A Cytological Study of a Pleomorphic Strain of Azotobacter With the Electron and Phase Microscopes and the Robinow Nuclear-Staining Technique

Abstract

L6hnis and Smith (1923) pointed out that not less than seven different morphological types can be developed and stabilized from Azotobacter cultures. This extreme transmutation of cells has been questioned by subsequent investigators (Lewis, 1937, 1941). Nevertheless, peculiar morphological phases do occur in Azotobacter cultures and no satisfactory explanation has been given as to their function. Several new techniques, as well as some new concepts, have been developed since these earlier workers debated the significance of the morphological cell types in Azotobacter species. The purpose of the investigation now being reported was to see whether phase microscopy, electron microscopy, and nuclear staining might add information concerning the cytology of Azotobacter. Also, recent discussions of sexuality and peculiar reproductive processes in bacteria (Dienes, 1946; Lederberg, 1948) encouraged the authors of this paper to search for nuclear arrangements among large bacteria, such as in the genus Azotobacter. MATERIALS AND METHODS About 20 strains of organisms representing all species of the genus Azotobacter were observed. One culture, however, was chosen for this report so that results would be comparable. The organism selected for this study was Azotobacter agile (University of Illinois strain ZN 350). This species was grown on several different media, but the results in this report are from cells grown on Ashby's nitrogen-free medium. Cultures ranging in age from 3 hours to 60 days were examined. Each culture was observed with phase microscopy after light nigrosin background staining, according to a method previously described (Eisenstark and McMahon, 1949), to determine morphology and to view internal structures at different ages. Electron microscopy was done with the R.C.A. type EMC instrument. Cultures were prepared by suspension of cells in distilled water followed by centrifugation and resuspension. With full recognition that this method may produce slightly distorted cells,

A Study of Large Bodies in Azotobacter Agile

“…Tests were made by nuclear staining and phase microscopy to determine a possible pattern of changes of chromatinic bodies in the development of various forms. Distinct bodies could be seen in all phases; however, there did not seem to be the definite pattern of change that has been described for other species of bacteria (Robinow, 1942;Bissett, 1948;Tulsne, 1949a;Dienes, 1950a;Smith 1950;Eisenstark et al, 1950). Instead of the typical dumbbell-shaped units found in rod bacteria, dots along the edge of the cell seemed to be joined in a circle by a fine thread in most cases (figures 11 and 12).…”

The Azotobacteriaceae

“…L6hnis and Smith (13) have gone farthest in this respect and described a complex "life cycle" comprising many different types of organisms as stabilized stages. This has not been confirmed by later investigators (14,15,16,17,18,19,20,21), who have mostly explained L6hnis and Smith's results as due to latent contaminants that often are difficult to eliminate from the cultures of Azotobacter; this explanation, indeed, would hardly account for the reported regeneration of normal azotobacter from some of the aberrant forms, e.g., the so-called "fungoid form" studied by Lbhnis and Smith. Yet the morphology of Azotobacter in reliably pure cultures is remarkably variable, and general agreement seems to exist that the following cell types occur:…”

“…These have played an important role in discussions on pleomorphism in the azotobacter and were described as "gonidangia" by L3hnis and Smith (13). Their nature is not yet clear but upon the whole they seem to arise under conditions of nutrition different from those in simple nitrogen-free media, or generally under the influence of an unfavorable environment (16,19,20,21,22). Den Dooren de Jong (19) has given a very detailed description of the many bizarre cell types of A. chroococcum, including sometimes enormously swollen cells that arise in media with combined, especially organic, nitrogen.…”

“…Cells of azotobacter often contain granular inclusions, partly fat and volutin (18,24), partly bodies of a still unknown nature. Chromatinie material stainable by Robinow's method is certainly present at least at some stages of growth (20,21,22,23), and Bisset and Hale (25) claim the existence of a vesicular nucleus in A.…”

The Effect of X-Rays on the Survival of Bacteria and Yeast

“…Although A. agile cells, which are 3 to 4 ,& in diameter, are much larger than the usual bacterial cells, their cytologic organization appears superficially similar to that of smaller bacteria. Chromatinic bodies which undergo orderly division and distribution to the daughter cells during cell division have been demonstrated by nuclear staining techniques (Pochon et al, 1948;Eisenstark et al, 1950), although the details of the nuclear structure are not fully understood (Jensen, 1954).…”