ANTIMICROBiAL AGENTS AND CHEMOTHERAPY, May 1978, p. 842-844 0066-4804/78/0013-0842$02.00/0 Copyright X) 1978 American Society for Microbiology
Vol. 13, No. 5
Printed in U.S.A.
Evaluation of a Dispensing Instrument (Dynatech MIC-2000) for Preparing Microtiter Antibiotic Plates and Testing Their Potency During Storage PHILIP R. B. McMASTER,* E. ARTHUR ROBERTSON, FRANK G. WITEBSKY, AND JAMES D. MAcLOWRY Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20014
Received for publication 20 December 1977
The MIC-2000 96-channel dispenser was evaluated for accuracy and repeatability of dispensing 50-pl volumes of water. It was found to perform within the manufacturer's specifications for accuracy of ±5%, but not within the limits for repeatability of ±1%. Overall, instrument performance was found to be satisfactory for the antimicrobial susceptibility testing technique which has been implemented in this laboratory. A method is suggested for simple evaluation of the volumes dispensed. A variety of antibiotics may be stored in Trypticase soy broth in Microtiter plates without loss of potency for periods of at least 2 weeks at -20°C and 4 months at -700C.
Advances in the technology of antimicrobial susceptibility testing of bacteria have made routine quantitative testing in hospital laboratories possible (1-9). The Dynatech MIC-2000 system (Dynatech Laboratories, Inc., Alexandria, Va.) was designed to facilitate such procedures in a routine laboratory (Instruction Manual 96Channel Dispenser). Few studies are available to document the performance of this system. Accordingly, the Dynatech MIC-2000 dispenser, which is an integral part of the whole system, was evaluated to determine the accuracy and reproducibility of the fluid quantity it dispensed into each of the 96 wells of a Microtiter plate. In addition, a selected battery of antibiotics was dispensed, frozen, and stored at -20 and -70°C. These antibiotics were examined at subsequeint intervals to determine their potency. MATERIALS AND METHODS Apparatus. As described in the Instruction Manual provided with the instrument, the MIC-2000 dispenser (Dynatech) is a manually fed dispenser which transfers accurate preset volumes of up to 96 different aqueous solutions from test tubes (18 by 150 mm) to individual wells of a modified Microtiter plate. Although the instrument will dispense 50- or 100-pl amounts, only the 50-pl volume was evaluated in this study. Dispensed volumes. For testing the amount dispensed into each well, a metal container filled with water was inserted into the pressure chamber in place of the test tube rack. To assure that the instrument was accurately primed, successive jets of water were dispensed into the base of the drawer plate holder at least 10 times, and allowed to drain away, before
collection in the Microtiter plates was started. The instrument was then operated to dispense 50 pl once into each well of a series of Microtiter plates. After dispensing, the trays were covered with Scotch Tape to prevent evaporation until the volume in the wells could be measured later in the same day. To measure the volume in each well, a 50-pl disposable capillary pipette was attached to a plastic suction tube. The fluid in a well was then aspirated into the pipette, and the length of the water column was measured to the nearest millimeter. This method of measuring the volume was evaluated by placing 50 Al of water in each of 20 wells with a Lang-Levy pipette. The contents of each well were aspirated into the disposable capillary pipette. Length of the fluid column was measured to the nearest millimeter, and it measured 76 mm in each instance. After each measurement, no visible fluid remained in the well. Altogether, 11 plates were analyzed. These were selected from five runs made over a period of 4 weeks. In the course of these studies, an occasional error in sampling a well occurred, in which case no measurement was obtained for the volume in that particular well of that particular plate. Consequently, the volume in each of the corresponding wells of the 11 plates was
