Isolation and Identification of Azotobacter (1).pptx

Isolation and Identification of Azotobacter
This presentation covers the isolation and identification of Azotobacter, a free-living nitrogen-fixing bacterium. It includes its characteristics, culture methods, biochemical tests, and significance in agriculture for soil fertility and plant growth promotion.

1. Isolation and Identification of Azotobacter spp from Soil sample Kiran, Nawang, Kritisha, Laxmeey, Nikki

2. O BJECTIVE To become aware of the role of microbes in maintaining our environment. To learn various techniques of isolation of Azotobacter spp To learn how could we identify Azotobacter spp. B y Morphological means

3. Introduction Gram negative, free-living, aerobic soil dwelling, 2-4 micrometer oval or spherical bacteria that form thick-walled cysts and may produce large quantities of capsular slime. Found in neutral and alkaline soils in water six species in the genus Azotobacter. aerobic, free-living soil microbes

4. INTRODUCTION They are aerobic, free-living soil microbes that play an important role in the nitrogen cycle in nature, binding atmospheric nitrogen, which is inaccessible to plants, and releasing it in the form of ammonium ions into the soil (nitrogen fixation). Azotobacter respires aerobically, receiving energy from redox reactions, using organic compounds as electron donors, and can use a variety of carbohydrates, alcohols, and salts of organic acids as sources of carbon.

5. INTRODUCTION Cells of the genus Azotobacter are relatively large for bacteria (2–4 μm in diameter). They are usually oval, but may take various forms from rods to spheres. In microscopic preparations. The cells can be dispersed or form irregular clusters or occasionally chains of varying lengths. In fresh cultures, cells are mobile due to the numerous flagella. Later, the cells lose their mobility, become almost spherical, and produce a thick layer of mucus, forming the cell capsule. The shape of the cell is affected by the amino acid glycine, which is present in the nutrient medium peptone.

6. Distribution Azotobacter species are ubiquitous in neutral and weakly basic soils, but not acidic soils. They are also found in the Arctic and Antarctic soils, despite the cold climate, short growing season, and relatively low pH values of these soils. In dry soils, Azotobacter can survive in the form of cysts for up to 24 years.

7. Uses Azotobacter species play an important role in maintaining soil Nitrogen status. Soils usually contain large amounts of total P in different available forms, including insoluble forms such as tri-calcium P (Ca3PO4)2, aluminum P (Al3PO4), and iron P (Fe3PO4). Unfortunately, compared to the other major nutrients, P is by far the least mobile and available nutrient to plants in most soils, even if the total soil P content is well beyond plant needs. However, these forms may be converted to soluble P by soil phosphate-solubilizing microorganisms Screening of various salt-tolerant strains of Azotobacter has revealed that some strains are able to colonize the rhizosphere successfully and promote plant growth under stress conditions like droughts and salinity. Cysts of Azotobacter are used for Biotechnogical studies in resisting changes.

8. Principle Nitrogen fixing bacteria mostly isolated from Rhizosphere for plants. Rhizosphere is that area around the roots that are influences by roots exudates. Nitrogen fixation in diazotrophs catalyzed by nitrogenase, consisting of two proteins dinitrogenase reductase and dinitrogenase. Also, high respiration rate to experience limited oxygen exposure

9. Principle Azotobacters and similar bacteria turn nitrogen into ammonia through the process of nitrogen fixation, after which the ammonia is turned into proteins. The occurrence of this organism has been reported from the rhizosphere of a number of crop plants such as rice, maize, sugarcane, bajra, vegetables and plantation crops The strain A. paspali is a highly specific bacteria and was first isolated from the rhizosphere of the subtropical grass, Paspalum notatum ; Isolated the bacteria from the rhizosphere soil by making a series of dilution of soil from 10-1 to 10-7 on Azotobacter media and incubation for 48 h at 30ºC.

10. Principle Non-symbiotic nitrogen fixating bacteria uses mannitol as a carbon source and atmospheric nitrogen as a nitrogen source. Azotobacters are chemoorganotrophic, using sugars, alcohols and salts of organic acids for growth. Dipotassium phosphate provides buffering to the medium Composition of Ashby’s media

11. Principle Azotobacter are large gram negative motile rods which are oval or coccoidal in shape (pleomorphic) They are beneficial nitrogen fixers, their contribution to nitrogen enrichment is limited as they also fix ammonia in the soil. Hence, if ammonia is present in the medium, nitrogen fixation is suppressed.

12. Procedure Enrichment of Ashby’s mannitol agar A 40.7 grams of medium was suspended in 1000 ml purified / distilled water. B It was Heated just to boiling. C It was then Sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes. D Then Cooled to 45-50°C. E The content was Mixed well and poured into sterile Petri plates.

13. Procedure 2) Serial Dilution 10 gm of soil sample with 100 ml of dilution blank was made to 10-1. 1 ml from this tube was transferred to 9 pm of next blank to make 10-2. S erial dilution performed until 10-9. 15-20 ml of sterile molten plate agar was poured to petriplates. The content mixed well and distributed even Streaking performed and the plates were incubated for 1 week at 28-30 *C Ater Idenfitication, the development of colony was seen Gram staining, Gram staining and Hanging drop method.

14. Observation Colony Morphology Sample Media used Colony shape Colony size Colony elevation Colony margin Colony Opacity Consistency Soil A Ashby’s mannitol Agar Circular Less than 2mm Convex Undulating Opaque Paste like

15. Observation Gram staining Sample Reagent’s used Type of Staining Colour Shape Arrangement Inferences SOil A Crystal Violet, Gram’s Iodine, Decolorizer, Safranin Gram Staining Dark Pick Ovoid, Spherical (Coccoid shape) Cluster Gram Negative Coccoid shaped bacteria

16. Observation Capsule staining Motility Sample Reagent used Type of staining Results Inference Soil A Sample Reagents used Type of Technique Results Inference Soil A

17. Reference Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1. Kloepper J.W., Beauchamp C.J. A review of issues related to measuring colonization of plant roots by bacteria. J. Microbiol. 1992;38:1219–1232 Andjelković, S., Vasića, T., Radovića, J., Babića, S., Markovića, J., Zornića, V., et al. (2018). Abundance of azotobacter in the soil of natural and artificial grasslands. Solut. Proj. Sustain. Soil Manage. 172.