streptomycin in body fluids (Stebbins and Robinson, 1945). A slide-cell technique

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1 ASSAY OF STREPTOMYCIN BY THE PAPER-DISC PLATE METHOD Y. H. LOO, P. S. SKELL, AND H. H. THORNBERRY University of Illinois, Urbana, Illinois JOHN EHRLICH Parke, Davis and Company, Detroit, Michigan J. M. McGUIRE Eli Lilly and Company, Indianapolis, Indiana G. M. SAVAGE The Upjohn Company, Kalamazoo, Michigan J. C. SYLVESTER Abbott Laboratories, North Chicago, Illinois Received for publication September 7, 1945 The potential importance of streptomycin (Schatz et at., 1944) as a therapeutic agent has made the study of this antibiotic an urgent problem. In order to expedite progress the Abbott, Eli Lilly, Parke Davis, and Upjohn research laboratories have co-operated in a joint attack on the problem, and the Departments of Chemistry and Horticulture of the University of Illinois have contributed to certain phases of the work. One of the first problems to be investigated was that of developing an assay method which would be satisfactory, not only for beers, but also for solutions of streptomycin containing organic solvents and reagents. Waksman and coworkers have used the agar-dilution method in defining the currently accepted Escherichia coli streptomycin unit (Schatz et al., 1944). The agar-diffusion method has been recommended for an assay of potency (Waksman, 1945) and has been applied to the determination of streptomycin in body fluids (Stebbins and Robinson, 1945). A slide-cell technique for the assay of body fluids also has been described (Heilman, 1945). Since considerable difficulty was encountered in obtaining consistent results with the agar-diffusion method, it seemed desirable to make our experiences available to other workers in the field. In this paper we are presenting studies on the important sources of variation and the procedure currently employed in our laboratories for the assay of streptomycin. PROCEDURE Medium. Bacto-streptomycin assay agar, dehydrated, was prepared by the Difco Laboratories, Inc., in collaboration with our group, to provide a supply of a standard, uniform medium for the assay of streptomycin.1 The medium is prepared for use by dissolving 25.5 g per 1,000 ml in doubly distilled water. After sterilization the medium may be stored at 2 to 4 C until used. 1 The authors gratefully acknowledge the co-operation of Mr. H. G. Dunham of the Difco Laboratories, Inc., in making this medium available. 701

2 702 LOO ET AL. The medium as prepared for use has the following composition: Peptone g Beef extract g Yeast extract g Agar g Distilled water, q.s... 1,000.0 ml ph after sterilization :1 0.1 Organism. A strain of Bacillus subtilis sensitive to streptomycin is employed as the test organism. A stock spore suspension is prepared by cultivation of the organisms on agar or in submerged culture. When microscopic examination reveals good sporulation, the cells are separated from the medium, suspended in sterile 0.05 M potassium phosphate buffer, ph 7.0, and the suspension pasteurized to kill the vegetative cells. A viable spore count is made by plating. The stock spore suspension is stored at 2 to 4 C and used as needed. Preparation of plates. To petri dishes (pyrex dishes, 100 mm in diameter, selected for uniform flat bottoms2) are added 20.0 ml of sterle assay agar, preferably with an automatic dispensing pipette. The agar is allowed to harden with the petri dish tops tilted or removed. While the plates are still warm, 4.0 ml of seeded agar is added and distributed evenly over the surface. The volume of spore suspension used to seed the medium depends on the concentration of viable spores. The amount to be used is best determined by actual test in the assay procedure, that quantity being selected which gives large, sharp zones. Usually a concentration of approximately 250,000 spores per ml in the seeded agar gives the desired results. The spores are added to the liquefied agar at 60 C, and. the seeded medium is maintained at that temperature until all the plates are poured. The 'plates are stored at 2 to 4 C as soon as they have hardened and may be kept for several days with no effect on the assay. It is essential that the plates be prepared on an absolutely level table top. A section of heavy plate glass or marble supported on adjustable legs and equipped rwith a level indicator serves adequately for this purpose. Preparation of the sample. All samples are diluted on the basis of their estimated potency to fall on the standard curve. The initial dilution of liquid samples is made with an equal volume of 0.2 M potassium phosphate buffer, ph 7.9, and all subsequent dilutions with 0.1 M buffer. Solid samples are dissolved directly in 0.1 M buffer. Samples of submerged-culture beers are not clarified before dilution. Setting up the assay. Filter paper discs' are placed, flat side down, on the agar plates. As each disc is placed, an ml sample is immediately (within 5 seconds) pipetted onto it. Since the disc rapidly absorbs moisture from the agar it is exceedingly important that the sample be applied in the shortest posp. sible time. As the sample is being delivered the disc is gently pressed to the agar with the tip of the pipette. Four to six discs may be placed on one plate sym- 2Pressed petri dishes with flat bottoms are being developed by the Corning Glass Works. The use of such dishes will be of considerable advantage. 3Schleicher and Schuell no. 740E i-inch filter paper discs.

