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1028 Subpart A—Susceptibility Discs

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1028 Subpart A—Susceptibility Discs § 460.1 21 CFR Ch. I (4±1±96 Edition) 460.137 Methicillin concentrated stock solu- Neomycin: 30 mcg. tions for use in antimicrobial suscepti- Novobiocin: 30 mcg. bility test panels. Oleandomycin: 15 mcg. 460.140 Penicillin G concentrated stock so- Penicillin G: 10 units. lutions for use in antimicrobial suscepti- Polymyxin B: 300 units. bility test panels. Rifampin: 5 mcg. 460.146 Tetracycline concentrated stock so- Streptomycin: 10 mcg. lutions for use in antimicrobial suscepti- Tetracycline: 30 mcg. bility test panels. Tobramycin: 10 mcg. 460.149 Tobramycin concentrated stock so- Vancomycin: 30 mcg. lutions for use in antimicrobial suscepti- bility test panels. The standard discs used to determine 460.152 Trimethoprim concentrated stock the potency shall be made of paper as solutions for use in antimicrobial suscep- described in § 460.6(d). Each antibiotic tibility test panels. compound used to impregnate such 460.153 Sulfamethoxazole concentrated stock solutions for use in antimicrobial standard discs shall be equilibrated in susceptibility test panels. terms of the working standard des- ignated by the Commissioner for use in AUTHORITY: Sec. 507 of the Federal Food, determining the potency or purity of Drug, and Cosmetic Act (21 U.S.C. 357). such antibiotic. SOURCE: 39 FR 19181, May 30, 1974, unless (b) Packaging. The immediate con- otherwise noted. tainer shall be a tight container as de- fined by the U.S.P. and shall be of such Subpart AÐSusceptibility Discs composition as will not cause any change in the strength, quality, or pu- § 460.1 Certification procedures for an- rity of the contents beyond any limit tibiotic susceptibility discs. therefor in applicable standards, except (a) Standards of identity, strength, that minor changes so caused that are quality, and purity. Antibiotic suscepti- normal and unavoidable in good pack- bility discs are round flat discs that aging, storage, and distribution prac- have a diameter of one-fourth inch and tice shall be disregarded. Each imme- are made of clear absorbent paper con- diate container may contain a des- taining antibiotic compounds. They are iccant, and each may be packaged in capable of absorbing moisture rapidly combination with containers of suit- and the antibiotic is evenly distrib- able discs of drugs other than those de- uted. The thickness is sufficient to as- scribed in paragraph (a) of this section. sure rigidity and to have permitted the Such other discs shall be suitable only complete absorption of an adequate if the manufacturer and packer have volume of antibiotic solution (approxi- submitted to the Commissioner infor- mately 0.02 milliliter). The identity of mation of the kind described in § 431.17 each disc is signified either by a color of this chapter, and such information or by means of an identifying sign. The has been accepted by the Commis- absorbent paper and dye or ink used sioner. must not affect either bacterial growth (c) Labeling. Each package of discs or the antibiotic. Each disc shall have shall bear on its label or labeling, as a uniform potency that is equivalent to hereinafter indicated, the following: that contained in a standard disc pre- (1) On the outside wrapper or con- pared with one of the following quan- tainer and the immediate container: tities of antibiotic drugs: (i) The batch mark. Ampicillin: 10 mcg. (ii) The name and potency of each Bacitracin: 10 units. disc in the batch according to the fol- Carbenicillin: 50 mcg. lowing: Cefamandole:30mcg. Cefoxitin: 30 mcg Content of antibiotic in Cephalothin: 30 mcg. Name of disc micrograms or units per disc Chloramphenicol: 30 mcg. Clindamycin: 2 mcg. Ampicillin-class disc ............... 10 mcg. ampicillin. Colistin: 10 mcg. Bacitracin disc ....................... 10 units bacitracin. Carbenicillin disc .................... 50 mcg. carbenicillin. Erythromycin: 15 mcg. Cefamandole disc .................. 30 mcg. cefamandole. Gentamicin: 10 mcg. Cefoxitin disc ......................... 30 mcg. cefoxitin. Kanamycin: 30 mcg. Cephalosporin-class disc ....... 30 mcg. cephalothin. Methicillin: 5 mcg. Chloramphenicol disc ............ 30 mcg. chloramphenicol. 1028 VerDate 17<JUL>96 11:55 Jul 29, 1996 Jkt 167069 PO 00000 Frm 01023 Fmt 8010 Sfmt 8010 C:\CFR\21V5.001 pfrm13 Food and Drug Administration, HHS § 460.1 A. PREPARATION OF CULTURE MEDIUM AND Name of disc Content of antibiotic in micrograms or units per disc PLATES Colistin disc ........................... 10 mcg. colistin. 1. Melt previously prepared and sterilized Erythromycin disc .................. 15 mcg. erythromycin. Mueller-Hinton agar medium and cool to 45°± Gentamicin disc ..................... 10 mcg. gentamicin. 50° C. Kanamycin disc ..................... 30 mcg. kanamycin. 2. For the purpose of testing certain fas- Lincomycin-class disc ............ 2 mcg. clindamycin. tidious organisms such as streptococci and Neomycin disc ....................... 30 mcg. neomycin. Haemophilus species, 5 percent defibrinated Novobiocin disc ..................... 