used; for example, a photometric method. In some cases it may be possible to get an adequate inoculum density in the tube even without incubation.

NOTE: Extremes in inoculum density should be avoided. Undiluted overnight broth culture should never be used for streaking plates.

C. INOCULATING THE PLATES

1. Dip a sterile cotton swab on a wooden applicator into the properly diluted inoculum. Remove excess inoculum from the swab by rotating it several times with firm pressure on the inside wall of the test tube above the fluid level.

2. Streak the swab over the entire sterile agar surface of a plate. Streaking successively in three different directions is recommended to obtain an even inoculum.

3. Replace the plate top and allow the inoculum to dry for 3 to 5 minutes.

4. Place the susceptibility discs on the inoculated agar surface and with sterile forceps, or needle tip flamed and cooled between each use, gently press down each disc to insure even contact. Space the discs evenly so that they are no closer than 10 to 15 millimeters to the edge of the petri dish and sufficiently separated from each other to avoid overlapping zones of inhibition. (Spacing may be accomplished by using a disc dispenser or by putting the plate over a pattern to guide the placement of discs.) Within 30 minutes, place the plate in an incubator under aerobic conditions at a constant temperature in the range of 35°-37° C. 5. Read the plate after overnight incubation or, if rapid results are desired, the diameters of the zone of inhibition may be

readable after 6 to 8 hours incubation. In the latter case, the results should be confirmed by also reading the results after overnight incubation.

NOTE: Microbial growth on the plate should be just or almost confluent. If only isolated colonies are present the inoculum was too light and the test should be repeated.

Modifications of the inoculation procedure described in 1-3 above, such as the use of the agar overlay method described in Barry, A. L., Garcia, F., and Thrupp, L. D.: "An Improved Single-disk Method for Testing the Antibiotic Susceptibility of Rapidlygrowing Pathogens." Amer. J. Clin. Pathol. 53:149-58, 1970, a copy of which is on file with the Office of the Federal Register, may be used if the procedure is standardized to produce results with the control cultures that are equivalent to those obtained with the recommended cotton swab streak method.

D. READING THE PLATES

Measure and record the diameter of each zone (including the diameter of the disc) to the closest millimeter, reading to the point of complete inhibition as judged by the unaided eye. Preferably, read from the underside of the plate without removing the cover, using a ruler, calipers, transparent plastic gage, or other device. A mechanical zone reader may be used. If blood agar is used, measure the zones from the surface with the cover removed from the plate.

E. INTERPRETATION OF ZONE SIZES

Interpret the susceptibility according to the following table:

Diameter (millimeters) of zone of inhibition

G. LIMITATIONS OF THE METHOD The method of interpretation described in E above applies to rapidly growing pathogens and should not be applied to slowly growing organisms. The latter show larger zones of inhibition than those given in the table. Susceptibility of gonococci to penicillin, and of slow-growing strains, e.g., Bacteroides species and fastidious anaerobes to any antibiotic, should be determined by the broth-dilution or agar-dilution method unless specifically standardized diffusion tests are used.

(d) Requests for certification; samples. (1) In addition to complying with the requirements of § 431.1 of this chapter, a person who requests certification of a batch of antibiotic susceptibility discs shall submit with his request a statement showing the batch mark, the number of packages of each size in such batch, and, unless it was previously submitted, the date on which the latest assay of the antibiotic used in making such batch was completed, the potency of each disc, the quantity of each ingredient used in making the batch, the date on which the latest assay of the drug comprising such batch was completed, and a statement that each ingredient used in making the batch conforms to the requirements prescribed therefor by this section.

(2) Such person shall submit in connection with his request results of the tests and assays made by him on an accurately representative sample of the batch for potency.

(3) Such person shall submit in connection with his request an accurately representative sample of the batch consisting of one disc for each 5,000

discs in the batch, but in no case less than 36 discs collected by taking single discs at intervals throughout the entire time of packaging the batch so that the quantities packaged during the intervals are approximately equal.