measured on 9 to 11 occasions. Antibiotics. A selected battery of antibiotics was dissolved in buffer as recommended (4), except for streptomycin which was dissolved in distilled water. These were then mixed with Trypticase soy broth (TSB) to make the desired final concentrations. The specific antibiotics used and the concentrations tested appear in Table 2. After each solution was prepared, it was frozen at -70°C. When all solutions were prepared, they were thawed, put in test tubes, and placed in the pressure chamber rack. The solutions were then dispensed into Microtiter plates. Some of -the plates were tested immediately for antibiotic potency, and others were either frozen at -20 or at -70°C. After 842
DYNATECH MIC-2000 PLATE DISPENSER
VOL. 13, 1978
having been frozen at -20°C, selected plates were thawed and the antibiotics were tested at intervals up to 14 days. Those frozen at -70°C were examined at intervals up to 112 days. To test the activity of the antibiotics in the wells, 20-pl samples from each well were aspirated by using an Eppendorf pipette and disposable plastic tips. Paper disks (no. 740-E, 6.35-mm diameter, Schleicher and Schuell, Keene, N.H.) were placed on a MuellerHinton agar plate that had been seeded with a layer of Staphylococcus aureus (NIH-289). The inoculum was prepared by making a 1:1,000 dilution in TSB of an overnight culture in TSB. A 5-ml amount of this inoculum was flooded on the plate and allowed to stand for 1 min, and then the excess was aspirated. The plates were allowed to dry for approximately 30 min with the lids ajar. After a disk was placed on the seeded agar, a sample from one of the wells was placed on the disk. A 20-pl fraction was discharged onto that disk before placement of the next disk on the agar. Duplicate samples were obtained from each well. The plates were then incubated at 37°C for 18 h. The diameter of the zone of inhibition of growth was measured to the nearest 0.1 mm with vernier calipers. Commercial 1-,ug oxacilhin disks were used as a control for each run. RESULTS
The volume of fluid dispensed into each well examined first. Since 50 pl extended for 76 within the capillary pipette, each measurement was expressed in millimeters of pipette occupied, rather than by converting that measurement to a volume. The mean length occupied by the volume of fluid dispensed into all the wells of all the plates was 77.0 mm, with a standard deviation of 3.4 mm. This falls within the manufacturer's specified limits of ±5% for accuracy. However, none of the wells met the manufacturer's specified limits of ±1% repeatability. The coefficients of variation ranged from 1.4 to 10.5%. Table 1 shows a statistical summary of the system's repeatability. The overall coefficient of variation (calculated over all days, all plates, and all positions) was only 4.4%. An analysis of variance indicates that random variawas mm
843
TABLE 2. Stability studies offrozen antibioticsa SD of
CV of
zone
size
zone M
(jAg/ml)
Mean diam of zone size
Ampicillin Ampicillin Ampicillin Ampicillin
2 10 20 200
13.8 24.3 28.4 38.5
1.3 1.0 0.9 1.5
9.4 4.1 3.2 3.9
Kanamycin Kanamycin Kanamycin
50 200 800
10.5 17.1 22.3
1.0 1.0 1.0
9.5 5.8 4.5
Cephalothin Cephalothin Cephalothin
20 100 200
24.8 32.8 35.9
1.1 1.1 1.1
4.4 3.4 3.1
1
1.5 1.1 1.0
9.0 3.6 2.3
Concn
Antibiotic
(nn
Simsizee
Penicillin G Penicillin G Penicillin G
200
16.7 30.3 42.8
Gentamicin Gentamicin
12 48
11.7 17.0
0.7 1.0
6.0 5.9
Lincomycin
10 20
11.2 14.3
1.2 1.2
10.7 8.4
1000
20.5
1.2
5.9
Tobramycin Tobramycin
10 50
9.8 16.5
2.2 1.1
22.4 6.7
Clindamycin Clindamycin Clisdamycin
2 10 20
12.0 18.5 21.7
1.1 1.5 1.3
9.2 8.1 6.0
Oxacilhin
10
22.1
1.1
5.0
40 400
25.1 35.7
0.8 1.2
3.2 3.4
5
14.3
1.0
6.6
10
Streptomycin
Carbenicillin Carbenicillin
Erythromycin
a Combined data from antibiotics stored for 2 weeks at -20°C and for 4 months at -70°C. SD, Standard deviation; CV, coefficient of variation.
TABLE 1. Evaluation of dispensed volumea Variation
Mean (mm)
SDb (mm)
Among all plates Plate with lowest mean Plate with highest mean (also highest CV) Plate with lowest CV
76.99 74.70 79.06
3.4 2.5 4.2
77.24
2.5
Least amt
Greatest
4.4 3.3 5.3
63 68 65
96 81 96
3.2
72
84
CV
Among wells 74 1.4 1.0 75.64 Well with least 73 10.5 9.2 88.50 Well with greatest in a column aVolume is expressed as length in millimeters of a fluid capillary pipette. b SD, Standard deviation. c CV, Coefficient of variation.
