CHAPTER 4 RESULTS

Azotobacter is Gram-negative, pleomorphic aerobic bacterium which produces catalase,

oval, spherical or rods that form thick-walled cysts and may produce large quantities of capsular
slime. Azotobacter are the most intensively investigated heterotrophic group possessing the highest
respiratory rates. Members of these genera are mesophilic, which require optimum temperature of
about 28˚C. Azotobacter, a free-living microbes, acts as plant growth promoting rhizobacteria
(PGPR) in the rhizosphere of almost all crops. Azotobacter has its significant role in nitrogen
fixation. It is beneficial for the production of biofertilizer that can provide a healthy life to the plant
which have been consumed in whole world wide.

The present study is emphasized on the isolation of Azotobacter from different soil rhizospheres.
Morphological and biochemical studies were also conducted to show their reaction performance
in different media for detecting confirmed Azotobacter. Inhibition and resistance pattern to certain
antibiotics were also optimized by assaying Antibiotic sensitivity profiling. Along with it, the
strains were also tested for their growth on different temperatures and pH. Different results of these
experiments are reported as follows.

4.1. COLLECTION OF SAMPLES

Fig. 4.1. Collection of soil samples.

30 Soil samples were collected from different locations (Fig.4.1) of Sardar Vallabhbhai Patel
University of Agriculture & Technology, Modipuram, Meerut and Hapur (Uttar Pradesh) from
cultivated land. Samples were withdrawn at a depth of 0-10 cm, collected into polyethylene bags,
and stored at 4°C.

4.2. ISOLATION OF AZOTOBACTER

Total 30 soil samples were collected from different location of Meerut and Hapur (Table
3.1) that were used for isolation of Azotobacter strains on their selective Azotobacter (Mannitol)
agar media in the laboratory. After 3-5 days of incubation period creamy translucent, mucoid,
circular bacterial colonies were observed on 30 Azotobacter mannitol agar plates (Fig.4.2). These
plates were selected positively for Azotobacter tests according to their colonies morphology.
Samples are listed in the table 4.1.

Fig.4.2 Isolation of Azotobacter from soil sample on Azotobacter mannitol agar medium.
Table 4.1 Showing the presence or absence of smooth/translucent mucoid circular
colonies of Azotobacter on mannitol agar medium.

Creamy/translucent, mucoid
Soil Samples Location and circular Azotobacter
colonies growth

A-1 HAPUR +

A-2 HAPUR +

A-3 HAPUR +

A-4 HAPUR +

A-5 HAPUR +

A-6 MEERUT +

A-7 HAPUR +

A-8 HAPUR +

A-9 HAPUR +

A-10 HAPUR +

A-11 HAPUR +

A-12 MEERUT +

A-13 HAPUR +

A-14 HAPUR +

A-15 HAPUR +
 A-16 HAPUR +

A-17 HAPUR +

A-18 HAPUR +

A-19 HAPUR +

A-20 HAPUR +

A-21 MEERUT +

A-22 MEERUT +

A-23 MEERUT +

A-24 HAPUR +

A-25 HAPUR +

A-26 HAPUR +

A-27 HAPUR +

A-28 HAPUR +

A-29 HAPUR +

A-30 HAPUR +

IARI NEW DELHI +

Note :(+) Presence of Azotobacter colonies.

4.3. IDENTIFICATION

4.3.1. GRAM STAINING

All the Azotobacter isolates collected form 30 soil samples were subjected to Gram staining
procedure described earlier in Materials and Methods section 3.6.1.

All the Azotobacter strains are stained with Gram staining and gave favorable results (Fig.4.3 and
Table 4.2).

Fig.4.3. Pink, rod-oval shaped bacteria observed after Gram staining.

All the colonies from different strains were subjected to stain by the method of Gram staining,
smear was prepared and then the protocol was followed till observing it under microscope at 40X
to 100X. All the strains were appeared to be pink, rod-oval shaped, signifying that Azotobacter is
a Gram negative bacteria (Table 4.2).
Table 4.2 Gram staining of native Azotobacter isolates.

