Azotobacter spp.
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The document describes isolating and identifying Azotobacter species from a soil sample. It discusses enriching the soil in Ashby's Mannitol Broth for a week to activate nitrogen-fixing bacteria. Colonies were then streaked on the broth and incubated for 4-6 days. Gram staining identified gram-negative cells with a red color and capsule staining showed transparent capsules around violet cells, indicating motility. The colonies were creamy white, raised, circular and mucoid, consistent with Azotobacter.
Azotobacter spp.
- 1. ISOLATION AND IDENTIFICATION OF AZOBACTER spp. FROM SOIL SAMPLE BY- SANJU SAH ST. XAVIER’S COLLEGE, MAITIGHAE, KATHMANDU DEPARTMENT OF MICROBIOLOGY
- 2. Objectives • To isolate and identify Azotobacter species from soil . • To study the structure ,colony morphology and classification of the species by gram staining and capsule staining.
- 3. Introduction Nitrogen-fixing bacteria can be isolated from rhizosphere plants, and one of the N2- fixing bacteria is Azotobacter. The genus Azotobacter was discovered in 1901 by Dutch microbiologist and botanist Martinus Beijerinck, who was one of the founders of environmental microbiology. The Azotobacter species is strictly aerobic ,non spore forming, encapsulated and motile with petritrichous flagella (tail like projections all over its surface) and produces copious amount of extra cellular slime ,hence are sticky in nature. Although non spore forming ,they do form thick walled cyst during unfavorable conditions. They are quite large (2mm or more) and occur either in pairs or clumps or sometimes in chains.
- 5. The genus Azotobacter, which belongs to the family Pseudomonadaceae from the subclass γ-Proteobacteria, comprises seven species: Azotobacter vinelandii, A. chroococcum, A. salinestris, A. nigricans, A. beijerinckii, A. paspali and A. armeniacus. Azotobacter improves corn, barley, wheat, cabbage ,potato and rice yields. Although there is a considerable amount of experimental evidence of these positive effects on plant growth, mechanisms involved are not fully understood. The ability to fix N2 was the main feature leading to the use of Azotobacter as a biofertilizer in the past.
- 6. Azotobacter can be isolated in lab by growing on a medium that does not contain any N2 but is rich in inorganic salts and simple carbohydrates from which they derive energy like Jenson’s media, Ashby’s mannitol agar, etc. Different species of Azotobacter on the basis of type of pigmentation A. chroococcum: varies from rod to lozenge(rhombus or diamond shape) shaped cells,colonies change from transparent to kilky and produce a water soluble brown pigment which in some strains becomes black as the culture gets older.
- 7. A.beijerinckia: similar to A. chroococcum in morphology and size, non motile, produces yellow or cinnamon colored, water insoluble pigment A. vinelandii:produces water soluble pigment that show up green under UV light,large ovoid rod cells frequently in pairs,motile with petritrichous flagella A. paspali: similar to A. vinelandii in producing pigment
- 8. Characteristics Gram negative bacteria capable to form cyst White Transparent Viscous Member of genus were recognized by production of blue diffusible pigment Most colonies which turn dark brown after 5-7 days of incubation on a manntiol N free agar . Members of genus Azospirillum produced slimy, glistening,smooth,whitish,weakly convex, 2-10 mm in diameter colonies. however ,slightly difference in size and sliminess of colonies can be observed among different species
- 9. Principle The Azotobacter species is strictly aerobic, non spore forming, encapsulated and motile with petritrichous flagella (tail like projections all over its surface) and produces copious amount of extra cellular slime, hence are sticky in nature. Although they are non spore forming, they do form thick walled cyst during unfavorable conditions. They are quite large (2mm or more) and occur either in pairs or clumps or sometimes in chains. These free living diazoterophic bacteria have the highest metabolic rate compared to any other micro-organism. They have unique ability to fix nitrogen aerobically. They can grow on any medium having suitable PH that contains an organic carbon source, minerals (especially phosphate) , some trace elements (in particular molybelenum) and no combined nitrogen. So generally Ashbys mannitol agar is used for the isolation. Manitol Agar is yellow colored, clear to slightly opalescent gel with a slight precipitate forms in petriplates. Its PH is about 8.10- 8.50
- 10. These bacteria utilize atmospheric nitrogen gas for their cell protein synthesis. This cell protein is then mineralized in soil after the death of Azotobacter cells thereby contributing towards the nitrogen availability of the crop plants. Azotobacter spp. is sensitive to acidic pH, high salts, and temperature. Azotobacter has beneficial effects on crop growth and yield through, biosynthesis of biologically active substances, stimulation of rhizospheric microbes, producing phyopathogenic inhibitors.
- 11. Materials Required 1. Soil sample 2. Manitol Broth and Agar 3. Petriplates 4. Inoculating loops 5. Test tubes
- 12. Procedure • Enrichment in Ashby’s Mannitol Broth 1. Sterilize 50 ml of broth in a conical flask. 2. Inoculate 5gm of soil sample to this broth. 3. Shake well to completely dissolve the soil. 4. Incubate at 28-30°C for 1 week. • Isolation of Azobacter spp. 1. Remove the pellicle in the broth but do not agitate the media. 2. Take a loopful of suspension and streak it on surface of the media. 3. Incubate at 28-30°C for 4-6 days. 4. Note the development of characteristics colonies 5. Perform Gram staining, capsule staining and hanging drop technique for motility.
- 14. In order to activate the organism present in the soil sample, and to isolate Azotobacter from soil sample, Ashby’s Mannitol Broth was prepared first and foremost. As Ashby’s Mannitol Broth is free from any Nitrogenous components, the Azotobacter spp. can utilize atmospheric nitrogen without hindrace since, the bacteria itself os free nitrogen living bacteria for the proper growth of organism, it was placed in shaking incubator with abundance air. Azotobacter forms a thin pellide layer in broths surface in which a loopful of organism was streaked in Mannitol Broth previously and after the proper incubation of 5 days at 28c . Colony morphology was observed. The colony morphology includes creamy white colour, raised elevation with circular shape and mucoid consistency.
- 15. Gram staining further identified the cell to be gram negative with re color. Transparent capsule with violet coloured cells were visualized while performing capsule staining as for the motility test, the cells showed swirling movement hence it was motile.
- 16. Why the azotobacter shows glistening and slimy colonies? Due to the capability of these microorganisms to metabolize the sucrose. • The pellicle or ring on the liquid surface of the enrichment was formed not only by the aerobic N – fixing microorganisms, but also by bacteria unable to fix nitrogen. This fact was probably due to the high amount of soil inoculated in the enrichment solution.
- 17. Practical significances • Isolation of Bacterial spp.from soil sample. • Azotobacter spp.is very important biofertilizer. Precautions • Pellide layer formed shouldnot be distributed while taking loopful of organisms. • Medium components should be added in proper amount.
- 18. Reference • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4138622/ • https://pdfs.semanticscholar.org/0075/380552940ae9a4ab3c16e0fc0 788e5074f99.pdf • Pelczar M. Jr., 1957, Manual of Microbiological Methods. • Mazinani Z, Aminaafshar M, Asgharzadeh A, Chamani M - Different methods for isolation and preliminary identification of Azotobacter, Intl J Agri Sci and Res, 2012,3(1);1-8