface. After the agar has each plate. To prepare the seed layer, hardened, place 6 cylinders described in add the suggested inoculum of the test § 436.100(a)(1) on the inoculated agar organism suspension to a sufficient surface so that they are at approxi- amount of agar, which has been melted mately 60° intervals on a 2.8—centi- and cooled to 48° C–50° C. Swirl the meter radius.
Media to be used (as Milliliters of media to Suggested listed by medium be used in the base volume of number in and seed layers standard- § 436.102(b)) ized Incubation Antibiotic Test or- inoculum to Tempera- ganism be added to ture for the Base Seed Base Seed each 100 plates layer layer layer layer milliliters of seed agar
Milliliters Degrees C. Amoxicillin ...... 11 11 21 4 C 0.5 32±35 Amphotericin B ...... None 19 None 8 E 1.0 29±31 Ampicillin ...... 11 11 21 4 C 0.5 32±35 Bacitracin ...... 2 1 21 4 B 0.3 32±35 Bacitracin ...... 2 1 21 4 L 0.3 32±35 Bleomycin ...... 35 35 10 6 X 1.0 32±35 Carbenicillin ...... 9 10 21 4 W 2 0.5 36±37.5 Cefactor ...... 2 1 21 5 A 0.05 36±37.5 Cefadroxil ...... 2 1 21 4 A 0.05 36±37.5 Cefamandole ...... 2 1 21 5 A 0.06 36±37.5 Cefazolin ...... 2 1 21 4 A 0.05 32±35 Cefotaxime ...... 2 1 21 5 A 0.1 36±37.5 Cefoxitin ...... 2 1 21 5 A 0.1 36±37.5 Cephalexin ...... 2 1 21 4 A 0.05 32±35 Cephaloglycin ...... 2 1 21 4 A 0.2 32±35 Cephaloridine ...... 2 1 21 4 A 0.1 32±35 Cephalothin ...... 2 1 21 4 A 0.1 32±35 Cephapirin ...... 2 1 21 4 A 0.08 32±35 Cephradine ...... 2 1 21 4 A 0.05 32±35 Clindamycin ...... 11 11 21 4 C 1.5 36±37.5 Cloxacillin ...... 2 1 21 4 A 0.1 32±35 Colistimethate, sodium ...... 9 10 21 4 F 0.1 36±37.5 Colistin ...... 9 10 21 4 F 0.1 36±37.5 Cyclacillin ...... 11 11 21 4 C 0.5 36±37.5 Dactinomycin ...... 5 5 10 4 H (1) 36±37.5 Dicloxacillin ...... 2 1 21 4 A 0.1 32±35 Dihydrostreptomycin ...... 5 5 21 4 H (1) 36±37.5 Erythromycin ...... 11 11 21 4 C 1.5 32±35 Gentamicin ...... 11 11 21 4 D 0.03 36±37.5 Kanamycin B ...... 5 5 21 4 H (1) 36±37.5 Methicillin ...... 2 1 21 4 A 0.3 32±35 Mitomycin ...... 8 8 10 4 H 0.5 36±37.5 Nafcillin ...... 2 1 21 4 A 0.3 32±35 Natamycin ...... None 19 None 8 E 0.8 29±31 Neomycin ...... 11 11 21 4 A 0.4 32±35 Neomycin ...... 11 11 21 4 D 1.0 36±37.5 Netilmicin ...... 11 11 20 5 D 0.25 36±37.5 Novobiocin ...... 2 1 21 4 D 4.0 34±36 Nystatin ...... None 19 None 8 T 1.0 29±31 Oleandomycin ...... 11 11 21 4 D 1.0 36±37.5 Oxacillin ...... 2 1 21 4 A 0.3 32±35 Paromomycin ...... 11 11 21 4 D 2.0 36±37.5 Penicillin G ...... 2 1 21 4 A 1.0 32±35 Penicillin V ...... 2 1 21 4 A 1.0 32±35 Plicamycin ...... 8 8 10 4 A 0.1 32±35 Polymyxin B ...... 9 10 21 4 F 0.1 36±37.5 Rifampin ...... 2 2 21 4 H 0.1 29±31 Sisomicin ...... 11 11 21 4 D 0.03 36±37.5 Streptomycin ...... 5 5 21 4 H (1) 36±37.5 Ticarcillin ...... 38 38 21 4 Y 1.5 36±37.5
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Media to be used (as Milliliters of media to Suggested listed by medium be used in the base volume of number in and seed layers standard- § 436.102(b)) ized Incubation Antibiotic Test or- inoculum to Tempera- ganism be added to ture for the Base Seed Base Seed each 100 plates layer layer layer layer milliliters of seed agar
Milliliters Degrees C. Vancomycin ...... 8 8 10 4 H (1) 36±37.5 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 standard stock solution. Store the stock solutions and standard response line working standard stock solution under solutions. For each antibiotic listed in refrigeration and do not use longer the table in this paragraph, select the than the recommended storage time. working standard drying conditions, Further dilute an aliquot of the work- solvent(s), concentrations, and storage ing standard stock solution to the time for the standard solutions and proper concentrations to prepare the proceed as follows: If necessary, dry standard response line solutions. The the working standard as described in reference concentration of the assay is § 436.200; dissolve and dilute an accu- the mid concentration of the response rately weighed portion to the proper line. concentration to prepare the working