3 ASSAY OF STREPTOMYCIN BY PAPER-DISC PLATE METHOD metrically arranged around the center. All discs should be equidistant and not less than 10 mm from the edge. A standard is included on each plate. Two to four replicates of each unknown are run on one or more plates z 25 w zo 24 N wo 22- c) 21~ 20[ 20 FIG ~ 19 I liq *-0 I UNITS PER ML TYPICAL DAILY STANDARD CURVE OF STREPTOMYCIN STANDARD JR4S2 ON DIFCo STREPTOmyYCIN ASSAY AGAR WITH 30 C INCUBATION Plates containing the streptomycin standard are prepared daily, four to eight replicates of each of six dilutions of the standard being used. The concentration range of the standard depends on the other conditions of the procedure and is chosen7to obtain a range of zone diameters of from 20 to 30m. Under optimum conditions a concentration as low as 0.5 unit per ml (zone diameter 16 to 18 mm) can be determined. Not more than four to six plates are removed from the refrigerator at one

4 704 LOO ET AL. time. These are handled as rapidly as possible and placed in the incubator as soon as they are completed (usual elapsed time, 15 minutes). They may be inverted during refrigeration and incubation. Incubation of plates. The plates are incubated at 30 C for a minimum of 15 hours. After this time no significant change in the size of the zone occurs up to 30 hours of incubation. At 37 C the zone size is reduced, and on long incubation the organism tends to overgrow the cleared area. However, the zones develop more rapidly at 37 C, and the readings may be made after 4 to 6 hours of incubation, thus affording a shorter assay time. Estimation of potency. The diameters of the zones of inhibition are measured to the closest one-quarter millimeter, and replicates are averaged. A daily curve is prepared from the dilutions of the standard by plotting the zone diameters in mm against the concentration in units per ml. The potency of the unknown samples is determined from the standard curve. A typical standard curve is illustrated in figure 1. STUDIES AND DISCUSSION In the development of a practical assay it was found that many factors exert a considerable influence on the range, sensitivity, and precision of the method. It is obvious that the inherent antibacterial activity of streptomycin, the growth rate of the organism, or the rate of diffusion of streptomycin in the agar may be affected by one or more of these factors. Further studies revealed that certain of the factors are of critical importance in obtaining consistent results. A brief discussion of these studies is presented here. Composition of assay medium. Different brands and even different lots of 'the same brand of meat extract and peptone were found to produce marked differences in zone size. Variation in the concentration of these components was found to have the same effect. The addition of glucose to the medium caused a reduction in the area of inhibition. The very marked effect of variation in the ph of the assay agar is shown in figure 2. Since the sensitivity increases with the alkalinity of the medium, it was desired to use a medium of the highest practical ph and a value of 7.9 j 0.1 was adopted. ph of sample. The zone size obtained with a solution of constant streptomycin concentration increases markedly with increasing ph of the solution. Since the assay agar has very little buffer capacity, it is necessary to control the ph of the samples to be assayed. For this purpose it is convenient to adjust the ph of the samples by making all dilutions in phosphate buffer, ph 7.9 d 0.1, to a final concentration of 0.1 M buffer. The ph of the samples applied to the discs must be in the range 7.6 to 8.0. Solutions containing 0.1 N acid must be diluted at least 1:20 with buffer if this ph is to be reached. At lower dilutions the samples should be adjusted to ph 7.6 to 8.0 with sodium hydroxide before dilution. It is essential to have a constant buffer concentration because zone diameters increase with concentration of buffer. Obviously the standard must be prepared in the same way.

