ANTIMICROBIAL AGENTS AND CHEMOTItERAPY, July 1979, p. 43-45 0066-4804/79/07-0043/03$02.00/0
Vol. 16, No. 1
Chloramphenicol Bioassay ROBERT M. BANNATYNE* AND ROSE CHEUNG Department of Bacteriology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 Received for publication 27 April 1979
An accurate plate diffusion bioassay for chloramphenicol is described, in which the fast-replicating Beneckea natriegens and 1.5% salt agar are used. Zones of inhibition were well defined after 3 h, and the limit of sensitivity of the method was around 2 pg/ml. The concurrent presence of gentamicin did not influence the assay. The assay is simple to carry out and duplicate assays can be performed with as little as 100 pug of capillary blood.
Chloramphenicol is enjoying a renewed and enforced popularity. The apparition of ampicillin resistance in Haemophilus influenzae (3), the recognition of the importance of Bacteroides fragilis in intra-abdominal and intracerebral infections (7), and the reawakened interest in the drug for the treatment of neonatal meningitis (2, 4) have combined to uncover fresh indications for its use. This increase in the use of chloramphenicol has created a requirement for methods of measuring serum concentrations of the drug in order to ensure that therapeutic levels are being attained and toxic levels are being avoided. Existing methods for dete iing serum chloramphenicol have the disadvantages of technical complexity (8, 15), low sensitivity (6, 12), glowness, or the need for sophisticated equipment (9, 11, 13, 14). In the modification of the standard diffusion bioassay described here, Beneckea natriegens, a marine bacterium with an extremely short doubling time (5), is used to provide a simple, fast, and accurate method for determining serum chloramphenicol levels in as little as 40 p1 of capillary blood.
0.05 ml of the suspension was subcultured into 2.5 ml of fresh broth which was incubated at 370C for 3 h. The turbidity of the suspension was then adjusted to a light transmission of 25% at 550 nm with a Bausch and Lomb Spectronic 20 spectrophotometer (approximately 5 x 10' cells per ml). This suspension was incorporated in the agar medium of the assay plates. Sterile melted Trypticase soy agar supplemented with 1.5% NaCl, pH 8.0, was cooled to 450C and augmented with 1 volume each of adjusted B. natriegens suspension and 0.5% tetrazolium per 100 volumes (total volume). The agar was well mixed without excess agitation and dispensed in 20-mi volumes with a wide-bore serological pipette into sterile plastic petri dishes (150 by 15 mm; Fisher Scientific Co. or Falcon Plastics) placed on a special heated level surface (Bio-
Tech Co., Lynden, Ontario, Canada). After the plates had set, they were held at- 40C for at least 1 h. Before use the cooled plates were dried at 37°C for 10 min to remove excess surface moisture, and 22 wells were cut with a 4.0-mm cork borer in a formation which minimized the variation in zone size as a result of unevenness of the agar depth. Before the wells were filled, any excess moisture in them was removed with a sterile disposable pipette. Preparation of stock drug solution for use in dose-response curve. One gram of chloramphenicol powder (Parke, Davis & Co., Brockville, Ontario, Canada) was dissolved in 95% ethanol to a concentration of 6,400 pug/ml. This stock solution was stored at -70°C until needed, when it was further diluted in pooled normal human serum, heat inactivated, and filtered through a 0.2-pm membrane filter (Nalgene Labware Div., Nalge/Sybron Corp., Rochester, N. Y.) to concentrations of 5, 10, 20, 40, 60, 80, and 100 pg/ ml. These standard solutions retained their potencies for up to 7 days if stored at 4.00C. Dose-response curve. Heparinized capillary tubes (100 p1; Instrumentation Laboratory) were filled with each of the standards and, using a microrubber bulb to expel the fluid, the wells were filled in duplicate (40 ul each). Plates were incubated on a level surface at 370C for 3 h, after which zone sizes were distinct and measurable. Zones of inhibition were measured in two diameters by using a calibrating viewer (Johns Scientific). The standard curve was obtained by plotting the
MATERIALS AND METHODS Media and Cultures. B. natriegens (ATCC 14048) with a tube dilution minimal- inhibitory concentration for chloramphenicol of 1.56 ug/ml was used as the assay organism. It was susceptible to ampicillin (4 pg/ ml), carbenicillin (1 pg/ml), polymyxin B (2 pg/ml), tetracycline (1 pg/ml), and nalidixic acid (1 pg/ml) but relatively resistant to gentamicin (10 ug/ml), tobramycin (20 pg/ml), trimethoprim (20 pg/ml), sulfamethoxazole (100 pg/ml), cephaloridine (20 pg/ml), cephalexin (40 ug/ml), clindamycin (20 pg/ml), erythromycin (10 ug/ml), nitrofurantoin (10 pg/ml), and vancomycin (25 pg/ml). Cultures were maintained at 220C on Trypticase soy agar (Baltimore Biological Laboratory) slants supplemented with 1.5% sodium chloride
and were subcultured monthly. Before use the organisn was grown overnight in brain heart infusion broth (Difco Laboratories) with (1.5%) NaCl at 37°C, and
43
44
ANTIMICROB. AGENTS CHEMOTHER.
