CHAPTER-1

INTRODUCTION

1.1 Background of study

According to the World Health Organization, inappropriate prescription and the improper
use of antibiotics has led to the development of antibiotic resistance in many bacterial
pathogens. Due to this reason, the current leading problem of the world is that the
emergence of drug resistant strains of pathogens is more rapid than the rate of discovery of
new drugs and antibiotics. Serious infection causing bacteria have become resistant to
commonly used antibiotics and become a major global healthcare problem in the 21st
century.

Today, many scientists and pharmaceutical industries concentrate on the isolation and
screening of Actinomycetes from different unexplored habitats, for the production
of antibiotics. The increasing problem of antibiotic resistance demands the discovery of
new antibacterial agents effective against those resistant to currently available antibiotics.
Actinomycetes are distributed widely, however only a small percentage of globe and a
small proportion of Actinomycetes have been screened. So there is a need to screen
Actinomycetes, a potential antibiotic producer from different habitats for antimicrobial
activity with the hope of discovering new strains capable to produce antibiotics against the
multi-drug resistant pathogenic microorganisms. Thus, the present study was undertaken
to isolate Actinomycetes from the soil samples of Biratnagar and to assess their anti-
bacterial activities.

1.2 Introduction to Actinomycytes

Soil is a reservoir of a tremendous variety of microorganism. The microorganism which

are native to soil plays a dynamics roles. Among such microorganism Actinomycetes is one
which play important role in decomposition of organic matter, recycling of mineral
compounds, composting, humus formation, production of antibiotics and cause some types
of diseases.

1
Actinomycetes are Gram-positive, but several species have complex cell wall structures
that make the Gram staining un-suitable. They are also considered a transitional group
between bacteria and fungi because Actinomycetes morphologically share characteristics
with both these groups, though taxonomically they are placed together with bacteria in the
class Schizomycetes in the order Actinomycetales. Actinomycetes have prokaryotic nuclei,
are susceptible to antibiotics, and have cell walls that contain muramic acid much like
bacteria. However, Actinomycetes also form filaments or hyphae, similar to many forms of
hyphae fungi.

Characteristic similar to fungi

 Extensive branching mycelium
 Aerial mycelium and asexual spores (conidia) on their hyphae
 Growth in the broth media results in pellets or clumps not turbidity

Characteristic similar to bacteria

 Size similar to bacteria, diameter of the hyphae rarely exceeds one micron
 Staining reaction and physiology similar to bacteria i.e. Gram positive
 Some have flagella similar to bacteria
 Cell wall composition similar to bacteria, there is no chitin and cellulose
 Susceptible to bacterial viruses, antibacterial agents and not antifungal agents

Actinomycetes have two main forms of reproduction; spore formation and hyphae
fragmentation. During reproduction, Actinomycetes can form conidiophores,
sporangiospores, and oidiospores. In reproducing through hyphae fragmentation, the
hyphae formed by Actinomycetes can be a fifth to half the size of fungal hyphae, and bear
long spore chains.

Actinomycetes can be found mostly in soil and decaying organic matter, grow in pH range
of 6.5 to 8 as well as in living organisms such as humans and animals (water logging is
unfavorable for their growth). They form symbiotic nitrogen fixing associations with over
200 species of plants, and can also serve as growth promoting or bio-control agents, or
cause disease in some species of plants. Actinomycetes can be found in the human
urogenital tract as well as in the digestive system including the mouth, throat, and
2
gastrointestinal tract without causing disease in the host. They also have wide medicinal
and botanical applications, and are used as a source many antibiotics and pesticides.

Many species of Actinomycetes produce antimicrobial compounds under certain conditions

and growth media. Streptomycin, actinomycin, and streptothricin are all medically
important antibiotics isolated from Actinomycetes bacteria. Almost two-thirds of the
natural antimicrobial drug compounds used currently are produced by different species of
Actinomycetes.