5 ASSAY OF STREPTOMYCIN BY PAPER-DISC PLATE METHOD 705 Volume of sample. The volume of the sample applied to the disc is of critical importance, as shown in table 1. These data indicate the necessity for measuring the sample accurately. The volume applied should be sufficient to saturate the disc, yet not so large that the paper will not absorb rapidly the amount delivered from the pipette. A volume of ml is satisfactory with the Schleicher and Schuell paper discs. Accurate and rapid delivery of this amount of sample is best obtained by the use of a calibrated pipette. Transfer of the sample by FIG , 25 wi z 24 0 N 23 U t 20 I- IL 19 4 _ IS!!le Is ph u UNITS PER ML INFLUENCE OF ph OF ASSAY AGAR UPON ZONE SIZE WHEN Six LEVELS OF THE SAME STANDARD, ALL AT ph 7.8, ARE ASSAYED means of a wire loop was early found to be unsatisfactory because of differences in surface tension of beers, extracts, and solutions containing organic solvents. Influence of salts.' Since Foster and Woodruff ( ) reported that phosphate and other salts suppressed the activity of streptothricin against Escherichia coli in nutrient broth, a study was made of the effect of salts on the antibacterial activity of streptomycin. For this purpose the regular assay medium was used, and the streptomycin samples were made up in aqueous solutions containing various concentrations of the salts. The addition of phosphate to the streptomycin solutions caused a marked increase in the size of the zones of inhibition (compare controls with and without phosphate in table 2). An,X I5

6 706 LOO ET AL. enhancing effect was also shown by sodium chloride, acetate, bicarbonate, and sulfate, potasium chloride and bicarbonate, and lithium chloride and sulfate. However, in the presence of 0.1 M phosphate buffer the effect of these salts is TABLE 1 Effect of variation in the volume of sample applied to the disc on the diameter of the zone of inhibition VOLUME 01 STREPOMYCn SOLUTION APPLIED TO DISC RNG si (MEAN ow 13) ml mm TABLE 2 Influence of phosphate buffer and sodium chloride on assay of streptomycin Each sample contained identical concentrations of streptomycin CONCENTRATION O SAPLE COMONENT ANTACTERIL ACTIVI Sodium chloride* Phosphate buffer Average of three days Above control per cenot moarity units/mlt Po ceo Control Control * In the absence of streptomycin, 0.05 to 5.00 per cent sodium chloride produced no inhibition when assayed directly. t Zone sizes were converted to units per ml by reading off the corresponding values from a typical streptomycin standard phosphate curve, as for example figure 1. minimized except at high concentrations (table 2). This is another advantage afforded by the use of phosphate buffer in the assay samples. Influence of organic reagente. The presence in the diluted samples of either 60 per cent methanol, 40 per cent ethanol, 40 per cent acetone, 10 per cent dioxan, or 5 per cent pyridine has little or no effect on the assay values. Dioxan at a

7 ASSAY OF STREPTOMYCIN BY PAPER-DISC PLATE METHOD 707 level of 25 per cent gives a marked decrease in potency, and pyridine at a level of 10 per cent completely inhibits the growth of the test organism. Picric acid, acetic acid, and a variety of sulfonic acids do not interfere with the assay of streptomycin. Comparison of paper discs and penicylinders.4 Paper discs were found to have certain advantages over penicylinders as a means of applying the sample to the plate. Solutions containing organic solvents (methanol, ethanol, acetone, dioxan) give high and erratic results when assayed by the penicylinder method, whereas these substances have little or no effect on the paper-disc assay. The discs also avoid errors inherent in the cylinder method such as variable diffusion from inconsistently applied cylinders and leakage from chipped or dented cylinders. The placing of the discs on the plates is simple and convenient. The plates can be' handled with no special precautions and incubated in an inverted position. One critical point in the use of the paper discs is the necessity of applying the sample rapidly and accurately to the discs. However, this technique is easily mastered. The test organism. Other species of bacteria including Serratia marcescens, Aerobacter aerogenes, Escherichia coli, Proteus vulgaris, and Bacillus mycoides have been tested for use in the assay of streptomycin. Of the organisms tested B. subtilis has proved to be the most satisfactory. Assay of beers. It was found in the assay of submerged-culture beers that removal of the mycelium by centrifugation or filtration results in a reduction of the potency of the sample. This reduction does not occur if the samples' are acidified to ph 2 before clarification. We routinely assay submerged-culture beers unclarified, since the values thus obtained check closely with the values for acidified, clarified beers. The streptomycin standard. The streptomycin standard used in our laboratories is a partially purified preparation which was arbitrarily assigned a potency. The streptomycin unit which was decided upon approximates the activity of Waksman's5 E. coli streptomycin unit (Schatz et al., 1944). Subsequent comparisons of our standard with other streptomycin preparations indicate that our unit is reasonably close to that used by other laboratories. A large quantity of the standard preparation was dispensed into small vials, lyophile-dried, and stored at 2 to 4 C. This material has been used for all streptomycin assays in our laboratories. Although factors other than those discused in this report may influence the assay values, it is possible to obtain consistent results if the known variables are rigidly controlled. No attempt has been made to investigate all the factors which influence the assay but rather to determine the conditions necessary for a usable assay for streptomycin. Statistical analysis of the method. An analysis of the accuracy of the method 4This is a trade name for a glass cylinder in common use for the assay of penicillin. 6 The authors gratefully acknowledge the courtesy and co-operation of Dr. Selman A. Waksman, Microbiologist, the New Jersey Agricultural Experiment Station, New Brunswick, New Jersey, who assayed for us samples of our standard and other preparations.

