Vol. 14, No. 5
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1978, P. 761-764 0066-4804/78/0014-0761$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Improved Susceptibility Disk Assay Method Employing an Agar Overlay Technique BLONDELL ANDERSON Antibiotic Residue Branch, National Center for Antibiotics Analysis, Food and Drug Administration, Washington, D.C. 20204 Received for publication 3 August 1978
Susceptibility disks impregnated with 35 different antibiotics were assayed by the current Code ofFederal Regulations method and by a new method employing an agar overlay. The new method, a simplified analytical procedure, exhibited improvement in zone defmition and an increase in dose response. Assays performed by the new method were accurate and precise. The agar overlay technique appears to be superior to the current Code of Federal Regulations method. Under the antibiotic certification program, susceptibility disks are tested for potency and uniformity before being distributed on the market. However, the agar diffusion assays presently employed in the performance testing exhibit certain qualities that can and should be improved. A review of the analytical procedures reveals many of the variables that may contribute to differences in analytical results, some of which are clearly described by Lorian (2). For example, the nutrient media used in the assays may vary from batch to batch, creating differences in the quality of zone definition. Differences in the quality of the paper used in making the disks may affect the diffusion characteristics of the antibiotic, significantly changing potency determinations (3). Attempts have been made by this laboratory to eliminate many of the existing variables of the disk assay procedures. The investigations described in this paper were conducted to develop an improved disk assay procedure that employs an agar overlay much like that described by Barry et al. in their modification of the Kirby-Bauer procedure (1). Attempts were made to improve zone clarity and zone definition, to increase the response to standard doses of the drug, and to provide a simpler assay procedure. MATERIALS AND METHODS Petri dishes (20 by 150 mm) were prepared, each with a 42-mi nutrient agar base layer. The bacterial inoculum was carried in an 8-ml agar overlay layer. The agar layer was formulated to contain 15 g of granulated agar in 1 liter of distilled water and was sterilized at 121°C for 15 min, with a final pH between 6.9 and 7.0. Details of the individual assay method by the new technique are outlined in Table 1. The guidelines for the Code of Federal Regulations (CFR) assays are found in Section 460 of that document (4). Three filter paper disks, 0.25 inch (ca. 6.3 mm) in diameter (no. 740 E; supplied by Schleicher and
Schuell Co.), were impregnated with the antibiotic at three different doses and placed on the solidified agar surface. The petri dishes were incubated overnight (16 to 18 h) at temperatures of 32 or 37°C. The diameters of the zones of inhibition were measured with the aid of a model 3011 Bausch and Lomb Balopticon overhead projector. The slope of the regression line was calculated as follows: b = n xy - (x)(y) n X2
(X)2
where x is the logarithm of each standard dose (a log dose), y is the average response to each log dose, and n is number of standard doses. Two plates were prepared daily by both method A (CFR) and method B (agar overlay) for each antibiotic. Each plate contained three replicate disks of three standard drug concentrations. The data were collected from a 4-day test period for each antibiotic. Test cultures. The test organisms and their CFR numbers were: Staphylococcus aureus ATCC 6538P, 1; S. aureus ATCC 13150, 3; Sarcina lutea ATCC 9314, 4; Bacillus subtilis ATCC 6633, 5; Staphylococcus epidermidis ATCC 12228, 6; Bordetella bronchiseptica ATCC 4617, 7; Klebsiella pneumoniae ATCC 10031, 10; Streptococcus faecalis ATCC 14506, 11; and Pseudomonas aeruginosa ATCC 25619, 12. All suspensions were prepared as directed in Section 460.6b of the CFR (4). Nutrient media. The media used were: CFR medium A, antibiotic medium 1 (seed agar); medium C, antibiotic medium 11 (neomycin agar); medium G, antibiotic medium 10 (polymyxin-seed agar).
RESULTS Results of the assay of 35 antibiotics by two agar diffusion procedures indicate major differences between method A and method B. Results outlined in Table 1 show that the average slope value for each antibiotic was greater by the new method than by the CFR method. Each slope value represents the average of 24 determinations. 761
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VOL. 14, 1978
AGAR OVERLAY METHOD FOR ANTIBIOTIC DISK ASSAY
An example of this increased dose response is illustrated by the graph in Fig. 1. Zone clarity and defmition were improved in most assays. Improvements in zone clarity were particularly evident with carbenicillin, clindamycin, lincomycin, chloramphenicol, and oleandomycin. Changes in zone clarity with carbenicillin and clindamycin could be seen easily with the unaided eye. Improvement in zone definition was shown most dramatically through projection and magnification of the zones. The clarity of the zone definition is illustrated in Fig. 2.
A
B
N
24 i8 3 0mm 20 26 22 FIG. 1. Standard dose response lines of carbenicillin disk assay, illustrating significant increases in regression or slope values (b) and increases in the inhibition zone size. Line A, CFR method; line B, new agar overlay technique.
