Unformatted text preview: elor of Science (Honors) dissertation, University of Sydney, 1980). The
thermoresistant antigen 7., is shared by Azomonas and Azotobacter
(Tchan et al., 1980). All species of the genus are sensitive to streptomycin (0.2 rig/ml).
Their sensitivities to chlortetracycline, oxytetracycline, polymyxin,
sulfanilamide, penicillin and neomycin vary depending upon strain.
Only A. insignis is entirely sensitive to penicillin (Thompson and
Skerman, 1979). The members of this genus are resistant to phenol, benzoate and
mercuric chloride to varying degrees. Most strains tolerate sodium
ﬂuoride up to 0.01 M. The resistance of A. agilis to iodoacetate can be
used for selective isolation of this species (Thompson and Skerman,
1979). A. agilis and A. insignis are aquatic bacteria and A. macrocytogenes
is found in soil, although no information is available as to whether A.
macrocytogenes also has an aquatic habitat. Thus it appears that the
genus is ecologically heterogeneous. Only A. macrocytogenes is resistant
to desiccation (five out of seven strains survived desiccation for at least
1 month (Thompson and Skerman, 1979)), as the other members have
no such need. All species have a salt tolerance of up to 1%, suggesting
that the aquatic members are capable of living in contaminated water FAMILY ||. AZOTOBACTERACEAE 231
Characteristics differentiating the species of the genus Azomonas"
1. 2. A. 3. A. macro-
Characteristics A. agilis insignis cytogenes
Presence of enlarged cells in me- — — +
dia with ethanol
Peritrichous + — —
Lophotrichous —- + —
Monotrichous — — +b
Gram-reaction — — v
Formation of colony-retained — — +
Brown-black, on benzoate me— — d —
Blue—white ﬂuorescent, on + — d
Utilization as carbon sources:
Mannitol — — +
Maltose cl — +
Malonate + + — where concentrations of organic matter and mineral salts can be
relatively high. Enrichment and Isolation Procedures The enrichment and isolation techniques depend upon the ability of
Azomonas to grow by ﬁxing nitrogen, using organic matter as the
energy source. Nonselective enrichment methods (Tchan, 1952). These methods are
the same as those described for the genus Azotobacter. Selective enrichment methods (Thompson and Skerman, 1979). Media
containing either 1% sodium benzoate (Derx, 1951b; V. Jensen, 1955)
or 1 mM iodoacetate are selective for A. agilis. Incorporation of 1%
benzoate into nitrogen-free sucrose medium having a pH of 6.0, and
incubation of the cultures at a decreased incubation temperature (10—
12°C) favors the growth of A. macrocytogenes. No selective enrichment method is available for A. insignis. Isolation methods. The enrichment cultures are inspected daily.
When macroscopic growth becomes apparent, the culture is examined
by phase-contrast microscopy to detect the presence of Azomonas cells
(ovoid cells 2.5 X 5.0 pm, or sometimes ﬁlamentous forms). Growth
will usually occur within 2—5 days. Positive cultures are streak plated
on nitrogen-free agar medium with glucose as the carbon source. For
selective isolation, nitrogen-free agar media with appropriate selective " Symbols: see standard deﬁnitions.
’7 Rarely, two ﬂagella may occur at one pole.
C See Procedures for Testing Special Characters. substances should also be used. Sometimes nitrogen-free iron-deﬁcient
agar can be used to advantage to encourage pigment production by
some species. Maintenance Procedures For routine maintenance, Azomonas strains should be subcultured at
monthly or bimonthly intervals on Winogradsky’s agar medium* with glucose.
Paraffin oil preservation method (Tchan, unpublished data) and ly- ophilization method. These methods and their shortcomings are the
same as those described for the genus Azotobacter. Procedures for Testing Special Characters Methods for determining cell morphology, Gram—reaction, ﬂagellar
arrangement, production of homopolysaccharides, pigment production
and acid production from organic substrates are the same as those
described for the genus Azotobacter. Differentiation of the genus Azomonas from other genera See Table 4.48 in the chapter on the genus Azotobacter for charac—
teristics that can be used to differentiate Azomonas from morphologi-
cally or physiologically similar genera. Taxonomic Comments The genus Azomonas can be separated from Azotobacter by the lack
of cyst formation. However the taxonomic position of A. macrocytogenes
has presented difﬁculties since the initial description of the organism
by H. Jensen (1955). The early statement regarding the formation of
the microcyst was not precise. Jensen in his initial description indi-
cated: “Formation of cysts could not be seen with certainty but took
place possibly at 5—10°C where a number of smaller round cells with
clearly visible cell walls were seen . . . ." However, electron microscopic
investigation (Tchan, 1968) did not reveal the presence of typical
Azotobacter microcysts, although vegetative cells escaping from capsu- * Deﬁned in preceding article on Azotobacter. lar~1ike material were demonstrated. Baillie et a1. (1962) moved A.
macrocytogenes from Azotobacter and placed it in the genus Azomonas.
In the eighth edition of Bergey’s Manual of Determinative Bacteriology
(1974), A. macrocytogenes was classiﬁed in Azomonas, a noncyst form-
ing genus. The existence of microcysts in A. macrocytogenes was again proposed
by Thompson and Skerman (1979). Microcyst—like cells were produced
at 28°C by four out of seven strains examined. In addition, the ability
of A. macrocytogenes to survive desiccation was used by these authors
as supporting evidence for the presence of microcysts. However, the
productionof unusually large cells, the relative tolerance to acid con—
ditions, the monotrichous ﬂagellation and the unusual tendency to
Gram-positiveness differentiate this organism from Azotobacter. Nu—
merical analysis (Thompson and Skerman) showed that A. macrocyto—
genes fused at a low hierarchical level with A. paspali. At a higher level
the A. macrocytogenes-A. paspali group fused with the two other Azo— ...
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