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www.wjpr.net Vol 8, Issue 9, 2019.
Kundu et al. World Journal of Pharmaceutical Research
723
ISOLATION, CHARACTERIZATION, TAXONOMIC STUDIES AND
BIOLOGICAL ACTIVITIES OF AN ANTIBIOTIC PRODUCING SOIL
BACTERIUM FROM MANGROVE REGION OF SUNDARBANS,
WEST BENGAL, INDIA
Bikas Kumar Kundu1* and Prasanta Kumar Sircar
2
1Microbiology Department, Maulana Azad College, Kolkata, India.
2Department of Botany, Ballygunge Science College, University of Calcutta, India.
ABSTRACT
The aim of this study was to characterize, taxonomic studies and
biological activities of antibiotic producing soil bacterium isolated
from Mangrove region of Sundarban, West Bengal, India, designated
as IIIM-A. For the taxonomic placement of the organism,
morphological studies of the isolated organism were done primarily
under light microscope growing the isolated organism on the solid
media and in liquid media. The nature of growth, colony morphology,
colour and nature of mycelia and diffusible pigments were studied in
different bacteriological media. Physiological characters of the
organism were determined by the studies of melanin pigment formation, proteolysis, urease
activity, starch hydrolysis; H2S gas production, utilization of different carbon sources and
antagonistic activity in addition to the study of cell wall composition. Tube dilution technique
and agar cup assay were employed to determine the antimicrobial activities against gram
positive bacteria, gram negative bacteria and few species of fungi as test organisms.
KEYWORDS: Soil, Bacteria, Mangrove, Sundarban, West Bengal, India, Characterization,
Taxonomy, Antagonism.
INTRODUCTION
The Actinomycetes is a large group of microorganisms consisting of a variable number of
genera and species. The group was considered as a transition group between fungi and
bacteria but recent evidences prove that the group is more closely related to bacteria.
World Journal of Pharmaceutical Research SJIF Impact Factor 8.074
Volume 8, Issue 9, 723-738. Research Article ISSN 2277– 7105
Article Received on
27 May 2019,
Revised on 17 June 2019,
Accepted on 07 July 2019,
DOI: 10.20959/wjpr20199-15320
*Corresponding Author
Dr. Bikas Kumar Kundu
Microbiology Department,
Maulana Azad College,
Kolkata, India.
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Morphological features of Actinomycetes undoubtedly play an important role for the
classification of this group but generic identification based on morphology alone may be
controversial in some cases. Waksman and Henrici (1943), suggested a comprehensive
system of classification in which the Actinomycetes were subdivided into four genera and
formation of a new genus Streptomyces where division was solely based on morphological
characters like production of aerial mycelium and catenulate spores. The color of aerial and
substrate mycelium was used for the separation of species. Waksman (1961) proposed a
system of classification for the genus Streptomyces with series based on color and growth of
substrate and aerial mycelium.[15][16]
Biochemical characters drew more attention for the classification of Actinomycetes. Pridham
and Gottlieb (1948) emphasized on the utilization of acetate, malate, pyruvate, propionate
and succinate as species characters.[12][13]
Krasilinikov (1960) did not agree to accept the
characters such as proteolysis, starch hydrolysis, sucrose hydrolysis, cellulose utilization and
nitrate reduction as taxonomic criteria because the characters are found in almost all
Actinomycetes, but utilization of urea, creatine and certain amino acids, casein, tyrosine and
xanthine could have more significance in classification. Quantitative production of antibiotics
(Krasilinikov, 1960),[4][5][6][7]
production of sulphide on peptone agar (Tresner and Danga,
1958)[13]
were useful criteria for Actinomycetes classification. Cell wall composition and
whole cell hydrolysate proved to be the practical use for the classification of
Actinomycetes.[8]
This group was subdivided into nine broad groups based on the distribution
of amino acids and sugars in the cell wall (Cumming, 1962). Presence of DL-dap
(Diaminopimelic acid) and LL-DAP in peptidoglycan and sugar compositions of
polysaccharides in the cell wall were used as an important marker for classification of
Actinomycetes (Kroppenstedt and Krtzner, 1976). Lipid pattern was considered to separate
Streptomyces from Nocardioform group.[9]
Present study deals with the characterization of a bacterial strain isolated from Mangrove
region of Sunderbans, India considering morphological, cultural, physiological and
biochemical characteristics and generic assignment with taxonomic placement of the strain
on the basis of determined characters.
