Contamination of Azotobacter chroococcum by Gram-negative Bacterial Rods

Abstract

IN a recent investigation, it proved virtually impossible to obtain a pure culture of Azotobacter chroococcum. After initial isolation from soil in flasks containing 2 per cent mannitol and 0.02 per cent sodium phosphate in tap water, progressive subcultures were made on solid nitrogen–free medium containing washed agar and the same percentages of mannitol and phosphate in distilled water—a recognized procedure for purifying Azotobacter¹. Characteristic colonies of Azotobacter were obtained in about a month, and stained smear preparations of these colonies revealed only the characteristic large cells of the organism. If, however, colonies were inoculated into peptone water (in which Azotobacter will not grow²) a faint haze due to growth of small Gram-negative rods appeared after 24–48 hr. incubation at 22° C. A more abundant growth of these rods was obtained in 24 hr. on nutrient agar containing 0.1 per cent whole milk, whereas on this medium the growth of Azotobacter was very poor after three to four days. Subculture of Azotobacter on solid nitrogen-free medium (above) was therefore extended up to periods of four months, and 0.1 per cent sodium silicate, 0.002 per cent iron (FeCl₃) and boron, 0.00004 per cent sodium molybdate, soil extract and hay-infusion were incorporated in some cultures to heighten the resistance of Azotobacter. In practically all cases, however, the contamination persisted. On the one or two occasions on which it appeared to be absent, growth of Azotobacterwas very poor, marked by absence of the characteristic mucilage, and ceased within seven to fourteen days; nor could it be restored by incorporation of any of the above substances. Fourteen strains of contaminants were isolated the cultural characters of which proved conclusively they were not pleomorphic forms of Azotobacter, the majority being strains of Pseudomonas (fluorescent and non-fluorescent) with one or two species of Achromobacter. In the absence of Azotobacter, they grow well up to four weeks in flasks of the above liquid mannitol medium, reducing its reaction to a pH of 4.0 in a fortnight although growth was still vigorous. Their source of nitrogen in such cultures is obscure. They grew in liquid media, distilled water with 1 per cent glucose and ammonia (0.1 per cent (NH₄)₂HPO₄) as the sole source of nitrogen; but only feebly in 1 per cent glucose only. Incorporation of whole killed cultures, extracts or nitrates of cultures improved growth of Azotobacter on solid nitrogen-free media and improved its mucilage production.