[39 FR 19181, May 30, 1974, as amended at 41 FR 7093, Feb. 17, 1976; 41 FR 35061, Aug. 19, 1976; 44 FR 10376, Feb. 20, 1979; 44 FR 20666, Apr. 6, 1979]

§ 460.6 Tests and methods of assay for potency of antibiotic susceptibility discs. (a) Culture media. Use ingredients that conform to the standards prescribed by the United States Pharmacopeia or The National Formulary. In lieu of preparing the media from the individual ingredients, they may be made from a dehydrated mixture which, when reconstituted with distilled water, has the same composition as such media. Minor modification of the specified individual ingredients is permissible if the resulting media possess growth-promoting properties at least equal to the media described. (1) Medium A:

(b) Preparation of test organism suspensions—(1) Suspension 1. Staphylococcus aureus (ATCC 6538P)1 is maintained and grown on medium A. Wash the organisms from an agar slant, incubated for 24 hours at 32° C. to 35° C., with 3.0 milliliters of sterile sodium chloride solution onto the agar surface of a Roux bottle containing 300 milliliters of medium A. Spread the suspension of organisms over the entire agar surface with the aid of sterile glass beads. Incubate 24 hours at 32° C. to 35° C. Wash the resulting growth from the agar surface with about 50 milliliters of sterile sodium chloride solution. Standardize this stock suspension by determining the dilution that will permit 20 percent light transmission. Store the stock suspension in the refrigerator (1 week) and use the indicated dilution prepared daily.

(2) Suspension 2. Follow the procedure described for suspension 1, except standardize the bulk suspension so that a 1:10 dilution in saline solution gives 20 percent light transmission. In this case, the bulk suspension, and not the 1:10 dilution of it, is used for the inoculum.

(3) Suspension 3. The test organism is

Staphylococcus aureus (ATCC 13150). Follow the procedure described for suspension 1, but determine how much the bulk suspension should be diluted to obtain a suspension permitting 80 percent light transmission. Use the indicated dilution prepared daily for the inoculum for the plates.

(4) Suspension 4. Sarcina lutea (ATCC 9341)1 is maintained on agar slants of medium A and transferred to fresh slants approximately every 2 weeks. This culture is incubated overnight at 26° C., and then stored in the refrigerator. Prepare an inoculum for the plates as follows: Streak an agar slant heavily with the test organism and incubate for 24 hours at 26° C. Wash the growth from the slant with

1Available from: American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.

3 milliliters to 4 milliliters of medium D, and transfer to the surface of a Roux bottle containing 300 milliliters of medium A. Spread the suspension evenly over the entire surface with the aid of sterile glass beads. Incubate for 24 hours at 26° C. Wash the growth from the agar surface with 15 milliliters of medium D. If an aliquot of this bulk suspension when diluted 1:10 with medium D gives 10 percent light transmission, the bulk suspension is satisfactory for use. It may be necessary to adjust the bulk suspension by dilution so that an aliquot of the adjusted suspension when diluted 1:10 will give the desired 10 percent light transmission. The adjusted bulk suspension only, and not the 1:10 dilution of it, is used in preparing the inoculum. Store the stock suspension in the refrigerator and use for 2 weeks.

(5) Suspension 5. Bacillus subtilis (ATCC 6633) is maintained on agar medium A and transferred to a fresh slant every month. To prepare the spore suspension, inoculate a fresh slant of agar medium A with the test organism and incubate at 37° C. for 16 hours to 24 hours. Wash the culture from the slant with 3 milliliters of sterile sodium chloride solution onto the surface of a Roux bottle containing 300 milliliters of agar medium B. Incubate for 5 days at 37° C. Suspend the growth in 50 milliliters of sterile saline solution, centrifuge, and decant the supernatant liquid. Reconstitute the sediment and heat-shock the suspension by heating for 30 minutes at 70° C. Store the spore suspension in the refrigerator. It may be kept several months. Light transmission is not used for standardization.

(6) Suspension 6. Staphylococcus epidermidis (ATCC 12228)' is maintained on medium A and transferred to a fresh slant once a week. Inoculate a fresh slant of medium A with the test organism and incubate at 32° C. to 35° C. for 24 hours. Wash the culture from the slant with 3 milliliters of sterile sodium chloride solution onto the surface of a Roux bottle containing 300 milliliters of medium A. Incubate at 32° C. to 35° C. for 24 hours. Wash the resulting growth from the agar surface with about 50 milliliters of sterile sodium chloride solution.