CHAPTER V TAXONOMIC STUDIES OF THE SELECTED ISOLATE...
Transcript of CHAPTER V TAXONOMIC STUDIES OF THE SELECTED ISOLATE...
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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).
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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
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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.
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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.
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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
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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.
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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
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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.
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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.
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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.
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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.
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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:
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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.
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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.
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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.
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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.
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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
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Figure 11: Antimicrobial spectrum of promising isolate C9 by submerged
fermentation in selected medium
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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
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119
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