78 96
844
McMASTER ET AL.
tion and position-to-position differences accounted for the largest portion of the observed variability, although there was a smaller but statistically significant contribution from plateto-plate differences. Linear-regression analysis of the data indicated that there was a minimal increase in average measured volume on repeated runs. This increase averaged 0.1 mm in the length of the column of fluid per well per run from plate 1 to 11, with a total increase of 1.1 mm. The probability that such a difference would be observed by chance is 0.002. Whereas the overall coefficient of variation was 4.4%, individual positions had coefficients of variation ranging from 1.4 to 6.2%, excluding a single well whose value was 10.5% (Table 1). At selected intervals after the antibiotics had been frozen in the Microtiter plates, a sample plate was thawed. The potency of the thawed antibiotics was evaluated by measuring the diameter of the zone of inhibition of bacterial growth. The mean of the diameter of the zones of inhibition obtained with each concentration of antibiotic stored at -20 and -70°C were within 1 standard deviation of each other. Accordingly, the data obtained at both temperatures were combined for the sake of brevity and simplicity and are presented in Table 2. A regression analysis was performed on the data to determine if the zone size produced by the antibiotics changed during storage. The analysis indicated a minimal increase in the diameter of the inhibition zone during the study. This increase was statistically significant in only tl*ee instances and even then only at the 5% level. A similar control analysis was performed by using the zone size produced by commercial oxacillin disks each day the stored antibiotics were tested. The mean of all these 36 oxacillin tests was 29.1 mm, and the standard deviation was 1.7 mm. Here, too, the regression analysis showed a trend of increasing zone size with time. The rate of this increase was calculated to average 0.01 mm per trial. Despite its small magnitude, this was statistically significant at the 1% level.
DISCUSSION The MIC-2000 96-channel dispenser was found to deliver a reasonably constant volume of fluid within runs and from day to day. Presumably by correcting the delivery system of the one well that was consistently high and variable in its volume on our instrument, the system would give more repeatable results overall. The instrument's delivery pattern may be readily checked from time to time by the method described above. The time required is approxi-
ANTIMICROB. AGENTS CHEMOTHER.
mately 90 min to check the volumes in the 96 wells of 1 plate. When the instrument is used as part of the whole MIC-2000 system, small volumes of bacterial inoculum are added to each well. Under these circumstances, there is only a minimal dilution of the solution dispensed into the wells, so that exact volume dispensed is not critical. However, when a larger volume of inoculum is used, as occurs when using a dropping pipette, then the accuracy of the initial volume of antibiotic solution in the well becomes much more significant. Although the drift from run to run seems to be so small as to be unimportant, it could theoretically become significant over a period of prolonged use. It is probably advisable, therefore, to make such volume measurements periodically. The potency of the antibiotics tested remained stable during storage at least for 2 weeks at -20°C and for 4 months at -70°C. The small increase in the inhibition zone diameter was accompanied by a parallel increase in zone size produced by commercial oxacillin disks. This change in the control antibiotic zone size suggests that the small increase in the zone size of the stored antibiotic solutions did not reflect an increase in potency, but rather a subtle change of undetermined nature over time. LITERATURE CITED 1. Barry, A. L, F. Garcia, and L. D. Thrupp. 1970. An improved single disc method for testing the antibiotic susceptibility of rapidly growing pathogens. Am. J. Clin. Pathol. 64:149-158. 2. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turek. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45:493-496. 3. Foster, J. W., and H. B. Woodruff. 1943. Microbiological aspects penicillin. I. Methods of assay. J. Bacteriol. 46:187-202. 4. MacLowry, J. D., M. J. Jaqua, and S. T. Selepak. 1970. Detailed methodology and implementation of a semi-automated serial dilution microtechnique for antimicrobial susceptibility testing. Appl. Microbiol. 20:46-53. 5. MacLowry, J. D., and H. H. Marsh. 1968. Semiautomatic microtechnique for serial dilution-antibiotic testing in the clinical laboratory. J. Lab. Clin. Med. 72:685-687. 6. Marymont, J. H., Jr., and R. M. Wentz. 1966. Serial dilution antibiotic sensitivity testing with the microtitrator system. Am. J. Clin. Pathol. 45:548-551. 7. Schwartz, L. H., and B. A. Brown. 1954. An improved rapid method of tube dilution antibiotic sensitivity testing. Antibiot. Chemother. 4:333-337. 8. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 9. Vincent, J. G., and H. W. Vincent. 1944. Filter paper disc modification of the Oxford cup penicillin determination. Proc. Soc. Exp. Biol. Med. 55:162-164.
