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CHAPTER V

TAXONOMIC STUDIES OF THE SELECTED ISOLATE C9

Selection of media for taxonomic studies:

Culture media used for taxonomic studies on actinomycetes comprise:

1) Media used for characterization and identification of species, consisting

of both synthetic and organic forms; the synthetic media have found

extensive application in the study of the morphology, physiology and

cultural properties of the organisms. While the organic media are used for

obtaining supplementary cultural evidence.

2) Media used for obtaining maximum yield of the metabolite of interest.

Waksman et al., 1948 recommended the inclusion of the following media for

characterization of actinomycetes.

1. At least three synthetic media, preferably sucrose, sodium nitrate, salt

agar or sucrose ammonium salt, agar, glucose or glycerol asparagine agar

and calcium malate or citrate agar.

2. Two or three organic media such as nutrient agar, yeast extract malt

extract agar, potato glycerol glutamate agar, or oatmeal agar.

3. Three or four complex natural media such as potato plug, gelatin or milk.

4. Peptone iron yeast extract agar for H2S production.

5. Tyrosine medium for tyrosine reaction.

6. A synthetic medium for carbohydrate utilization.

In the present work, morphological studies and colour determination of

the selected isolate was studied in accordance with the International

Streptomycetes Project (ISP) procedures (Shirling and Gottlieb, 1966).

92

The following media as recommended by the ISP (Shirling and Gottlieb,

1966) were used for morphological studies and colour determinations.

1. Yeast extract malt extract agar (ISP – 2)

2. Oat meal agar (ISP - 3)

3. Inorganic salts starch agar medium (ISP – 4)

4. Glycerol asparagine agar medium (ISP – 5)

The following biochemical tests were carried out employing the

prescribed media:

1. Nitrate reduction test

2. Carbon source utilization

3. Catalase production test

4. Urease test

5. H2S production test

6. Starch hydrolysis test

7. Casein hydrolysis test

8. Gelatin hydrolysis test

9. Citrate utilization test

10. IMViC tests

11. Ability to produce bioactive metabolite factors

12. Sodium chloride tolerance test

13. Growth temperature range

14. Growth pH range

15. Oxidase activity test

93

Preparation of inocula:

In general, the agar media favouring abundant sporulation are those with

C/N ratio as jowar starch agar, oatmeal agar (ISP) and starch casein agar

medium.

In the present study starch casein agar was used for isolation of

actinomycetes from soil samples. These slants were inoculated from the stock

culture and incubated at 28oC for 7 to 10 days for maximum sporulation. Spore

suspension was prepared by transferring a few loopful of spores from these slants

into sterile distilled water and shaken thoroughly. Fresh spore suspension was

prepared for each test. For gelatin liquefaction, starch hydrolysis and casein

hydrolysis, a loopful of spores taken from the stock culture was used for

inoculation. For all other tests spore suspensions prepared as above, were used

employing equal volumes of the suspension in each case.

Preparation of media:

Detailed composition of all the media employed in this work were given

in the Appendix-II, unless otherwise stated, 20 ml of each sterile medium was

poured into each Petri dish of 9 cm diameter. After holding the dish for 24 h at

28oC, these were inoculated with the organism under investigation, distributing

the inoculum uniformly and incubated at 28oC.

94

BIOCHEMICAL TESTS

1. Nitrate reduction test:

Nitrate broth of 5ml was inoculated with a loopful of spores of selected

isolate and incubated at 28oC for 7 days. Controls were also run without

inoculation. On 7th day, the clear broth was tested for the presence of nitrate in

the following way:

Regent a) α – naphthylamine test solution:

α - naphthylamine : 5.0 g

Conc. H2So4 : 8.0 ml

Distilled water : 1.0 l

- naphthylamine was added to dilute sulphuric acid and stirred until

solution was effected.

Reagent b) Sulphanilic acid

Sulphanilic acid : 8.0 g

Conc. H2So4 : 48.0 ml

Distilled water : 1.0 l

Sulphuric acid was added to 500 ml of water. Then sulphanilic acid was

added, followed by water to make upto volume.

