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Microbiological assay of individual antibiotics in mixtures of antibiotics depends on the use of selective inactivation and/or of test bacteria with differential susceptibility. Controlled experiments revealed that streptomycin in concentrations of 20 and 40 mug/ml did not influence a disk diffusion assay of penicillin with Sarcina lutea (ATCC 9341) as the test organism. In the case of penicillin concentrations less than or equal to 1 IU/ml, addition of 80 mug of streptomycin per ml influenced the penicillin assay significantly. Clinical use of streptomycin resulting in levels above 40 mug/ml usually did not occur; therefore penicillin could be assayed as though streptomycin were not present. We observed additionally that S. lutea was unable to grow on agar plates prepared with semicarbazide hydrochloride.

Antibiotics that inhibit the biosynthesis of the cell wall, such as vancomycin, penicillin, d-cycloserine, and bacitracin, stimulate the incorporation of lysine into lipids that are extractable with n-butanol-6 M pyridinium acetate. Approximately 93% of this lysine is in lysylphosphatidylglycerol (LPG). The remaining lysine is incorporated in another as yet uncharacterized lipid. Because the lysine in the latter lipid is released by mild alkaline hydrolysis, it is not the C(55)-isoprenyl-pyrophospho-N-acetyl-muramyl pentapeptide. Vancomycin and penicillin stimulate the incorporation of lysine into both LPG and the minor lipid fractions, whereas treatment with d-cycloserine results in an increase only in LPG. Antibiotics that inhibit protein synthesis do not influence the incorporation of lysine into the lipid fractions. Analysis of the extracted lipids indicate that the incorporation of radioactive lysine into LPG is due to an enhancement in synthesis of LPG from phospholipids in the cytoplasmic membrane.

An analytical procedure was devised which can distinguish members of the streptothricin family of antibiotics. It is based upon an analysis of the hydrolysis products of the antibiotics using a Technicon amino acid autoanalyzer under special conditions. The various fragments including the different streptolidine-amino sugar compounds were well resolved. A basic water-soluble antibiotic discovered in our laboratories and named LL-BL136 was compared to other members of this group by this technique. It was not differentiated from the antibiotic SF-701 reported by Tsuruoka. The autoanalyzer results along with other physicochemical data permitted a structure proposal for this antibiotic, which is the N-methyl-desformimino derivative of antibiotic LL-AC541.

Under well-established culture conditions, Pseudomonas reptilivora produced several antibiotics that have been purified by solvent extraction, chromatography in Sephadex G-25, electrophoresis, and paper chromatography in different solvent systems. Activity has been monitored at the different steps of isolation and purification by measurement of the inhibition of the growth of Staphylococcus aureus by the cylinder-plate method, as well as by bioautography of chromatograms and electropherograms. Three antibiotics have been isolated and named A, B(1), and B(2). The B(1) and B(2) activities were studied in greater detail than A. The B(1) substance was crystallized, and its chemical properties were found to coincide with those of YC 73 or fluopsin C described by Egawa et al. and Itoh et al., respectively.

The myxomycete Physarum gyrosum was cultured in its plasmodial stage on agar plates containing 0.025 M phosphate buffer at pH 6.5, 2% bakers' yeast, and 0.2% glucose and was supplemented with live Escherichia coli. Extracts of these plasmodia contained several antibiotic substances. Antibiotic materials were partially purified by dialysis of the agar medium-mold mixture, evaporation of the dialyzate, and butanol extraction of the residue. Further purification in two paper and two thin-layer chromatographic systems gave one product which was pure in six thin-layer chromatographic systems. Antibiotic activity against some gram-positive and gram-negative bacteria and yeasts was demonstrated with partially purified extracts and a paper-chromatographically separated fraction. One pure antibiotic was effective against Bacillus cereus.

Two paper strips, each containing different antimicrobial agents, were placed on plates on Mueller-Hinton agar to permit antibiotic to enter the agar. A filter membrane was placed on this plate, and the microorganisms were planted on the membrane. After 6 h of incubation at 37 C, the membrane was transferred to antibiotic-free Mueller-Hinton agar containing triphenyltetrazolium hydrochloride and incubated for 18 h at 37 C. Specific growth patterns were indicative of additive (indifferent), synergistic, or antagonistic effects of the drug combination used. Trimethoprim and sulfamethoxazole proved to act synergistically against 85% of Escherichia coli, 86% of Klebsiella, and 89% of Proteus mirabilis strains tested. A few strains resistant to either drug were susceptible to their combination. The technique was useful against organisms with widely differing susceptibilities to the two antimicrobial agents tested.

Butirosin, a new aminoglycosidic antibiotic complex, was produced by submerged fermentation with each of two strains of Bacillus circulans. A paper-disc, agar-diffusion assay which employs Escherichia coli (P-D 04863) as the test organism has been developed. Shaken-flask and stirred-jar fermentations in a medium containing glycerol, soybean meal, meat peptone, ammonium chloride, and calcium carbonate reach titers of 500 to 700 mug of butirosin base per ml. Butirosin is active against several gram-positive and gram-negative bacteria including Pseudomonas aeruginosa.