BANNATYNE AND CHEUNG
average zone diameter (in millimeters) against the concentration of the standards (in micrograms per milliliter) on two-cycle 70-division semilog graph paper and joining these points with the best-fit straight line. Reproducibility. Assay precision was assessed on the basis of 13 specimens spiked to contain from 2.5 to 90 ,ug of chloramphenicol per ml, and the correlation between the prepared and the assayed concentrations was calculated (1). Determinations were made twice (i.e., two sets of duplicates) on different plates and with a different set of standards. Within-sample standard deviation (SD) of the paired results was calculated by the equation SD = (: d2/2n)'/2, where d is the difference in micrograms per milliliter between the actual and observed values on the same specimen and n is the number of specimens.
RESULTS A composite dose-response curve derived from measurements on 100 separate curves is shown in Fig. 1. The curve was linear over the normal range for chloramphenicol serum levels at recommended doses. The lower limit of sensitivity was of the order of 2.0 ug/ml. The indicated ranges of the zone diameters at each of the standard concentrations were within acceptable limits. The precision of the assay technique is shown in Fig. 2. The closeness of the paired values to the line of identity emphasizes the high degree of reproducibility. The within-sample standard deviation of each pair of results was 1.19 ,tg/ml. With the zones of inhibition sharpened by added tetrazolium, plates could easily be read after 3 h of incubation. However, if desired, the assay could be reread after overnight incubation, with a slight shift of the curve, but no change in sample value above 5.0 ,ug/ml. Only tetracycline (data not shown) altered zone sizes when 12.0
104 E co -.
O-
91 D
Q
u
z 0
0*',
0-~~~~~0
74 z
*ActualIsam,ple values @ 0Observd sample values
6C 2
/~~~~~~
54 00
z
2
ua
2(
0
0
10 20 30 40 50 60 70 80 90 100 CHLORAMPHENICOL CONCENTRATION (vg/mi)
FIG. 2. Reproducibility of assay results for chloramphenicol. Paired points represent the mean result of two sets of duplicate assays performed on one spiked serum sample plotted against the expected results on the same specimen. One value is plotted on the ordinate, and the other is plotted on the abscissa. The line of identity is included for reference.