Procedure:

To 1 ml of the broth under examination and 1 ml of control, two drops of

sulphanilic acid followed by two drops of α – napthylamine solution were added.

The presence of nitrate was indicated by a pink, red or orange colour and absence

of colour change was considered as nitrite negative. In the latter case, the

presence or absence of nitrate in the broth under examination was confirmed by

adding a pinch of zinc dust after the addition of the reagents. When the

unreduced nitrite, if present gave a pink, red or orange colour.

Result: This test gave positive result with the selected isolate.

95

2. Carbon source utilization:

The ability of different actinomycete isolate in utilizing various carbon

compounds as source of energy was studied by following the method

recommended by ISP. Chemically pure carbon sources certified to be free of

admixture with other materials were used. The following carbon sources (at 1%

level) were used for this test. D – glucose, L (+) – arabinose, sucrose, D-xylose,

meso-inositol, D-mannitol, D-fructose, L (+) rhamnose, raffinose, galactose and

salicin. The inoculated tubes were incubated at 28oC and observed on 7th day and

14th day. Acid production from above carbon sources were studied

Table 24: The results of carbon source utilization with the selected isolate

The results were recorded as follows:

Acid production from

arabinose

galactose

glucose

mannitol

raffinose

salicin

xylose

sucrose

rhamnose

meso-inositol

fructose

_

+

+

_

_

+

_

+

_

_

+

Positive utilization (+): When growth on tested carbon was significantly better

than on the basal medium without carbon

Utilization negative (-): When growth was similar to or less than growth on

basal medium without carbon.

96

3. Catalase production Test:

During aerobic respiration in the presence of oxygen, microorganisms

produce hydrogen peroxide (H2O2) which is lethal to the cell. The enzyme

catalase present in some micro organisms breaks down hydrogen peroxide to

water and oxygen as shown below:

2 H2O2 2 H2O + O2

and helps them in their survival. Catalase test is performed by adding H2O2 to

trypticase soyagar slant culture. Release of free oxygen gas (O2) bubbles is a

positive catalase test.

Procedure:

Preparation of trypticase soyagar (pH 7.3) slants

Composition of tripticase soyagar

Trypticase - 15.0 gm

Phytone - 5.0 gm

Sodium chloride - 5.0 gm

Agar - 15.0 gm

Distilled water - 1.0 litre

Trypticase soyagar slants were inoculated with the selected isolate. An

uninoculated trypticase soyagar slant was kept as control. Then these tubes were

incubated at 35oC for 24-48 h. While holding the inoculated tube at an angle,

allow 3-4 drops of hydrogen peroxide to flow over the growth of each slant

culture.

Results: A catalase positive culture will produce bubbles of oxygen within one

minute after addition of H2O2. Release of free oxygen gas bubbles was observed

in the selected isolate, thus showing catalase positive test.

97

4. Urease activity test:

Urea is a major organic waste product of protein digestion in most

vertebrates and is excreted in the urine. Some microorganisms have the ability to

produce the enzyme urease. The urease is a hydrolytic enzyme which attacks the

carbon and nitrogen bond amide compounds (eg: urea) with liberation of

ammonia as shown below:

H2N

C = 0 + H2O 2NH3 + CO2

H2N Ammonia

Urea Hydrolysis

Urease test is performed by growing the test organisms on urea broth or

agar medium containing the pH indicator phenol red (pH 6.8). During incubation,

microorganisms possessing urease will produce ammonia that raises the pH of

the medium/broth. As the pH becomes higher, the phenol red changes from a

yellow colour (pH 6.8) to a red or deep pink colour. Failure of the development

of a deep pink colour due to no ammonia production is evidence of a lack of

urease production by the micro organisms.

Results: Development of a deep pink colour due to ammonia production was

observed. Urease production was observed by the selected isolate.

