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A Turbidimetric Assay for Terramycin
A Turbidimetric Assay for Terrarnycin* By R. C. KERSEY A rapid and reliable assay method applicable t o both impure and purified preparations of terramycin is described. T h e method described involves a turbidimetric procedure utilizing Klebsiella pneumoniae P. C . I. 602 as the test organism. An incubation period of only three and one-half hours is required. following assay method affords a n accuconvenient a n d rapid method for determining the potency of terramycin. This antibiotic is produced b y a n actinomycete isolated from soil and given the name Streptomyces rimosus by Finlay, et al., (1). Terramycin has a broad bacterial spectrum which includes both Gram-positive a n d Gram-negative organisms a n d has a low toxicity in t h o . T h e procedure embodies the test organism and media for the streptomycin turhidimetric assay as recommended by the Food a n d Drug Administration (2). HE
Trate,
PROCEDURE Culture and Inoculum-Stock cultures of Klebsiella pneumoniae P . C. 1. 602 are carried on agar slants of the following composition: Beef extract 1.5 Gm., yeast extract 3.0 Gm., peptone 6 Gm., agar 15'Gm., distilled water 1 liter. The PH of the medium is adjusted t o 7.0. The assay inoculum is prepared by transferring the organism to fresh agar slants and incubating for six hours a t 37". The growth from two or three slants is suspended in sterile distilled water and 5.0 ml. of the suspension added t o each of two Roux bottles containing the agar described above. The Roux bottles are incubated at 37" for six hours, the growth harvested and sufficient sterile distilled water added to give a light transmission reading of 80% using a 650 mp filter in a photoelectric colorimeter. This suspension may be stored for two weeks. Assay Medium.-The medium used in the assay is composed of: Peptone 5.0 Gm., yeast extract 1.5 Gm., beef extract 1.5 Gm., sodium chloride 3.5 Gm., glucose 1.0 Gm., KHzPOd 1.32 Gm., KzHPOa 3.68 Gm., distilled water 1 liter. The medium is adjusted t o pH 7.0 and sterilized. This medium may be obtained in dehydrated form.' Standard Solution.-A stock solution is prepared by dissolving 25.0 mg. of crystalline terramycin base in 100 ml. of 0.1 N HCl. This stock solution may be stored in the refrigerator for a period of five days with no loss in potency. Final dilutions of the standard are prepared daily by diluting the stock solution t o 2.5 pg./ml. with distilled water. *Received March 23 1950 from the Biochemical Research Laboratories, C h k PBkr and Co., Inc., Brooklyn. N. Y. 1 Baltimore Biological Laboratories, Inc., Baltimore, Md., Antibiotic Assay Broth.
Samples.-Liquid samples are diluted with watcr to contain from 0.5 to 2.5 pg./ml. of terramycin. Dry samples of the free base, which is not readily soluble in water, are more easily handled by first dissolving in a small amount of 0.1 N HCl and then making the required dilution with water. Urine samples are centrifuged if turbid and the proper dilutions made with water. Samples of whole blood are centrifuged or allowed to clot and dilutions made on the clear serum. ' Assay Techniques.-The general assay procedure is based on McMahan's turbidimetric assay of penicillin (3). The preparation of the standard curve and sample series is described below. A standard curve (0.05 to 0.5 pg./tube) is prepared by pipetting, with a micro-pipette (3), the following amounts of the standard terramycin solution into a series of 15 x 100-mm. Pyrex test tubes: 0.20, 0.18, 0.16, 0.14, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02 ml. In a further series the diluted samples (0.5 t o 2.5 pg./ml.) are pipetted as follows: 0.20, 0.16, 0.12, 0.08, 0.04 ml. The small volume of sample employed (0.02 to 0.20 ml.) makes an insignificant difference in the final volume of medium in the tube (7 ml.) and hence no precaution need be taken t o adjust all the tubes to the same volume. A flask containing sufficient sterile assay broth to fill all tubes in the test series is inoculated with 6 ml. of the standardized Klebsiella pneumoniae cell suspension per 100 ml. of broth. A Brewer automatic pipetting machine is used to dispense 7 ml. of the inoculated medium to each tube, the tubes covered with inverted stainless steel trays and incubated a t 37" for three and one-half hours in a water bath. The racks are removed at the end of the incubation period, and steamed for ten minutes in an Arnold sterilizer t o prevent additional growth from taking place during the reading of the test. Turbidity readings are made with a Lumetron colorimeter. Model 402E, and light transmission is read directly from the galvanometer. The sensitivity of the colorimeter is adjusted so that a galvanometer deflection of 80 divisions is obtained between the high and low points of the standard curve. Galvanometer readings are plotted against potency and the points connected in a smooth curve as illustrated in Fig. 1. Calculations of a typical assay are given in Table I. RESULTS
A reliable assay is dependent upon: ( a ) consistency of values when potencies are calculated from various portions of the standard curve (see Table I, Column c / a ) ; ( b ) recovery experiments; and (c), reproducibility of assays on successive days. Data are presented in Table I, Column c / a demonstrating that the small variations noted at different portions of the standard curve are within the limits of other turbidimetric assays. Recovery experiments with human and dog urine demonstrate that urines do not interfere with the method (see Table 11).