In industrial microbiology, Actinomycetes make its position in the top as a potential source
of antibiotics. An antibiotic is a metabolic product of one microorganism that in very small
amount is detrimental or inhibitory to other microorganisms. Among antibiotics produced
by fermentation technology, ¾ of all known products are synthesized with the use of
Actinomycetes. The Streptomyces spp. especially important which can produce a great
many antibiotics and other classes of biologically active secondary metabolites and cover
around 80% of total antibiotics produced. Some important antibiotic producers are listed
in table.

Actinomycetes Antibiotics produced

Micromonospora purpurea Gentamycin
Actinoplane liguriae Gradimycin
Streptomyces spp Streptomycin, tetracyclin, puromycin,
neomycin, vancomycin etc
Nocardia orientalis Vancomycin
Nocardia spp Rifmycin

Name of some Actinomycetes species are as follows:

A. bovis A. marimammalium
A. bowdenii A. meyeri
A. canis A. naeslundii
A. cardiffensis A. nasicola
A. catuli A. neuii

3
 A. coleocanis A. odontolyticus
A. dentalis A. oricola
A. denticolens A. radicidentis
A. europaeus A. radingae
A. funkei A. slackii
A. georgiae A. streptomycini
A. gerencseriae A. suimastitidis
A. graevenitzii A. suis
A. hongkongensis A. turicensis
A. hordeovulneris A. urogenitalis
A. howellii A. vaccimaxillae
A. humiferus A. viscosus
A. hyovaginalis A. israelii

Streptomyces

The genus Streptomyces includes aerobic, Gram-positive, filamentous bacteria that

produce well-developed vegetative hyphae (between 0.5-2.0 µm in diameter) with
branches. They form a complex substrate mycelium that aids in scavenging organic
compounds from their substrates. Although the mycelia and the aerial hyphae that arise
from them are non-motile, mobility is achieved by dispersion of spores. Spore surfaces
may be hairy, rugose, smooth, spiny or warty. In some species, aerial hyphae consist of
long, straight filaments, which bear 50 or more spores at more or less regular intervals,
arranged in whorls. Each branch of a verticil produces, at its apex, an umbel, which carries
from two to several chains of spherical to ellipsoidal, smooth or rugose spores. Some
strains form short chains of spores on substrate hyphae.

On agar plates, it has dusty appearance, compact nature and has certain color. It produces
typical musty odor of soil.

Nocardia

Develops a rudimentary mycelium that soon fragments into short rod like cells.

4
Micromonospora

Bear single conidia at the tip of specialized hyphal branches.

Roles / function of Actinomycetes

1. Degrade or decompose all sorts of organic substances like cellulose,

polysaccharides, protein, fats, organic acids etc.

2. They decompose or degrade the more resistant and indecomposable organic

substances or matter and produce a number of dark to brown pigments which
contribute to the dark color of soil humus.

3. They are also responsible for subsequent further decomposition of humus in soil.

4. They are responsible for earthy or musty odor or smell of fleshy ploughed soils.

5. Many genera species and strains of Actinomycetes produce or synthesize number

of antibiotics.

1.3 Antibiotics
An antibiotic, also called an antibacterial, is a type of antimicrobial drug used in the
treatment and prevention of bacterial infections. They may either kill or inhibit the growth
of bacteria. A limited number of antibiotics also possess antiprotozoal activity. Antibiotics
are not effective against viruses such as the common cold or influenza; drugs which inhibit
viruses are termed antiviral drugs or antivirals rather than antibiotics.

Sometimes the term antibiotic (which means "opposing life") is used to refer to any
substance used against microbes. However, the difference between antibiotics and
antimicrobials is that the former is produced naturally, while the latter is synthetic
(although both maintain the same goal of killing or preventing the growth of
microorganisms). Some sources distinguish between antibacterial and antibiotic;
antibacterial are used in soaps and disinfectants, while antibiotics are used as medicine.

Antibiotics revolutionized medicine in the 20th century. However, their effectiveness and
easy access have also led to their overuse, prompting bacteria to develop resistance. This

5
has led to widespread problems, so much as to prompt the World Health Organization to
classify antimicrobial resistance as a "serious threat that is no longer a prediction for the
future, it is happening right now in every region of the world and has the potential to affect
anyone, of any age, in any country".