The potency of antibiotic can be determined by chemical, physical and biological means. An
assay is made to determine the ability of an antibiotic to kill or inhibit the growth of living
microorganisms. Species and strains of microorganisms have varying degrees of
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susceptibility to different antibiotics. The susceptibility of an organism to a given antibiotic
may change, especially during treatment. Therefore, microbiologists should make an accurate
microbiological diagnosis to determine the susceptibility of an organism to antibiotic. The
susceptibility of an organism to antibiotic can be determined by the tube -dilution technique
or the paper -disc-plate technique.[11]
1. Tube dilution technique
By the tube dilution technique, one can determine the smallest amount of antibiotic required
to inhibit the growth of the organisms in vitro. This amount is referred to as the minimal
inhibitory concentration (MIC). In this technique increasing amounts of antibiotic under
examination are placed in a series of culture tubes containing a suitable amount broth
medium and incubated with the test organism keeping a control group of tubes having no
antibiotic. After incubation, the concentration of antibiotic required to inhibit the growth of
the organism is determined by the absence of growth.
2. Paper-disc-plate technique
The paper-disc-plate technique is the most commonly used technique for determining
susceptibility of microorganisms to antibiotic. Small paper discs of the same size
impregnated with known amounts of antibiotic are placed upon the surface of an inoculated
plate. After incubation the plates were observed for any zones of inhibition surrounding the
discs. A zone of inhibition around the disc indicates that the organism is inhibited by the
antibiotic which diffused into the agar from the disc.
All incubations were made at 37°C for bacterial species, 30°C for fungal species, sterilized at
15lb for 20 minutes, inoculated aseptically where necessary, observations were made with
five replica unless stated otherwise. Readings were taken 3 days of incubations.
MATERIALS AND METHODS
In course of our screening programs a strain of soil bacteria isolated from Mangrove region
of Sundarbans, India, showed antimicrobial activity against gram positive and gram negative
bacteria but not against fungi. So we were intrigued to characterize and identify the strain in
our laboratory for taxonomic placement in order to assign proper systematic position of the
organism. The identification was based on the criteria suggested by International
Streptomyces Project (ISP), other characters were also studied.[9]
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All incubations were made at 32°-35°C unless stated otherwise. Observations were made
with 5 replicas.
Morphological Characteristics
Morphological characteristics of the organism were studied on the solid and liquid culture
media. Nutrient broth and agar medium (modified after Waksman, 1961) and Glucose Yeast
extract broth and agar medium were used for morphological studies.
Plates were prepared with media and sterile square cover slips were dipped at an angle of 45°
into the solidified medium. The organism was spread along the cover slip-medium interface.
A number of replicas were prepared and after incubation one set was observed daily.
Morphological characteristics of the organism were also studied growing the organism in
submerged condition.
Cultural Characteristics
Plates were made with respective agar media (Table – 1). The organism was streaked on the
solidified agar medium and incubated at 32°-35°C for 14 days. The nature of growth,
morphology of colony, colour and nature of mycelium and formation of diffusible pigments
were noted. All compositions of the media were in g/L. The agar media contained 20.0g of
agar, sterilized at 15lb pressure for 20 minutes.
Physiological characteristics
1) Melanin formation
Melanin formation (Melanoid pigments) was studied on agar slants.
Melanin formation medium (Waksman, 1961)
Components of the medium Amount in g/L
Yeast extract 1.0
L-Tyrosine 1.0
NaCl 8.5
Bacteriopeptone-Yeast extract-Iron agar medium (Shirling and Gottlieb, 1966)
Components of the medium Amount in g/L
Bacteriopeptone (Difco) 15.0
Protease peptone (Difco) 5.0
Ferric ammonium citrate 0.5
Dipotassium phosphate 1.0
pH of the medium was adjusted at 7.0-7.2
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Agar slants of these media were inoculated and incubated for 8 days. Formation of diffusible
pigments were noted.