Vol. 13, No. 5
Printed in U.S.A.
Evaluation of a Dispensing Instrument (Dynatech MIC-2000) for Preparing Microtiter Antibiotic Plates and Testing Their Potency During Storage PHILIP R. B. McMASTER,* E. ARTHUR ROBERTSON, FRANK G. WITEBSKY, AND JAMES D. MAcLOWRY Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20014
Received for publication 20 December 1977
The MIC-2000 96-channel dispenser was evaluated for accuracy and repeatability of dispensing 50-pl volumes of water. It was found to perform within the manufacturer's specifications for accuracy of ±5%, but not within the limits for repeatability of ±1%. Overall, instrument performance was found to be satisfactory for the antimicrobial susceptibility testing technique which has been implemented in this laboratory. A method is suggested for simple evaluation of the volumes dispensed. A variety of antibiotics may be stored in Trypticase soy broth in Microtiter plates without loss of potency for periods of at least 2 weeks at -20°C and 4 months at -700C.
Advances in the technology of antimicrobial susceptibility testing of bacteria have made routine quantitative testing in hospital laboratories possible (1-9). The Dynatech MIC-2000 system (Dynatech Laboratories, Inc., Alexandria, Va.) was designed to facilitate such procedures in a routine laboratory (Instruction Manual 96Channel Dispenser). Few studies are available to document the performance of this system. Accordingly, the Dynatech MIC-2000 dispenser, which is an integral part of the whole system, was evaluated to determine the accuracy and reproducibility of the fluid quantity it dispensed into each of the 96 wells of a Microtiter plate. In addition, a selected battery of antibiotics was dispensed, frozen, and stored at -20 and -70°C. These antibiotics were examined at subsequeint intervals to determine their potency. MATERIALS AND METHODS Apparatus. As described in the Instruction Manual provided with the instrument, the MIC-2000 dispenser (Dynatech) is a manually fed dispenser which transfers accurate preset volumes of up to 96 different aqueous solutions from test tubes (18 by 150 mm) to individual wells of a modified Microtiter plate. Although the instrument will dispense 50- or 100-pl amounts, only the 50-pl volume was evaluated in this study. Dispensed volumes. For testing the amount dispensed into each well, a metal container filled with water was inserted into the pressure chamber in place of the test tube rack. To assure that the instrument was accurately primed, successive jets of water were dispensed into the base of the drawer plate holder at least 10 times, and allowed to drain away, before
collection in the Microtiter plates was started. The instrument was then operated to dispense 50 pl once into each well of a series of Microtiter plates. After dispensing, the trays were covered with Scotch Tape to prevent evaporation until the volume in the wells could be measured later in the same day. To measure the volume in each well, a 50-pl disposable capillary pipette was attached to a plastic suction tube. The fluid in a well was then aspirated into the pipette, and the length of the water column was measured to the nearest millimeter. This method of measuring the volume was evaluated by placing 50 Al of water in each of 20 wells with a Lang-Levy pipette. The contents of each well were aspirated into the disposable capillary pipette. Length of the fluid column was measured to the nearest millimeter, and it measured 76 mm in each instance. After each measurement, no visible fluid remained in the well. Altogether, 11 plates were analyzed. These were selected from five runs made over a period of 4 weeks. In the course of these studies, an occasional error in sampling a well occurred, in which case no measurement was obtained for the volume in that particular well of that particular plate. Consequently, the volume in each of the corresponding wells of the 11 plates was
