Farzana Yasmin Shomi
Department of Microbiology, Stamford University Bangladesh, 51, Siddeswari Road, Dhaka-1217, Bangladesh
Md. Borhan Uddin
Department of Microbiology, Stamford University Bangladesh, 51, Siddeswari Road, Dhaka-1217, Bangladesh
Tamanna Zerin*
Department of Microbiology, Stamford University Bangladesh, 51, Siddeswari Road, Dhaka-1217, Bangladesh
Azotobacter sp., N2 -fixation, N2 -free media, N2 -fixing bacteria, biofertilizer.
Stamford University Bangladesh, Siddeswari, Dhaka
Crop-Soil-Water Management
Nitrogen fixing bacteria
Sample collection and processing. The sample was collected from the garden of Stamford University Bangladesh, Siddeswari, Dhaka on March, 2020. For soil collection, a sterile beaker, a marking pen, and a spatula were used. Sufficient amount of soil sample was collected from one point in the garden in a sterile beaker and tagged it. The sample was collected from the top 6 cm of the soil profile. This layer is the preferred zone of most of the microbial activities, where most of the bacterial population is concentrated.
Processing of soil sample. 1.0 g of soil sample was transferred in a sterile beaker containing 99 ml of autoclaved normal saline and homogenized. One ml of homogenized soil sample was transferred into 9 ml sterile normal saline and serial dilution was carried out from 10-1 to 10-8 dilutions.
Screening of nitrogen-fixing bacteria from soil. Serially diluted bacterial cultures (100 µl) were inoculated on Jensen’s media, a nitrogen-free media and incubated at 37oC until growth was observed. Their colony morphology was noted and pure colonies were obtained by repeating subculture through streaking on Jensen’s medium. Isolated colonies were streaked on Jensen’s media containing Bromothymol Blue (BMB) and Yeast Extract Mannitol Agar (YEMA) media containing Congo red to determine nitrogen fixation capacity. The Petri plates were kept in the incubator at 370C until the growth was observed. Followed by incubation, the colony characteristics were noted.
Determination of nitrogen-fixation capacity of isolated bacteria using Nessler’s reagent. The isolates were grown in peptone water, shaken at 100 rpm, at 30°C for 3, 5, 7 and 9 days. Following incubation, the broth was centrifuged at 4,000 rpm for 20 minutes and the supernatant was reserved. After treatment with 0.5 ml of Nessler’s reagent to 1 ml of supernatant, the sample develops color from yellowish to brown if ammonia is present there. The color intensity of the solution was found to correspond to the amount of ammonia present in the sample.
Morphological and biochemical characterization of nitrogen-fixing bacteria. The isolate that showed nitrogen-fixing capacity using Nessler’s reagent was subjected to morphological identification through gram staining and biochemical characterization include oxidase, catalase, triple sugar iron (TSI), motility indole urea (MIU), citrate, methyl red (MR), voges-proskauer (VP), and starch hydrolysis tests.
Stamford Journal of Microbiology, 2021. Vol. 11, Issue 1, p. 11-13 ISSN: 2074-5346 (Print); 2408-8846 (Online)
Journal