2) Proteolytic activity (Gordonand Mihm, 1959)
Gelatin slabs containing 20g gelatin in 100mL distilled water were inoculated and incubated
for 7 days. The cultures were kept at 4°C for one hour and observed for
solidification/liquefaction.
3) Urease activity (Nitsch and Kutzner, 1969).[10]
Components of the medium Amount in g/L
Peptone 1.0
NaCl 5.0
Glucose 1.0
Phenol red 0.012
Urea 20.0
Agar 3.0
pH of the N/45 Phosphate buffer was adjusted to 6.8.
(Urea was dissolved in a small amount of buffer and sterilized by Seitz filtration and
incorporated into the medium).
The semisolid slabs were inoculated and the colour change from phenol red to darker red due
to liberation of NH3 from urea was noted for five consecutive days and compared to the
uninoculated control set without urea.
4) Hydrolysis of starch
Starch agar (Waksman, 1961)
Components of the medium Amount in g/L
Soluble starch 10.0
NaNO3 0.3
K2HPO4 0.3
NaCl 0.5
MgCO3 1.0
The starch agar plates were spot inoculated and after 14 days of inoculation the plates were
flooded with 2.0mL of Lugol’s iodine solution (3.0g KI and 2.0g Iodine dissolved in 300mL
distilled water). Hydrolysis of starch was noted by the formation of clear zones around the
colonies.
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5) Production of H2S
Lead acetate agar (Kuster and Williams, 1964)
Components of the medium Amount in g/L
Bactopeptone 15.0
Protease peptone 5.0
Dextrose 1.0
Sodium thiosulphate 0.08
Lead acetate 0.2
The slants were inoculated with the organism and after 7 days of growth the formation of
dark brown pigment in the medium was noted.
6) Antagonistic property (Waksman, 1945)
Antimicrobial activity was tested against bacteria and fungi by cup assay method. Organism
was grown in nutrient broth medium (Waksman, 1961, modified) for 6 days. Bacillus subtilis
was placed in the nutrient agar medium and Saccharomyces cerevisiaewas placed in PDA
medium. Cups were cut in the middle of the plate, 0.1mL of the spores free culture filtrate
was put into the cup and incubated for 7 days. Inhibition of bacterial and fungal growth
around the agar cups indicated the antagonistic activity.
7) Utilization of carbon sources (Shirling and Gottlieb, 1966)
The carbon sources were filtered through a 0.45μ millipore filter before added to the
sterilized basal mineral salt agar medium.
Basal mineral salt agar
Components of the medium Amount in g/L
(NH4)2SO4 2.64
KH2PO4 2.38
K2HPO4 5.65
MgSO4.7H2O 1.0
The medium containing different carbon sources were plated and inoculated with the
organism. Plates containing D-glucose was considered as positive control.
Analysis of cell wall
Cell wall composition of the organism was analyzed according to the method of Boone and
Pine (1968).[1]
Culture was grown for 6 days in 50ml of modified medium of Waksmam,
1961 containing 0.5% peptone and 0.25% beef extract in 250ml conical flask. Mycelia were
collected by centrifugation and thrice with sterile water. About 0.5gm of mycelia was
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extracted with 5ml of 0.1 (N) NaOH in tightly sealed screw capped tubes for one hour in
water bath. The mixture was cooled, centrifuged and washed with sterile water. The alkali
extract was discarded and the cell wall was suspended in 1.0ml of water and 0.7ml was
utilized for the detection of cell wall sugars and 0.3ml was utilized for the detection of cell
wall amino acids.
Sugars
A sample of 0.7ml was made to 1.0ml with 2 (N) HCl and taken in a tightly sealed screw-
capped and placed in a boiling water bath for 2 hours. The hydrolyzed materials were
transferred to a small beaker and dried on the water bath adding sterile water time to time for
evaporation of acid-water. Finally the material was suspended in 0.5ml sterile water and used
for chromatography.
Amino acids
The rest sample of 0.3ml was hydrolyzed with 6 (N) HCl and dried in the same way as
mentioned in sugars.
