CHAPTER 5

IDENTIFICATION OF BACTERIA BY
BIOCHEMICAL TESTS
 CHAPTER -V

IDENTIFICATION OF BACTERIA BY
BIOCHEMICAL TESTS

INTRODUCTION

In the present study the analysis of molecular diversity in freshwater bacterial

communities would typically involve a combination of classical and molecular
techniques.

Bacteria, archaea, fungi, algae, protozoan and protists are integral part of
microbial diversity and these seem to be an unnoticed normal resource that deserves
greater attention. Microbial diversity is the variety that exists among
microorganisms and their environments. Microorganisms are found in all
ecosystems. The bacteria are remarkable in the abilities to live in environments that
are hospitable for life and the greatest among energy sources. (Ganesan and
Muthuchelian, 2009)

Freshwater bacteria are a very diverse assemblage of prokaryote organisms,

varying in their morphology, physiology and ecological preferences. Bacteria may
be conveniently grouped into a number of natural assemblages based characteristics
such as cell shape, spore forming capabilities and whether they are aerobic /
anaerobic or Gram positive / Gram negative. (Sigee, 2005)

The morphological and biochemical method of identification of bacteria is

the classical method of characterization of bacteria. Classical identification of
individual bacterial species in environmental samples typically involves isolation,
laboratory culture and then taxonomic characterization.

The classification of bacteria into families, genera and species is based on a

wide range of phenotypic characteristics (Holt et al, 1994). These include culture
conditions, colony morphology, biochemical characteristics and detailed
morphology.

100
 In addition, there are thousands of microorganisms which live in water and
are transported. Water receives microorganisms from air, soil, sewage, organic
waste, dead plants and animals etc. It is obvious that at times almost any organisms
may be found in water, those finding unfavorable condition die and those finding
favorable environment grow, multiply to increase their population. It is believed that
certain acceptable amount of pollution to water without any adverse effect on itself
gets purified due to natural biological cycles and self purification capacity of water.

Indicator organisms are commonly used to assess the bacteriological quality

of water. Faecal Streptococci are the most commonly used bacterial indicators of
faecal pollution. They are found in water that is contaminated with faecal wastes of
human and animal origin. The ratio between faecal eoliforms and faecal streptococci
gives a faecal index, which indicates the origin of pollution.

Microbiological studies are of great importance both from the point of view
of monitoring and maintaining a proper aquatic environment as well as optimum
utilization of the available and added nutrient for any biological production. The
greatest need in limnological studies is bacteriological information, which has
immense applications in productivity studies. The microbiological examination of
water has significance in case of pollution studies (Mathivanan, et al., 2007).

India is likely to face increasing environmental problems in general and

water pollution in particular. Recent realizations of pollution hazards in aquatic
system give importance to the prodigious amount of research. The confluence of
industrial effluents in the nearby river system conglomerates the entire aquatic biota
and undergoes self purification during its course depending on the intensity of its
pollutant contents.

Now a day’s water pollution is widely spreading throughout the world and is
leading to occurrence of new diseases because of various human activities. Water
pollution is the biggest menace of urbanization, industrialization and modem
agricultural practices which has led to alteration in physical, chemical and biological
properties of water bodies as well as that of the environment. It directly or indirectly
affects the life processes of flora and fauna of the water body.

101
 These pathogenic bacteria are some time useful because of their ability to
produce pigments, enzymes, any chemicals and antimicrobial compounds. These
compounds like primary and secondary metabolites produced by microbes are useful
in food, agricultural, medicinal and industrial purposes.

In India most of the microbiological laboratories are depending on the

conventional methods to identify and study the diversity of bacteria. So in the
present study diversity of some selected bacteria was studied by applying both
conventional and molecular methods. The conventional methods includes,
phenotypic characterization (colony morphologies, Gram staining, endospore
staining etc.), biochemical characteristics (nutrient requirements - sugars, enzymatic
activities, and /or metabolic activities). But in conventional methods the
characteristics are not static and can change with stress or evolution (Ocham et al.,
2005). So in this regard the selected cultures are subjected to biochemical and
molecular works to withstand prolonged bacterial study

102
LITERATURE REVIEW

Microbial world is invisible to unaided human eyes. Thus what goes on in

their domain is not easily perceived, unless some direct measurements and analyses
are carried out. Comprising of bacteria, fungi, yeasts, protozoans, phytoplankton and
other micro fauna within 500 jxm sizes, microbial communities perform immense
tasks mainly to keep themselves perpetuating in their ecosystems.