1.4 Significance of study

The study assessed the current status of presence of antibiotics producing Actinomycetes
in the soil sample of Biratnagar. It is hope that the result obtained from this study will help
to get idea about the presence of Actinomycetes spp. and the antibiotics activities of isolated
Actinomycetes.

1.5 Area of study

The study was focused on different sites around Biratnagar municipality. Actinomycetes
spp varied depending on the texture and cultivation status of soil so five different soil
sample from different locality of Biratnagar was taken for study and sampling.

6
 CHAPTER 2

OBJECTIVE

2.1 General Objective

 To isolate the Actinomycetes spp. from soil sample of Biratnagar.
 To study antibacterial activity of isolated organism.

2.2 Specific objective

 To differentiate Actinomycetes spp from non-Actinomycetes bacteria.
 To carry out the process of submerged fermentation for the extraction of crude
antibiotics.
 To study the antibiotics activity from isolated Actinomycetes spp

7
 Chapter-3
REVIEW OF LITERATURE
The availability of antibiotic producing Actinomycetes varied depending on the texture and
cultivation status of the soil, the highest number found in regularly cultivated lacustrine
soil. Out of the total isolated colonies producing antibiotics; most of them belonged to
genus Streptomyces. One of the isolated colony largely inhibited the growth of E. coli and
Staphylococcus aureus with zone of inhibition more than 20 mm for both during primary
screening. (Rai et al.,2016 )

Among 70-90% growth obtained on agar plate, it is of Streptomyces spp. Streptomyces spp.
form a well-developed mycelium that doesn’t divide into segments. Mature colonies
consist of aerial hyphae called sporophores bearing numerous conidia in chain like
arrangement (5-50 or more conidia per chain). The conidia and sporophores are often
pigmented. On agar plates, Streptomyces spp. colony has dusty appearance, compact nature
and has certain color. It produces typical musty order of soil. Nocardia develops a
rudimentary mycelium that soon fragments into short rod like cells and Micromonospora
bear single conidia at the tip of specialized hyphal branches (Manandhar er al., 2013)

A large proportion of the Actinomycetes inhabiting natural substrates have the capacity to
inhibit the growth of bacteria and other microorganisms. The ability of these Actinomycetes
to exert an inhibiting effect upon microorganisms is highly specific. This selectivity
depends, not only on the strain of organism, but also on the medium in which it is grown
and conditions of growth. Antagonistic Actinornycetes produce a variety of antibiotics that
vary in chemical nature, in antimicrobial action, in toxicity to animals, and in their
chemotherapeutic potentialities. The antibiotics that have, so far, been isolated from
Actinomycetes vary in the degree of purification. Some are crude preparations, whereas
others have been crystallized, and considerable information has been gained concerning
their chemical nature. They include: actinomycitin, actinomyces lysozyme, actinomycin,
micromonosporin, streptothricin, streptomycin, and mycetin. Some Actinomycetes produce
more than one antibiotic substance. Some antibiotics are produced by several different
organisms. There is some evidence, however, that, although the same general type of

8
substance may be formed by the various cultures, it may not possess exactly the same
antibiotic spectrum; this suggests the possibility of variations in the chemical structure of
the agent (Waksman et al.,2010 )

Freshwater Actinomycetes appear to have immense potential as a source of antibacterial

compounds ( Gebreyohannesa et al.,2013 )

A new procedure for isolating Actinomyces of definite groups was developed (for soil
sample) i.e. preliminary exposure of soil suspensions to UV light. With the use of the
procedure Actinomycetes belonging to different genera were isolated. There was a marked
decrease after the irradiation in isolation of cultures belonging to Streptomyces, a genus
most widely distributed in nature while isolation of cultures belonging to other genera,
promising as sources of novel antibiotics, increased. Micromonospora, Amycolatopsis and
Nocardia proved to be the most stable to the effect of UV light. With the use of the
procedure it is possible to increase 2-3-fold isolation of cultures belonging to
Micromonospora, a genus known as a producer of many antibiotics including those used
clinically (Galatenko et al.,1990 )

Mycelium color and diffusible pigment of Actinomycetes isolates were determined on the
basis of morphological characterization, and most of the Actinomycetes isolates showed
positive results for catalase, starch utilization, and casein utilization and others also, while
the isolates showed negative results for indole and Voges–Proskauer tests (Hotam et
al.,2013).