SAMPLE CODE SHAPE COLOUR NATURE

A-1 Rod-Oval Pink Gram negative

A-2 Rod-Oval Pink Gram negative

A-3 Rod-Oval Pink Gram negative

A-4 Rod-Oval Pink Gram negative

A-5 Rod-Oval Pink Gram negative

A-6 Rod-Oval Pink Gram negative

A-7 Rod-Oval Pink Gram negative

A-8 Rod-Oval Pink Gram negative

A-9 Rod-Oval Pink Gram negative

A-10 Rod-Oval Pink Gram negative

A-11 Rod-Oval Pink Gram negative

A-12 Rod-Oval Pink Gram negative

A-13 Rod-Oval Pink Gram negative

A-14 Rod-Oval Pink Gram negative

A-15 Rod-Oval Pink Gram negative

A-16 Rod-Oval Pink Gram negative

A-17 Rod-Oval Pink Gram negative

 A-18 Rod-Oval Pink Gram negative

A-19 Rod-Oval Pink Gram negative

A-20 Rod-Oval Pink Gram negative

A-21 Rod-Oval Pink Gram negative

A-22 Rod-Oval Pink Gram negative

A-23 Rod-Oval Pink Gram negative

A-24 Rod-Oval Pink Gram negative

A-25 Rod-Oval Pink Gram negative

A-26 Rod-Oval Pink Gram negative

A-27 Rod-Oval Pink Gram negative

A-28 Rod-Oval Pink Gram negative

A-29 Rod-Oval Pink Gram negative

A-30 Rod-Oval Pink Gram negative

IARI Rod-Oval Pink Gram negative

All Azotobacter strains can also be characterized by observing the morphology of the strains with
naked eyes and also by setting up biochemical reactions.

After the conformation of the pure strains of Azotobacter, selective media slants were prepared
and pure strains of Azotobacter were streaked and preserved for future uses (Fig.4.4).
 Fig.4.4 Isolation of pure culture of Azotobacter on Azotobacter mannitol agar slants.

4.3.2. RYU’S TEST

The Azotobacter isolates were tested for ryu’s test. The isolates were treated with 3% KOH
(Potassium Hydroxide) solution as mentioned in Materials and Methods section 3.6.2. The
isolates with ryu’s test forms string which shows that it is a gram negative bacteria (Fig.4.5). This
ryu’s test is a fast method to identify weather our bacteria is a gram negative or gram positive.

Fig.4.5 String formation in Ryu’s test.

4.4. BIOCHEMICAL TESTS
Different biochemical tests (Triple sugar iron agar, Citrate utilization, Methyl red, Voges-
Proskauer, Catalase, Oxidase, Nitrate reduction, Urease, Starch hydrolysis, Motility tests) were
performed for characterization of 30 different Azotobacter isolates as mentioned in Materials and
Methods section 3.8. The isolates showed good results in every biochemical tests. Some isolates
gave positive results while some gave negative as well. The results of different biochemical tests
are represented in Table 4.4.

4.4.1. TRIPLE SUGAR IRON AGAR TEST

The Triple sugar iron agar (TSI) test is a microbiological test roughly named for its ability
to test microorganism’s ability to ferment sugars and to produce hydrogen sulphide.

If the organism ferments glucose but does not ferment lactose and/or sucrose, then the slant
becomes red and butt remains yellow. If the organism in addition to glucose ferments lactose
and/or sucrose, the fermentation product formed on the slant will more than neutralize the alkaline
amines rendering the slant acidic (Yellow). If the organism is non-fermenter, instead of sugars,
peptone is utilized as an alternate source of energy under aerobic condition on the slant which
makes it alkaline indicated by the red color while there is no change in the color of the butt. The
formation of CO2 and H2 is indicated by the presence of bubbles or cracks in the medium or by the
separation of the agar from sides or bottom of the tube. The production of H2S requires an acidic
condition and is indicated by blacking of the butt of the medium in the tube.

After 48-72 hours of incubation, TSI slants were observed and following results were observed as
described in Table 4.3. Color change of slant from red to yellow indicates sugar fermentation.
Color change of butt into black indicates H2S production and Formation of bubbles/cracks
indicates gas production as shown in Fig.4.6. The best results of TSI are given below. A part shows
isolates with yellow butt and slants, while B part of the figure indicates isolates with red butt and
slants. C part indicates isolates with yellow but and red slants while D part few isolates shows H2S
production.
 A B

C D

Fig 4.6. Triple sugar iron agar test results.