In the detection, identification and quantification of target organisms some

approaches are solely based on a single technique whereas other strategies take
advantage of a combination of different methods. For example, to identify
Escherichia coli reliance can be placed on a one-day-cultivation on chromogenic
media. The traditional cultivation techniques are usually sensitive but the
identification is often not as reliable as desired. Methods based on molecular biology
tend to be sensitive and yield reliable identification, but cultivation techniques
always show viable organisms whereas molecular methods often reveal dead or
inactivated target organisms/nucleic acid.

Tanprasert and Reed, (1997), performed detection and identification of

bacterial contaminants from Strawberry runner explants. The biochemical tests such
as Gram's stain, motility, oxidase, and gelatinase, and carbon source utilization
tests were performed for 30 isolates and identified, the majority bacteria such as
Pseudomonas fluorescens (A, F, and G types). P. corrugata, P. tolaasii, P.
paucimobilis, Xanthomonas campestris, Xanthomonas spp., and Enterobacter
cloacae were also identified.

Ganesan and Muthuchelian, (2009) used some of the biochemical tests to

screen the high density of bacteria and then subjected to molecular work for further
study. Studied isolation, identification and comparative study of fungal and bacterial
strains found in organic and inorganic soils of different agricultural fields, and they
did the preliminary identification of bacteria by biochemical and colony
morphology.

Kolawole et al., (2009), isolated 6 bacterial species were viz., Staphylococcus

aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaricus, Bacillus

103
subtiilis and Staphylococcus epidermidis in selected lime treated river water of Agba
River, Nigeria. All these isolates are isolated by using serial dilution and pour plate
methods. The media used were nutrient agar, Eosine Methylene Blue (EMB) and
Methyl Red Voges - Proskauer broth. Total bacterial and total coliform counts were
carried out and isolates were subjected to biochemical tests and tentative
identification done using the Bergey’s Manual of Determinative Bacteriology.

A large number of microorganisms are found in water which falls under the
group of bacteria, fungi, protozoa, nematodes and actinomycetes. A large amount of
animal viruses are also transmitted through water. A majority of bacteria like E. coll,
Pseudumonas sp, Alginomonas spp, Xanthomonas sp, Mycobacterium sp,
Salmonella sp, Yersinia sp, Chromobacterium sp, etc were found in polluted water
due to increased soil runoff, sewage, industrial disposal and faecal contamination.
Eutrophic lakes support luxuriant growth of bacteria, fungi, algae. (Dubey and
Maheshwari, 2006)

Biochemical tests that investigate the enzymatic activities of cells are

powerful tests in the identification of bacteria. In the research lab, a microbiologist
would be begin by examining the cell morphology, colony morphology, Gram stain
reaction, and environment from which the bacterium was isolated. Once these
properties are known, a specific series of tests, defined by a specific flow chart,
would be performed to aid in the identification of the genus and species of the
bacterium. As many as 50 - 100 tests may have to be performed in order to
positively identify the bacterium.

In order to identify the unknown bacteria, examination of seven

characteristics of the unknown bacteria is necessary. These seven characteristics are:
1. colony morphology, 2. cell morphology, 3. Gram stain reaction, 4. oxygen
requirements for growth, 5. carbon source utilization, 6. presence of endospores in a
culture and 7. Motility and finally comparison was performed based on Bergy’s
manual.

