(8) Medium H:

Peptone

Yeast extract_.

Beef extract_.

Sodium chloride.

Dextrose----

Agar.

Distilled water, qs-

pH 6.0 to 6.2 after sterilization.

9.4 gm. 4.7 gm.

2.4 gm.

10.0 gm.

10.0 gm.

23.5 gm. 1,000.0 ml.

(b) Preparation of test organism suspensions (1) Suspension 1. Staphylococcus aureus (ATCC 6538P) 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.

2

(4) Suspension 4. Sarcina lutea (ATCC 9341) is maintained on agar slants of medium A and transferred to fresh slants approximately every 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 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 heatshock 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. Standardize this stock suspension by determining the dilution that will give 80 percent light transmission. Store the stock suspension in the refrigerator (1 week) and use the indicated

dilution prepared daily for the inoculum for the plates.

(7) Suspension 7. Bordetella bronchiseptica (ATCC 4617) is maintained on medium F and transferred to a fresh slant every 2 weeks. To prepare a stock suspension inoculate a fresh slant of medium F and incubate at 37° C. for 16 hours to 24 hours. Wash the culture from this slant with 3 milliliters of sterile distilled water onto the surface of a Roux bottle containing 300 milliliters of medium F, and incubate 24 hours at 37° C. Wash off the growth with 50 milliliters of sterile distilled water and standardize the resulting stock suspension by determining the dilution that will give 50 percent light transmission. Store the stock suspension in the refrigerator (2 weeks), and use the indicated dilution prepared daily for the inoculum for the plates.

(8) Suspension 8. Saccharomyces cerevisiae (ATCC 9763) is maintained on slants of medium H and transferred once a week. After transfer, the culture is incubated at 37° C. for 24 hours and then kept refrigerated. Wash the organism from a freshly incubated agar slant with 3 milliliters of sterile saline solution onto the agar surface in a Roux bottle containing 300 milliliters of medium H. Spread the suspension of organisms over the entire agar surface with the aid of sterile glass beads. Incubate for 24 hours at 37° C. and then wash the resulting growth from the agar surface with about 25 milliliters of sterile saline solution. Store the suspension in the refrigerator and use for 1 month.

(9) Suspension 9. Follow the procedure described for suspension 1, except 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.

(10) Suspension 10; Klebsiella pneumoniae (A.T.C.C. 10031), noncapsulated, 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 overnight at 32° C.-35° C. Wash the culture from the slant with 3 milliliters of sterilized U.S.P. saline T.S. onto the surface of a Roux bottle containing 300 milliliters of medium A. Incubate at

32° C.-35° C. for 24 hours. Wash the resulting growth from the agar surface with about 50 milliliters of sterilized U.S.P. saline T.S. If an aliquot of this bulk suspension when diluted 1:9 with saline solution gives 40 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 1:9 will give the desired 40 percent light transmission. The adjusted bulk suspension (not the 1:9 dilution) is used in preparing the inoculum. Store the suspension in the refrigerator and use for no more than 1 week.

(11) Suspension 11. Streptococcus fecalis (A.T.C.C. 14506) is maintained on medium E and transferred to a fresh agar slant once a week. After transfer, the culture is incubated at 37° C. for 24 hours and then kept refrigerated. Transfer from a freshly incubated agar slant to a tube containing 10 milliliters of culture medium described in § 147.3 (b)(1). Incubate the broth culture for 16 to 18 hours at 37° C. and store in the refrigerator. This culture may be used for no more than 1 week.

The light transmission values referred to in this paragraph were determined with a Lumetron Model 400-A photoelectric colorimeter at a wavelength of 650 millimicrons. If other instruments are used, different light transmission readings will probably be obtained. The values given are to be used as guides in this paragraph.

(c) Preparation of plates-(1) Base layer. Depending on the particular antibiotic in the discs to be tested, add 42 milliliters of the appropriate medium prescribed in subparagraph (3) of this paragraph to each Petri dish (20 millimeters x 150 millimeters) and allow to harden on a flat, level surface and dry slightly by raising the tops on one side.

(2) Seed layer. Add the appropriate amount of inoculum, as prescribed by subpargaraph (3) of this paragraph, to the seed agar which has been melted and cooled to 48° C. Swirl the flasks to obtain a homogeneous suspension. Add 8 milliliters of the appropriate seed agar, as specified in subparagraph (3) of this paragraph, to each plate, spread evenly over the hardened base layer, and allow to harden and dry on a flat level surface.

(d) Preparation of control discs. Use round, blank discs having a diameter of 1/4-inch made of clear-white paper weighing 30 milligrams ±4 milligrams per square centimeter, and which will absorb 2.5 to 3.0 times its own weight of distilled water. The paper shall contain no material that either enhances or inhibits the activity of any antibacterial agent impregnated thereon. In addition, the paper shall contain no materials which will affect the pH of any solvent placed on it or buffer any solution placed on it. The following methods shall be used to determine the suitability in this regard of any paper proposed for this use: Weigh 2 grams of paper or paper discs into a clean, glass-stoppered, 250-milliliter flask. Add 30 milliliters of freshly boiled and cooled distilled water (the pH of which has been determined). Stopper and shake vigorously for 1 hour on a shaking machine. Filter through a medium-porosity sintered glass filter.

1.0

1.0

Determine the pH of the filtrate. Take

To

the two 10-milliliter aliquots. To one add 0.05 milliliter of 0.01 N HCl. the second aliquot add 0.05 milliliter of 0.01 N NaOH. Determine the pH of each solution. The paper shall be satisfactory for use, if (1) the pH of the paper filtrate was not more than ±0.3 pH units different from the pH of the distilled water used; (2) the pH of the acidified aliquot was lowered by at least 1.0 pH units; (3) the pH of the alkalinized aliquot was raised by at least 1.5 pH units. Place blank discs on aluminum or stainless steel wire mesh which is supported in a manner to allow circulation of air above and below the discs. Prepare the desired number of discs for each point on the standard curve by accurately adding 0.02-milliliter-increments of the appropriate standard stock solution to each disc, using a suitable pipette. Dry discs in circulating air or under vacuum. Discs may be stored for