In this TSI test total 25 strains shows positive test (A-1, A-2, A-4, A-5, A-7, A-8, A-9, A-11, A-
12, A-13, A-15, A-16, A-17, A-19, A-20, A-21, A-22, A-23, A-24, A-25, A-27, A-28, A-29, A-
30, IARI) while remaining strains shows negative test i.e. they produce red butt. From this we can
conclude that the 25 strains are similar to each other and ferment the three sugars.

Table 4.3 Triple sugar iron agar results for different isolates of Azotobacter.

SAMPLE COLOUR OF ACID CO2/H2 H2 S TSI +VE/

CODE SLANTS/BUTT PRODUCTION PRODUCTION PRODUCTION -VE

A-1 Yellow/Yellow Yes Yes/Yes No +ve

A-2 Yellow/Yellow Yes Yes/Yes No +ve

SAMPLE COLOUR OF ACID CO2/H2 H2 S TSI +VE/
CODE SLANTS/BUTT PRODUCTION PRODUCTION PRODUCTION -VE

A-3 Red/Red No No/No No -ve

A-4 Yellow/Yellow Yes Yes/Yes Yes +ve

A-5 Yellow/Yellow Yes Yes/Yes Yes +ve

A-6 Red/Red No No/No No -ve

A-7 Red/Yellow Yes Yes/Yes No +ve

A-8 Red/Yellow Yes Yes/Yes No +ve

A-9 Yellow/Yellow Yes Yes/No No +ve

A-10 Red/Red No No/No No -ve

A-11 Yellow/Yellow Yes Yes/Yes No +ve

A-12 Yellow/Yellow Yes Yes/Yes No +ve

A-13 Red/Yellow Yes No/No No +ve

A-14 Red/Red No No/No No -ve

A-15 Yellow/Yellow Yes Yes/Yes No +ve

A-16 Yellow/Yellow Yes Yes/Yes No +ve

A-17 Red/Yellow Yes Yes/Yes No +ve

A-18 Red/Red No No/No No -ve

A-19 Red/Yellow Yes Yes/Yes No +ve

A-20 Red/Yellow Yes No/Yes No +ve

A-21 Red/Yellow Yes No/Yes No +ve

A-22 Red/Yellow Yes No/Yes Yes +ve

A-23 Red/ Yellow Yes Yes/Yes Yes +ve

A-24 Yellow/Yellow Yes Yes/Yes No +ve

SAMPLE COLOUR OF ACID CO2/H2 H2 S TSI +VE/
CODE SLANTS/BUTT PRODUCTION PRODUCTION PRODUCTION -VE

A-25 Red/Yellow Yes Yes/Yes No +ve

A-26 Red/Red No No/No No -ve

A-27 Red/Yellow Yes Yes/Yes No +ve

A-28 Yellow/Yellow Yes Yes/Yes No +ve

A-29 Red/Yellow Yes No/Yes No +ve

A-30 Red/Yellow Yes No/Yes No +ve

IARI Red/Red No No/No No +ve

4.4.2. CITRATE UTILIZATION TEST

Citrate Utilization test is used to detect the ability of an organism to utilize sodium citrate
as a sole source of carbon and ammonium salt as a sole source of nitrogen as mentioned in
Materials and Methods section 3.8.2.

The test organism is cultured in a medium containing sodium citrate, an ammonium salt and
bromothymol blue indicator. The organisms use citrate (the only source of carbon) and ammonia
(the only source of nitrogen). The citrate utilization is followed by alkaline reaction (change of the
color from light green to blue) and the growth in the medium is indicated by appearance of
turbidity.

Fig. 4.7 Citrate utilization test for Azotobacter isolates, color changes from green to blue
indicates positive test.
In this citrate utilization test all the 31 strains including IARI shows positive results i.e. they utilize
citrate and convert forest green color into blue as shown in Fig. 4.7.

4.4.3. METHYL RED (MR) TEST

Methyl Red (MR) test determines whether the microbe performs mixed acids fermentation
when supplied glucose. Types and proportion of fermentation products produced by anaerobic
fermentation of glucose is one of the key taxonomic characteristics which help to differentiate
various genera of enteric bacteria.
In mixed acid fermentation, three acids (acetic, lactic and succinic) are formed in significant
amounts. The mixed acid pathway gives 4 mol of acidic products (mainly lactic and acetic acid),
1 mol of neutral fermentation product (ethanol), 1 mol of CO2, and 1 mol of H2 per mol of glucose
fermented.
These large amounts of acid results significant decrease in the pH of the medium below 4.4. This
is visualized by using pH indicator, methyl red (p-dimethylaminoaeobenzene-O-carboxylic acid),
which is yellow above pH 5.1 and red a t pH 4.4. The results are given in the figure 4.8.