Urease

98

5. Hydrogen sulfide production test:

Hydrogen sulfide (H2S) commonly called “rotten egg” gas because of the

copious amounts liberated, when eggs decompose, by certain bacteria such as

Proteus vulgaris through reduction (hydrogenation of sulphur containing amino

acids (cystine, cysteine and methionine) or through the reduction of inorganic

sulphur compounds such as thisulfates (S2O32-), sulfates (SO4

2-) or sulfites

(SO32). The hydrogen sulfide production can be detected by incorporating a

heavy metal salt containing (Fe2+) or (Pb2-) ion as H2S indicator to a nutrient

culture medium containing cysteine and sodium thiosulfate as the sulfate

substrates.

The inoculated peptone-yeast extract-iron agar slants were incubated for 7

days. Hydrogen sulfide colourless gas when produced reacts with the metal salt

(ferrous sulfide) forming visible insoluble black ferrous sulfide percipitate).

Observations:

The inoculated tubes were examined for the presence or absence of black

colouration and they were compared with uninoculated controls.

Results: No Colour change in the medium was observed. So the selected isolate

gave negative test for hydrogen sulphide production test.

99

6. Starch hydrolysis (amylase production test):

Amylase is an exoenzyme that hydrolyses (cleaves) starch, a

polysaccharide (a molecule which consists of eight or more monosaccharide

molecules) into maltose, a disaccharide (double sugars, i.e. composed of two

monosaccharide molecules) and some monosaccharides such as glucose. These

disaccharides and monosaccharides enter into the cytoplasm of the bacterial cell

through the semipermeable membrane and there by used by the endoenzymes.

Starch is a complex carbohydrate (polysaccharide) composed of two

Constituents-amylase, a straight chain polymer of 200-300 glucose units, and

amylopectin, a large branched polymer with phosphate groups.

Amylase production is known in some bacteria while well-known in

fungi. Amylases commercially produced from various aspergilli. They are used

in the initial steps in several food fermentations processes to convert starch to

fermentable sugars. They are also used to partially predigest the foods for young

children, to clarify fruit juices and in the manufacture of corn and chocolate

scruples.

The ability to degrade starch is used as a criterion for the determination of

amylase production by a microbe. In the laboratory, it is tested by performing the

starch test to determine the absence or presence of starch in the medium by using

iodine solution as an indicator. Starch in the presence of iodine produces a dark

blue colouration of the medium and a yellow zone around a colony in a blue

medium indicates amylolytic activity.

Results: On addition of iodine solution, it has not produced as dark blue

colouration of the medium. So the selected isolate gave negative test for starch

hydrolysis.

100

7. Casein hydrolysis test:

Casein is the major protein found in milk. It is a macromolecule

composed of aminoacids linked together by peptide bonds, CO-NH. Some

microorganisms have the ability to degrade the protein casein by producing

proteolytic enzyme, called proteinase (caseinase) which breaks, the peptide bond

CO-NH by introducing water into the molecule, liberating smaller chains of

amino acids called peptides, which are later broken down into free amino acids

by extracellular or intracellular peptidases which are transported through the cell

membrane into the intracellular amino acid pool for use in the synthesis of

structural and functional cellular proteins.

Casein hydrolysis can be demonstrated by supplementing nutrient agar

medium with milk. The medium is opaque due to the casein in colloidal

suspension. Formation of a clear zone adjacent to the bacterial growth, after

inoculation and incubation of agar plate cultures, is an evidence of casein

hydrolysis.

Results: No Clear zone is formed around the growth zone. So the selected isolate

gave negative test for casein hydrolysis.

101

8. Gelatin hydrolysis test:

Proteins are organic molecules composed of aminoacids, in other words

proteins contain carbon, hydrogen, oxygen and nitrogen, though some proteins

contain sulphur too. Amionoacids are linked together by peptide bonds to form a

small chain (a peptide) or a large molecule (polypeptide) of protein.

