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.

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.

13. RESULT AND DISCUSSION
.

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