Fig. 4.8 Methyl red test for Azotobacter isolates, color changes into red indicates positive
test.

In this test total 20 strains of Azotobacter (A-1, A-2, A-4, A-5, A-7, A-8, A-11, A-12, A-15, A-16,
A-17, A-19, A-21, A-22, A-23, A-24, A-25, A-27, A-28, A-30) shows positive results i.e. these
strains turns red in color after adding methyl red. While rest of the strains gave negative results
and do not change their color after adding methyl red.

4.4.4. VOGES-PROSKAUER (VP) TEST

Voges-Proskauer is a double eponym, named after two microbiologists working at the

beginning of the 20th century. They first observed the red color reaction produced by appropriate
culture media after treatment with potassium hydroxide. It was later discovered that the active
product in the medium formed by bacterial metabolism is acetyl methyl carbinol, a product of the
butylene’s glycol pathway as mentioned in Materials and Methods section 3.8.4.
Pyruvic acid, the pivotal compound in the fermentative degradation of glucose, is further
metabolized through various metabolic pathways, depending on the enzyme systems possessed by
different bacteria. One such pathways result in the production of acetion (acetyl methyl carbinol),
a neutral-reacting end product. In the presence of atmospheric oxygen and 40% potassium
hydroxide, acetoin is converted to diacetyl, and alpha-naphthol serves as a catalyst to bring out a
red complex as given in Fig. 4.9.

Fig. 4.9 VP test for Azotobacter isolates, color changes into red indicates positive test.
In VP test total 16 strains (A-1, A-2, A-4, A-5, A-7, A-8, A-10, A-11, A-12, A-15, A-16, A-19,
A-25, A-27, A-28, A-30, ) gave positive results while rest gave negative results. From this we
can conclude that all the strains are not similar to each other.

4.4.5. CATALASE TEST

This test is used to demonstrate the presence of catalase, an enzyme that catalyses the release
of O2 and H2O from hydrogen peroxide (H2O2). It is used to differentiate between catalase
producing bacteria and non-catalase producing bacteria as mentioned in the Materials and
Methods section 3.8.5.

In this test all the strains performed well except few. Some isolates showed highest no. of bubble
formation while some showed little bubble formation. The bubble formation takes place and the
test for Azotobacter was considered as positive test. The result for this test is shown in Fig. 4.10.

Fig. 4.10 Catalase test showing bubble formation.

Here 15 strains (IARI, A-5, A-8, A-12, A-16, A-17, A-18, A-19, A-20, A-21, A-22, A-25, A-28,
A-29, and A-30) out of 31 gave positive results. While rest of the strains did not produced bubbles.
4.4.6. OXIDASE TEST

The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme
of the bacterial electron transport chain.

When present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p-phenylenediamine)

to (indophenols) purple color end product. When the enzyme is not present, the reagent remains
reduced and is colorless as mentioned in the Materials and Methods section 3.8.6.

In the given test we tested our different Azotobacter isolates on whatman filter paper and found
that some of the isolates were showing positive results and some were showing negative. Those
with positive results gave blue color at the end as given in the Fig. 4.11.

Fig. 4.11 Oxidase test for Azotobacter.

Here 22 strains (IARI, A-1, A-2, A-3, A-4, A-5, A-6, A-7, A-8, A-9, A-10, A-11, A-12, A-13,
A-14, A-15, A-16, A-17, A-19, A-20, A-24, A-26) shows similar results i.e. they gave blue color
as an end product.

4.4.7. NITRATE REDUCTION TEST

Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on

the basis of their ability to produce nitrate reductase enzyme that hydrolyze nitrate (NO3–) to nitrite
(NO2–) which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous
oxide and ammonia (NH3) depending on the enzyme system of the organism and the atmosphere
in which it is growing as mentioned in Materials and Methods section 3.8.7.
Heavy inoculum of test organism is incubated in nitrate broth. After four hours incubation, the
broth is tested for reduction of nitrate (NO3–) to nitrite (NO2–) by adding sulfanilic acid reagent and
α- naphthylamine.