Gelatin is a protein produced by hydrolysis of collagen, a major

component of connective tissue and tendons in humans and other animals. It

dissolves in warm water (gels) when cooled below 250C.

Large protein molecules are hydrolyzed by exoenzymes and the smaller

products of hydrolysis are transported into the cell. Hydrolysis (liquefaction) of

gelatin in the laboratory can be demonstrated by growing microorganisms in

nutrient gelatin. Once the degradation of gelatin occurs in the medium by an

exoenzymes, it can be detected by observing liquefaction, (i.e. even very low

temperature 4oC will not restore the the gel characteristic) or testing with a

protein-precipitating material (i.e. flooding the gelatin agar medium with the

mercuric chloride solution and observing the plates for clearing around the line

of growth.

The refrigerated isolate inoculated gelatin tubes were examined to see

whether the medium is solid or liquid and the flooded plates for any clearing,

around the line of growth.

Results: No liquefaction of gelatin by the isolate was observed. The selected

isolate gave negative test for gelatin hydrolysis.

102

Citrase

9. Citrate utilization test:

Citrate test is used to differentiate among enteric bacteria on the basis of

their ability to utilize citrate as the sole carbon source. The utilization of citrate

depends on the presence of an enzyme citrase produced by the organism that

breaks down the citrate to oxaloacetic acid and acetic acid.

These products are later converted to pyruvic acid and carbondioxide

enzymatically as shown below.

Citric acid Oxaloacetic acid + acetic acid

enzymes

Pyruvic acid + CO2

The citrate test in performed by inoculating the microorganisms into an

organic synthetic medium, Simmon‟s Citrate agar, where sodium citrate is the

only source of carbon and energy. Bromothymol blue is used as an indicator.

When the citric acid is metabolized, the CO2 generated combines with sodium

and water to form sodium carbonate an alkaline product, which changes the

colour of the indicator from green to blue and this constitutes a positive test.

CO2 + 2Na+ + H2O Na2CO3

(produced during citric acid Alkaline pH

metabolism) (blue Colour)

Bromothymol blue is green when acidic (pH 6.8 and below) and blue

when alkaline (pH 7.6 and higher).

Results: In Simmon‟s citrate agar slants which were inoculated with the isolate,

if was observed that there is no change in the colour of the medium. It was

confirmed that the isolate was a citrate-negative where the medium remains

green.

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IMVIC TESTS

The IMVIC tests consist of four different tests: (i) Indole production (ii)

Methyl-red (iii) Voges-Proskauer and (iv) Citrate Utilization. The IMVIC Tests

were designed to differentiate Gram negative intestinal bacilli (Family

Enterobacteriaceae) particularly Escherichia Coli and Enterobacter-Klebsiella

group, on the basis of their biochemical properties and enzymatic reactions in the

presence of specific substrates.

10 (a) Indole production test

Tryptophan, an essential amino acid, is oxidized by some bacteria by the

enzyme tryptophanase, resulting in the formation of indole, pyruvic acid and

ammonia. The indole test is performed by inoculating a bacterium into tryptone

broth, the indole produced during the reaction is detected by adding Kovac‟s

reagent (dimethyl aminobenzaldehyde) which produces a cherry-red reagent

layer as illustrated.

Tryptophan Indole + Pyruvic acid + NH3

Indole + Kovac‟s Reagent Rosindole + H2O

(Cherry-red compound)

Results: Tryptone broth was inoculated with the isolate and one tube was kept as

an uninoculated comparative control. After 48 h of incubation, there was no

development of a cherry (deep) red colour in the top layer of the tube. It was

confirmed that the isolate was indole-negative due to absence of red colouration.

Tryptophanase

HCl

Butanol

104

10 (b) Methyl-red and Voges-Proskauer tests:

The Methyl-red (MR) and the Voges-Proskauer (VP) tests are used to

differentiate two major types of facultatively anaerobic enteric bacteria that

produce large amounts of acid and those that produce the neutral product acetoin

as end product. Both these are performed simultaneously because they are

physiologically related and are performed on the same medium MR-VP broth.