Detection
Both samples each of 0.1ml were spotted on Whatman number one paper separately and two
dimensional descending chromatography was performed. Primary migration of sugars and
amino acids was done with phenol: water (80ml 90% phenol: 20ml water) at 30°C for 6
hours. After complete evaporation of phenol and water the chromatography papers were
subjected to second dimensional run at the same temperature in n Butanol : Acetic acid :
Water (25 :6 : 25 v/v). For detecting sugars, the chromatograms were dipped in 25ml acetone
containing 0.1ml 50% AgNO3 and dried at room temperature and again dipped in 0.5 (N)
KOH in 90% Ethanol. On drying for 2 to 3 minutes dark brown spots appeared and the
chromatograms were dipped in 1.0% Sodium thiosulphate to remove dark background.
Whereas amino acids were detected by spraying the chromatograms with 0.2% ninhydrin
solution in nButanol and finally dried at 80°C for 5 minutes.
Antimicrobial action
Preparation of inocula
Bacterial inocula were prepared from 24 hours broth culture of each test organism in their
respective media. Bacteria were inoculated in 250ml Erlenmayer flask containing 50ml broth
medium consisting of ingredients as required for growth. The inoculated flasks were
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incubated at 37°C for 24 hours at 120 r.p.m. for agitation. The prepared bacterial broth
cultures were employed in antimicrobial activity tests. The fungal inocula were prepared
from the spore suspensions or cell suspensions. The fungal spores/cell from the solid culture
were suspended in sterile distilled water to make the inocula for antimicrobial susceptibility
tests.
Preparation of serially diluted antibiotic fractions
Stock solution of antibiotic compound was prepared with weighed amount of lyophilized
antibiotic in sterile distilled water (1000μg/ml). From the stock solution, diluted fractions of
antibiotic were prepared. The concentrations of antibiotic in the diluted fractions were
500μg/ml, 200μg/ml, 100μg/ml, 50μg/ml, 20μg/ml and 10μg/ml. After the determination
inhibitory concentration range, the fractions were again serially diluted within the range and
assay was performed to obtain the approximate MIC against test organisms.
Microorganisms employed for the antimicrobial activity
Gram -ve and gram +ve bacterial species including the species of Mycobacterium and some
species of fungi were employed in this test (the list of microbial species is given in Table –
5).
Methods employed for the determination of MIC
Tube dilation technique was employed for the determination of antimicrobial activities and
MIC. Increasing amounts of antibiotics, 10μg/ml, 20μg/ml, 50μg/ml, 100μg/ml, 200μg/ml
and 500μg/ml, were placed in a series of culture tubes containing 5ml suitable broth medium
inoculated with 0.1ml of the test organism. In each case, control set of culture tubes was kept
where only antibiotic was absent. The inoculated culture tubes were incubated on the rotary
shaker (120 r.p.m.) at 37°C temperature for bacterial species and 30°C for fungal species.
After 72 hours of incubation, the concentration of antibiotic required to inhibit the growth of
the microorganisms was determined by colorimetric studies.
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Fig. 1: Submerged 2 days culture of organism IIIMA showing branched mycelia with
well developed hyphae (×1000). Grown in nutrient broth medium composing 0.5%
peptone and 0.25% beef extract, incubated at 35°C.
Fig. 2: Submerged 7 days culture of organism IIIMA showing branched aerial mycelia
which are sporulated to form coccoid spores (×1000). Grown in nutrient broth medium
composing 0.5% peptone and 0.25% beef extract, incubated at 35°C.
Fig. 3: Substrate mycelia of organism IIIMA showing fragmentation. The
microorganisms grown on cover slip – solid medium interface (×1000). Grown in
nutrient broth medium composing 0.5% peptone and 0.25% beef extract, incubated at
35°C for 7 days.
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Fig. 4: Aerial mycelia of the organism IIIMA showing chains of spores. The
microorganisms grown on cover slip – solid medium interface (×1000). Grown in
nutrient broth medium composing 0.5% peptone and 0.25% beef extract, incubated at
35°C for 14 days.