In the give test Azotobacter strains shows good and positive results as given in Fig. 4.12.

Fig. 4.12 Nitrate reduction test showing positive results.

Total 13 strains (A-2, A-4, A-6, A-8, A-13, A-14, A-15, A-16, A-18, A-20, A-24, A-28, and A-
29) gave positive results i.e. these strains are converting nitrate to nitrite and turning into red color.

4.4.8. UREASE TEST

Urea is a diamide of carbonic acid. It is hydrolyzed with the release of ammonia and carbon
dioxide. Many organisms especially those that infect the urinary tract, have a urease enzyme which
is able to split urea in the presence of water to release ammonia and carbon dioxide. The ammonia
combines with carbon dioxide and water to form ammonium carbonate which turns the medium
alkaline, turning the indicator phenol red from its original orange yellow color to bright pink as
mentioned in Materials and Methods section 3.8.8.

In this total 13 strains (A-1, A-11, A-20, A-23, A-24, A-25, A-26, A-27, A-28, A-29, A-30) gave
positive results i.e. these strains are producing bright pink color while splitting urea in presence of
water and release ammonia and carbon dioxide as shown in Fig. 4.13.
 Fig. 4.13 Urease test showing positive results.

4.4.9. STARCH HYDROLYSIS TEST

Bacteria have the ability to hydrolyse starch, as they can produce the amylase enzyme. While
the starch forms dark blue color with iodine, its hydrolyzed products do not acquire such dark blue
color with iodine.

Starch agar is a differential medium that tests the ability of an organism to produce certain
exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch. Starch
molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade
starch into subunits that can then be utilized by the organism as given in Materials and Methods
section 3.8.9.

The result of this test is shown as the starch forms dark blue color with iodine. Total 9 strains (A-
1, A-4, A-6, A-10, A-11, A-12, A-23, A-26, A-27) were found positive for hydrolyzing starch
while rest shows negative results. From this result as given in Fig. 4.14 we can conclude that all
these 9 strains shows similar activities for hydrolyzing starch and are found similar from those
who gave negative results.
 Fig. 4.14 Starch hydrolysis test of Azotobacter showing mixed results.

4.4.10. MOTILITY TEST

This test is used to identify the motility of the bacteria by means of its flagella. The location
of the flagella varies with bacterial species. Non-motile bacteria do not possess flagella. The semi-
solid agar method permits the isolation of motile and non-motile strains from some cultures which
were non-motile with the hanging drop technique.

The semi-solid agar method is particularly advantageous to use with testing of highly pathogenic
organisms and routine testing, because the results are cumulative and macroscopic.

The motility test shows a positive result by showing the growth around the stabbed line. All the
31 strains shows positive results in this test as given in the Fig. 4.15.
 Fig. 4.15. Motility test for Azotobacter isolates showing positive results.

In the given pictures, these isolates shows the best motility. By this we can say that Azotobacter is
motile in nature.

Table 4.4 Result of all biochemical test for Azotobacter.

SAMPLE
CODE
BIOCHEMICAL TESTS

TSI CU MR VP CT OT NR UT SH MT

A-1 + + + + _ + _ + + +

A-2 + + + + _ + + _ _ +

A-3 _ + _ _ _ + _ _ _ +

A-4 + + + + _ + + _ + +

A-5 + + + + + + _ _ _ +

A-6 _ + _ _ _ + + _ + +

A-7 + + + + _ + _ _ _ +

A-8 + + + + + + + _ _ +
A-9 + + _ _ _ + _ _ _ +

A-10 _ + _ + _ + _ _ + +

A-11 + + + + _ + _ + + _

A-12 + + + + + + _ _ + _

A-13 + + _ _ _ + + _ _ +

A-14 _ + _ _ _ + + _ _ _

A-15 + + + + _ + + _ _ +

A-16 + + + + + + + _ _ +

A-17 + + + _ + + _ _ _ _

A-18 _ + _ _ + _ + _ _ _

A-19 + + + + + + _ _ _ +

A-20 + + _ _ + + + + _ _

A-21 + + + _ + _ _ _ _ _

A-22 + + + _ + _ _ _ _ +

A-23 + + + _ _ _ _ + + _

A-24 + + + _ _ + + + _ _

A-25 + + + + + _ _ + _ _

A-26 _ + _ _ _ + _ + + _

A-27 + + + + _ _ _ + + _

A-28 + + + + + _ + + _ +
 A-29 + + _ _ + _ + + _ _

A-30 + + + + + _ _ + _ _

IARI + + _ _ + + _ _ _ _

TSI= Triple sugar iron agar test, CU= Citrate utilization test, MR= Methyl red test, VP= Voges-Proskauer, CT=
Citrate test, OT= Oxidase test, NR= Nitrate reduction test, UT= Urease test, SH= Starch hydrolysis test, MT= Motility
test.