According to Petti et al„ (2005), when common microorganisms present

with uncommon phenotypes, when unusual microorganisms are not present in
reference databases, or when databases are out of date, reliance on phenotypes can

104
compromise accurate identification. Technologist bias or inexperience with an
unusual phenotype or isolates may similarly compromise identification when results
of biochemical tests are interpreted to fit expectations

Su

105
MATERIALS AND METHODS
MATERIALS:
Table - 9: Media Used for Isolation and Biochemical Tests
Media Compositions Quantity
Nutrient agar media Peptone 5g
Beef extract 3g
Sodium chloride 5g
Agar 15g
Distilled water 1000ml
pH: 6.9-7.2±0.2
Brilliant Green Lactose Peptone 5g
Broth Beef extract 3g
Bile salt 5g
Lactose 5g
Brilliant green 0.065g
Distilled water 1000ml
pH: 7.0±0.2
Lactose Broth Peptone 5g
Beef extract 3g
Lactose 5g
Phenol red 0.05g
Distilled water 1000ml
pH:7.0±0.2
Salmonella-Shigella Peptone 5g
medium Beef extract 3g
Lactose 10g
Sodium citrate 8.5g
Bile salt 8.5g
Sodium thiosulphate 8.5g
Ferric chloride lg
Neutral red 0.025g
Brilliant green o.33g
Agar 15g
Distilled water 1000ml
pH: 6.9±0.2
Simons citrate agar Ammonium dihydrogen phosphate lg
medium Dipotassium phosphate lg
Sodium chloride 5g
Magnasium sulphate 0.2g
Bromothymol blue 0.8g
Agar 15g
Distilled water 100ml
pH:6.9±0;2
Peptone Broth Peptone 10g
Distilled water 1000ml
pH:7.0±0.2
MR-VP broth Peptone 7g
Dextrose 5g
Potassium phosphate 5g
Distilled water 1000ml
pH:6.9±0.2

106
 Gelatin Broth Peptone 5g
Beef extract 3g
Gelatin 120g
Distilled water 1000ml
pH:6.8±0.2
Starch agar medium Starch 20g
Beef extract 3g
Peptone 5g
Distilled water 1000ml
pH:6.8±0.2
Urea agar medium Peptone lg
Sodium chloride 8g
Potassium dihydrogen phosphate 2g
Agar 15g
Distilled water 1000ml
Glucose lg
Urea 2%
Phenol red 6ml
pH:7.3±0.2
SIM Agar Medium Peptone 30g
Beef extract 3g
Ferrous ammonium sulphate 0.2g
Sodium thiosulphate 0.025g
Agar 3g
Distilled water 1000ml
pH:7.3±0.2
EMB agar medium Peptone lOg
Lactose 5g
Dipotassium hydrogen phosphate 2g
Eosin Y 0.4g
Methylene blue 0.65g
Agar 15g
Distilled water 1000ml
pH:7.2±0.2
Tripticase nitrate broth Trypticase 20g
Nitrate lg
Disodium phosphate 2g
Dextrose lg
Agar lg
Distilled water 1000ml
pH:7.2±0.2
Carbohydrate Trypticase/Peptone 10g
fermentation medium Carbohydrate 5g
Sodium chloride 15g
Phenol red 0.018g
Distilled water 1000ml
pH:7.2±0.2
Tryptone Broth Peptone / Tryptone 10g
Distilled Water 1000ml
pH: 7.0±0.2

107
Stains: Crystal Violet, Safranin, Malachite Green, Methylene Blue (Aneja, 2003)

Reagents: Grams Iodine, Kovac’s reagents, Methyl Red, VP reagents (I and II)
(Voges - Proskauer) Reagent A-sulfanilic acid and Reagent B-Dimethyl a-
naphthalamine. [(Aneja, (2003) and APHA, (1998)]

METHODS:
Identification of bacterial isolates:
Tentative identification of bacterial cultures was done by using following
methods using manual Aneja, (2003), APHA, (1998) and Bergey’s Manual.

Colony characteristics:

The isolates were preliminarily identified based on morphological

characteristics like colour, size, margin, form, elevation and texture. .