Opposite results are usually obtained for the Methyl-Red and Voges-Proskauer

tests, i.e. MR+, VP- OR MR-, VP+. In these tests, if an organism produces large

amounts of organic acids: formic acid, acetic acid, lactic and succinic acid (end

products) from glucose, the medium will remain red (a positive test) after the

addition of methyl red a pH indicator (i.e. pH remaining below 4.4). In other

organisms, methyl red will turn yellow (a negative test) due to elevation of pH

above 6.0 because of the enzymatic conversion of the organic acids (produced

during the glucose fermentation) to non-acidic end products such as ethanol and

acetoin (acetyl methyl carbinol).

MR-VP broth tubes were inoculated with the isolate. After 48 h of

incubation, to one tube 5 drops of methyl red indicator was added, to the second

tube 12 drops of V-P reagent I and 2-3 drops of V-P reagent II were added and

one tube was kept as uninoculated comparative control.

Results: In the MR test, in the isolate inoculated broth, the colour of methyl red

turned to yellow, is a negative test. In the VP test, in the isolate inoculated broth,

there is no change in colouration, is a negative test.

105

11. Ability to produce antibiotic factor:

The antibiotic producing capacity of the selected isolate was studied again

for confirmation by submerged fermentation technique in PM2 medium. The

antimicrobial spectra of the selected isolate were checked again by agar well

diffusion method.

Table 25: Antimicrobial spectrum of promising isolate by submerged

fermentation in selected medium

Microorganism

Antimicrobial activity (Diameter of

zone of Inhibition in mm)

20 µl 30µl 40µl 50µl

Bacillus megaterium 11 13 15 17

Bacillus subtilis 30 32 33 35

Staphylococcus aureus 21 25 26 28

Klebsiella pneumoniae 32 33 34 35

Pseudomonas fluorescens 24 24 25 28

Proteus vulgaris 31 34 35 37

Escherichia coli 10 10 11 12

106

Table 26: Antimicrobial spectrum of promising isolate by submerged

fermentation in PM2 medium

Microorganism

Antimicrobial activity (Diameter of

zone of Inhibition in mm)

20 µl 30µl 40µl 50µl

Bacillus megaterium 23 27 27 27

Bacillus subtilis 24 26 28 29

Staphylococcus aureus 23 26 27 28

Klebsiella pneumoniae 26 26 24 24

Pseudomonas fluorescens 26 31 33 33

Proteus vulgaris 25 26 28 28

Escherichia coli 21 21 24 26

12. Sodium chloride tolerance test:

This test was carried out on Bennett‟s agar medium. Fifty ml of sterile

molten Bennett‟s agar medium each contained different concentrations of sodium

chloride (2%, 5%, 7% and 10.5%) was cooled to 40-45 oC and poured into sterile

petriplates (6” dia) and allowed to solidify. The isolate was streaked on the agar

medium incubated at 28 oC for 7 days and the presence or absence of growth was

noted.

Note: Results of this test were presented in Table 28.

13. Growth temperature range:

The starch-casein agar medium slants were inoculated with the selected

isolate and incubated at 12 oC, 25 oC, 28 oC, 37 oC, 42 oC and 50 oC. The extent of

growth was recorded on 4th day and 8th days.

Note: Results of this test were presented in Table 28.

107

14. Growth pH range:

The starch-casein agar medium slants which were previously adjusted to

various pH range starting from pH 5.2, pH 8.0, pH 9.0 and pH 10.5 and they

were incubated at 28o C for 7 days and the presence or absence of growth was

noted.

Note: Results of this test were presented in Table 28.

15. Oxidase activity of the selected isolate:

To differentiate certain group of bacteria, oxidase activity is one of the

tests. Certain bacteria are oxidase negative as found in most of the numbers of

family. Enterobacteriaceae, while pseudomonas shows oxidase positive. To

perform the test, a dye dimethyl-p-phenylene diamine hydrochloride is used. It

donates the electron to cytochrome C, becomes oxidised, and produces a colour.