252
SCIENTIFIC EDITION
253
TABLEI.-TERRAMYCIN ASSAYAND CALCULATIONS Volume of Standard. M1.
Terramycin/ Tube, fig.
Galvanometer Reading
0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00
0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00
90 85 80 79 70 60 50 39 28 20 13
Volume of Sample, M1. (a)
0.20 0.16 0.12 0.08 a
-
Galvanometer Reading
(a) 84 73 60 42
Terramycin Calculated from Curve, fig.
Terramycm,
a.per
MI. Diluted Sample
(C)
(
0.430 0.325 0.250 0.165
2.15 2.03 2.09 2.06
Aver3
4
2.08
d X dilution = potency of sample.
TABLE 11.-RECOVERY OF TERRAMYCIN FROM BLOOD AND URINE= om Calculated, fig./Ml.
Human Control Dog Control Human Control Dog Control 4
Urine 50 0 50 0 Blood 25 0 25
0
Found, fig./Ml.
52 0 54 0 25.5 0 27 0
Recovery,
Fig. 1.-Terramycin
%
104
015 a2 025 a3 au a4 OM as MICROGRAMS OF TERRAMYGIN PER TUBE
QI
dose response curve.
FERENT
108
...
...
108
od
TABLE III.-REPRODUCIBILITYOF ASSAYSON DIF-
...
102
ou
Terramycin base Terramycin HCl a
DAY^
1st Day
a./M1. 2nd Day
3rd Day
915 861
930 861
915 847
Average of three assays.
...
SUMMARY
All values are an average of three assays.
Reproducibility of the assay on different days is demonstrated by the data in Table 111. Representative samples of terramycin base and hydrochloride were analyzed on each of three successive days. Assays were reproducible within 2%. The method has been applied successfully on widely different types of samples including fermentation broths, samples from the recovery process, blood, and urine. The applicability of the method t o other biological fluids is being investigated.
A method is described for the turbidimetric assay of terramycin employing Klebsiella pneumonrae P. C. I. 602 as the assay organism. An incubation period of three and one-half hours is required. The method is rapid, reliable, and applicable to both impure and purified preparations. REFERENCES (1) (2) graph (3)
Finlay A. C. el al. Science 111 85(1950). Fedem1 Register, bct. 28, '1948: section 141.101, para. (1).
McMahan, J. R., J . B i d . Chem., 153, 249(1944).
Trate,
PROCEDURE Culture and Inoculum-Stock cultures of Klebsiella pneumoniae P . C. 1. 602 are carried on agar slants of the following composition: Beef extract 1.5 Gm., yeast extract 3.0 Gm., peptone 6 Gm., agar 15'Gm., distilled water 1 liter. The PH of the medium is adjusted t o 7.0. The assay inoculum is prepared by transferring the organism to fresh agar slants and incubating for six hours a t 37". The growth from two or three slants is suspended in sterile distilled water and 5.0 ml. of the suspension added t o each of two Roux bottles containing the agar described above. The Roux bottles are incubated at 37" for six hours, the growth harvested and sufficient sterile distilled water added to give a light transmission reading of 80% using a 650 mp filter in a photoelectric colorimeter. This suspension may be stored for two weeks. Assay Medium.-The medium used in the assay is composed of: Peptone 5.0 Gm., yeast extract 1.5 Gm., beef extract 1.5 Gm., sodium chloride 3.5 Gm., glucose 1.0 Gm., KHzPOd 1.32 Gm., KzHPOa 3.68 Gm., distilled water 1 liter. The medium is adjusted t o pH 7.0 and sterilized. This medium may be obtained in dehydrated form.' Standard Solution.-A stock solution is prepared by dissolving 25.0 mg. of crystalline terramycin base in 100 ml. of 0.1 N HCl. This stock solution may be stored in the refrigerator for a period of five days with no loss in potency. Final dilutions of the standard are prepared daily by diluting the stock solution t o 2.5 pg./ml. with distilled water. *Received March 23 1950 from the Biochemical Research Laboratories, C h k PBkr and Co., Inc., Brooklyn. N. Y. 1 Baltimore Biological Laboratories, Inc., Baltimore, Md., Antibiotic Assay Broth.