It has been reported that 1g of soil when plated, harbours up to 10 billion microorganisms,
of which about 4.2 × 106 CFU/g (dry weight) are accounted for by bacteria species. If the
soil is collected from an area that is relatively steep and not quite in the shade. The
steepness of the terrain, exposure to the sun rays and lack of vegetation covering of the soil
(bare) may causes leaching of Actinomycetes and therefore have partly contributed to the
observed low Actinomycetes CFU/g of dry soil (Agadagba et al.,2014).

In bacteria, several sugars can be used as carbon sources. Although glucose is often an
excellent carbon and energy source for microbial growth, it is infrequently utilized as the
major carbon and energy source in secondary metabolite fermentation. When incubated in

9
media containing glucose and another carbon source, bacteria metabolize first glucose that
represses the transcription of genes required for the utilization of the secondary carbon
sources. When glucose is exhausted, the metabolism of the second carbon source is
activated, and generally to his correlates with the onset of antibiotic production. This
phenomenon is referred to as carbon catabolite repression and is mediated via components
of the phosphenol pyruvate: carbohydrate phosphor-transferase system, which transports
and phosphorylates carbohydrates (Grasso et al.,).

10
 CHAPTER 4

MATERIALS AND METHODS

4.1 Materials

The list of materials: equipments, chemicals, media and reagent are listed in appendix .

4.2 Methods

4.2.1 Sampling site

The study was conducted by collecting soil sample from different sites of Biratnagar.

4.2.2 Collection of sample

During the study, 5 different soil sample were collected aseptically from different sites at
a depth of 10 cm using standard methods in a sterile plastic bag. The collected samples was
then transferred within half hour to research laboratory of microbiology, Department of
Microbiology, Mahendra Morang Adarsha Multiple College, Biratnagar where the entire
research work was carried out.

4.2.3 Processing of sample

The aseptically collected soil sample was left for a week at room temperature for air dry
transferring it in a sterile petri plate.

From each sample, 1g of soil was beaded in test tube containing 9ml sterile distilled water
and shaken well. This was then serially diluted by using two fold serial dilution methods
up to 10-6. These test tubes was considered as stock cultures for different soil sample sites.
From each tubes 0.1 ml of sample was taken and spread plate technique was carried out in
Starch casein agar plate and the plate were incubated at 28°C for 7 days for the isolation of
Actinomycetes.

11
4.3 Microbial examination of soil sample

4.3.1 Isolation and identification of Actinomycetes

After growth observed on SCA plate selected colonies were inoculated on Nutrient agar
medium for each discrete colony presented in specimen to obtain well isolated pure
colonies incubated at 28 C for 3 to 4 days.

Isolated colonies were subjected to Gram’s staining, Biochemical test: Catalase test,
Oxidase test, MR, VP, Urease, Citrate SIM to identify the Actinomycetes based on the
Bergey’s Manal of Determination of Bacteriology, 1994.

4.3.2 Carbohydrate fermentation test of Actinomycetes strains

1. Broth media was prepared by mixing all ingredients of phenol red carbohydrate
(glucose, sucrose, mannitol, maltose, lactose, fructose) in 100 mL of
distilled/deionized water and heating gently to dissolved it.
2. 13 x 100 mm test tubes were filled with 4-5 ml of phenol red carbohydrate broth.
3. A Durham tube was introduced to detect gas production.
4. The prepared test media (at 121°C for 15 minutes) was autoclave to sterilize. (Note:
While using arabinose, lactose, maltose, salicin, sucrose, trehalose, or xylose,
autoclave at 121°C for only 3 minutes.
5. Aseptically each test tube were inoculated with the respective isolated
microorganism using an inoculating needle or loop and incubated for 24-36 hrs and
result were noted.