Procedure:

The selected isolate was inoculated on trypticase soyagar plate and

growth was observed on 7th day. Few drops of oxidase reagent dimethyl-p-

phenyline diamine hydrochloride were placed on the colonies, in such a way, it

should cover the growth.

Results: The selected isolate has produced oxidase as indicated by oxidation of

reagent to a deep pink colour. The colonies first became pink, then changed to

dark red and finally turned to black, it indicates that the selected isolate was

oxidase positive.

108

Morphology:

The aerial mycelium of isolate C9 and Psuedonocardia compacta were

white and appeared powdery they are characterized by acropetal budding of

substrate and aerial mycelium leading to segmented hyphae. The colonies are

composed of aerial hyphae which bear mostly apical and sometimes apical

swellings or spores. The substrate mycelium was yellowish brown for both the

cultures.

Based on the biochemical and physiological characteristics, our isolate

was identified as Psuedonocardia species and designated as RC 1714 and

deposited with MTCC Acession number MTCC 10601. Our isolate was

compared with the data based on cultural characteristics biochemical and

physiological properties of reported species of Psuedonocardia which revealed

that our isolate was similar in some aspects with Psuedonocardia compacta.

Hence a detailed comparison of the morphological, physiological and

biochemical properties of our isolate C9 and Psuedonocardia compacta was

carried out.

Our isolate C9 and P. compacta were similar with respect to the following

biochemical properties. They were both negative for H2S, gelatin, starch and

casein hydrolysis, NaCl tolerance and positive in ability to reduce nitrate.

Both the cultures did not utilize arabinose, meso inositol, rhamnose and

xylose. Our isolate C9 and P. compacta were different in the following aspects.

109

Table 27: Similarities and differences between our isolate C9 and

P. compacta

Biochemical and physiological properties isolate C9 P.compacta

H2S production - -

Gelatin hydrolysis - -

Starch hydrolysis - -

Casein hydrolysis - -

Nitrate reduction + +

Sodium chloride tolerance at 7% - -

Acid production from

L(+)arabinose - -

Meso- inositol - -

Rhamnose - -

D(+)xylose - -

D- fructose + +

D-glucose + +

Sucrose + -

Galactose + -

Salicin + -

D- mannitol - +

Urease production + -

Isolate C9 utilized glucose fructose, sucrose, galactose, and salicin, while

P. compacta utilized glucose and fructose only, mannitol was utilized by

P. compacta while our isolate did not.

The two cultures were similar to each other in morphology and most of

the physiological and biochemical properties. However, some differences were

observed in the utilization of sucrose, galactose, mannitol and salicin. In view of

dissimilarities observed in the utilization of the above compounds, we proposed

to assign our isolate C9 as a new variant of P. compacta and designated it as P.

compacta var.tirumala.

110

IDENTIFICATION AND CHARACTERIZATION OF THE

SELECTED ISOLATE

Identification and characterization of micro-organisms play a key role as

it expands the scope for industrially important products. In the present

investigation, a criteria laid down by the International streptomycetes project

were followed for the identification and characterization of the selected isolate.

To establish the novelty or otherwise of the present isolate with the

reported organisms in the literature, the various morphological, cultural and

biochemical characteristics of the isolate were compared with the description of

the numerous Pseudonocardia sp and other species cited in the literature. The

literature survey includes.

Bergey‟s Manual of Determinative Bacteriology (Buchanan and Gibbons,

1974), Bergey‟s Manual of systematic Bacteriology (Williams, 1992-93), the

Actinomycetes – Vol – II by Waksman 1961 (Waksman, 1961), Reports of the

international streptomyces project (ISP) (Shirling and Gottlieb, 1968; 1969;

1972) and the information collected from the following journals: Journal of

General Microbiology, Journal of Bacteriology, the journal of antibiotics,

(Japan), International journal of systematic Bacteriology, Hindustan antibiotics

Bulletin, Indian Journal of microbiology, journal of biotechnology, and

bioengineering, biological and microbiological abstracts and chemical abstracts.