Samples.-Liquid samples are diluted with watcr to contain from 0.5 to 2.5 pg./ml. of terramycin. Dry samples of the free base, which is not readily soluble in water, are more easily handled by first dissolving in a small amount of 0.1 N HCl and then making the required dilution with water. Urine samples are centrifuged if turbid and the proper dilutions made with water. Samples of whole blood are centrifuged or allowed to clot and dilutions made on the clear serum. ' Assay Techniques.-The general assay procedure is based on McMahan's turbidimetric assay of penicillin (3). The preparation of the standard curve and sample series is described below. A standard curve (0.05 to 0.5 pg./tube) is prepared by pipetting, with a micro-pipette (3), the following amounts of the standard terramycin solution into a series of 15 x 100-mm. Pyrex test tubes: 0.20, 0.18, 0.16, 0.14, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02 ml. In a further series the diluted samples (0.5 t o 2.5 pg./ml.) are pipetted as follows: 0.20, 0.16, 0.12, 0.08, 0.04 ml. The small volume of sample employed (0.02 to 0.20 ml.) makes an insignificant difference in the final volume of medium in the tube (7 ml.) and hence no precaution need be taken t o adjust all the tubes to the same volume. A flask containing sufficient sterile assay broth to fill all tubes in the test series is inoculated with 6 ml. of the standardized Klebsiella pneumoniae cell suspension per 100 ml. of broth. A Brewer automatic pipetting machine is used to dispense 7 ml. of the inoculated medium to each tube, the tubes covered with inverted stainless steel trays and incubated a t 37" for three and one-half hours in a water bath. The racks are removed at the end of the incubation period, and steamed for ten minutes in an Arnold sterilizer t o prevent additional growth from taking place during the reading of the test. Turbidity readings are made with a Lumetron colorimeter. Model 402E, and light transmission is read directly from the galvanometer. The sensitivity of the colorimeter is adjusted so that a galvanometer deflection of 80 divisions is obtained between the high and low points of the standard curve. Galvanometer readings are plotted against potency and the points connected in a smooth curve as illustrated in Fig. 1. Calculations of a typical assay are given in Table I. RESULTS
A reliable assay is dependent upon: ( a ) consistency of values when potencies are calculated from various portions of the standard curve (see Table I, Column c / a ) ; ( b ) recovery experiments; and (c), reproducibility of assays on successive days. Data are presented in Table I, Column c / a demonstrating that the small variations noted at different portions of the standard curve are within the limits of other turbidimetric assays. Recovery experiments with human and dog urine demonstrate that urines do not interfere with the method (see Table 11).
252
SCIENTIFIC EDITION
253
TABLEI.-TERRAMYCIN ASSAYAND CALCULATIONS Volume of Standard. M1.
Terramycin/ Tube, fig.
Galvanometer Reading
0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00
0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00
90 85 80 79 70 60 50 39 28 20 13
Volume of Sample, M1. (a)
0.20 0.16 0.12 0.08 a
-
Galvanometer Reading
(a) 84 73 60 42
Terramycin Calculated from Curve, fig.
Terramycm,
a.per
MI. Diluted Sample
(C)
(
0.430 0.325 0.250 0.165
2.15 2.03 2.09 2.06
Aver3
4
2.08
d X dilution = potency of sample.
TABLE 11.-RECOVERY OF TERRAMYCIN FROM BLOOD AND URINE= om Calculated, fig./Ml.
Human Control Dog Control Human Control Dog Control 4
Urine 50 0 50 0 Blood 25 0 25
0
Found, fig./Ml.
52 0 54 0 25.5 0 27 0
Recovery,
Fig. 1.-Terramycin
%
104
015 a2 025 a3 au a4 OM as MICROGRAMS OF TERRAMYGIN PER TUBE
QI
dose response curve.
FERENT
108
...
...
108
od
TABLE III.-REPRODUCIBILITYOF ASSAYSON DIF-
...
102
ou
Terramycin base Terramycin HCl a
DAY^
1st Day
a./M1. 2nd Day
3rd Day
915 861
930 861
915 847
Average of three assays.
...
SUMMARY
All values are an average of three assays.
Reproducibility of the assay on different days is demonstrated by the data in Table 111. Representative samples of terramycin base and hydrochloride were analyzed on each of three successive days. Assays were reproducible within 2%. The method has been applied successfully on widely different types of samples including fermentation broths, samples from the recovery process, blood, and urine. The applicability of the method t o other biological fluids is being investigated.
A method is described for the turbidimetric assay of terramycin employing Klebsiella pneumonrae P. C. I. 602 as the assay organism. An incubation period of three and one-half hours is required. The method is rapid, reliable, and applicable to both impure and purified preparations. REFERENCES (1) (2) graph (3)
Finlay A. C. el al. Science 111 85(1950). Fedem1 Register, bct. 28, '1948: section 141.101, para. (1).
McMahan, J. R., J . B i d . Chem., 153, 249(1944).