Vol. 10, No. 4 Printed in U.S.A.
ANTIMIcROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1976, p. 776-777 Copyright C 1976 American Society for Microbiology
New Medium for In Vitro Susceptibility Studies with Amphotericin B C. J. UTZ, S. WHITE, AND S. SHADOMY* Division ofInfectious Diseases, Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Received for publication 1 July 1976
Antibiotic medium 20 FDA and antibiotic medium 3 FDA were compared to determine if antibiotic medium 3 would be suitable for in vitro susceptibility testing with amphotericin B.
Many of our previous in vitro studies involving susceptibility testing of amphotericin B against clinical isolates of pathogenic fungi were performed using a broth medium originally recommended by Groves and Randall for use in microbiological assay of this drug (1). This medium, known as antibiotic medium 20 FDA (M-20), more recently has been recommended for routine use in susceptibility testing with both amphotericin B and nystatin (3). Unfortunately, commercial production of M-20 has been discontinued. With the nonavailability of M-20, it became necessary to choose another medium that would yield diagnostically similar results in in vitro susceptibility tests. A somewhat similar medium, antibiotic medium 3 FDA (Difco Laboratories, M-3) was selected. The main differences between these two media are in the total amounts of yeast extract, dextrose, and tryptone. M-3 contains 5 g of yeast extract per liter less and 10 g of dextrose per liter less than does M-20, whereas M-20 contains 10 g of tryptone per liter. All other constituents of the two media are similar (Table 1). A brief study was undertaken to compare the two media and to determine if M-3 would be suitable for in vitro susceptibility testing with amphotericin B. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations were determined in both media for a variety of pathogenic yeasts in parallel using a previously described broth dilution method (3). Concentrations of amphotericin B ranged from 50 to 0.05 jig/ml. The drug was initially dissolved in 100% (CH3)2SO, and a stock concentration of 100 ,ug/ ml was then prepared in 5% (CHA)2SO and maintained at -20°C. Five clinical isolates each of Cryptococcus neoformans and Candida albicans and four of Candida parapsilosis were tested. Saccharomyces cerevisiae ATCC 9763 was tested along with each isolate as a control. MIC end points were determined after 48 h of incubation at 30°C. The MIC was defined as the 776
TABLE
1. Compositions of antibiotic media M-3 and M-20 M-3 (g/liter) M-20 (g/liter) Component Beef extract 1.5 1.5 Yeast extract 1.5. 6.5 Peptone 5.0 5.0 Tryptone 10.0 Dextrose 1.0 11.0 NaCl 3.5 3.5 K2PO4 3.68 3.68 NaHPO4 1.32 1.32 Final pH 7.0 6.6
TABLE 2. In vitro susceptibility tests with amphotericin B in two nonsynthetic media Antibiotic medium M-20
M-3
Isolate MIC a
MFCa
(gI/ml)
(/1g/ml)
mgl)
MFC ml)
0.05 0.39 0.39 0.05 0.20
0.10 0.39 0.39 0.20 0.20
0.05 0.20 0.05 0.05 0.05
0.05 0.20 0.05 0.05 0.05
0.39 0.39 0.78 0.39 0.78
1.56 0.78 1.56 0.78 1.56
0.20 0.20 0.20 0.20 0.20
0.20
0.20 0.39 0.20 0.39
0.39 0.20 0.39 0.10
0.39 0.39 6.25 0.39
0.05 0.05 0.20 0.05
12.5 0.20 12.5 0.05
0.39-0.78
0.39-0.78
0.05
0.05
MIC
Cryptococcus neoformans 9.1 9.7 9.17 9.18 9.49
Candida albicans 7.2 7.7 7.18 7.31 7.66
Candida parapsilosis 7.1 7.3 7.6 7.21
Saccharomyces cerevisiae 14 tests a
MIC, Minimal inhibitory concentration; MFC, minimal fungicidal concentration.
VOL. 10, 1976
lowest concentration of drug that inhibited growth as measured by the absence of a visible "button" at the bottom of each tube. MFC were determined by subculturing, on Sabouraud agar, all negative tubes. Results with this study indicate that M-3 can be used for susceptibility testing with yeastlike organisms and amphotericin B (Table 2). The MIC and MFC results in all 14 individual tests with the control organism S. cerevisiae were constant, 0.05 ,ug/ml, indicating uniformity of results. Most MIC and MFC values measured for the clinical isolates in M-3 were somewhat lower than those measured in M-20. Differences between MIC values measured in the two media were significant (t test, P < 0.001), but differences in MFC values were not. With only two isolates, Candida parapsilosis 7.1 and C. parapsilosis 7.6, was a higher MFC measured in M-3 than in M-20. These differences were not critical in determination of clinical susceptibility. Differences between inhibitory
NOTES
777
and fungicidal end points measured in the two media most likely could be attributed to a lesser production of acid in M-3, with a subsequent reduction in decay of active drug, because of the lower carbohydrate content of this medium. The adverse effect of an acidic pH on the activity of amphotericin B is the primary reason why unbuffered synthetic media, such as yeast nitrogen base, are unsuitable for in vitro testing with polyene compounds (2). LITERATURE CITED 1. Grove, D. C. and W. A. Randall. 1955. Assay methods of antibiotics. Medical Encyclopedia, Inc., New York. 2. Shadomy, S. 1974. Laboratory evaluation of antifungal agents, p. 27-36a. In 14th National Medicinal Chemistry Symposium. Division of Medicinal Chemistry, American Chemical Society, University of New Hampshire, Durham, N. H. 3. Shadomy, S., and A. Espinel-Ingroff. 1974. Susceptibility testing of antifungal agents, p. 569-574. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.
ANTIMIcROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1976, p. 776-777 Copyright C 1976 American Society for Microbiology
New Medium for In Vitro Susceptibility Studies with Amphotericin B C. J. UTZ, S. WHITE, AND S. SHADOMY* Division ofInfectious Diseases, Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Received for publication 1 July 1976
Antibiotic medium 20 FDA and antibiotic medium 3 FDA were compared to determine if antibiotic medium 3 would be suitable for in vitro susceptibility testing with amphotericin B.
Many of our previous in vitro studies involving susceptibility testing of amphotericin B against clinical isolates of pathogenic fungi were performed using a broth medium originally recommended by Groves and Randall for use in microbiological assay of this drug (1). This medium, known as antibiotic medium 20 FDA (M-20), more recently has been recommended for routine use in susceptibility testing with both amphotericin B and nystatin (3). Unfortunately, commercial production of M-20 has been discontinued. With the nonavailability of M-20, it became necessary to choose another medium that would yield diagnostically similar results in in vitro susceptibility tests. A somewhat similar medium, antibiotic medium 3 FDA (Difco Laboratories, M-3) was selected. The main differences between these two media are in the total amounts of yeast extract, dextrose, and tryptone. M-3 contains 5 g of yeast extract per liter less and 10 g of dextrose per liter less than does M-20, whereas M-20 contains 10 g of tryptone per liter. All other constituents of the two media are similar (Table 1). A brief study was undertaken to compare the two media and to determine if M-3 would be suitable for in vitro susceptibility testing with amphotericin B. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations were determined in both media for a variety of pathogenic yeasts in parallel using a previously described broth dilution method (3). Concentrations of amphotericin B ranged from 50 to 0.05 jig/ml. The drug was initially dissolved in 100% (CH3)2SO, and a stock concentration of 100 ,ug/ ml was then prepared in 5% (CHA)2SO and maintained at -20°C. Five clinical isolates each of Cryptococcus neoformans and Candida albicans and four of Candida parapsilosis were tested. Saccharomyces cerevisiae ATCC 9763 was tested along with each isolate as a control. MIC end points were determined after 48 h of incubation at 30°C. The MIC was defined as the 776
TABLE
1. Compositions of antibiotic media M-3 and M-20 M-3 (g/liter) M-20 (g/liter) Component Beef extract 1.5 1.5 Yeast extract 1.5. 6.5 Peptone 5.0 5.0 Tryptone 10.0 Dextrose 1.0 11.0 NaCl 3.5 3.5 K2PO4 3.68 3.68 NaHPO4 1.32 1.32 Final pH 7.0 6.6
TABLE 2. In vitro susceptibility tests with amphotericin B in two nonsynthetic media Antibiotic medium M-20
M-3
Isolate MIC a
MFCa
(gI/ml)
(/1g/ml)
mgl)
MFC ml)
0.05 0.39 0.39 0.05 0.20
0.10 0.39 0.39 0.20 0.20
0.05 0.20 0.05 0.05 0.05
0.05 0.20 0.05 0.05 0.05
0.39 0.39 0.78 0.39 0.78
1.56 0.78 1.56 0.78 1.56
0.20 0.20 0.20 0.20 0.20
0.20
0.20 0.39 0.20 0.39
0.39 0.20 0.39 0.10
0.39 0.39 6.25 0.39
0.05 0.05 0.20 0.05
12.5 0.20 12.5 0.05
0.39-0.78
0.39-0.78
0.05
0.05
MIC
Cryptococcus neoformans 9.1 9.7 9.17 9.18 9.49
Candida albicans 7.2 7.7 7.18 7.31 7.66
Candida parapsilosis 7.1 7.3 7.6 7.21
Saccharomyces cerevisiae 14 tests a
MIC, Minimal inhibitory concentration; MFC, minimal fungicidal concentration.
VOL. 10, 1976
lowest concentration of drug that inhibited growth as measured by the absence of a visible "button" at the bottom of each tube. MFC were determined by subculturing, on Sabouraud agar, all negative tubes. Results with this study indicate that M-3 can be used for susceptibility testing with yeastlike organisms and amphotericin B (Table 2). The MIC and MFC results in all 14 individual tests with the control organism S. cerevisiae were constant, 0.05 ,ug/ml, indicating uniformity of results. Most MIC and MFC values measured for the clinical isolates in M-3 were somewhat lower than those measured in M-20. Differences between MIC values measured in the two media were significant (t test, P < 0.001), but differences in MFC values were not. With only two isolates, Candida parapsilosis 7.1 and C. parapsilosis 7.6, was a higher MFC measured in M-3 than in M-20. These differences were not critical in determination of clinical susceptibility. Differences between inhibitory
NOTES
777
and fungicidal end points measured in the two media most likely could be attributed to a lesser production of acid in M-3, with a subsequent reduction in decay of active drug, because of the lower carbohydrate content of this medium. The adverse effect of an acidic pH on the activity of amphotericin B is the primary reason why unbuffered synthetic media, such as yeast nitrogen base, are unsuitable for in vitro testing with polyene compounds (2). LITERATURE CITED 1. Grove, D. C. and W. A. Randall. 1955. Assay methods of antibiotics. Medical Encyclopedia, Inc., New York. 2. Shadomy, S. 1974. Laboratory evaluation of antifungal agents, p. 27-36a. In 14th National Medicinal Chemistry Symposium. Division of Medicinal Chemistry, American Chemical Society, University of New Hampshire, Durham, N. H. 3. Shadomy, S., and A. Espinel-Ingroff. 1974. Susceptibility testing of antifungal agents, p. 569-574. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.