,ig/ml was added to sera containing therapeutic concentrations of chloramphenicol (>5 ,ug/ml). No such effect was observed with trimethoprim (6.0 ,ug/ml), clindamycin (3 Itg/ml), gentamicin (24 ,ug/ml), or polymyxin B (30 I,g/ml). When ampicillin or carbenicillin (B. natriegens minimal inhibitory concentrations, 4.0 and 1 lig/ml, respectively) were known to be present, the addition of penicillinase to the serum proved effective. DISCUSSION
B. natriegens is a marine bacterium (5). Its unusually short generation time (9.8 min), its preference for salt, and its high degree of susceptibility to chloramphenicol combine to make it an ideal seed strain for the rapid assay of this drug. The nature of the standard curve and the 80high degree of reproducibility of the technique attest to the accuracy and sensitivity of this 40method for chloramphenicol assay. This assay is economical with blood (only 40 pl is required), 020 and results with this system were available in 3 h, i.e., in time to adjust subsequent chloram0 10 phenicol doses even when the drug is adminis0 tered every 4 h. Technically simple, it avoids the need for sophisticated equipment and is thus within the capabilities of even small laboratories. 2 In a preliminary comparison with the hemolysisr inhibition assay of Louie et al. (10), we experiI I I I III enced difficulties with indistinct zones in their 6 7 8 9 10 11 12 13 14 16 17 18 19 20 method, and others (9) have encountered probDIAMETER OF ZONES OF INHIBITION (mm) FIG. 1. Bioassay for chloramphenicol. This repre- lems in this method with colony maintenance sents a composite dose response curve, with ranges, and plate preparation. The resistance of B. naderived from mean values for 100 separate dose-re- triegens to the common antibiotics likely to be partnered with chloramphenicol and the inhibisponse curves. 100 -
=
E -
60-
z
z
0
z
4
._-
O
I
I
15
I
CHLORAMPHENICOL BIOASSAY
VOL. 16, 1979
tory effect of the 1.5% NaCl in the assay medium combined to limit interference in the assay due to other antibiotics. Among the legitimate indications for chloramphenicol therapy are now included salmonellosis, bacterial meningitis due to ampicilhin-resistant H. influenzae (3), intra-abdominal and intracerebral infections with B. fragilis (7), neonatal meningitis (2, 4), and certain rickettsial or chlamydial diseases (16). The treatment of these diseases with chloramphenicol has created a requirement for an assay system that ensures that therapeutic levels are being attained and that toxic levels are being avoided. We believe that the B. natriegens bioassay method can fulfill this role. LUTERATURE CITED 1. Bannatyne, R. M*, and R. Cheung. 1977. Microassay for amphotericin B. Antimicrob. Agents Chemother. 11:44-46. 2. Black, S. B., P. Levine, and N. R. Shinefleld. 1978. The necessity for monitoring chloramphenicol levels when treating neonatal meningitis. J. Pediatr. 92:235236. 3. Clymo, A. B., and I. A. Harper. 1974. Ampicillin-resistant Haemophilus influenzae meningitis. Lancet i:453454. 4. Dunkle, L A. 1978. Central nervous system chloramphenicol concentration in premature infants. Antimicrob. Agents Chemother. 13:427-429. 5. Eagon, R. G. 1962. Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J. Bacteriol. 83:736-737. 6. Glazko, J. J., L M. Wolf, and W. A. Dill. 1949. Bio-
45
chemical studies on chloramphenicol. I. Colorimetric methods for determination of chloramphenicol and re-
lated nitro compounds. Arch. Biochem. 23:411-418. 7. Gorbach, S. L, and J. G. Bartlett. 1974. Anaerobic infections. N. Engl. J. Med. 290:1177-1184, 1237-1245, 1289-1294. 8. Joslyn, D. A., and M. Galbraith. 1950. A turbidimetric method for assay of antibiotics. J. Bacteriol. 59:711716. 9. Koop, J. R., B. Brodsky, A. Lau, and T. R. Beam, Jr. 1978. High-performance liquid chromatographic asay of chloramphenicol in serum. Antimicrob. Agents Chemother. 14:439-443. 10. Louie, T. J., F. P. Tally, J. G. Bartlell, and S. L Gorbach. 1976. Rapid microbiological assay for chloramphenicol and tetracyclines. Antimicrob. Agents Chemother. 9:874-878. 11. Nilsson-Ehle, I., G. Kahimeter, and P. Nilsson-Ehle. 1978. Detennination of chloramphenicol in serum and cerebrospinal fluid with high-presure liquid chromatography. J. Antimicrob. Chemother. 4:169-176. 12. Randall, W. A., A. Kirshbaum, J. K. Nielsen, and D. Wintermere. 1949. Diffusion plate assay for chloramphenicol and aureomycin. J. Clin. Invest. 28:940-942. 13. Resnick, G. L., D. Gorbin, and D. Sandberg. 1966. Determination of serum chloramphenicol utilizing gasliquid chromatography and electron capture spectrometry. Anal. Chem. 38:582-585. 14. Robinson, L R., R. Selig8ohn, and S. A. Lerner. 1978. Simplified radioenzymatic assay for chloramphenicol. Antimicrob. Agents Chemother. 13:25-29. 15. Whitlock, C. M., Jr., A. D. Hunt, and S. G. Tushman. 1951. A single simple turbidimetric method for the assay of aureomycin, chloramphenicol, penicillin, streptomycin and terramycin in capillary blood and other body fluids. J. Lab. Clin. Med. 37:155-161. 16. Wilaon, W. R. 1977. Tetracyclines, chloramphenicol, erythromycin and clindamycin. Mayo. Clin. Proc. 52:
635-640.
