Microdilution and broth dilution techniques were compared for the susceptibility testing of 50 clinical yeast isolates to 5-fluorocytosine and amphotericin B. Good correlation between methods was obtained with all isolates except Cryptococcus neoformans.Several broth dilution techniques for the susceptibility testing of yeasts to 5-fluorocytosine (5-FC) and amphotericin B (AmB) have been described elsewhere (5,9, 11-13). These methods are cumbersome to perform, and the development of more rapid and simple procedures is desirable.This report describes the comparison of a microdilution and broth dilution technique for the susceptibility testing of yeasts to both 5-FC and AmB. Comparisons were made between tests incubated for 24 and 48 h at 25 and 35°C. Stability of the drugs at -70°C is also discussed.Organisms. Fifty yeast isolates were obtained from clinical specimens in the mycology laboratory at Hartford Hospital and identified by standard methods (2-4). These included 14 strains of Cryptococcus neoformans, 22 strains of Candida albicans, 15 Candida species not albicans, 4 strains of Torulopsis glabrata, and 1 strain each of Geotrichum candidum and Trichosporon species. The yeasts were maintained at room temperature on Sabouraud dextrose agar slants.Drugs. 5-FC was supplied as a pure powder by Hoffman-LaRoche, Inc., Nutley, N.J. The drug was dissolved in sterile distilled water with the aid of gentle heat from a bunsen burner, sterilized through a 0.45-,um filter, and frozen at -70°C in 1.5-ml portions of 1,000 jig/ml. AmB (Fungizone) was obtained from E.R. Squibb and Sons, Inc., Princeton, N.J., rehydrated with 100% dimethyl sulfoxide, and frozen at -70°C in 1.5-ml portions of 1,000 ,ug/ml.Methods. The broth dilution technique for 5-FC was performed in yeast nitrogen broth as described by Shadomy (11,12). AmB was tested in Sabouraud dextrose broth (5, 7, 9). Duplicate twofold dilutions of each drug were prepared in 1-ml volumes on the day of testing and inoculated with 50 pl of a 105-cells-per-ml yeast suspension in the appropriate liquid medium. The final density was 5 x 103 cells per ml. One set of tubes was incubated at 35°C, and another was incubated at 25°C. The microdilution technique was performed in microtiter U-bottom trays (Flow Laboratories, Rockville, Md.) with a semiautomatic pipetter and diluter (Cooke Laboratory Products, Alexandria, Va.) to prepare the twofold serial dilutions. The trays were individually wrapped in plastic and stored at -70°C. Trays were removed from the freezer 0.5 h before use and thawed at room temperature. The same suspension as used for broth dilution was used for microdilution, yielding a final inoculum density of 104 cells per ml. Duplicate trays were sealed with tape; one tray was incubated at 25°C and another was incubated at 35°C. The minimum inhibitory concentration (MIC) was read at 24 and 48 h as the least amount of drug which inhibited visual growth. A C. albicans no. 6631 control was provided by Hoffman-LaRoche and run each day of testing with both 5-FC and A...
The ratio between the concentrations of different antimicrobial agents varies widely during therapy and often bears no resemblance to the ratios assessed by in vitro methods to evaluate the effectiveness of multiple antimicrobial therapy. The authors examined an alternative method using patient serum that reflects the actual antibiotic levels achieved in the patient. Previous investigators have shown that the ratio of the concentration of free drug (drug-f) to the minimal bactericidal concentration (MBC) in broth approximates the serum bactericidal titer (SBT) when pooled normal human serum is used as the diluent. Theoretically, when two antimicrobial agents are administered, the SBT should be equivalent to (drug-f A/MBC drug A) + (drug-f B/MBC drug B). SBT and drug level determinations were performed on peak and trough serum specimens from ten patients with endocarditis or osteomyelitis who were receiving multiple antimicrobial therapy. The serum dilution synergy method predicted additive interactions twice as often as the checkerboard and kill curve, and predicted synergy less frequently than the kill curve. The checkerboard predicted antagonism four times more often than the other methods and provided equivocal results in four of ten cases. The suggested method may offer an alternative procedure to assess antimicrobial interactions, which is based on antibiotic levels actually achieved in vivo instead of the arbitrary concentrations often used in in vitro tests.