Motility test:
Clean cavity slide was taken and placed on the table with depletion upper side
4
A cover slip was taken and wax was applied on four comers
4
A loopful of culture was transferred exactly at the center of the cover slip
4
Cavity slide was placed on the cover slip and pressed gently
4
The preparation was lifted gently so that the culture drop is suspended in the form of
hanging drop
1
Edge was observed under 45X objective
Gram staining:
Thin smear of bacterial culture was made on clean glass slide
4
Air dried and heat fixed
4
Smear was covered with crystal violet for 30 seconds
4
Slide was washed with distilled water
4
Smear was covered with Grams iodine solution for 60 seconds
4
Slide was washed with 95% ethyl alcohol and then distilled water
4
Again the smear was covered with safranin for 30 seconds
4
Washed with distilled water and blot dried
4
Air dried and observed under microscope

108
Endospore staining:

Thin smear of bacterial culture was made on clean glass slide j

Air dried and heat fixed
i
Smear was covered with malachite green
i
Slide was steamed for 5 minutes by adding more stain to the smear for time
to time
i
Slide was washed slowly under tap water
I
Again the smear was covered with safranin for 30 seconds
I
Smear was washed with distilled water and air dried
I
Observed under microscope

Biochemical test for bacterial isolates:

1. Catalase test:
Nutrient agar medium was prepared
1
The medium was poured into culture tubes and flasks
i
It was sterilized by autoclaving at 151b pressure for 15 minutes
I
The nutrient agar slants were inoculated with test organisms
I
An inoculated nutrient agar slant was kept as control
i
The cultures were incubated at 35°C and 3-4 drops of hydrogen peroxide was added
on the growth of each slant culture
i
The culture was observed for the appearance or absence of gas bubbles.

109
2. Oxidase test:

A piece of filter paper was divided into three equal section and labeled with the name
of organism

4
A loop full of culture was rubbed on the moistened filter paper using a sterile loop

4
The color of the smear was checked exactly 15-30 seconds after rubbing the cells on
the reagent moistened filter paper

4
A deep blue color indicates positive reaction

4
Light violet or purple color developed within 10 seconds was recorded as negative.

3. OF test:

Fermentation media was prepared

4
The media and glassware’s were sterilized in autoclave at 151b pressure for 15minutes

4
Each of specified fermentation tubes of media was labeled with the name of the
organism to be inoculated

4
A layer of oil was added on the top of the media present in test tube

4
Incubated at 35°C for 24- 48 hours and observed for color change

110
4. Carbohydrate fermentation test:

Fermentation medium was prepared with specific carbohydrate such as glucose,

fructose, maltose, lactose and sucrose were used

4
The media was sterilized using autoclave at 151b pressure for 15minutes

4
Each of specified fermentation tubes of media was labeled with the name of the
organism to be inoculated

4
Four types of sugars fermentation broth was inoculated with test organism and one
uninoculated tube with each fermentation broth was kept as comparative control

4
The tubes were observed for the change in color (due to production of acid) or change
in color and appearance of bubbles (due to production of acid and gas)

5. IMViC test:

a) Indole production test:

1% tryptone broth was prepared and sterilized using autoclave at 151bs for 15 minutes
4
The tryptone broth was inoculated with test organism and an uninoculated tube was
kept as control
4
The tubes were incubated at 35°C for 48hours; 1ml of Kovac s reagent was added
48hrs of incubation
4
The tubes were shaken gently after intervals of 10 to 15minutes
4
The tubes were allowed to stand for few minutes to permit the reagent to come to the
top
4
The tubes were observed for cherry red layers in the top layer

111
b) Methyl-Red and Voges-Proskauer test;
MRVP broth was prepared and sterilized using autoclave
4
5ml of the broth was poured into each tube
4
The tubes were inoculated with test organism
4
All the tubes were incubated at 25°C for 48hrs
4
5 drops of methyl red indicator was added to the tubes of each set
4
The change in color of methyl red was observed for MR test
4
12 drops of VP reagent -1 and 2-3 drops of VP reagent- II was added to the other set
of tubes
4
The tubes were gently shaken for 30 seconds with the caps off to expose the media to
oxygen
4
The tubes were kept aside for 15-30minutes and observed for change in colour for the
VP test

c) Citrate utilization test:

Simmon’s citrate agar media was prepared and sterilized using autoclave
4
5ml of media was poured into the culture tubes and slants were prepared
4 '

