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Isolation of Azotobacter

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Niki Yazdani

on 20 February 2014

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Transcript of Isolation of Azotobacter

Isolation Streak Plates

Motility Test
Facts about
Azotobacter
Aerobic Gram-Negative Organism
Large and ovoid morphology
Thick-walled cysts
Fix atmospheric Nitrogen
Do not have symbiotic relations
Motile
Free-living in soil and water
Distinguished by pigmentation
Samples
Two samples of soil were collected

Purpose
To confine and determine species from
Azotobacter
by using soil samples, Mannitol Salts enrichment media, and multiple identification tests .

Azotobacter
Isolation
and Biofilm Project

Common
Azotobacter
Species
Environmental Isolation
of Azotobacter
Prepared Mannitol Salts Broth (N-free)
Niki Yazdani and Lauren McGehee
Part 1:
Azotobacter
Isolation
A. chroococcum
A. vinelandii
A. aerogenes
Recipe:
Mannitol 10.5 g
K2HPO4 .458 g
MgSO4 .14 g
CaSO4 .09 g
NaCl .14 g
CaCO3 3.5 g
dH2O 1000 mL

1
2
Motility deep for our four colonies
All four colonies tested positive
Wet mounts also positive
Mannitol Test
Used Mannitol test to see if the colonies are able to ferment Mannitol as a carbon source
All tubes changed from red to yellow indicating a positive test
Oxidase Test
Oxidase test was used to determine if ATP is generated by cytochromes
Oxidase reagent was combined with water
Colonies were placed on reagent
Positive result for all colonies
Table of Results
Results
We narrowed down our colonies to 3 species:
A. vinelandii
A. armeniacus
A. chroococcum
Since the test results are similar, we couldn't distinguish between A. armeniacus and A. chroococcum
A. vinelandii
A. armeniacus
A. chroococcum
Conclusion
Biofilm Growth
Biofilm grown from deionized water and soil samples
Sample sat for a number of days
Biofilm Analysis
LSL to EMB
Part 2: Biofilm Isolation
API 20E & 20NE
API 20E: Enterics = lactose fermenters
API 20NE: Nonenterics = non fermenters
Rapid ID Results
API 20E
Pseudomonas luteola
Opportunistic pathogen


API 20 NE
Enterobacter cloacae
Nosocomial
pathogen
Works Cited
3
4
g-proteobacteria
Motile
Opaque and convex morphology
Motile
Blunt rods
Randomly placed granules
Mannitol and caproate as carbon source
Yellow-green fluorescent
Many carbon sources
Motile
Easily cultured and grown
Colony 1
Colony 2
Colony 3
Colony 4
Gram Stain
Gram Stain
with capsules
Gram Stain
Gram Stain
UV Light
Gram Stain
Part 3: Rapid Identification Systems
Wet Mount
Bergey, David H., and John Holt. Bergey's Manual of
Determinative Bacteriology. Philadelphia: Lippincott, 2000. 552-72. Print.

Identified three possible species of
Azotobacter
Better identification if we did more tests
More conclusive results
Different samples
Better technique
If we didn't have snow days
"
Azotobacter sp.
cells." 2009. JPEG file.


"Biofilm Formation." 2011. JPEG file.


"
Azotobacter vinelandii
in the phase microscope at 400X."
2007. JPEG file.


"
Azotobacter chroococcum.
" Tchan & New (1984) in
Bergey's Manual of Systematic Bacteriology, vol. 1, N.R.
"Rough and smooth colony types of
P. luteola
after 48
hours." 2005. JPEG file.
"
Enterobacter cloacae
EMB." 2006. JPEG file.
Full transcript