Disc Plate Method of Microbiological Antibiotic Assay: I. Factors Influencing Variability and Error 1

Several factors are investigated that normally cause variation in zone diameters in conventional disc plate diffusion assay procedures. Of these factors the most serious is the unequal exposure of the individual plates at top or bottom of stacks to temperatures above and below room temperature. This unequal temperature exposure is avoided by novel handling and incubation procedures. A major variable, but one which can be controlled, is the varying time interval between pouring seeded agar and the time of applying the pads with antibiotic to the plates. This influence of time of setting and the effects of several other sequential operations are combined into a composite variable. This variable is then accounted for and normalized by interposing "external" reference plates set with a reference solution in the sequence of approximately 100 plates. No "internal" reference zones are employed. Such factors as volume of agar poured, wedge shape of agar in a dish, volumetric errors in dilutions, and timing considerations are studied and discussed. The results of this study form the basis for a test protocol which is presented in a following paper.     

Bleomycin, an Antitumor Antibiotic: Improved Microbiological Assay and Tissue Distribution Studies in Normal Mice

A microbiological assay was developed for bleomycin, an antitumor antibiotic reported to be active in human trials. The assay bacterium was a strain of Escherichia coli which is resistant to ethionine. Studies revealed relatively high concentrations of bleomycin in the blood and urine of mice after a single dose, < 0.33 ld(10), injected intraperitoneally.     

Microbiological Assay for Estimating Salivary Concentrations of the Cariostatic Antibiotic Actinobolin

A logarithmic-ratio microbiological assay has been developed for the estimation of concentrations of actinobolin (or inhibitory equivalents) in human saliva. The utility of this assay has been demonstrated by presenting data that show concentrations of this antibiotic (or inhibitory equivalents) detected in saliva or saline after incubation at 37 C for periods as long as 6 hr. The results of some statistical analyses of the assay data have been included.     

Modified Microbiological Assay for Rapid Estimation of Antibiotic Concentrations in Human Sera

Antibiotic concentrations in human sera were estimated in 5 to 6 hr by a modified microbiological assay. By using Staphylococcus aureus and Streptococcus pyogenes as the assay organisms, the seeded assay plates were preincubated for 2 to 6 hr and then were stored at 4 C until used for assay. Paper discs saturated with the specimen were placed on the preincubated assay plates with reference discs saturated with known concentrations of antibiotic. After 5 to 6 hr of incubation, zones of antibacterial activity were measured and compared with a standard curve for estimation of antibiotic concentration. Results from this rapid assay method compared favorably with those from the commonly used 24-hr assay.     

Improved Method of Assay for Choline

A modified assay for choline is described which is shorter, yields more consistent responses, and requires considerably less time, space, and equipment than the existing assays.     

Semiautomated Microbiological Method for the Assay of Quaternary Ammonium Compounds

A semiautomated method has been developed for quantitatively assaying the activity of detergent disinfectants. Automation permitted a high level of reproducibility, which in turn allowed a meaningful comparison between the activities of some types of quaternary ammonium compounds.     

Simplified, Accurate Method for Antibiotic Assay of Clinical Specimens

Large glass plates are used for this modified agar-well diffusion assay method, allowing up to 81 replications on a single plate. With a specially designed agar punch, it is possible to prepare the small agar wells very quickly. The saving in serum resulting from fewer replications of standards with the large plates, and the small volume of the agar wells, makes it economically feasible to use pooled human serum for the standard antibiotic solutions. Methods are described for preparing the standard solutions, and for providing controls for the deterioration of standards and unknowns. Procedures for preparing and maintaining the commonly used assay organisms are presented. Serum specimens are tested directly rather than diluting them to a narrow range of antibiotic concentrations. This is possible because of a procedure for calculations that recognizes the curvilinear relationship between zone sizes and antibiotic concentrations. Adaptation of this method to a number of the commonly used antibiotics is described. With this method, it has been possible to test large numbers of clinical specimens in a minimal time, and with accuracy consistently better than 10%.     

Microbiological Assay for Organic Compounds in Seawater. II: Distribution of Adenine, Uracil, and Threonine

Biochemically deficient strains of Serratia marinorubra have been isolated with specific requirements for adenine, uracil, and threonine. Standard curves for dose to growth response have been obtained showing a linear sensitivity from 0.5 to 4.0 mg of adenine per liter of seawater, 0.1 to 2.0 mg of uracil per liter of seawater, and 0.5 to 10 mg of threonine per liter of seawater. These mutants have been used to test for the presence of their required metabolites in natural seawater samples from the Gulf of Mexico and adjacent bays. Of the three compounds under investigation, adenine was found in 10 samples, uracil in 2 samples, and threonine in none. The possible significance of these findings to the marine environment is discussed.     

用蚀斑法滴定痘苗病毒的准确性及其精确数学模型的探讨

用蚀斑法滴定病毒是确定感染病毒颗粒存在数量的一种较准确方法。本实验表明,痘苗病毒吸附4h后仍有大量病毒粒子未能吸附到细胞单层,进而测定出病毒接种量、维持液加量和所测病毒滴度间具有一种互为消长的非线性相关性。因而设计了几种检测方法,其准确性均优于常规痘苗病毒蚀斑测定法。利用装配有Mathematic软件包的计算机在痘苗病毒接种量、维持液加量和所测病毒滴度间建立了曲线拟合模型和曲面拟合模型。通过曲线拟合模型推断病毒感染滴度为常规法滴定值的近5倍。     

Disc-Agar Diffusion Microbiological Assay Procedure for Determining Serum and Urine Levels of Sulfacytine and Other Sulfonamides

A reasonably precise, reproducible, and sensitive microbiological procedure for directly assaying sulfacytine and other sulfonamides as antibacterially active drugs has been developed by appropriately modifying the standard disc-agar diffusion technique. Blood and urine levels as low as 3 mug/ml may be determined through the use of a strain of Escherichia coli and a chemically defined agar medium devoid of sulfonamide antagonists. Results indicate that this assay method should be a useful adjunct to the Bratton-Marshall colorimetric procedure, by permitting the direct measurement of antibacterially active drug in clinical specimens.     

An Improved Microbiological Procedure for Detection of Streptomycin Residues in Milk and Animal Tissues

A method is described which allows detection of 0.025 µg streptomycin sulfate per ml. This represents an improvement of sensitivity by 8 times when compared with the currently used method. By adding penicillin to the assay medium in subinhibitory concentrations, a synergistic effect of streptomycin and penicillin is exerted towards the test organism, Bacillus subtilis, resulting in an increased sensitivity to streptomycin.     

The Structures of Myocotrienins I. and II,a Novel Class of Ansamycin Antibiotic

Endopolygalacturonase I [EC 3.2.1.15], the major component of endopolygalacturonases causing silver-leaf symptoms, was purified from culture liquids of Stereum purpureum by column chromatographies on CM-52 and Sephadex G-100. The purified enzyme was homogeneous on Polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation coefficient (S_20,W) was determined to be 3.21 S, and the molecular weight was estimated to be 40,000 by gel filtration, 41,000 by SDS-polyacrylamide gel electrophoresis and 44,000 by sedimentation equilibrium. The enzyme had an isoelectric point of pH 8.5. The optimal pH of the enzyme was 3.5 for trigalacturonic acid, 4.0 for tetragalacturonic acid, and 4.5 for pectic acid. The enzyme was stable in the range of pH 4.0 to 9.0 and up to 70%C for 30 min. The amount of the enzyme which was required to induce silver-leaf symptoms on apple trees was 20 μg/tree.