763
The agar overlay technique also exhibited an increase in the inhibition zone size for all the antibiotics tested except oxacillin. The decrease in nutrients may have permitted the antibiotic to diffuse more rapidly, thereby enhancing its ability to inhibit bacterial growth. Figure 1 illustrates the increase in the inhibition zone size of the antibiotics.
DISCUSSION In a two-layer agar diffusion assay system for susceptibility disks, the upper (seed) layer is important in the diffusion process because it forms a surface directly in contact with the antibiotic-impregnated disks. Because there may be some constituents in a medium which might interfere with the formation of a zone, the use of a nutrient-free layer may permit the antibiotic to diffuse more freely. The agar overlay technique eliminated the need of nutrient media for both layers of the two-layer system. Good quality control is needed to minimize problems associated with variations that could occur in batches of media for either nutrient layer. The agar overlay technique, however, eliminates some of these problems. Because the new technique employs only three types of nutrient media, the need for quality control of many different media is minimized. Improved zone clarity and zone definition in the new method results in more accurate measurement of zone diameters and in greater accuracy of potency determinations. The increase in response to standard doses minimizes the variability in potency determinations and results in greater assay precision, clearly demonstrating the superiority of the new method over that currently being used.
~~~~~~~ m _...... |h. FIG. 2. Differences in zone definition with carbenicillin and Pseudomonas aeruginosa ATCC 25619. (A) Media system A (CFR method), polybase agar = base layer, polyseed agar = seeded layer. (B) New agar overlay technique, seed agar = base layer, agar-agar = seeded layer.
764
ANDERSON
ACKNOWLEDGMENTS I thank the members of the Antibiotic Residue Branch, National Center for Antibiotics Analysis, Food and Drug Administration, Washington, D.C., for their help in collecting data and giving guidance in the preparation of this paper. Special thanks are extended to Gordon Carter, Ira Zimmerman, and Larry Ouderkirk. LITERATURE CITED 1. Barry, A. L., F. Garcia, and L. D. Thrupp. 1970. An improved single-disk method for testing antibiotic sus-
ANTIMICROB. AGENTS CHEMOTHER. ceptibility of rapidly-growing pathogens. Am. J. Clin. Pathol. 53:149-158. 2. Lorian, V. 1966. Antibiotics and chemotherapeutic agents in clinical and laboratory practice. Charles C Thomas, Publisher, Springfield, Ill. 3. Rippere, R. A. 1978. Effects of paper on performance of antibiotic-impregnated disks. J. Pharm. Sci. 67:367-371. 4. United States Government Printing Office. 1975. Code of Federal Regulations. Title 21, Section 460. United States Government Printing Office, Washington, D.C.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1978, P. 761-764 0066-4804/78/0014-0761$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Improved Susceptibility Disk Assay Method Employing an Agar Overlay Technique BLONDELL ANDERSON Antibiotic Residue Branch, National Center for Antibiotics Analysis, Food and Drug Administration, Washington, D.C. 20204 Received for publication 3 August 1978
Susceptibility disks impregnated with 35 different antibiotics were assayed by the current Code ofFederal Regulations method and by a new method employing an agar overlay. The new method, a simplified analytical procedure, exhibited improvement in zone defmition and an increase in dose response. Assays performed by the new method were accurate and precise. The agar overlay technique appears to be superior to the current Code of Federal Regulations method. Under the antibiotic certification program, susceptibility disks are tested for potency and uniformity before being distributed on the market. However, the agar diffusion assays presently employed in the performance testing exhibit certain qualities that can and should be improved. A review of the analytical procedures reveals many of the variables that may contribute to differences in analytical results, some of which are clearly described by Lorian (2). For example, the nutrient media used in the assays may vary from batch to batch, creating differences in the quality of zone definition. Differences in the quality of the paper used in making the disks may affect the diffusion characteristics of the antibiotic, significantly changing potency determinations (3). Attempts have been made by this laboratory to eliminate many of the existing variables of the disk assay procedures. The investigations described in this paper were conducted to develop an improved disk assay procedure that employs an agar overlay much like that described by Barry et al. in their modification of the Kirby-Bauer procedure (1). Attempts were made to improve zone clarity and zone definition, to increase the response to standard doses of the drug, and to provide a simpler assay procedure. MATERIALS AND METHODS Petri dishes (20 by 150 mm) were prepared, each with a 42-mi nutrient agar base layer. The bacterial inoculum was carried in an 8-ml agar overlay layer. The agar layer was formulated to contain 15 g of granulated agar in 1 liter of distilled water and was sterilized at 121°C for 15 min, with a final pH between 6.9 and 7.0. Details of the individual assay method by the new technique are outlined in Table 1. The guidelines for the Code of Federal Regulations (CFR) assays are found in Section 460 of that document (4). Three filter paper disks, 0.25 inch (ca. 6.3 mm) in diameter (no. 740 E; supplied by Schleicher and
Schuell Co.), were impregnated with the antibiotic at three different doses and placed on the solidified agar surface. The petri dishes were incubated overnight (16 to 18 h) at temperatures of 32 or 37°C. The diameters of the zones of inhibition were measured with the aid of a model 3011 Bausch and Lomb Balopticon overhead projector. The slope of the regression line was calculated as follows: b = n xy - (x)(y) n X2
(X)2
where x is the logarithm of each standard dose (a log dose), y is the average response to each log dose, and n is number of standard doses. Two plates were prepared daily by both method A (CFR) and method B (agar overlay) for each antibiotic. Each plate contained three replicate disks of three standard drug concentrations. The data were collected from a 4-day test period for each antibiotic. Test cultures. The test organisms and their CFR numbers were: Staphylococcus aureus ATCC 6538P, 1; S. aureus ATCC 13150, 3; Sarcina lutea ATCC 9314, 4; Bacillus subtilis ATCC 6633, 5; Staphylococcus epidermidis ATCC 12228, 6; Bordetella bronchiseptica ATCC 4617, 7; Klebsiella pneumoniae ATCC 10031, 10; Streptococcus faecalis ATCC 14506, 11; and Pseudomonas aeruginosa ATCC 25619, 12. All suspensions were prepared as directed in Section 460.6b of the CFR (4). Nutrient media. The media used were: CFR medium A, antibiotic medium 1 (seed agar); medium C, antibiotic medium 11 (neomycin agar); medium G, antibiotic medium 10 (polymyxin-seed agar).