measured on 9 to 11 occasions. Antibiotics. A selected battery of antibiotics was dissolved in buffer as recommended (4), except for streptomycin which was dissolved in distilled water. These were then mixed with Trypticase soy broth (TSB) to make the desired final concentrations. The specific antibiotics used and the concentrations tested appear in Table 2. After each solution was prepared, it was frozen at -70°C. When all solutions were prepared, they were thawed, put in test tubes, and placed in the pressure chamber rack. The solutions were then dispensed into Microtiter plates. Some of -the plates were tested immediately for antibiotic potency, and others were either frozen at -20 or at -70°C. After 842
DYNATECH MIC-2000 PLATE DISPENSER
VOL. 13, 1978
having been frozen at -20°C, selected plates were thawed and the antibiotics were tested at intervals up to 14 days. Those frozen at -70°C were examined at intervals up to 112 days. To test the activity of the antibiotics in the wells, 20-pl samples from each well were aspirated by using an Eppendorf pipette and disposable plastic tips. Paper disks (no. 740-E, 6.35-mm diameter, Schleicher and Schuell, Keene, N.H.) were placed on a MuellerHinton agar plate that had been seeded with a layer of Staphylococcus aureus (NIH-289). The inoculum was prepared by making a 1:1,000 dilution in TSB of an overnight culture in TSB. A 5-ml amount of this inoculum was flooded on the plate and allowed to stand for 1 min, and then the excess was aspirated. The plates were allowed to dry for approximately 30 min with the lids ajar. After a disk was placed on the seeded agar, a sample from one of the wells was placed on the disk. A 20-pl fraction was discharged onto that disk before placement of the next disk on the agar. Duplicate samples were obtained from each well. The plates were then incubated at 37°C for 18 h. The diameter of the zone of inhibition of growth was measured to the nearest 0.1 mm with vernier calipers. Commercial 1-,ug oxacilhin disks were used as a control for each run. RESULTS
The volume of fluid dispensed into each well examined first. Since 50 pl extended for 76 within the capillary pipette, each measurement was expressed in millimeters of pipette occupied, rather than by converting that measurement to a volume. The mean length occupied by the volume of fluid dispensed into all the wells of all the plates was 77.0 mm, with a standard deviation of 3.4 mm. This falls within the manufacturer's specified limits of ±5% for accuracy. However, none of the wells met the manufacturer's specified limits of ±1% repeatability. The coefficients of variation ranged from 1.4 to 10.5%. Table 1 shows a statistical summary of the system's repeatability. The overall coefficient of variation (calculated over all days, all plates, and all positions) was only 4.4%. An analysis of variance indicates that random variawas mm
843
TABLE 2. Stability studies offrozen antibioticsa SD of
CV of
zone
size
zone M
(jAg/ml)
Mean diam of zone size
Ampicillin Ampicillin Ampicillin Ampicillin
2 10 20 200
13.8 24.3 28.4 38.5
1.3 1.0 0.9 1.5
9.4 4.1 3.2 3.9
Kanamycin Kanamycin Kanamycin
50 200 800
10.5 17.1 22.3
1.0 1.0 1.0
9.5 5.8 4.5
Cephalothin Cephalothin Cephalothin
20 100 200
24.8 32.8 35.9
1.1 1.1 1.1
4.4 3.4 3.1
1
1.5 1.1 1.0
9.0 3.6 2.3
Concn
Antibiotic
(nn
Simsizee
Penicillin G Penicillin G Penicillin G
200
16.7 30.3 42.8
Gentamicin Gentamicin
12 48
11.7 17.0
0.7 1.0
6.0 5.9
Lincomycin
10 20
11.2 14.3
1.2 1.2
10.7 8.4
1000
20.5
1.2
5.9
Tobramycin Tobramycin
10 50
9.8 16.5
2.2 1.1
22.4 6.7
Clindamycin Clindamycin Clisdamycin
2 10 20
12.0 18.5 21.7
1.1 1.5 1.3
9.2 8.1 6.0
Oxacilhin
10
22.1
1.1
5.0
40 400
25.1 35.7
0.8 1.2
3.2 3.4
5
14.3
1.0
6.6
10
Streptomycin
Carbenicillin Carbenicillin
Erythromycin
a Combined data from antibiotics stored for 2 weeks at -20°C and for 4 months at -70°C. SD, Standard deviation; CV, coefficient of variation.
TABLE 1. Evaluation of dispensed volumea Variation
Mean (mm)
SDb (mm)
Among all plates Plate with lowest mean Plate with highest mean (also highest CV) Plate with lowest CV
76.99 74.70 79.06
3.4 2.5 4.2
77.24
2.5
Least amt
Greatest
4.4 3.3 5.3
63 68 65
96 81 96
3.2
72
84
CV
Among wells 74 1.4 1.0 75.64 Well with least 73 10.5 9.2 88.50 Well with greatest in a column aVolume is expressed as length in millimeters of a fluid capillary pipette. b SD, Standard deviation. c CV, Coefficient of variation.