Vol. 16, No. 1
Chloramphenicol Bioassay ROBERT M. BANNATYNE* AND ROSE CHEUNG Department of Bacteriology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 Received for publication 27 April 1979
An accurate plate diffusion bioassay for chloramphenicol is described, in which the fast-replicating Beneckea natriegens and 1.5% salt agar are used. Zones of inhibition were well defined after 3 h, and the limit of sensitivity of the method was around 2 pg/ml. The concurrent presence of gentamicin did not influence the assay. The assay is simple to carry out and duplicate assays can be performed with as little as 100 pug of capillary blood.
Chloramphenicol is enjoying a renewed and enforced popularity. The apparition of ampicillin resistance in Haemophilus influenzae (3), the recognition of the importance of Bacteroides fragilis in intra-abdominal and intracerebral infections (7), and the reawakened interest in the drug for the treatment of neonatal meningitis (2, 4) have combined to uncover fresh indications for its use. This increase in the use of chloramphenicol has created a requirement for methods of measuring serum concentrations of the drug in order to ensure that therapeutic levels are being attained and toxic levels are being avoided. Existing methods for dete iing serum chloramphenicol have the disadvantages of technical complexity (8, 15), low sensitivity (6, 12), glowness, or the need for sophisticated equipment (9, 11, 13, 14). In the modification of the standard diffusion bioassay described here, Beneckea natriegens, a marine bacterium with an extremely short doubling time (5), is used to provide a simple, fast, and accurate method for determining serum chloramphenicol levels in as little as 40 p1 of capillary blood.
0.05 ml of the suspension was subcultured into 2.5 ml of fresh broth which was incubated at 370C for 3 h. The turbidity of the suspension was then adjusted to a light transmission of 25% at 550 nm with a Bausch and Lomb Spectronic 20 spectrophotometer (approximately 5 x 10' cells per ml). This suspension was incorporated in the agar medium of the assay plates. Sterile melted Trypticase soy agar supplemented with 1.5% NaCl, pH 8.0, was cooled to 450C and augmented with 1 volume each of adjusted B. natriegens suspension and 0.5% tetrazolium per 100 volumes (total volume). The agar was well mixed without excess agitation and dispensed in 20-mi volumes with a wide-bore serological pipette into sterile plastic petri dishes (150 by 15 mm; Fisher Scientific Co. or Falcon Plastics) placed on a special heated level surface (Bio-
Tech Co., Lynden, Ontario, Canada). After the plates had set, they were held at- 40C for at least 1 h. Before use the cooled plates were dried at 37°C for 10 min to remove excess surface moisture, and 22 wells were cut with a 4.0-mm cork borer in a formation which minimized the variation in zone size as a result of unevenness of the agar depth. Before the wells were filled, any excess moisture in them was removed with a sterile disposable pipette. Preparation of stock drug solution for use in dose-response curve. One gram of chloramphenicol powder (Parke, Davis & Co., Brockville, Ontario, Canada) was dissolved in 95% ethanol to a concentration of 6,400 pug/ml. This stock solution was stored at -70°C until needed, when it was further diluted in pooled normal human serum, heat inactivated, and filtered through a 0.2-pm membrane filter (Nalgene Labware Div., Nalge/Sybron Corp., Rochester, N. Y.) to concentrations of 5, 10, 20, 40, 60, 80, and 100 pg/ ml. These standard solutions retained their potencies for up to 7 days if stored at 4.00C. Dose-response curve. Heparinized capillary tubes (100 p1; Instrumentation Laboratory) were filled with each of the standards and, using a microrubber bulb to expel the fluid, the wells were filled in duplicate (40 ul each). Plates were incubated on a level surface at 370C for 3 h, after which zone sizes were distinct and measurable. Zones of inhibition were measured in two diameters by using a calibrating viewer (Johns Scientific). The standard curve was obtained by plotting the
MATERIALS AND METHODS Media and Cultures. B. natriegens (ATCC 14048) with a tube dilution minimal- inhibitory concentration for chloramphenicol of 1.56 ug/ml was used as the assay organism. It was susceptible to ampicillin (4 pg/ ml), carbenicillin (1 pg/ml), polymyxin B (2 pg/ml), tetracycline (1 pg/ml), and nalidixic acid (1 pg/ml) but relatively resistant to gentamicin (10 ug/ml), tobramycin (20 pg/ml), trimethoprim (20 pg/ml), sulfamethoxazole (100 pg/ml), cephaloridine (20 pg/ml), cephalexin (40 ug/ml), clindamycin (20 pg/ml), erythromycin (10 ug/ml), nitrofurantoin (10 pg/ml), and vancomycin (25 pg/ml). Cultures were maintained at 220C on Trypticase soy agar (Baltimore Biological Laboratory) slants supplemented with 1.5% sodium chloride
and were subcultured monthly. Before use the organisn was grown overnight in brain heart infusion broth (Difco Laboratories) with (1.5%) NaCl at 37°C, and
43
44
ANTIMICROB. AGENTS CHEMOTHER.