Cell wall composition:

The cell wall composition and whole cell sugar pattern of the isolates

were determined using the following procedure:

111

Procedure:

All the strains were grown in 25 ml of yeast extract malt extract medium

for about 48h on a rotary shaker at 28oC. After sufficient growth has been

obtained, this inoculum was transferred into 75ml of the same media contained in

500 ml conical flasks. These flasks were kept on a rotary shaker at 28oC for

about 5 days. The cells were harvested at maximum growth. The cells were

collected by centrifugation and washed three times with distilled water.

These cells were then analyzed for the composition of amino acids and

sugars. The cells were separated into the screw capped tubes. One tube was used

to analyze amino acids and the other tube for sugars.

Amino acids:

About 10mg of cells were hydrolyzed for 2h with 1ml of 6N HCl in a

closed screw capped tube held at 100oC. After cooling, contents were filtered

through a filter paper. The solid material on the paper was washed with 3 drops

of distilled water. The liquid hydrolysate was dried three consecutive times on a

steam bath in watch glass to remove the HCl. The residue was taken up in 0.3ml

of distilled water for chromatography.

Sugars:

10mg of cells were hydrolyzed for 2 hrs with 1ml of 2N HCl in a closed

screw capped tube held at 100oC and the rest of the procedure is same as for

amino acids.

112

Chromatography:

Amount of about 5-10 l of each sample was spotted on Wahatman No.1

Chromatography paper. The solvent system used for amino acids was butanol:

acetic acid : water (4:1:5) and descending chromatography technique was used.

The solvent system used for carbohydrates was butanol: pyridine: water: toluene

(5:3:3:4) and descending chromatography technique was used.

Developing reagents:

For amino acids: 0.24% W/V Ninhydrin in 95% V/V Ethyl alcohol, were

sprayed, the after spraying the paper was dried at 65oC for 25 min and spots were

recorded.

For sugars: 0.1 ml of Aniline in 100 ml of 0.1 N oxalic acids was used. After

spraying the paper was dried at 65oC for 2-5 min and spots were recorded.

Result: Cell wall composition analysis revealed that our isolate possessed type

IV cell wall, which was inferred by the presence meso-DAP, arabinose and

galactose as characteristic sugars.

The isolation of various antibiotics from actinomycetes species in the

middle of this century stimulated extensive screening for antimicrobial

compounds and every new antibiotic was produced by a new species.

To establish the novelty the following literature was referred. Bergey‟s

Manual of Determinative Bacteriology (Buchanan et al., 1974), Bergey‟s Manual

of Systematic Bacteriology (Williams et al., 1992-93) and all other relevant

journals.

113

Identification of the Selected Isolate:

Micromorphology:

The aerial mycelium developed moderately on most of the media. The

aerial hyphae were short and extensively branched. The sporophores appeared

straight. The spore chains araised by successive acropetal formation designated

as Pseudonocardia type (Henssen and Schnepf, 1967). The spore chains arised

terminally or laterally. The colonies were found to be white in colour and the

aerial mycelium was powdery forming a thick cover. Our strain grows slowly on

a variety of media and the aerial mycelium appears after 7-10 days of

growth.Good growth was observed in starch casein agar and glycerol aspargine

agar media. Moderate growth was observed on yeast extract malt extract agar

and belongs to the rectus-flexible (RF) type.

Figure 10: Scanning electron microscopy of pseudonocordia species

The data on cultural characteristics, physiological and biochemical

properties and antimicrobial spectrum, carbon utilization pattern are given in

Tables 28,29&30.

