Isolation of diazotroph using Burk's medium.

1. How we Characterise Diazotrophs from
rhizosphere?
Dr. Pooja Sharma,
Department of Biotechnology,
MMDU Mullana, Ambala-133207

2. INTRODUCTION
1. To fulfill the world population a huge amount of chemical
nitrogen is used for enhancement of crop production.
2. Due to excessive use of chemical nitrogen soil getting exposed
with harmful effect of acidification, water contamination, air
pollution and various mysterious disease that were not found in
19th century.
3. One of the alternative method of synthetic fertilizer is
diazotrophs. They are ecofriendly nitrogen fixing bacteria.

3. 1. Sample collected from different soil rhizosphere
2. All the samples store in sterilized bags and keep at 4°C for
further investigation
3. Serial dilution method for isolation of nitrogen fixing bacteria.
4 Pour 1.0 gm of soil in 99.0ml of sterilized water keep at shaker
for overnight at 37°C
Methodology

4. 5. Take 100 microliter bacteria on nitrogen deficient medium
(Burks) and spread properly.
6. After Spreading Keep the plates at 30°C for 24 to 48 hrs. to
isolate different N fixing bacteria.
7. Master plates of each region for further evaluation.
Cont.

5. 1. Ammonium Production test
Freshly grown colony culture were inoculated in 2ml peptone water in each
tube and incubated for 48-72 hr at 28± 2°C and few drop of Nessler’s
reagent was added in tube. Development of brown to yellow colour was a
positive test for Ammonium production.
Preliminary Test for confirmation
of nitrogen fixing bacteria

6. 2. Indole Acetate Assay
Each bacterial colony will grow in 2.0ml tryptophan broth at 37°C for 48hrs.
After that add 1.0ml of kovac reagent in each tubes. Development of cherry red
colour was positive test for tryptophan.
3. Gram Staining
Slide will prepared from bacterial culture with the help of sterilized platinum
loop using gram stain. Gram positive showing blue colour and gram negative
showing pink colour.
4. Catalase Test
Pour one drop of H2O2 on glass slide and add bacterial growth on it, mix well
using sterilized tip. If froth is there in the mixture. Colony will be catalase
positive.

7. THANK YOU