Simmon’s citrate agar slants were inoculated with test organism

4
The uninoculated tubes were kept as control
4
The tubes were incubated at 370C for 48hours
4
Slant culture was observed for the growth and coloration of the medium

112
6. Gelatin hydrolysis:
Gelatin media was prepared and sterilized using autoclave at 121°C
4
Stab inoculation was made using inoculating loop, from each culture into its
appropriately labelled deep tube of nutrient gelatin
4
The tubes were incubated at 370C for 4 to 7 days
4
After incubation, the tubes were placed in refrigerator at 4°C for 15minutes
4
The tubes were observed for liquification

7. Starch hydrolysis test:

Starch agar media was prepared and sterilized using autoclave at 151bs for ISminutes
I
The media was poured into Petri plate and allowed to solidify
4
The test organism was inoculated on to the plate with a sterile transfer loop
4
The plate was incubated at 35°C for 48hrs
4
After incubation the plate was flooded with Gram’s iodine
4
Plate was observed for clear zone around the test organism

8. Urease test:
Urease agar medium was prepared and sterilized using autoclave
4
The medium was allowed to solidify in the slanting position to form a slope
4
The slants were inoculated with test organism
4
The tubes were incubated at 37°C for 24 to 48hrs
/

4
The slants were observed for colour

113
9. Hydrogen sulphide production test:
SIM agar medium was prepared and sterilized using autoclave
4
SIM agar tubes were labelled with the name of the organism to be inoculated
4
Each organism were inoculated into its appropriately labelled tubes by means of stab
inoculation
4
The tubes were incubated at 35° to 36°C for 48hours
4
The tubes were observed for the presence or absence of black colouration along the
line of stab inoculation

10. Nitrate production test:

Trypticase nitrate broth was prepared

4
Broth was sterilized along with glassware using autoclave at 151bs for 15minutes
I
The broth was cooled and poured into the test tube
4
The test organism was inoculated using sterile loop
1
The tubes were incubated at 37°C for 24-48hours
4
After incubation 2-3 drops of reagent A-sulfanilic acid and equal amount of reagent
B-Dimethyl a-naphthalamine was added to the broth culture
4
The broth was observed for the appearance of red colour
4
Absence of red colour showed negative effect of nitrate reduction
4
It was confirmed by adding pinch of zinc dust and the tube was shaken vigorously
4
Appearance of red color confirmed the zinc had reduced the nitrate and the organism
was negative for nitrate reduction.

114
RESULTS AND DISCUSSIONS

From the bacteriological parameters of the present it was confirmed that

there are bacterial diversity but not that extent compared to other rivers mentioned in
literature review expect rod shaped bacteria (both gram positive and negative rods).
The diversity was found less. The well grown, good cultures were subjected to
biochemical test and are tabulated in the table - 10. In the present work we aimed to
grow the culture which can withstand for prolonged sub culture. Some of the
cultures which are survived after prolonged sub culturing are subjected to
biochemical tests. For any biochemical tests bacterial cultures should be long
survival culture. The cultures which are subjected to biochemical tests are tabulated
in table - 10. After biochemical tests some of the good cultures such as those
showed maximum number of positive results for different biochemical tests and
based on colony morphology are subjected to molecular work.

Conventional methods for microbial identification and diversity study

require the recognition of differences in morphology, growth, enzymatic activities
and metabolism to define genera and species. (Petti et al., 2005)

According to Clarridge. (2004), the genotypic identification of

microorganisms by 16S rRNA sequence analysis has emerged as a more objective,
accurate and reliable method for bacterial identification and diversity study, with
added capability of defining taxonomical relationships among bacteria. But it is not
perfect or suit to study the phenotypic and biochemical characteristics. Therefore, in
our work, we first performed conventional methods to study the diversity of bacteria
based on phenotypic and biochemical characteristics.