BANNATYNE AND CHEUNG
average zone diameter (in millimeters) against the concentration of the standards (in micrograms per milliliter) on two-cycle 70-division semilog graph paper and joining these points with the best-fit straight line. Reproducibility. Assay precision was assessed on the basis of 13 specimens spiked to contain from 2.5 to 90 ,ug of chloramphenicol per ml, and the correlation between the prepared and the assayed concentrations was calculated (1). Determinations were made twice (i.e., two sets of duplicates) on different plates and with a different set of standards. Within-sample standard deviation (SD) of the paired results was calculated by the equation SD = (: d2/2n)'/2, where d is the difference in micrograms per milliliter between the actual and observed values on the same specimen and n is the number of specimens.
RESULTS A composite dose-response curve derived from measurements on 100 separate curves is shown in Fig. 1. The curve was linear over the normal range for chloramphenicol serum levels at recommended doses. The lower limit of sensitivity was of the order of 2.0 ug/ml. The indicated ranges of the zone diameters at each of the standard concentrations were within acceptable limits. The precision of the assay technique is shown in Fig. 2. The closeness of the paired values to the line of identity emphasizes the high degree of reproducibility. The within-sample standard deviation of each pair of results was 1.19 ,tg/ml. With the zones of inhibition sharpened by added tetrazolium, plates could easily be read after 3 h of incubation. However, if desired, the assay could be reread after overnight incubation, with a slight shift of the curve, but no change in sample value above 5.0 ,ug/ml. Only tetracycline (data not shown) altered zone sizes when 12.0
104 E co -.
O-
91 D
Q
u
z 0
0*',
0-~~~~~0
74 z
*ActualIsam,ple values @ 0Observd sample values
6C 2
/~~~~~~
54 00
z
2
ua
2(
0
0
10 20 30 40 50 60 70 80 90 100 CHLORAMPHENICOL CONCENTRATION (vg/mi)
FIG. 2. Reproducibility of assay results for chloramphenicol. Paired points represent the mean result of two sets of duplicate assays performed on one spiked serum sample plotted against the expected results on the same specimen. One value is plotted on the ordinate, and the other is plotted on the abscissa. The line of identity is included for reference.
,ig/ml was added to sera containing therapeutic concentrations of chloramphenicol (>5 ,ug/ml). No such effect was observed with trimethoprim (6.0 ,ug/ml), clindamycin (3 Itg/ml), gentamicin (24 ,ug/ml), or polymyxin B (30 I,g/ml). When ampicillin or carbenicillin (B. natriegens minimal inhibitory concentrations, 4.0 and 1 lig/ml, respectively) were known to be present, the addition of penicillinase to the serum proved effective. DISCUSSION
B. natriegens is a marine bacterium (5). Its unusually short generation time (9.8 min), its preference for salt, and its high degree of susceptibility to chloramphenicol combine to make it an ideal seed strain for the rapid assay of this drug. The nature of the standard curve and the 80high degree of reproducibility of the technique attest to the accuracy and sensitivity of this 40method for chloramphenicol assay. This assay is economical with blood (only 40 pl is required), 020 and results with this system were available in 3 h, i.e., in time to adjust subsequent chloram0 10 phenicol doses even when the drug is adminis0 tered every 4 h. Technically simple, it avoids the need for sophisticated equipment and is thus within the capabilities of even small laboratories. 2 In a preliminary comparison with the hemolysisr inhibition assay of Louie et al. (10), we experiI I I I III enced difficulties with indistinct zones in their 6 7 8 9 10 11 12 13 14 16 17 18 19 20 method, and others (9) have encountered probDIAMETER OF ZONES OF INHIBITION (mm) FIG. 1. Bioassay for chloramphenicol. This repre- lems in this method with colony maintenance sents a composite dose response curve, with ranges, and plate preparation. The resistance of B. naderived from mean values for 100 separate dose-re- triegens to the common antibiotics likely to be partnered with chloramphenicol and the inhibisponse curves. 100 -