RESULTS Results of the assay of 35 antibiotics by two agar diffusion procedures indicate major differences between method A and method B. Results outlined in Table 1 show that the average slope value for each antibiotic was greater by the new method than by the CFR method. Each slope value represents the average of 24 determinations. 761
762
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VOL. 14, 1978
AGAR OVERLAY METHOD FOR ANTIBIOTIC DISK ASSAY
An example of this increased dose response is illustrated by the graph in Fig. 1. Zone clarity and defmition were improved in most assays. Improvements in zone clarity were particularly evident with carbenicillin, clindamycin, lincomycin, chloramphenicol, and oleandomycin. Changes in zone clarity with carbenicillin and clindamycin could be seen easily with the unaided eye. Improvement in zone definition was shown most dramatically through projection and magnification of the zones. The clarity of the zone definition is illustrated in Fig. 2.
A
B
N
24 i8 3 0mm 20 26 22 FIG. 1. Standard dose response lines of carbenicillin disk assay, illustrating significant increases in regression or slope values (b) and increases in the inhibition zone size. Line A, CFR method; line B, new agar overlay technique.
763
The agar overlay technique also exhibited an increase in the inhibition zone size for all the antibiotics tested except oxacillin. The decrease in nutrients may have permitted the antibiotic to diffuse more rapidly, thereby enhancing its ability to inhibit bacterial growth. Figure 1 illustrates the increase in the inhibition zone size of the antibiotics.
DISCUSSION In a two-layer agar diffusion assay system for susceptibility disks, the upper (seed) layer is important in the diffusion process because it forms a surface directly in contact with the antibiotic-impregnated disks. Because there may be some constituents in a medium which might interfere with the formation of a zone, the use of a nutrient-free layer may permit the antibiotic to diffuse more freely. The agar overlay technique eliminated the need of nutrient media for both layers of the two-layer system. Good quality control is needed to minimize problems associated with variations that could occur in batches of media for either nutrient layer. The agar overlay technique, however, eliminates some of these problems. Because the new technique employs only three types of nutrient media, the need for quality control of many different media is minimized. Improved zone clarity and zone definition in the new method results in more accurate measurement of zone diameters and in greater accuracy of potency determinations. The increase in response to standard doses minimizes the variability in potency determinations and results in greater assay precision, clearly demonstrating the superiority of the new method over that currently being used.
~~~~~~~ m _...... |h. FIG. 2. Differences in zone definition with carbenicillin and Pseudomonas aeruginosa ATCC 25619. (A) Media system A (CFR method), polybase agar = base layer, polyseed agar = seeded layer. (B) New agar overlay technique, seed agar = base layer, agar-agar = seeded layer.
764
ANDERSON
ACKNOWLEDGMENTS I thank the members of the Antibiotic Residue Branch, National Center for Antibiotics Analysis, Food and Drug Administration, Washington, D.C., for their help in collecting data and giving guidance in the preparation of this paper. Special thanks are extended to Gordon Carter, Ira Zimmerman, and Larry Ouderkirk. LITERATURE CITED 1. Barry, A. L., F. Garcia, and L. D. Thrupp. 1970. An improved single-disk method for testing antibiotic sus-
ANTIMICROB. AGENTS CHEMOTHER. ceptibility of rapidly-growing pathogens. Am. J. Clin. Pathol. 53:149-158. 2. Lorian, V. 1966. Antibiotics and chemotherapeutic agents in clinical and laboratory practice. Charles C Thomas, Publisher, Springfield, Ill. 3. Rippere, R. A. 1978. Effects of paper on performance of antibiotic-impregnated disks. J. Pharm. Sci. 67:367-371. 4. United States Government Printing Office. 1975. Code of Federal Regulations. Title 21, Section 460. United States Government Printing Office, Washington, D.C.