114

Table 28: Physiological and biochemical properties:

S.No Reaction Response Result

1. Nitrate reduction Colour changes from brown to

orange

Positive

2. Catalase production Production of free oxygen gas

bubbles

Positive

3. H2S production No Colour change in the

medium

Negative

4. Urease production Colour changes from yellow to

deep pink

Positive

5. Starch hydrolysis Colour of the medium does not

changes to dark blue

Negative

6. Casein hydrolysis No clear zone is formed around

the growth zone

Negative

7. Gelatin hydrolysis No liquifaction of gelatin Negative

8. Milk coagulation and

peptonization

No Coagulation and no

peptonization

Negative

9. Growth temp. range

a) at 12oC

b) at 25oC

c) at 28oC

d) at 37oC

e) at 42oC

f) at 50oC

-

+

+++

++

+

-

Growth between

280 -37o C

10. Growth pH range

a) at pH 5.2

b) at pH 8.0

c) at pH 9.0

d) at pH 10.5

+

+++

++

+

11. Tolerance to NaCl

Growth on

NaCl 2% W/V

NaCl 5% W/V

NaCl 7% W/V

NaCl 10% W/V

NaCl 13% W/V

+

+

-

-

-

The following grades were used to indicate the extent of growth

throughout the present investigation.

-: no growth, +: poor growth, ++: moderate growth, +++: good growth.

115

The following Table-29 shows the carbon source utilization pattern.

Table 29: Carbon source utilization pattern

Utilization Carbon Sources

Positive D-glucose (++),

D-fructose (+++),

sucrose (+++),

galactose (++),

salicin (++)

Negative mannitol (-),

raffinose (-),

xylose (-), rhamnose (-),

meso-inositol (-), L (+)

arabinose (-)

Table 30: Antimicrobial spectrum of the culture filtrate in selected medium

Test organism

Diameter of zone of Inhibition in mm

20 µl 30 µl 40 µl 50 µl

A) Gram positive bacteria

Bacillus megaterium

Bacillus subtilis

Staphylococcus aureus

11.0

30.0

21.0

13.0

32.0

25.0

15.0

33.0

26.0

17.0

35.0

28.0

B) Gram negative bacteria

Pseudomonas fluorescens,

Klebsiella pnemoniae,

Proteus vulgaris

Escherichia coli.

24.0

32.0

31.0

10.0

24.0

33.0

34.0

10.0

25.0

34.0

35.0

11.0

28.0

35.0

37.0

12.0

Result: The culture filtrate exhibited significant broad spectrum antibacterial

activity

116

Figure 11: Antimicrobial spectrum of promising isolate C9 by submerged

fermentation in selected medium

117

Discussion:

The most significant characteristics of our isolate are summarized below:

The strain grew well on starch casein agar medium, glycerol aspargine

agar medium and moderate growth was observed on yeast extract-malt extract

agar and oat meal agar. The sporophores occurred as cylindrical rods, they are

arranged in chains. The aerial mycelium developed moderately to good on most

of the media. It was white in colour.

The strain was catalase, oxidase and urease positive. It didnot hydrolyze

starch, casein and gelatin. It did not coagulate and peptonize milk. It showed

strong nitrate reduction. It was negative for IMViC tests and H2S production. It

exhibited good growth at 28oC, no growth at 12oC and 50oC and showed poor

growth at 37oC. No growth was observed on meso-inositol, L (+) arabinose,

D-mannitol, D-xylose, D-rhamnose and on raffinose and moderate growth on D-

fructose, D-glucose, sucrose and on galactose. It exhibited good growth at pH 5.2

and pH 8.0 but moderate growth at pH 9.0 and at pH 10.5. It could tolerate 2%

W/V sodium chloride and 5% W/V sodium chloride but failed to grow at

7%, 10% & 13%w/v NaCl.

It exhibited excellent antibacterial activity against both Gram positive and

Gram negative organisms.

A detailed survey of the literature indicated that our strain belongs to the

genera Pseudonocardia species.

The culture was sent to IMTECH (Institute Of Microbial Technology) for

further characterization and identification.

Based on the biochemical and physiological characteristics, our isolate

was identified as Psuenocardia species and designated as RC1714 and deposited

with the MTCC accession number i.e. MTCC10601

118

119

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