=
E -
60-
z
z
0
z
4
._-
O
I
I
15
I
CHLORAMPHENICOL BIOASSAY
VOL. 16, 1979
tory effect of the 1.5% NaCl in the assay medium combined to limit interference in the assay due to other antibiotics. Among the legitimate indications for chloramphenicol therapy are now included salmonellosis, bacterial meningitis due to ampicilhin-resistant H. influenzae (3), intra-abdominal and intracerebral infections with B. fragilis (7), neonatal meningitis (2, 4), and certain rickettsial or chlamydial diseases (16). The treatment of these diseases with chloramphenicol has created a requirement for an assay system that ensures that therapeutic levels are being attained and that toxic levels are being avoided. We believe that the B. natriegens bioassay method can fulfill this role. LUTERATURE CITED 1. Bannatyne, R. M*, and R. Cheung. 1977. Microassay for amphotericin B. Antimicrob. Agents Chemother. 11:44-46. 2. Black, S. B., P. Levine, and N. R. Shinefleld. 1978. The necessity for monitoring chloramphenicol levels when treating neonatal meningitis. J. Pediatr. 92:235236. 3. Clymo, A. B., and I. A. Harper. 1974. Ampicillin-resistant Haemophilus influenzae meningitis. Lancet i:453454. 4. Dunkle, L A. 1978. Central nervous system chloramphenicol concentration in premature infants. Antimicrob. Agents Chemother. 13:427-429. 5. Eagon, R. G. 1962. Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J. Bacteriol. 83:736-737. 6. Glazko, J. J., L M. Wolf, and W. A. Dill. 1949. Bio-
45
chemical studies on chloramphenicol. I. Colorimetric methods for determination of chloramphenicol and re-
lated nitro compounds. Arch. Biochem. 23:411-418. 7. Gorbach, S. L, and J. G. Bartlett. 1974. Anaerobic infections. N. Engl. J. Med. 290:1177-1184, 1237-1245, 1289-1294. 8. Joslyn, D. A., and M. Galbraith. 1950. A turbidimetric method for assay of antibiotics. J. Bacteriol. 59:711716. 9. Koop, J. R., B. Brodsky, A. Lau, and T. R. Beam, Jr. 1978. High-performance liquid chromatographic asay of chloramphenicol in serum. Antimicrob. Agents Chemother. 14:439-443. 10. Louie, T. J., F. P. Tally, J. G. Bartlell, and S. L Gorbach. 1976. Rapid microbiological assay for chloramphenicol and tetracyclines. Antimicrob. Agents Chemother. 9:874-878. 11. Nilsson-Ehle, I., G. Kahimeter, and P. Nilsson-Ehle. 1978. Detennination of chloramphenicol in serum and cerebrospinal fluid with high-presure liquid chromatography. J. Antimicrob. Chemother. 4:169-176. 12. Randall, W. A., A. Kirshbaum, J. K. Nielsen, and D. Wintermere. 1949. Diffusion plate assay for chloramphenicol and aureomycin. J. Clin. Invest. 28:940-942. 13. Resnick, G. L., D. Gorbin, and D. Sandberg. 1966. Determination of serum chloramphenicol utilizing gasliquid chromatography and electron capture spectrometry. Anal. Chem. 38:582-585. 14. Robinson, L R., R. Selig8ohn, and S. A. Lerner. 1978. Simplified radioenzymatic assay for chloramphenicol. Antimicrob. Agents Chemother. 13:25-29. 15. Whitlock, C. M., Jr., A. D. Hunt, and S. G. Tushman. 1951. A single simple turbidimetric method for the assay of aureomycin, chloramphenicol, penicillin, streptomycin and terramycin in capillary blood and other body fluids. J. Lab. Clin. Med. 37:155-161. 16. Wilaon, W. R. 1977. Tetracyclines, chloramphenicol, erythromycin and clindamycin. Mayo. Clin. Proc. 52:
635-640.
