BAILLIE, A. , HODCKISS, W. & NORRIS, J. R. (1962). J. appl. Bact. 25 ( l ) , 316-119.

FLAGELLATION OF AZOTOBACTER SPP. AS DEMONSTRATED BY ELECTRON MICROSCOPY

BY ANN BAILLIE Bacteriology Department, University of G h g o w

W. HODGKISS Torry Research Station, Aberdeen

AND

J. R. NORRIS Bacteriology Department, University of Blnsgow

SUMMARY: The results of an electron microscope survey of' flagellation in the genus Azotobncter are presented and the implications of the findings are discussed.

ALTHOUGH INDIVIDUAL strains of Azotobacter have received considerable attention on account of their diverse biochemical activities and large cell size the genus as a whole is poorly known to the majority of bacteriologists. Its classification, however, presents problems which are of interest in the broader context of bacterial taxonomy and the genus is worthy of more than passing attention. In recent years new azoto- bacters have been described which, in contrast to the established species, are claimed to be polar flagellate. Work on the genus has been hampered by the absence of a systematic study of flagellation in the motile species. During the past few years we have examined over fifty strains of Azotobacter including representatives of each of the known species, using flagella staining techniques, and these have indicated that different types of flagellation do in fact exist. Such observations should be checked by electron microscopy (Hayward & Hodgkiss, 1961) and this paper records the results of an electron microscope survey using shadowing and phosphotungstic acid negative staining techniques.

METHODS Cultures. Twenty Azotobacter strains comprising type cultures and recently isolated

strains were examined. Cultures were grown on nitrogen-free agar of the following composition (% wlv): glucose, 1 ; K,HPO,, 0.1; MgS0,.7H,O, 0.02; CaCO,, 0.1; NaC1,0.02; Na,Mo04.2H,0, 0.0005; and agar, 2.0. The components were dissolved in distilled water and the medium was sterilized by autoclaving at 15 lb/inz for 10 min. Slope cultures were incubated a t 30" for 36 hr.

Preparation of specimens for electron microscopy. Cultures were harvested in distilled water, stored in suspension overqight in the refrigerator a t +5", washed three or more times in distilled water by slow centrifugation to remove capsular material, and finally resuspended in distilled water.

Flagellation of Azotobacter spp. "7

Shadow cast specimens. Droplets of the suspension were applied to formvar grids, allowed to dry in air and shadow cast with gold-palladium.

Negative stained preparations. A portion of the bacterial suspension was mixed with an equal volume of a 2% (w/v) ,solution of phosphotungstic acid adjusted to pH 7.4 with N KOH (Brenner & Horne, 1959). Small drops were then placed on formvar films which had been stabilized with carbon, partially blotted to remove excess phosphotungstic acid, and allowed to dry in air.

All preparations were examined in the Siemens Elmiskop I electron microscope using the single condenser system, with a 20Op condenser aperture, a 50p objective aperture and an accelerating voltage of 60 kV. Suitable fields were photographed a t a magnification of 8,000 or 10,000 diameters on Ilford N50 thin film half tone plates.

RESULTS Provided that steps are taken to remove capsular material, the flagella of Azotobacter spp. can be stained by conventional methods quite easily and they were readily visible under the electron microscope. The results obtained were fully in accord with visible light observations on stained material and flagellation type was constant for the various strains of each species. The illustrations show typical results.

Peritrichous flagellation was seen with Azotobacter vinelandii (Plate 1, a and b), A . agilis (Plate 1, c and d) and A . chroococcum (Plate 1, e and f). A . chroowccum had, as a rule, fewer flagella than the other species. A . insigne showed a polar arrangement of up to twelve flagella, which often appeared to arise from two distinct sites a t one end of the cell (Plate 2, a and b). A . macrocytogenes occurs in two phases (Jensen, H. L., 1955) and both showed polar flagellation with one, or sometimes two, flagella (Plate 2, c and d). A . beijerinckii is nonmotile and the strains which we have studied have no flagella.

DISCUSSION There is a great deal of confusion in the early literature on the genus Azotobacter as regards the type of flagellation encountered and the need to clarify the position for taxonomic purposes led Hofer (1944) to study cultures of A . chroococcum, A . beijerinckii, A . agilis and A . vinelandii-the four species then known-by direct staining and electron microscopy. He concluded that all of these organisms were peritrichous and peritrichous flagellation has, as a result, become established as one of the characteristics of the genus (cf. Breed, Murray & Smith, 1957).