30 mcg. novobiocin. human, horse, or sheep blood may be added Oleandomycin disc ................ 15 mcg. oleandomycin. to the above medium which is Penicillin-class disc ................ 10 units penicillin G. ``chocolatized'' when indicated. Penicillinase-resistant penicil- 5 mcg. methicillin. 3. To prepare the plates, pour the melted lin-class disc. medium into Petri dishes on a level surface Polymyxin B disc ................... 300 units polymyxin B. to a depth of 4 millimeters. Rifampin disc ......................... 5 mcg. rifampin. 4. Let the medium harden and allow to Streptomycin-class disc ......... 10 mcg. streptomycin. stand long enough for excess moisture to Tetracycline-class disc .......... 30 mcg. tetracycline. evaporate. (For this purpose plates may be Tobramycin disc .................... 10 mcg. tobramycin. placed in an incubator at 35°±37° C. for 15±30 Vancomycin disc .................... 30 mcg. vancomycin. minutes or allowed to stand somewhat longer at room temperature.) There should (iii) The statement ``Expiration date be no moisture droplets on the surface of the ÐÐÐÐÐ'', the blank being filled in medium or on the petri dish covers. The pH with the date that is 6 months after the of the solidified medium should be 7.2±7.4. month during which the batch was cer- Satisfactory plates may be used immediately tified, except that the blank may be or refrigerated. Plates may be used as long as the surface is moist and there is no sign filled in with a date that is 12, 18, 24, 30, of deterioration. 36, 42, 48, 54, or 60 months after the NOTE: Commercially prepared agar plates month during which the batch was cer- meeting the above specifications may be tified if the person who requests cer- used. tification has submitted to the Com- B. PREPARATION OF INOCULUM missioner results of tests and assays showing that such drug as prepared by 1. Select four or five similar colonies. 2. Transfer these colonies (obtained by him is stable for such longer period of touching the top of each colony in turn with time. If it is a packaged combination of a wire loop) in turn to a test tube containing discs of two or more drugs, its outside about 5 milliliters of a suitable liquid me- wrapper shall bear only one expiration dium such as soybean-casein digest broth, date, and that date shall be the date U.S.P. ° ° that is required for the shortest dated 3. Incubate the tube at 35 ±37 C. long enough (2 to 8 hours) to produce an organism discs contained in the package. suspension with moderate cloudiness. At (iv) The statement ``For laboratory that point the inoculum density of the sus- use only''. pension should be controlled by diluting it, (2) On the circular or other labeling or a portion of it, with sterile saline to ob- within or attached to the package, ade- tain a turbidity equivalent to that of a fresh- ly prepared turbidity standard obtained by quate directions for the use of such adding 0.5 milliliter of 1.175 percent barium discs, including the following rec- chloride dihydrate (BaCl22H2O) solution to ommended method: 99.5 milliliters of 0.36 N (1.0 percent) sulfuric acid. Other suitable methods for standardiz- STANDARDIZED DISC SUSCEPTIBILITY TEST ing inoculum density may be used; for exam- ple, a photometric method. In some cases it DIRECTIONS FOR USE may be possible to get an adequate inoculum density in the tube even without incubation. Quantitative methods that require the NOTE: Extremes in inoculum density measurement of zone sizes give the most pre- should be avoided. Undiluted overnight broth cise estimates of antibiotic susceptibilities. culture should never be used for streaking The following outline describes such a proce- plates. dure. Minor variations from this procedure may be used if the resulting procedure is C. INOCULATING THE PLATES standardized according to the results ob- 1. Dip a sterile cotton swab on a wooden tained in the laboratory from adequate stud- applicator into the properly diluted ies with control cultures. inoculum. Remove excess inoculum from the 1029 VerDate 17<JUL>96 11:55 Jul 29, 1996 Jkt 167069 PO 00000 Frm 01024 Fmt 8010 Sfmt 8010 C:\CFR\21V5.001 pfrm13 § 460.1 21 CFR Ch. I (4±1±96 Edition) swab by rotating it several times with firm was too light and the test should be re- pressure on the inside wall of the test tube peated. above the fluid level. Modifications of the inoculation procedure 2. Streak the swab over the entire sterile described in 1±3 above, such as the use of the agar surface of a plate. Streaking succes- agar overlay method described in Barry, A. sively in three different directions is rec- L., Garcia, F., and Thrupp, L. D.: ``An Im- ommended to obtain an even inoculum. proved Single-disk Method for Testing the 3. Replace the plate top and allow the Antibiotic Susceptibility of Rapidly-growing inoculum to dry for 3 to 5 minutes. Pathogens.'' Amer. J. Clin. Pathol. 53:149±58, 4. Place the susceptibility discs on the in- 1970,* a copy of which is on file with the Of- oculated agar surface and with sterile for- fice of the Federal Register, may be used if ceps, or needle tip flamed and cooled be- the procedure is standardized to produce re- tween each use, gently press down each disc to insure even contact.
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