Bacitracin, assay

A subsequent patent, U.S. Patent 2,828,246 described a commercial process for bacitracin production. A 1,230 gallon portion of a medium containing 10% soybean oil meal, 2.50% starch and 0.50% calcium carbonate having a pH of 7.0 was inoculated with a culture of bacitracin-producing bacteria of the Bacillus subtilis group and the inoculated medium incubated for a period of 24 hours with aeration such that the superficial air velocity was 12.1. An assay of the nutrient medium following the fermentation revealed a yield of bacitracin amounting to 323 units/ml. This was more than twice the yields previously obtained. 

Using an acrylamide-gel as support, Coombe- 0 has demonstrated the quantitative assay of neomycin with a recovery of 96% in the presence of bacitracin and polymixin. In this case quantitation was achieved by densitometry after staining the gel with naphthalene black. 

Titration with EDTA is used in the pharmacopoeial assays of bismuthsubcarbonate, calcium acetate, calcium chloride, calcium gluconate, magnesium carbonate, magnesium hydroxide, magnesium trisilicate, bacitracin zinc, zinc chloride and zinc undecanoate. 

Bacitracin Minimum fill, water, and assay Well-closed containers containing not more than 60 g controlled temperature... 

Volatile solvents and solutes, up to 0.3 ml, were removed by room temperature centrifugal vacuum (SpeedVac, Savant) in siliconized tubes pre-loaded with 200 pg BSA and 30 fig bacitracin. We found that trace amounts of mercaptoethanol seriously elevate the blanks in the standard radiolabeled isooctane partition assay (13), and residues of TFA require careful readjustment of the pH of the tissue culture medium. Therefore, evaporation was routinely extended for approximately 30 minutes beyond the time of apparent dryness to ensure good removal of solvent modifiers. Dried residues were dissolved in 0.85 ml of modified culture medium 199 (1) by one minute of ultrasonication through the walls of the plastic tube, then left to stand for ca. 30 minutes and finally vortex mixed. 

Resistance to bacitracin does not emerge rapidly in originally susceptible strains. The thermal and storage stability of bacitracin is enhanced by the presence of an equimolar concentration of Zn " and the antibacterial action is potentiated by an excess of Zn " ". It has been suggested that Zn + is also associated with the mechanism of antibacterial action. No chemical assay methods are yet available for bacitracin. For the determination of potency the cylinder plate method with Micrococcus flavus (ATCC 10 240) as test organism is used. ... 

A casual examination of the current literature indicates that there is considerable confusion regarding antibiotic assay standards with regard to the terms unit and microgram used to indicate potency. Bacitracin and polymyxin are defined in terms of antimicrobial units which are not directly expressible in terms of weight of the compounds since these antibiotics have not been isolated in pure form penicillin originally fell into this category until its crystallization. For others which have been isolated in pure... 

Good assay plate methods are available for penicillin, streptomycin, bacitracin, and polymyxin however, the newer broad spectrum antibiotics tend to give poorly defined zone edges on assay plates. 

Johnson et al. (323) described an antibiotic produced by B. subtilis, distinct from subtilin and particularly active against hemolytic Streptococci, and which they called bacitracin. The biological assay, for example the determination of antibiotic activity, has been described by Anker et al. (25) and by Hendlin (268). 

Under various conditions, whole cells of strain A-5 appear to produce only 5 to 10% of the quantity of bacitracin formed by cells of strain 10716 (Bernlohr and Novelli, i960, 1963 Snoke, 196O and Snoke and Cornell, 1965). However, the biosynthetic capacity of the two strains has not been directly compared to determine to what extent differences in conditions of culture and assay in different laboratories might be responsible for the apparent disparity in yield. Published information concerning possible variation in bacitracin yield among large numbers of strains of B. licheniformis is not available (Hickey, 1964). In view of possible associations between bacitracin production and sporu-lation (vide infra), it would be of interest to determine if asporogenous mutants form more, or less, antibiotic than do wild strains. 

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