RESULTS AND DISCUSSIONS
The microorganism
The microorganism produced both substrate and aerial mycelium. The substrate mycelium
were found penetrated into agar medium and ramified in it. The substrate mycelium was
branched and yellowish in colour, fragmented at maturity, fragments are of unequal in length
and bears chains of nonmotile coccoid spores. The microorganism formed whitish yellow to
brown aerial mycelia which were sporulated. Spores were brownish in colour, coccoid in
shape, grew in chains, chains were not curved, number of spores in chain varied from 5 to 30,
aerial spores were smaller than spores of substrate mycelia. There were no distinguishable
sporophore, and absence of sporangia, sclerotia, synnemata or other special structures. The
microorganism was aerobic, heterotroph, could grow on different media with varying colony
morphology, yellow or brown diffusible pigment was secreted on some media. The
microorganism was gram positive, non-acid fast, grew well from 15°C to 40°C with optimum
range 30°C to 35°C, melanoid pigments were not produced, could not decompose cellulose
and showed no urease activity but the microorganism could liquefy gelatin, could hydrolyze
starch, produced H2S and showed antagonistic activity against gram positive and gram
negative bacteria but not against fungi. The microorganism could utilize arabinose, glucose,
maltose, moderately utilize galactose and fructose but could not utilize urea, lactose, fucose,
sorbitol, mannitol and inositol. The cell wall contained meso-diaminopimelic acid, arabinose
as detectable sugar and the cell wall belongs to type IV.
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Taxonomic placement
The taxonomic placement of the strain was based on Bergey’s Manual of Determinative
Bacteriology, Group -22,1994 and Bergy’s Manual of Systematic Bacteriology,Vol-
4,1989,[3][18]
The strain can be excluded from the following groups of Actinomycetes by the characteristics
mentioned in each group.
1) Exclusion of Group II (Actinomycetes with multilocular sporangia)
i) Multilocular sporangia absent
ii) Aerial mycelia formed
iii) Filamentous phase present
2) Exclusion of Group III (Actinoplanetes)
i) Aerial mycelia formed
ii) Absence of single spore or sporangia on the hypha
3) Exclusion of Group IV (Streptomyces and related genera)
i) Substrate mycelia fragmented
ii) The strain contains meso-diaminopimelicacid and wall type IV
iii) Arabinose found as detectable sugar in the cell wall
iv) Spore chains on aerial mycelia not curles or spiral
4) Exclusion of Group V (Maduromycetes)
i) Substrate mycelia fragmented
ii) Arabinose detected in the cell wall
5) Exclusion of Group VI (Thermomonospora and related genera)
i) Single spore on the hypha not formed
6) Exclusion of Group VII (Thermoactinomycetes)
i) Absence of single or two spores on dichotomized hypha
7) Exclusion of Group VIII (Other genera)
i) Cell wall type IV
ii) Cell wall diagnostic sugar arabinose
Both substrate and aerial mycelia sporulated, mycelia fragmented at maturity, fragmented
hyphae of different length bearing short to long chains of spores, chain of spores on aerial
mycelia not curled or spiral, sporophore not distinguishable, no special structures like
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sporangia, sclerotia, synnemata etc., cell wall type Iv with arabinose as detectable sugar in the
cell wall.
………………….Nocardioform Actinomycetes
Extensively branched substrate mycelia growing on the surface and penetrating agar media,
substrate mycelia of older culture fragmented into coccoid elements, aerial mycelia visible
only microscopically, short to long chains of spores formed on aerial mycelia, no endospore,
sporangia, sclerotia or synnemata formed, gram positive, non-acid fast, colonies white in
young culture, brown in older culture, colonies velvety in appearance, raised and heaped,
yellow or brown diffusible pigment produced, cell wall contained meso -diaminopimelic acid
and arabinose belonging to type IV.
…………………..Nocardia
Among the known species of Nocardia the isolated strain might be close to Nocardia carnea
as per the characteristics of the strain IIIMA determined.
Antagonistic properties
The antimicrobial activities of the antibiotic substance produced by the strain were
determined and approximate MIC was determined (Table – 5). The strain showed strong
inhibitory activity against gram positive and gram negative bacteria but there was no
antifungal activity.
Table – 1: Cultural characteristics of the organisms.