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(c) Procedure for assay. For the stand- corrected diameters, including the av- ard response line, use a total of 12 erage of the 36 diameters of the ref- plates—three plates for each response erence concentration on 2-cycle line solution, except the reference con- semilog paper, using the concentration centration solution which is included of the antibotic in micrograms or units on each plate. On each set of three per milliliter as the ordinate (the loga- plates, fill three alternate cylinders rithmic scale), and the diameter of the with the reference concentration solu- zone of inhibition as the abscissa. The tion and the other three cylinders with response line is drawn either through the concentration of the response line these points by inspection or through under test. Thus, there will be 36 ref- points plotted for highest and lowest erence concentration zones of inhibi- zone diameters obtained by means of tion and nine zones of inhibition for the following equation: each of the four other concentrations of the response line. For each sample 3a + 2 b + c− e tested use three plates. Fill three alter- L = nate cylinders on each plate with the 5 standard reference concentration solu- tion and the other three cylinders with the sample reference concentration so- 3e + 2 d + c− a lution. After all the plates have incu- H = bated for 16 to 18 hours at the appro- 5 priate incubation temperature for each where: antibiotic listed in the table in para- L=Calculated zone diameter for the lowest graph (b) of this section, measure the concentration of the standard response diameters of the zones of inhibition line; using an appropriate measuring device H=Calculated zone diameter for the high- such as a millimeter rule, calipers, or est concentration of the standard re- an optical projector. sponse line; (d) Estimation of potency. To prepare c=Average zone diameter of 36 readings of the standard response line, average the the reference point standard solution; a, b, d, e=Corrected average values for the diameters of the standard reference other standard solutions, lowest to concentration and average the diame- highest concentration, respectively. ters of the standard response line con- centration tested for each set of three To estimate the potency of the sample, plates. Average also all 36 diameters of average the zone diameters of the the reference concentration for all four standard and the zone diameters of the sets of plates. The average of the 36 di- sample on the three plates used. If the ameters of the reference concentration average zone diameter of the sample is is the correction point of the response larger than that of the standard, add line. Correct the average diameter ob- the difference between them to the ref- tained for each concentration to the erence concentration diameter of the figure it would be if the average ref- standard response line. If the average erence concentration diameter for that zone diameter of the sample is lower set of three plates were the same as the than that of the standard, subtract the correction point. Thus, if in correcting difference between them from the ref- the highest concentration of the re- erence concentration diameter of the sponse line, the average of the 36 diam- standard response line. From the re- eters of the reference concentration is sponse line, read the concentrations 16.5 millimeters and the average of the corresponding to these corrected values reference concentration of the set of of zone diameters. Multiply the con- three plates (the set containing the centration by the appropriate dilution highest concentration of the response factor to obtain the antibiotic content line) is 16.3 millimeters, the correction of the sample. is +0.2 millimeter. If the average read- [39 FR 18944, May 30, 1974]
ing of the highest concentration of the EDITORIAL NOTE: For FEDERAL REGISTER ci- response line of these same three plates tations affecting § 436.105, see the List of CFR is 16.9 millimeters, the corrected diam- Sections Affected appearing in the Finding eter is then 17.1 millimeters. Plot these Aids section of this volume.