Derx (1951) described A . insigne, which closely resembled A . agilis but was apparently polar flagellate. This organism has since been isolated from Danish water courses by Jensen, V. (1955) and by Nagy (pers. comm.) and by one of US

(J.R.N.) from a Scottish river. Its flagellation is clearly polar in the sense that flagella are clustered at one end of the cell, and this is reflected in the distinctive, erratic, zigzag and circling type of movement which serves to distinguish this organism from the closely similar but peritrichous A . agilis. The consistent appear- ance of two tufts of flagella with different points of insertion gives an appearance which is not, in our experience, paralleled elsewhere in bacteriology.

I 1 8 Ann Baillie, W. Hodgkiss and J . R. Norris

A . macrocytogenes was described by Jensen, H. L. (1955), who drew attention t,o the fact that motile cells were apparently polar flagellate. Cells in the motile or ‘M’ phase were readily seen to possess one polar flagellum, but motile cells in the ‘0’ phase were not easy to examine for flagella on account of the large amount of slime associ- ated with the cells. A . wrocytogenes is a rare organism. Although subsequently re-isolated from the Danish soil in which it was f i s t found, it has only once been reported from another situation-a soil sample from the Flannan Islands (Norris & Baird, 1960). Both isolates showed one or sometimes two polar flagella in the ‘M’ and ‘0’ phases.

The demonstration of organisms with polar flagella in a ‘peritrichous’ ‘genus, although in conformity with the Adansonian approach to classification, is discon- certing to the traditional taxonomist who believes type of flagellation to be a character of prime importance in bacterial classification. Like Rubenchik (1959) and Leifson (1960) he may be inclined to remove the polar species to other, distant, genera. The weight of evidence, however, favours the retention of these six organisms in one closely knit group. All share the characteristics of active nitrogen fixing ability and large cell size. Norris (1960) demonstrated a common antigen present in each of them and a considerable sharing of antigens between some species such as A . agilis and A . insigne. A study of the saline soluble antigens (Norris & Nagy, 1960) also suggested that the group was a homogeneous one and provided antigenic support for the division into six species which is a t present recognized. The close similarity of the polar A . insigne and the peritrichous A . agilis is emphasized by the finding that several strains labelled A . q i l w which have existed in culture collections for many years are in fact A . insigne (Jensen, V., 1955; Norris & Jensen, 1958).

It is the purpose of this paper to illustrate the flagellation of the genus Azotobacter rather than to discuss in detail the classification of the genus, but we would like to record our own view on the taxonomy of these organisms. There are good grounds for dividing the genus Azotobacter into two sections, according to ability to produce microcysts (Winogradsky, 1938; Tchan, 1953; Jensen, V., 1955). We would support this and suggest that two genera should be recognized; Azotobacter, to include the microcyst-forming species Azotobacter chroococcum, Awtoba8cter beijerinckii and Azotobacter vinehndii, and Azomonm, to contain the species which do not form microcysts (Winogradsky, 1938; Jensen, V., 1955), in which both peritrichous ( A m m o m agilis) and polar (Azomonas insigne and Azomnas macrocytogenes) species would be included.

One of us (A.B.) is grltteful to the Department of Scientific and Industrial Research

The electron microscopy described in this paper wss carried out as part of the for a Vacation Studentship.

programme of the Department of Scientific and Industrial Research.

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Journal of Applied Bacteriology, Vol. 2 5 , Part 1

ANNE HAILLIE, w. HODQKISS AND J. R. 'NOXICIS -FLAGELLATION OF A z o m i 1 A C ~ 1 . E . x SPP. AS DEMONSTRATED BY ELECTRON MICROSCOPY. PLATE 2

Flagellation of Azotobacter spp. 119

DERX. H. G. (1951). Azotobacter insigne spec. nov., fixateur d’azote i flagellation polaire. Proe.

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EXPLANATION OF PLATES PLATE 1. Electron micrographs of Azotobacter cells showing flagellation: (a), (c) and (ej, phos- photungstic acid preparations; (b), (d) and (f), Au-Pd shadowed preparation, x 19,500 epprox. (a) and (b) Azotobacter uinelandii. (c) and (d) Azotobacter agilis. (e) and (f) Azotobacter chroococow~,r~

PLATE 2. photungstic acid preparations; (b) and (d), shadowed preparations, Y 12,500 approx. (a) and (b) Azotobacter insigne. ( c ) and (d) Azotobacter ,wmcrocytogerc.es.

Electron micrographs of Azotobacter cells showing flagellation: (a) and (c), phos-

(Received 8 January, 1962)