4.5. PHOSPHATE SOLUBILISING ACTIVITY TEST

Phosphate Solubilization is the activity by which, insoluble phosphate convert into soluble
form. Bacteria plays a major role in this activity, they solubilizes phosphate and provides free
phosphate to the plants. So they called phosphate solubilizing bacteria (PSB). Azotobacter is one
of PSB.

Fig. 4.16 Phosphate solubilizing test for Azotobacter showing negative results.

In this test none of the strains out of 31, solubilize phosphate. Hence showing negative results for
this test.
4.6. ANTIBIOTIC SENSITIVITY PROFILING TEST

The antibiotic sensitivity is the susceptibility of any bacteria to antibiotics. Because

susceptibility can vary even within a species, so antibiotic sensitivity test is usually carried out to
determine the susceptibility or resistivity of bacteria to a particular antibiotic. In Antibiotic
sensitivity test, the disc diffusion method was first performed in the year 1966. As mentioned in
Materials and Methods section 3.10.

Small wafers containing antibiotics are placed onto a plate upon which bacteria is growing. If the
bacteria are sensitive to the antibiotic, a clear zone of inhibition, is seen around the wafer indicating
poor growth. Present isolates were exposed to the octadisc having 8 different antibiotics residing
in wafer forms that are, Ciprofloxacin (Cf), Cloxacillin (Cx), Co-Trimoxazole (Co), Tetracycline
(T), Amoxyclav (Ac), Cephalexin (Cp), Clindamycin (Cd), and Erythromycin (E) Fig. 4.17. All
the isolates of Azotobacter were inhibited by antibiotic Ciprofloxacin with high clear zone
formation. All the isolates were resistant to Erythromycin and Amoxyclav as shown in table 4.5.

A B
 C D

Fig. 4.17 Octadisc showing clear zone for specific antibiotics.

Table 4.5. Antibiotic sensitivity profiling of Azotobacter.

ANTIBIOTICS

Cf Cp Ac T E Co Cx Cd

SAMPLES

A-1 2 3 R 5 R 3.3 R R

A-2 13 R R 6 R 12 R 6.5

A-3 8 1 R 8 R R 2 R

A-4 9 R R R R R R R

A-5 10 2 R R R R R R

A-6 7 R R R R R R R
ANTIBIOTICS

Cf Cp Ac T E Co Cx Cd

SAMPLES

A-7 5 R R R R 7 8 2

A-8 12 5.5 R 6.5 R 12 R 5.5

A-9 10 2 R 6 R 7.8 R 1

A-10 5 R R R R R R R

A-11 8 6.1 R R R R R R

A-12 4 R R R R 5 R R

A-13 7 4 R R R R R 1.4

A-14 5.8 R R R R R 2.6 R

A-15 8 R R R R 2 R R

A-16 4 1 R 6 R R R R

A-17 9 R R 1 R 2 3.7 R

A-18 10.2 R R R R R R R

A-19 13 R R 5 R 6 R R

A-20 4 R R R R R R R

A-21 7.1 3 R 1 R R 5 2

A-22 9 R R R R R R R

A-23 13 6 R 5 R 13 R 8
 ANTIBIOTICS

Cf Cp Ac T E Co Cx Cd

SAMPLES

A-24 3.9 R R 8 R 1.8 R R

A-25 5.9 R R 4 R R 7.4 R

A-26 12.1 8 R R R 1 6 4

A-27 11 R R R R R R R

A-28 6 R R 5 R 3 R R

A-29 13 7 R 7 R 13 6.5 6

A-30 8 R R R R 4 3.5 R

IARI 15 R R R R R R R

R=Resistant

4.7. pH AND TEMPERATURE TOLERANCE

4.7.1. pH TOLERANCE

In pH stress tolerance, the Azotobacter isolates were tested on Azotobacter agar media plate
with pH (6.0, 7.0, and 8.0) by spot inoculation method as given in the Fig.4.18.