A rapid method of antimicrobial susceptibility testing has been developed, which uses a modified microdilution procedure and an inoculum of 107 bacteria per ml. Results are determined within 4 h with an indicator conng of 2(piodophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride. The precipitation of a red fonnazan by bacteria uninhibited by antimicrobials is accelerated by the addition of phenazine methosulfate. Isolates are clified as resistant, indeterminate, or susceptible, based on growth in up to two antimicrobial concentrations which conform closely to concentrations which correlate with the millimeter breakpoints used in the Bauer-Kirby method. Results of testing 10 antimicrobial agents against 1,126 isolates were compared with results obtained when the Bauer-Kirby method and the agar dilution procedure were used as reference methods. Enterococci were excluded because of false resistance. Discrepancies were classified as very major (false susceptibility), major (false reLsitance), and minor (combinations of susceptibility or reistance with indeterminate results). The rapid method versus the agar dilution method yielded 2.3% very major, 0.7% major, and 2.9% minor discrepancies, for a total of 6.0%. Of 58 organim-antimicrobial agent combinations tested, 23 displayed 1% very major discrepancies between the rapid method and the agar dilution method. Six were not therapeutically important. The remainder involved Staphylococcus aureus, Staphylococcus epidermnidis, Acinetobacter op., and most organisms tested with chloramphenicol. It is suggested that adjustments in antibiotic concentrations and/or inoculum size may eliminate these discrepancies. The rapid method appeared economical when compared with Autobac 1 and the Bauer-Kirby procedure.
Dimethylsulfoxide (DMSO) , an alkyl sulfoxide, is a powerful solvent derived from a waste product of paper manufacture. Its properties and the clinical problems for which it has been tried have been described in a comprehensive review ( 1).Experimentally and clinically DMSO has been reported to penetrate skin rapidly and to cause no permanent tissue damage even in high concentrations (2). Effects on the collagen of the dermis have been claimed in the treatment of several dermatoses-psoriasis, scleroderma, keloids, radiation fibrosis, and eczema (3-5)-suggesting that a t least abnormal collagen is affected by DMSO.Burn therapy with DMSO has received only limited attention. DMSO alone and combined with Sulfamylon was applied intermittently in the treatment of partial and fullthickness burns in rats (6). Penetration of the eschar by DMSO was theoretically demonstrated by the addition of methylene blue. There was no increase in survival rates nor decrease in Pseudomonas organisms (7). Rabbit skin placed in DMSO for 24 hr produced skin that could be penetrated easily with a finger, indicating destruction (8). Tensile strength of rat tail tendons treated for 24 hr with varied concentrations of DMSO decreased when a concentration of 95% or higher was used. I t was concluded DMSO changed the physical properties of eollagen by causing lysis of its intermolecular bonds.With this background and because dried collagen from tendon swelled markedly in DMSO, we wondered if DMSO could alter the susceptibility of collagen to digestion by proteolytic enzymes that ordinarily did not attack it. By definition, only collagenase digests sclerocollagen while trypsin and similar pro-teolytic enzymes can digest collagen only after it is denatured. Here we have investigated the effect of DMSO on the tensile strength of normal and burned dog skin and on the capacity of proteolytic enzymes other than collagenase to digest both. Partially denatured beef tendon collagen is also used.Methods and Materials. Large swatches of fresh skin were excised from the abdomen of a dead dog and were mechanically defatted.Several 12 X 6 cm pieces were cut and burned to full thickness by passing them through a bunsen burner flame for 15 sec with the keratin side toward the flame. Sections of both burned and normal skin were then totally immersed in 50% and 100% DMSO at room temperature for 24 hr. All skin was then fashioned into 8 X 1-cm strips with small triangles cut from the middle of the long sides to leave a central isthmus of 3 mm. Their tensile strengths were tested on a Scott Tester, Model X-3,l with special clamps for holding the skin. To correlate anatomical changes, specimens of normal and normal treated, and burned untreated and treated were examined for microscopic differences after coloring with hematoxolin and eosin stain.The enzyme study was initiated by determining the effect of varied concentrations of DMSO on the activity of elastase and trypsin against substrates that they do attack. One milligram of elastase (Worthington) in 0.1 % concent...
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