115
 Stenotrophomonas

Bacillus sp______
Escherichia sp

Escherichia sp
Klebsiella sp

Klebsiella sp
Bacillus sp
Bacillus sp

Bacillus sp

Bacillus sp
Bacillus sp
Bacillus sp
Bacillus sp

Bacillus sp

Bacillus sp
E. coli

E. coli
E. coli
Buajayg
paimaapi XpAUBjuax

sp
UOipnpOJJ 0SB3J{1 I 1 I , + + I 1 + I + I I , + I I
uoipnpatf ajBJjijsj I + + + 1 + + + I 1+ 1+ I+ + + +
uoipnpoij , .................................................

asojang i + + + + + + + + i I + +
T able - 1 0 : B iochem ical C h aracterizatio n o f B acteria Identified in K av eri riv e r

+ + + + +
Carbohydrate
Fermentation

+ + + i + + + + + + + + + + I + + l

asopBq + + + 1 + ■ + ■ + + i + + + + ■ + I

asopiug 1 + + + + + + + + + + + + + + + + +

asoanin + + + + + + + + + + + + + + + + + +

uoijBziipjg
4- ■ + ■ + + + ■ + + ■ + 1 + 1 + + 1
Biochemical Tests

w*\\3
IMViC Tests

tlA ■ , , + - + .. ............................. , . . + , +

HW iii + , .++.+ , .+ ,,ii

uoipnpojj
■ , ■ , + , .++,+ , ,+ ,,,,
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VO

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4" 4-4-4- * *4* 1 *4- *4-4- *4" « 4* 4*

