**4. Isolation cultivation and axenic culture**

Selective enrichment cultures. For isolation purposes, the use of free carbon and nitrogen agar medium as a selective medium is recommended for recovering *Xanthobacter* from; soil, upper layers of marine or freshwater sediments, lake water, steam and root of aal types of plants. Because of slime formation by *X. autotrophicus* of agar plates free carbon and nitrogen source. Frequently, other oligotrophic organisms grow as contaminants in the slimy colonies of *Xanthobacter* easy to separate in nutrient agar the following basal medium can be used for autotrophic as well as heterotrophic growth except when urea is used as nitrogen source. No vitamins or additions of yeast extract as growth factor are required for most *X. autotrophicus*, enrichment 100 mg of yeast extract per liter to the mineral medium can reduce an extended lag time for autotrophic growth under free carbon and nitrogen-fixing. In order to demostrate its capacity for fixing N2 is important not to add any inorganic or organic nitrogen source. During isolation, a vitamin solution any mixture containing biotin can enrichment to stimulate is growth, absence of ammonium, or amino acids, peptide, protein as any organic nitrogen source [29, 30]. For heterotrophic growth, common carbon sources are used: 0.5% sugars, 0.3% (v/v) alcohols or 0.4–0.8% organic acids. For growth under non-N2-fixing conditions, 0.1% of ammonium chloride or sulfate is common. The

Xanthobacter autotrophicus *an Endophytic Beneficial Bacterium for Wheat and Other Plants… DOI: http://dx.doi.org/10.5772/intechopen.102066*

#### **Figure 2***. Phylogeny and taxonomy of* Xanthobacter *spp.*

exact composition of this medium is not critical, and good results have been obtained with free sucrose and nitrogen agar medium, for storge sterile soil is one the easy and best one to preserve viability for relative long period of time. *X. autotrophicus*, studied in more detail, most of the strains tested grow at pH 5.0–8.5 while pH recommend is about 6.8–7.2 and its temperature 30–37°C. The morphological features of *Xanthobacter* can be used initially for identification. Colony morphology depends on the type of carbon and nitrogen source and growth conditions. On most carbohydrates, the colonies of main species are large from 1 to 5 mm in diameter, smooth, convex, circular, filiform, opaque, and typically egg-yolk yellow color due to zeaxanthin dirhamnoside (see **Figure 2a**). The colonies become less yellow and less opaque as the amount of slime increases. The production of slime on nutrient agar plates frequently results in colonies resembling fried eggs [15]. Zeaxanthin dirhamnoside is water-insoluble, in contrast to the reddish/pinkish/brown pigment or to the yellow-green diffusing pigments with fluorescence of *Beijerinckia* and *Derxia* the other yellowish diazotroph isolated with well-known methods. The latter fact also makes it easy to distinguish *Xanthobacter* from *Derxia* colonies, which turn brown with age besides other morphological and biochemistry characteristics [5, 31]. *Xanthobacter* strains are sensitive to wide range of antibiotics, but the response depends on the method applied broth cultures or the use of Difco (Dispense-O-Disk minifilters). *X. autotrophicus* was sensitive to ampicillin, penicillin, chloramphenicol, erythromycin, novobiocin, and polymyxin B, but they were resistant to erythromycin and bacitracin. Few strains can grow on violet red-bile medium (Oxoid), deoxycholate medium (Oxoid), tellurate agar (Difco), and mineral medium supplemented with crystal violet red colonies [6, 32, 33].

#### **4.1 Methods of storage**

*Xanthobacter* cultures are grown on chemolithoautotrophic agar slants stored 1.5 years at 4°C after sealing the tubes tightly with parafilm. Also, liquid cultures grown under chemolithoautotrophic conditions mineral medium with 0.02% (w/v) yeast extract have been kept for more than 15 months at 4°C and, of course if glycerol is used as suspended solution at 40–60% (v/v) final, at –20°C and –75°C for more than 8 years. For long-term storage, cultures should be lyophilized on skim milk at 10% now sterile soil is an easy and safe technique [34, 35].