Media Colony morphology Soluble pigment
Nutrient agar (Waksman, 1961)
Round to convex, center
brownish, outer ring whitish,
outermost pale yellow and thin,
center colony raised, powdery,
heaped and velvety
Deep yellow
Glycerol Asparagines agar
(Pridham & Lyons, 1961)
Scanty growth, radiating, colony
not well organized Light yellow
Czapek’s medium agar
Around the small brown center a
thin whitish ring, outer ring
radiating and faint
Yellowish
Emerson’s agar (Waksman,
1961)
Round yellowish colony turned
white then brown, dotted brown
center surrounded by broad white
zone, outermost ring yellowish
Brown
Glucose asparagine agar
(Waksman, 1961)
Round yellowish colony turned
white then brown, dotted brown Yellowish brown
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center surrounded by broad white
zone, outermost ring yellowish
Egg albumin agar (Waksman,
1961) Very scanty growth None
Glucose yeast extract agar
(Waksman, 1961)
Raised, powdery, center brown,
whitish ring outside, margin
radiating, lensoid
Yellow
Cellulose agar (Waksman, 1959) None None
Inorganic salt starch agar
(Waksman, 1959)
Greyish brown center, outer ring
chocolate brown, radiating,
slightly raised
Light brown
Potato glucose agar (Waksman,
1961)
Uneven circles, raised, purplish
white ring, radiating None
Potato plug (Waksman, 1961) Growth throughout the surface of
the plug, no distinguished colony Deep yellow
Inorganic salt agar (Waksman,
1961) None None
Observations were made after incubation at 32°-35°C for 21 days.
Table – 2: Utilization of carbon sources by the organism.
Carbon source Utilization
Glucose +
Galactose +
Fructose +
Maltose +
Mannose +
Sucrose +
Arabinose +
Glycerol +
Lactose -
Carboxymethyl cellulose -
Fucose -
Mannitol -
Sorbitol -
Inositol -
Urea -
Utilization of carbon sources was investigated by using the procedure of Shirling and
Gottlieb (1966)
+: utilized
-: not utilized
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Table – 3: Physiological properties of the strain.
Physiological properties Response
Temperature range for growth (°C) 15-40
Optimum temperature (°C) 30-35
Gram nature Gram +ve.; non-acid fast
Formation of melanoid pigment (Shirling &
Gottlieb, 1966) -
Liquefaction of gelatin at 28°C (Gordon and
Mihm, 1962) +
Hydrolysis of starch (Lyons and Pridham, 1962) +
Decomposition of cellulose (Waksman, 1961) -
Production of H2S (Kuster and Williams, 1964) +
Urease activity (Nitsch and Kutzner, 1968) -
Antagonistic property (Waksman, 1965) + (Positive against species of bacteria but not
against species of fungi)
+: test positive; - : test negative
Results of cell wall analysis
Analysis of cell wall indicated L-Arabinose as diagnostic sugar and meso-DAP as diagnostic
amino acid present in the cell wall.
Table – 4: Comparison of isolated Nocardia sp. With related species.
Species of Nocardia Decomposition
of tyrosine
Production of
urease
Growth on sole carbon source (%,
w/v)
Arabinose
(1.0)
Inositol
(1.0)
Mannitol
(1.0)
N. amarae - + - + +
N. asteroids - + - - -
N. brasiliensis + + - + +
N. brevicatena - - - + +
N. carnea - - + - -
N. farcinia - + - - -
N. nova ND + - - -
N. otidiscaviarum - + - + +
N. pinensis - + ND ND ND
N. sterilae - - ND ND -
N. transvalensis - + - - -
N. vaccinii - + D + +
Isolated Nocardia sp. - - + - -
+ : test positive; - : test negative; ND : not determined; D : 11-89% strains positive
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Table – 5: Antimicrobial activities of antibiotic.
Test organisms MIC (μg/ml)
Gram positive bacteria
Bacillus subtilis >20
Bacillus licheniformis >20
Bacillus cereus >20
Staphylococcus aureus >20
Staphylococcus albus >20
Sarcinalutea >20
Micrococcus flavus >20
Mycobacterium phlei >50
Gram negative bacteria
Escherichia coli >10
Proteus sp. >20
Salmonella gallinarum >10
Pseudomonas sp. >20
Serratiamarcescens >20
Alkaligenesviscolactis >20
Klebsiella sp. >20
Providensia sp. >20
Fungi
Saccharomyces cerevisiae -
Penicilliumnotatum -
Aspergillus sp. -
Rhizopus sp. -
ACKNOWLEDGEMENTS
The study was supported by the Department of Botany, Calcutta University and
Microbiology Department, Maulana Azad College, Kolkata.
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