78 96
844
McMASTER ET AL.
tion and position-to-position differences accounted for the largest portion of the observed variability, although there was a smaller but statistically significant contribution from plateto-plate differences. Linear-regression analysis of the data indicated that there was a minimal increase in average measured volume on repeated runs. This increase averaged 0.1 mm in the length of the column of fluid per well per run from plate 1 to 11, with a total increase of 1.1 mm. The probability that such a difference would be observed by chance is 0.002. Whereas the overall coefficient of variation was 4.4%, individual positions had coefficients of variation ranging from 1.4 to 6.2%, excluding a single well whose value was 10.5% (Table 1). At selected intervals after the antibiotics had been frozen in the Microtiter plates, a sample plate was thawed. The potency of the thawed antibiotics was evaluated by measuring the diameter of the zone of inhibition of bacterial growth. The mean of the diameter of the zones of inhibition obtained with each concentration of antibiotic stored at -20 and -70°C were within 1 standard deviation of each other. Accordingly, the data obtained at both temperatures were combined for the sake of brevity and simplicity and are presented in Table 2. A regression analysis was performed on the data to determine if the zone size produced by the antibiotics changed during storage. The analysis indicated a minimal increase in the diameter of the inhibition zone during the study. This increase was statistically significant in only tl*ee instances and even then only at the 5% level. A similar control analysis was performed by using the zone size produced by commercial oxacillin disks each day the stored antibiotics were tested. The mean of all these 36 oxacillin tests was 29.1 mm, and the standard deviation was 1.7 mm. Here, too, the regression analysis showed a trend of increasing zone size with time. The rate of this increase was calculated to average 0.01 mm per trial. Despite its small magnitude, this was statistically significant at the 1% level.
DISCUSSION The MIC-2000 96-channel dispenser was found to deliver a reasonably constant volume of fluid within runs and from day to day. Presumably by correcting the delivery system of the one well that was consistently high and variable in its volume on our instrument, the system would give more repeatable results overall. The instrument's delivery pattern may be readily checked from time to time by the method described above. The time required is approxi-
ANTIMICROB. AGENTS CHEMOTHER.
mately 90 min to check the volumes in the 96 wells of 1 plate. When the instrument is used as part of the whole MIC-2000 system, small volumes of bacterial inoculum are added to each well. Under these circumstances, there is only a minimal dilution of the solution dispensed into the wells, so that exact volume dispensed is not critical. However, when a larger volume of inoculum is used, as occurs when using a dropping pipette, then the accuracy of the initial volume of antibiotic solution in the well becomes much more significant. Although the drift from run to run seems to be so small as to be unimportant, it could theoretically become significant over a period of prolonged use. It is probably advisable, therefore, to make such volume measurements periodically. The potency of the antibiotics tested remained stable during storage at least for 2 weeks at -20°C and for 4 months at -70°C. The small increase in the inhibition zone diameter was accompanied by a parallel increase in zone size produced by commercial oxacillin disks. This change in the control antibiotic zone size suggests that the small increase in the zone size of the stored antibiotic solutions did not reflect an increase in potency, but rather a subtle change of undetermined nature over time. LITERATURE CITED 1. Barry, A. L, F. Garcia, and L. D. Thrupp. 1970. An improved single disc method for testing the antibiotic susceptibility of rapidly growing pathogens. Am. J. Clin. Pathol. 64:149-158. 2. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turek. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45:493-496. 3. Foster, J. W., and H. B. Woodruff. 1943. Microbiological aspects penicillin. I. Methods of assay. J. Bacteriol. 46:187-202. 4. MacLowry, J. D., M. J. Jaqua, and S. T. Selepak. 1970. Detailed methodology and implementation of a semi-automated serial dilution microtechnique for antimicrobial susceptibility testing. Appl. Microbiol. 20:46-53. 5. MacLowry, J. D., and H. H. Marsh. 1968. Semiautomatic microtechnique for serial dilution-antibiotic testing in the clinical laboratory. J. Lab. Clin. Med. 72:685-687. 6. Marymont, J. H., Jr., and R. M. Wentz. 1966. Serial dilution antibiotic sensitivity testing with the microtitrator system. Am. J. Clin. Pathol. 45:548-551. 7. Schwartz, L. H., and B. A. Brown. 1954. An improved rapid method of tube dilution antibiotic sensitivity testing. Antibiot. Chemother. 4:333-337. 8. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 9. Vincent, J. G., and H. W. Vincent. 1944. Filter paper disc modification of the Oxford cup penicillin determination. Proc. Soc. Exp. Biol. Med. 55:162-164.
