Microbiological Aspects of Penicillin

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Abstract
The authors prefer Bacillus subtilis to Staphylococcus aureus as a test organism. The procedure consists of the following:[long dash](I) Prepn. of a spore inoculum of B. subtilis:[long dash]a) inoculation of slant culture into standard beef extract broth pH 6 to 7; b) preventing the formation of surface pellicle, and uniform cell suspension assured, by maintaining the liquid in constant agitation by securing flask to shaking machine; c) incubation at 30-37[degree] C from 4-8 days (until max. sporulation occurs); d) the whole culture is pasteurized at 60[degree] for 30 min. to destroy viable vegetative cells. The resulting heavy spore suspension, when stored cold, can be used for penicillin assays until depleted. Optimum density of inogulum is 15,000,000 spores per liter of melted agar.[long dash](II) Preparing the plates:[long dash]2% melted agar cooled to 50-60[degree] is seeded with spore suspension and shaken thoroughly. Melted agar can be stored in an oven at 55-60[degree] C. 13-ml. portions of melted seeded agar are pipetted into 100 [image] 15 mm. Petri dishes. It is preferable to store plates in the icebox for an hr. or more but not to allow plates to stand at room temp, for more than a few minutes.:[long dash](III) Setting up the cups:[long dash]The cups are small glass cylinders of, 12 mm.length, cut uniformly from plain pyrex glass tubing (8 mm. outside diam., 1 mm. wall thickness) by a motor-driven glass-cutting disc. Bevelled edges are not essential. The cylinders are sterilized with dry heat upright in petri dishes. All the cylinders are heated simultaneously on an electric hot plate, and set individually on the agar with forceps. Cups should not be warm enough to sink into the agar. Inverting plates tests whether or not the cups are sealed securely.[long dash](IV) Setting up the samples:[long dash]-Unknown penicillin solns. are diluted with sterile M/50 phosphate buffer at 6H 7, so that the final dilution contains 0.5-1 Oxford unit per ml. Penicillin must be kept cold. One person can assay about 150 previously diluted samples per day. Glassware for use in assays should be cleaned by heating to 80-90[degree] C in conc. H2SO4 with a few crystals of KN03 added daily. Penicillin is extremely unstable in the presence of certain metallic ions. The writers recommend use of 2 standard solns. of penicillin; if one loses its potency, the other remains for the day''s work. Cups are filled with a medicine dropper with 90[degree] angle outlet into one cup on each of 4 plates.[long dash](V) Measurement of inhibition zones:[long dash]Zones are measured to 0.5 mm. accuracy and are read against a standard curve, whose best range lies between 0.3 and 1.4 units per ml.