sis^[oapi|i ai)B[30

m . + + + ■+ + + . .+ .+ > + + + +

+ >+«+ + +'++'+■+•++'
aSBpiXQ

+ + + + + + + + + + + + + + + + + +
3SBIB)B3

+ + + ■ + + + + + + + + > + >

ajodgopug
io uoijBuuog
4-4-4- 4- 1 + + + ■ + ■ + +

T3
+ve Rod
+ve Rod

+ve Rod

+ve Rod

+ve Rod

+ve Rod
+ve Rod

+ve Rod

+ve Rod
+ve Rod
-ve Rod

-ve Rod

-ve Rod
-ve Rod

-ve Rod

-ve Rod
-veRod

i(So{oqdjioi\[ 2
KRD12

KRD16
KRD17
KRD14

KRD18
KRD13

KRD15
KRB10
KRD11
KRB4

KRB7
KRB2

KRB6

KRB8
KRB9
KRB3

KRB5
KRB1

■ow ajBiosi
 Stenotrophomonas
Stenotrophomonas
Stenotrophomonas

Pseudomonas sp
Pseudomonas sp
Pseudomonas sp

Escherichia sp
Escherichia sp
Escherichia sp
cu

Klebsiella sp
tfl

Bacillus sp

Bacillus sp
Bacillus sp

Bacillus sp

Bacillus sp
Bacillus sp
Bacillus sp
Bacillus sp

Bacillus sp
Bacillus sp

Bacillus sp
Bacillus sp
Bacillus sp
Bacillus sp
Bacillus sp
£

E. coli

E. coli

E. coli
-1

sp
sp
sp

1 1 4 1 + l + • ■ • ■ ' + 1 • + + 1 l t + l l i • ■ + ■ I

+ 1 + + + + • ■ + + + + ' + l + 1 + + + + ■ + -t- + + ' +

1 1 ' ' ' ■ ■ 1 ■ l ■ • ■ ' ' 1 I ' ' ■ ■ ■ ■ ■ l l i ' •

+ + + 1 + + + + + + + + • + + + + + + 1 ■ + + + + + + + +

+ + + 1 4 + + + + + + + + + ' + + + + ■ + + + + + ' + + +

+ + 4 + l + + + + + + ■ + 1 + + 4 • + ■ + + + + l + + +

+ ■ + + + + + l + + + + + + 4 + + + + + + + i + + + + + +

4* 4" + + + + + + + + + + + 4- 4* 4* + + + + + + + + + + + + +

+ + 4 i + l + + + + < + + + i + 4 4 t t + + + i + 1 + + 4

i i + i + l i ■ l ■ l 1 + i + - + • • l + 1 l i ■ + + 1 i

i ■ l i ■ + l ■ + i i ■ ■ + ■ ■ ■ + + l ■ l l i ■ ' • + ■

■ ' 1 ■ ' + l ■ + ■ 1 • ■ + ■ ■ i 4 4 ■ ■ i i ■ ' 1 + i

i + + + l + l l + + i i + l + l + + 1 +
■ ' ■ 4" + + ■ ■ +

+ + + + + I + + ■ + + + i • + 4~ + t i + i + + + + + + 1 +

i i + + + + i l l + + • + ■ + i + i + + + + l + l + + 1 +

4 + + ■ + i + + + + - + + + i + + + i l + + + ■ + t + + +

+ + + + + + + + + + + + + 4* + 4- + + + + + + + + + + + 4 +

+ + + + 4- + ■ + + + + + ■ + i ■ + + + + ■ + + + + ■ + + 4

■ • + + + 1 + ■ ■ + + l ■ i + + + ■ ■ + > + ■ + - + + ■ +
+ve Rod
+ve Rod
+ve Rod
+ve Rod

+ve Rod
+ve Rod

+ve Rod

+ve Rod

+ve Rod
+ve Rod

+ve Rod

+ve Rod

+ve Rod
+ve Rod
+ve Rod
-ve Rod
-ve Rod

-ve Rod

T3
-ve Rod
-ve Rod
-ve Rod
-ve Rod

-ve Rod
-ve Rod

-ve Rod

-ve Rod

-ve Rod

£
<£>
>i >i
K.RK27

KRK30

KRK32
KRK22

KRK24

KRK26

KRK28
KRK29
KRD20
KRD19

KRK25

KRK33
KRK23
KRD21

KRK31

KRT46
KRT47
KRT45
KRS34

KRS36
KRS37
KRS38

KRS40
KRS39

KRS42

KRS44
KRS35

KRS43

(Z>
§2
 i

+
KRT48 +veRod Bacillus sp

i
i

l +
KRT49 +ve Rod Bacillus sp

i
l

+
KRT50 +ve Rod Bacillus sp

t
i
t + l +
KRT51 +ve Rod Bacillus sp

i
KRT52 -ve Rod Escherichia sp
KRT53 i +veRod Bacillus s p

+ + + + 1 .+ +
+ + +
+ + + + 1 + +
KRT54 i +ve Rod Bacillus sp

i
i
i

+ • + I +
+ l I +
KRT55 -ve Rod E. coli

i
i i i i + i i + I
i l i 1 1 l i l l
+ l + l
1 1 l 1 ' ■ ■ 1 '

t
■ + ■ + ' l ■ ■ l

+ + + .+ + + + + +
+ + + + ' + + + +

+
+
+ + + + + + + + +
+ l + 1 + + + ■ +

+ l + ' + + + + +
+ + + + + + + + +

■+ + + + + + + + +
+
+ + + + I + + l +
+ + + + + + + + +
KRT56 +ve Rod Bacillus sp

Note: Where +ve and + = Positive, -ve and - = Negative, OF = Oxidative Fermentation Test, MR = Methyl Red Test, VP = Voges - Proskauer
Test, H2S Production = Hydrogen Sulphide Production Test

118
 All these bacterial isolates are identified tentatively. According to above
biochemical tests the similar bacterial isolates omitted and only about twenty four
bacterial isolates which are with stand prolonged subculture are subject to RAPD
analysis to study the molecular diversity of bacteria in Kaveri River Belt, Kodagu
District, Karnataka, India.

From the above table - 10. it was clear that in Kaveri River Belt majority of
bacteria is the Gram positive and rods. In this the dominating bacteria is the Gram
positive Bacillus sp. The present work also showed the presence of beneficial
bacteria in Kaveri River water. Bacteria isolated from Kaveri River water sources
showed great variety in the diversity of rod shaped bacteria only. About 80% of
bacteria is the Bacillus sp and remaining 20% is the Gram negative rod shaped
bacteria. The other bacteria such as Klebsiella sp, Escherichia sp. Pseudomonas sp
were also identified tentatively. In Kaveri River Belt and also in any of the river
water of India there is first time record of bacteria Stenotrophomonas sp. These are
animal as well as human pathogens. Thus consumption of water from these water
bodies showed the outbreak of intestinal disorders.