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§ 436.106 Microbiological turbidimetric the working standard as described in assay. § 436.200; dissolve and dilute an accu- Using the sample solution prepared rately weighed portion to the proper as described in the section for the par- concentration for the working standard ticular antibiotic to be tested, proceed stock solution. Store the working as described in paragraphs (a), (b), and standard stock solution under refrig- (c) of this section. eration and do not use longer than the (a) Preparation of working standard recommended storage time. Prepare stock solutions and standard response line the proper concentrations for the solutions. For each antibiotic listed in standard response line solutions by fur- the table in this paragraph, select the ther diluting an aliquot of the working working standard, drying conditions, standard stock solution. The reference solvent(s), concentrations, and storage concentration of the assay is the mid time for the standard solutions and concentration of the standard response proceed as follows: If necessary, dry line.
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§ N ber as listed in ...... N N N N Diluent (solution num- Distilled water Distilled water Distilled water 0.01 Distilled water Distilled water 0.1 Distilled water Distilled water 0.1 Distilled water Distilled water 0.1 Dimethyl sulfoxide 0.1 alcohol U.S.P. XX 13 Distilled water 15 Distilled water Working standard stock solutions ...... Initial solvent g. per ml.) µ Distilled water Ethyl alcohol (10,000 ...... 436.200) § as listed in (method number Drying conditions Not dried 5 ...... Not dried ...... Not dried Not dried Not dried Not dried Not dried Not dried Not dried Not dried Not dried ...... 1 ...... 1 ...... 1 ...... 1 ...... 1 ...... 5 ...... 1 ...... 6 ...... 1 ...... 1 2 ...... Antibiotic Use sterile equipment for all stages of this assay. The gramicidin working standard and the response line concentrations are used for assay of tyrothricin. 1 2 Amikacin Candicidin Capreomycin Chloramphenicol Chlortetracycline Cycloserine Demeclocycline Dihydrostreptomycin Doxycycline Gramicidin Kanamycin Lincomycin Meclocycline Methacycline Oxytetracycline Rolitetracycline Spectinomycin Streptomycin Tetracycline Tobramycin Troleandomycin Tyrothricin
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(b) Procedure for assay. For each anti- hours. The exact length of the incuba- biotic listed in the table in this para- tion period should be determined by ob- graph, select the test organism (as list- servation of growth in the reference ed in § 436.103(a)), nutrient broth (as concentration tube of the standard. Re- listed by medium number in move the tubes from the water bath § 436.102(b)), and suggested inoculum and add 0.5 milliliter of a 12-percent and proceed as follows: Place 1.0 milli- formaldehyde solution to each tube. liter (or 0.1 milliliter in the case of Determine the absorbance value of gramicidin and tyrothricin) of each each tube in a suitable photoelectric concentration of the standard response colorimeter, at a wavelength of 530 line (prepare as described in paragraph millimicrons. Set the instrument at (a) of this section) and of the sample solution in each set of three replicate zero absorbance with an uninoculated tubes (as described in § 436.100(b)(1)). blank composed of the same amounts Fifteen tubes are used for the five- of nutrient broth and formaldehyde point standard response line and three used in the assay. for each sample. To each tube add 9 NOTE: The amount of working standard and milliliters of the inoculated broth and sample solutions may be reduced as long as place immediately in a water bath at all other solutions used are reduced propor- the appropriate temperature for 2 to 4 tionately.
Suggested volume of standard- ized inoculum Test orga- Medium to be Incubation Antibiotic nism (nutrient added to tempera- broth) each 100 ture milliliters of medium (nutrient broth)
Amikacin ...... A 3 0.1 36±37.5 Candicidin 1 ...... E 13 0.2 27±29 Capreomycin ...... I 3 0.05 36±37.5 Chloramphenicol ...... J 3 0.7 36±37.5 Chlortetracycline ...... A 3 0.1 36±37.5 Cycloserine ...... A 3 0.4 36±37.5 Demeclocycline ...... A 3 0.1 36±37.5 Dihydrostreptomycin ...... I 3 0.1 36±37.5 Doxycycline ...... A 3 0.1 36±37.5 Gramicidin ...... K 3 1.0 36±37.5 Kanamycin ...... A 3 0.2 36±37.5 Lincomycin ...... A 3 0.1 36±37.5 Meclocycline ...... A 3 0.2 36±37.5 Methacycline ...... A 3 0.1 36±37.5 Oxytetracycline ...... A 3 0.1 36±37.5 Rolitetracycline ...... A 3 0.1 36±37.5 Spectinomycin ...... J 3 0.1 36±37.5 Streptomycin ...... I 3 0.1 36±37.5 Tetracycline ...... A 3 0.1 36±37.5 Tobramycin ...... A 3 0.15 36±37.5 Troleandomycin ...... I 3 0.1 36±37.5 Tyrothricin ...... K 3 1.0 36±37.5 1 Use sterile equipment for all stages of this assay.
