Azospirillum brasilense is a vastly studied plant growth-promoting rhizobacterium, which produces direct and indirect beneficial effects on several crops4. Historically, the culture of this bacterium has been performed using the semisolid NFb medium3, which is a nitrogen-free medium. Although other diazotrophs can grow in this culture medium2, A. brasilense can be isolated and identified by a typical growth after 120h of incubation3. Other authors have exhaustively described the NFb medium composition and growth of many diazotrophs in semisolid media1. Here, we show pictures of seven stages during the development of A. brasilense in the course of the incubation period.
A colony of A. brasilense was inoculated in a vial containing this culture medium. After 24h of incubation at 30°C, growth development can be observed by a halo formation around the colony as a ‘ghostly-balloon’ (Fig. 1A). The halo grows in size (Fig. 1B) and opens (Fig. 1C) after 48h of incubation, approximately. By aerotaxis, cells move to the specific place in the medium (Fig. 1D) where the rate of oxygen diffusion and bacterial respiration allows the nitrogenase enzyme to fix N2 without the irreversible inhibitory effect of oxygen2. Then, bacteria continue growing and form a typical sub-superficial whitish ‘veil-like’ pellicle after five days of incubation (Fig. 1E). As growth occurs, the alkalization of the culture medium can be observed by a change in color from green (Fig. 1F) to blue (Fig. 1G) due to the bromothymol blue redox dye. This culturing technique can also be used for other purposes, such as the inoculation of serial dilutions (Fig. 1F and G) to perform the estimation of the most probable number of this plant beneficial bacterium in commercial inoculants or the count of diazotrophs from environmental samples.
Growth development by Azospirillum brasilense in vials with semisolid NFb medium during the incubation period (a, b, c, d and e). Growth starts with the formation of a halo (white arrows) around the colony. Typical growth as a ‘veil-like’ pellicle (black arrows) under the medium surface (dotted circles) is formed. After six (f) and seven (g) days of incubation, it can be observed the change of color indicating the alkalization of the culture medium. Vials showed in a to e were inoculated with one colony of A. brasilense. Vials showed in f and g were inoculated with 50μl of the 10−2 dilution of an inoculant sample.
This work was partially supported by FONCYT 2008 PICT 1864 from the MINCyT and UBACyT project 20020090100255, Universidad de Buenos Aires (UBA) in Argentina.
Conflict of interestThe authors declare that they have no conflicts of interest.