All the isolates (A-1 to A-30) including IARI performed well and show good growth at pH 8. At
pH 7, isolates A-13, A-17, A-19 and A-20 did not show any growth. Isolates IARI, A-5, A-7, A-
27 and A-29 show maximum growth. If we look towards the isolates at pH 6 all performed well
except A-15, A-19 and A-20. Results are given in Table 4.6.
A B

Fig.4.18. Growth of Azotobacter at different pH

Table 4.6 Growth of Azotobacter at different pH.

DIFFERENT
pH
pH 6 pH 7 pH 8

SAMPLES

A-1 + ++ ++

A-2 +++ ++ +++

A-3 + ++ ++

A-4 ++ ++ ++

A-5 +++ +++ +++

A-6 ++ ++ ++

A-7 +++ +++ +

A-8 +++ ++ ++

A-9 ++ + +

A-10 + ++ +++

A-11 ++ ++ +++

A-12 +++ ++ ++

A-13 ++ _ ++

A-14 + ++ +

A-15 _ + ++
 SAMPLES pH 6 pH 7 pH 8

A-16 ++ ++ ++

A-17 ++ _ +++

A-18 + + ++

A-19 _ _ +

A-20 _ _ +

A-21 ++ ++ +

A-22 + ++ +++

A-23 ++ ++ +

A-24 ++ + ++

A-25 +++ ++ ++

A-26 ++ ++ +

A-27 +++ +++ +++

A-28 + ++ ++

A-29 +++ +++ +++

A-30 + + +

IARI +++ +++ +++

+++ = Best Growth, ++ = Better Growth, + = Good Growth, - = No Growth

4.7.2. TEMPERATURE TOLERANCE

In screening for temperature tolerance, the Azotobacter isolates were tested at different
temperatures viz. 28˚C, 33˚C, 38˚C, 43˚C, 44˚C and 45˚C for 48-72 hours to be examined as their
responses towards the growth in Azotobacter agar medium, which will uncover the maximum
temperature at which the isolates can grow. From the results it is clear that maximum strains were
seized to grow at 44˚C Fig.

Isolates were exposed to different temperatures and observed thoroughly. All isolates showed a
linear growth from 28˚C to 38˚C but started diminishing at high temperature, 43˚C and above. The
growth on plates started to be seen lesser at 44˚C, maximum strains exhausted Table 4.7.

A-1 A-2

28 ̊ C 33 ̊ C
 44 ̊ C

Table 4.7. Azotobacter growth at different temperatures.

TEMPERATURES

28˚C 33˚C 38˚C 43˚C 44˚C 45˚C

SAMPLES

A-1 +++ +++ +++ +++ + _

A-2 +++ +++ +++ +++ _ _

A-3 +++ +++ +++ +++ _ _

A-4 +++ +++ +++ + _ _

A-5 +++ +++ +++ +++ + _

A-6 +++ +++ +++ ++ _ _

A-7 +++ ++ ++ + _ _

A-8 +++ ++ ++ + + _

A-9 +++ +++ +++ + _ _

A-10 +++ +++ +++ + + _

A-11 +++ +++ +++ +++ _ _

A-12 +++ +++ +++ ++ + _

A-13 +++ +++ +++ ++ _ _

A-14 +++ +++ +++ + _ _

A-15 +++ +++ +++ ++ _ _

A-16 +++ +++ +++ ++ _ _

A-17 +++ ++ ++ ++ + _

A-18 +++ +++ +++ + + _

A-19 +++ ++ ++ + + _

A-20 +++ +++ +++ + _ _

A-21 +++ ++ ++ + _ _

A-22 +++ ++ ++ + _ _

A-23 +++ ++ ++ + ++ _

A-24 +++ +++ +++ + _ _

 A-25 +++ +++ +++ ++ _ _

A-26 +++ ++ ++ ++ _ _

A-27 +++ +++ +++ + + _

A-28 +++ +++ +++ ++ _ _

A-29 +++ +++ +++ ++ _ _

A-30 +++ +++ ++ + _ _

IARI +++ +++ +++ ++ + _

+++ = Best Growth, ++ = Better Growth, + = Good Growth, - = No Growth