8 708 LOO ET AL. is shown in figure 3, in which the standard deviation, standard error, and probable error, each expressed as a percentage of the mean, are plotted against the number of zones per asay. These curves were calculated from three typical groups of data from three of our laboratories, each on a different single sample. Each group contained ninety- 21 Q-A STANDARD DEVIATION 21 2C 0-0 STANDARD ERROR A G PROBABLE ERROR 19 z a- 16 A 6 z w I o0 10 z 9 o8\3 _ 6 0* e 241 a 0 I I-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ N UMBE R OF ZON ES PER AS SAY FIG. 3. A GRPHICAL. PRESENTATION OF THXE AccuJRAcy OF THE ME:THODsix individual zone diameters, which were converted to units per milliliter for calculation of the various statistics. These calculations were made by treating the ninety-six replicates successively as forty-eight 2-zone, twenty-four 4-zone, twelve 8-zone, sx16-zone, and three 32-zone assays. These curves allow us to select the 0umber of zones required to give any desired value of one of the statistics, e.g., a 4-zone assay is required if a standard

9 ASSAY OF STREPTOMYCIN BY PAPER-DISC PLATE METHOD 709 error of 8.5 per cent of the mean is to be expected. In other words, 95 per cent of all 4-zone replicate assays should fall within the range of M i 2e, or M d4 17 per cent. If a standard error as low as 3.0 per cent is demanded, a 32-zone assay is required, and 95 per cent of all 32-zone replicate assays should fall within the range of M i 6 per cent. The accuracy indicated by figure 3 may be expected for an assay taken from any part of the standard curve. If a standard is placed on each plate, and these values averaged, the point on the curve so established is evidently more accurate and hence becomes a "strong point" on the curve. For this reason, dilutions should be chosen so that the readings will fall as close as possible to this more accurate point. If the number of zones used to establish each point on the curve is identical, all points on the curve are equally reliable for assay purposes. SUMMARY A method is described for the quantitative determination of streptomycin by the filter paper disc, agar plate diffusion technique, using Bacillus subtilis as the test organism. The influence of various factors on the assay is reported and discussed. This procedure has proved satisfactory in the assay of surface and submerged-culture beers and of preparations obtained in isolation and purification processes. The authors gratefully acknowledge the co-operation of Drs. H. W. Anderson and H. E. Carter of the University of Illinois and the assistance of Mrs. Betty A. Adloff and Alma Whitman. REFERENCES FOSTER, J. W., AND WOODRUFF, H. B Microbiological aspects of streptothricin. II. Antibiotic activity of streptothricin. Arch. Biochem., 3, HEILMAN, D. H A method for estimating the concentration of streptomycin in body fluids. Proc. Staff Meetings Mayo Clinic, 20, SCHATZ, A., BUGIE, E., AND WAKSMAN, S. A Streptomycin, a substanceexhibiting antibiotic activity against gram-positive and gram-negative bacteria. Proc. Soc. Exptl. Biol. Med., 55, STEBBINS, R. B., AND ROBINSON, H. J A method for the determination of streptomycin in body fluids. Proc. Soc. Exptl. Biol. Med., 59, WAKSMAN, S. A Standardization of streptomycin. Science, 102,

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