16.13 Isolation of Azospirillum sp. from Soil and Plant Root 16.13.1 Introduction Azospirillum appear to have a worldwide distribution and occur in a large number in rhizospheric soils in association with the roots ofa variety of C3 and C4 plants. The Azospirillum are common habitant of tropical and temperate regions. Azospirillum have well-adapted and highly versatile metabolism that allow them to proliferate in carbon rich, nitrogen poor rhizosphere, thizoplane, and the endorhizosphere envi- ronments. Azospirillum prefers organic acids like malate, succinate, and lactate. ‘There are significant interspecies differences for the utilization of carbohydrates. It contains five species named Azospirillum brasilense, Azospirillum lipoferum, Azospirillum amazonense, Azospirillum halopraeference, and Azospirillum irakense. All species were proliferating in the rhizosphere and host root of numerous plants of Ally but not awys promote the growth ofthe hoa plan, and are commonly Known as plant-growth promoting bacteria Members of the genus Azospirillum are versatile toward Gram's nature, curved= rod shape, motile, oxidase positive, and exhibit acetylene reduction activity under microacrophilic conditions. They are capable of nitrogen synthesis since they can also grow on minimal media like nitrogen-free semisolid malate (NF). 8 SuAeAEME numMErS, Ley ast 16.13.2 Materials Soil sample, NF'b medium, root sample, dH1,O, sterile Petri plates and test tub ulating loop, dH,O, Bunsen burner, glassware marking pencil, etc Nefree malate medium for Azospirillum g/L: Malic acid 5, K.HPO, 0.5, KOH 4, 0.01, and 0.5% Ale, Bromophenol thymol blue (0.5% alcoholic solution) 2 ml, N 1, agar 1.8 (solid), pH 6.9-73, to 1 LdH,O, 16.13.3 Protocol 1. Collect the soil sample and prepare a 10-fold dilution series up to 10-*/plant root from the field. 2. Cut 0.5 em long root pieces and wash it with sterile dH,0. 3. Prepare semi solid agar medium and autoclave at 15 psi for 30 min. 4. When temperature cool down, transfer the medium into small screw cap bottles. . Place small pieces of washed roots/soil sample (a few microgram) on a semi-solid agar medium containing Na/Ca-malate as carbon source: 6. Incubate the plates for 2 days at 28°C-30°C. Pellicles of Azospirillum are seen medium at low partial pressure of O,, which favors the organism to grow ats state of N, fixation 7. Transfer Azospirillum serially from thin pellicle into fresh semi-solid medium to ‘obtain pure culture, 16,13.4 Result and Observations Development of white, dense, and undulating fine pellicle on the semi-solid malate medium is very characteristic of Azospirillum. It is polymorphic, vary ing in size, Gram-positive, containing -hydroxybutyrate granules. Cells have ‘one-half spiral turn and show spiral movement, Lateral and single polar flagella are present.16.14 Enumeration of Azospinlium by MPN Method 16.14.1 Introduction ‘The MPN method permits estimation of population density without an actual count of single cells or colonies. It is sometimes called the method of ultimate or extinetion dilution or end point dilution (Alexander 1965). The MPN technique is based on a determination of the presence or absence of microorganisms in several individual aliquots of each of several consecutive dilutions of soil. A prerequisite of the method is that microorganisms whose population is to be determined must be able to bring about some characteristic and readily recognizable transformation in the medium into which it is inoculated, or else the microorganism itself after it has undergone multiplication, must be easily recognizable in positive and negative tubes receiving certain quantity of inoculum, the MPN of organisms in that quantity of inocu- the sample. Azospirillum is a microaerophilic nitrogen fixer, A semi-solid N-free malate medium with bromothymol blue dye is used for enumeration. The presence of Azospirillum in the tube is indicated by 1. Change in dye color from green to blue 2. Formation of pellicle at the subsurface of medium 3. Reduction of acetylene to ethylene 16.142. Materials Glass culture tubes, soil sample, sterile pipettes, sterilized d11,0, and Petri plates. Nefree malate medium for Azospirillum g/L: Malic acid 5, K,HPO, 0.5, KOH 4, MgSO.,7H1,0 0.1, NaCl 0.02, CaCl, 0.01, FeSO,7H1,0 0.05, Na,MoO, 0.002, MnSO, 0.01, 0.5% alcoholic bromophenol thymol biue (0.5% alcoholic solution) 2 mL, NHC 1, and agar 18 (solid) pH 6.9-7.3, to LdHH,0. 16.14.3. Protocol Follow from the earlier experiment, 16.14.4. Result and Observations To calculate the MPN in the original sample, select as pl: The number of posi tubes in the least concentrated difution in which the greatest number of tubes is positive; and let p2 and p3 represent the numbers of positive tubes in the next two higher dilutions. Use the table of MV'N tor use With 1U-Told dilution and hve tubes per auubon. inen find the row of numbers in MPN table in which pl and p2 correspond to the values observed experimentally. Follow that row of numbers across the table to the column. headed by the observed value of p3. The figure at the point of intersection is the MPN. of organisms in the quantity of the original sample represented in the inoculums added in the second dilution (p2). Multiply this figure by the appropriate dilution factor to obtain the MPN for the original sample. Example: Suppose following observations were made Dilution ve 2010 In this series, pl=5, p2=3, and p3=1. For this combination ofp, p2, and p3, the MPN table gives asthe MPN of ongunismsin the quantity ofinoculumsapplied in the 10° (p2) dilution. Multiplying this number wit dilution factor 10° gives 1.1 «10° asthe MPN forthe original sample