L=Calculated absorbance value for the low- 3a+ 2 b+c− e L = est concentration of the standard re- 5 sponse line. H=Calculated absorbance value for the 3e+ 2 d +c− a H = highest concentration of the standard 5 response line. where:
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a, b, c, d, e=Average absorbance values for Replace the stopper and place the each concentration of the standard re- weighing bottle in a desiccator over a sponse line, lowest to the highest, re- desiccating agent, such as phosphorous spectively. pentoxide or silica gel, allow to cool to (c) Estimation of potency. To prepare room temperature, and reweigh. Cal- the standard response line, plot the av- culate the percent of loss. erage absorbance values for each con- (b) Method 2. Proceed as directed in centration of the standard response paragraph (a) of this section, except line on one-cycle semilogarithmic use a tared weighing bottle or weighing graph paper with the absorbance values tube equipped with a capillary-tube on the arithmetric scale and con- stopper, the capillary having an inside centrations on the logarithmic scale. diameter of 0.20–0.25 millimeter, and The response line is drawn either place it in a vacuum oven without re- through these points by inspection or moving the stopper. through points plotted for highest and (c) Method 3. Proceed as directed in lowest absorbance values obtained by paragraph (a) of this section, except means of the following equations. dry the sample at a temperature of 110° To estimate the potency of the sample, C. and a pressure of 5 millimeters of average the absorbance values for the mercury or less for 3 hours. sample and determine the antibiotic (d) Method 4. Proceed as directed in concentration from the standard re- paragraph (a) of this section, except sponse line. Multiply the concentration dry the sample at a temperature of 40° by the appropriate dilution factor to C. and a pressure of 5 millimeters of obtain the antibiotic content of the mercury or less for 2 hours. sample. (e) Method 5. Proceed as directed in paragraph (a) of this section, except [39 FR 18944, May 30, 1974, as amended at 40 ° FR 57797, Dec. 12, 1975; 41 FR 49483, Nov. 9, dry the sample at a temperature of 100 1976; 44 FR 22057, Apr. 13, 1979; 46 FR 3835, C. and a pressure of 5 millimeters of 3839, Jan. 16, 1981; 46 FR 16682, Mar. 13, 1981; mercury or less for 4 hours. 46 FR 33512, June 30, 1981; 46 FR 61072, Dec. (f) Method 6. Proceed as directed in 15, 1981; 47 FR 22515, May 25, 1982; 47 FR 23708, paragraph (a) of this section, except June 1, 1982; 48 FR 3960, Jan. 28, 1983; 53 FR dry the sample at a temperature of 40° 32607, Aug. 26, 1988] C. and a pressure of 5 millimeters of mercury or less for 3 hours. Subpart E—General Chemical (g) Method 7. Proceed as directed in Tests for Antibiotics paragraph (a) of this section, except dry the sample at a temperature of 25° § 436.200 Loss on drying. C. and a pressure of 5 millimeters of Use the method specified in the indi- mercury or less for 4 hours. vidual section for each antibiotic. (h) Method 8. Proceed as directed in (a) Method 1. In an atmosphere of paragraph (a) of this section, except about 10 percent relative humidity, transfer approximately 300 milligrams grind the sample, if necessary, to ob- of the sample to a tared weighing bot- tain a fine powder. When tablets, tro- tle equipped with a ground-glass stop- ches, or capsules are to be tested, use per; dry the sample at a temperature of four tablets, troches, or capsules in 25 ° C and a pressure of 5 millimeters of preparing the sample. Transfer about mercury or less for 4 hours, and then 100 milligrams of the sample to a tared dry the sample at a temperature of 100 weighing bottle equipped with a °C and a pressure of 5 millimeters of ground-glass stopper. Weigh the bottle mercury or less for 3 additional hours. and place it in a vacuum oven, tilting (i) Method 9. Use a suitable the stopper on its side so that there is thermogravimetric apparatus prepared no closure during the drying period. for vacuum operation. Rapidly and Dry at a temperature of 60° C. and a thoroughly grind a portion of the sam- pressure of 5 millimeters of mercury or ple and promptly transfer 5 to 10 milli- less for 3 hours. At the end of the dry- grams to the sample pan. Place the ing period, fill the vacuum oven with system under vacuum and allow it to air dried by passing it through a drying come to equilibrium before heating. agent such as sulfuric acid or silica gel. Obtain an accurate sample weight and
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