Nougang et al.. (2011) isolated faecal coliforms in urban streams in the

equatorial region of Cameroon (Central Africa) by using membrane filtration
technique. Identification of E. coli was done using biochemical tests.

Omezuruike et al., (2008) revealed that, the original source of any drinking
water is rich in aquatic microbes, some of which could be dangerous if they enter the
human body. Most common bacteria present in water bodies are Salmonella sp, E.
coli. Enterobacter sp, Klebsiella, etc. These are entered to the water bodies through
faecal contamination. This leads to the outbreak of intestinal disorders. In many
developing countries, availability of water has become a critical and urgent problem
and it is a matter of great concern to families and communities depending on non­
public water supply system.

Panneerselvam and Arumugam, (2012), isolated and identified bacteria from

Karai and Puliyandangal lake water in and around Ranipet area, Vellore district,
Tamilnadu state, India by using conventional method, such as swabbing and
streaking on nutrient agar, MacConkey agar, blood agar and EMB agar media. The

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pure colonies of bacteria were identified and characterizied using standard
microbiological techniques. The bacterial species viz, E. coli, K. pneumoniae, Vibrio
choleare, Proteus sp, Pseudomonas areomonas and S. aureus.

According to Chakrapani, (2005), water scarcity in India is widespread in

majority of the states. Even in states like Karnataka, where we are proud to say that
infrastructure is the best, there is water shortage affecting the school children also.
Around 65% of primary schools lack basic drinking water facilities. As the water
drawn from the well, rivers, and ponds are not adequately hygienic, water borne
diseases. Water quality characteristics of aquatic environments arise from a
multitude of physical, chemical and biological interaction (Arasu et al, 2007).
According to World Health Organization (WHO), there were estimated 4 billion
cases of diarrhea and 2.2 million deaths annually (WHO, 2002)

Omezuruike et al, (2008) revealed that the original source of any drinking
water is rich in aquatic microbes, some of which could dangerous if they enter the
human body. Most common bacteria present in water bodies are Salmonella,
Shigella, E.coli, Enterobacter, Klebsiella, etc.

To remove pathogen, drinking water should be treated properly. Because,

potable or drinking water is that water which should be free from pathogenic
microorganisms and chemicals that are deleterious to human health. Drinking water
has to be aesthetically accepted, being clear and colorless and without disagreeable
taste or odor.

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 PLATE-18

+ve = Positive
-ve = Negative

Biochemical tests on Bacterial Isolates of Kaveri River Water

121
 PLATE-19

+ve = Positive, -ve = Negative

Biochemical tests on Bacterial Isolates of Kaveri River Water

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 PLATE-20

A= Cultures of Bacillus sp, B= Cultures of Gram +ve (White colony), Gram —ve (yellow colony) of
Bacteria, C= Cultures of Gram +ve (White colony), Gram -ve (yellow colony) of Bacteria, D=Cultures of
Bacillus sp, E=Culture of Bacillus sp, F= Cultures of Gram +ve (Whitish yellow colony), Gram -ve
(yellowish orange colony) of Bacteria

Bacterial Colony Characteristic

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 PLATE-21

B: Gram - ve Rod (Stenotrophomonas sp)

C : Gram - ve Rod (Stenotrophomonas sp) D : Gram + ve Rod (Bacillus sp)

E: Gram - ve Rod Shaped Bacteria F: Gram - ve Rod Shaped Bacteria

Microphotography of Gram and Negative Rods shaped Bacteria

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 PLATE-22

G: Gram + ve Rod (Bacillus H: Gram + ve Rod (Bacillus

I: Gram + ve Rod (Bacillus sp) J: Gram + ve Rod (Bacillus sp)

K: Gram - ve Rod Shaped Bacteria L: Gram - ve Rod Shaped Bacteria

Microphotography of Gram Positive and Negative Rods shaped Bacteria

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 PLATE-23

M: Gram - ve Rod Shaped Bacteria N: Gram - ve Rod Shaped Bacteria

Microphotography of Gram Negative Rods shaped Bacteria

PLATE-23 a

O: Endospore of Bacillus sp P: Endospore of Bacillus sp

Microphotography of Endospore of Bacillus sp

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