4.3.3 Primary Screening test

Actinomycetes isolated and identified from different soil samples were screened for
antibiotic production. The test bacteria used for primary screening will be Escherichia coli,
Staphylococcus, Pseudomonas and Klebsiella.. Plate containing Mueller Hinton agar were
streaked with isolated colonies of Actinomycetes in the center along the length and then,
fresh sub cultured test organisms were streaked perpendicular to the Actinomycetes

12
isolates. Then the plates were incubated at 37°C for 24 hours. After incubation the zone of
inhibition formed was observed and recorded.

4.4 Submerged Fermentation and Extraction of Crude

Antibiotics
Based on the results of primary screening each isolates of Actinomycetes were subjected to
submerged fermentation in Yeast Malt Extract Broth (YMEB) with continuous shaking for
about two weeks at 28°C. The extract was then separated by centrifugation at 3000 rpm for
20 minutes. The pellets was discarded and supernatant was mixed with double volume of
chilled acetone and left overnight. Then the mixture was centrifuged at 4000 rpm for 20
minutes. The crude extract of antibiotic was filtered through membrane filter and washed
with tris-phosphate buffer (pH 7.4).

4.5 Well agar diffusion method

Extract obtained from submerged fermentation were tested for antibiotic activity against
test organism by well agar diffusion method. The process was like this:

A loop full of bacterial strain was inoculated in 30 ml of Nutrient broth in a conical flask
and incubated for 72 hrs to get active strain by using agar well diffusion method. After that
Muller Hinton Agar was prepared and before its solidification (at temper 50°𝐶) 0.25 ml of
test strains were inoculated in the media separately. Care was taken to ensure proper
homogenization. The experiment was performed under strict aseptic conditions. After the
medium solidified, a well was made in the plates with sterile borer (5mm).The extract
compound (10 μl) was introduced into the well and plates were incubated at 37°C for 72
hrs. All samples were tested in triplicates. Microbial growth was determined by measuring
the diameter of zone of inhibition. A control with standard antibiotic was kept for all test
strains and the control activity was deducted from the test and results were recorded.

4.6 Data analysis and Statistical tools

All laboratory data were analysed using Ms Excel 2013 and Ms-Word 2013.

13
 CHAPTER-5

RESULTS

15 Actinomycetes colonies were isolated and tested for it’s identification from 5 different
soil sample of Biratnagar. None of the isolated species produce antibiotics with four of the
tested organism i.e. Escherichiae coli, Klebsiella, Staphylococcus and Pseudomonas.
Maximum number of Actinomycetes were isolated from alluvial soil. Gram staining,
Catalase test, MR test, Citrate test were positive whereas Oxidase, VP, Urease test were
negative for isolated Actinomycetes. Moreover regarding carbohydrate fermentation test
most of the species showed positive test with glucose, fructose, maltose, mannitol and
lactose.

Number of colonies

9%

14%
Alluvial
Loamy
Clay
56%
Sandy
21%

Figure 1: Pie-chart of distribution of Actinomycetes in different soil type.

14
Table 1: Distribution of Actinomycetes among soil type.

S.No. Soil source Locality/ward Soil type No. of isolated

number colonies

01 Garden area (S1) Brt 13 Alluvial 4

02 Green field (S2) Brt 13 Loamy 3

03 Dumping site (S3) Brt 05 Alluvial 4

04 Uncultivate dry land (S4) Brt 03 Clay 2

05 River basin (S5) Brt 01 sandy 2

Picture 1: SCA plate with Actinomycetes colony

15
Table 2: Morphological and cultural characteristics of isolated Actionomycetes

Isolate Sample Colony appearance Color of colony Mycelium Gram’s

code No. staining

01 S1 Powdery, radiating Greyish brown P +ve

02 S1 Powdery, radiating Whitish brown P +ve

03 S1 Raised, compact whitish P +ve

04 S1 Discrete Greyish P +ve

05 S2 Radiating Yellowish P +ve

06 S2 Radiating Greyish white P +ve

07 S2 Rugose Whitish P +ve

08 S2 Radiating Greyish white P +ve

09 S3 Discrete Brown greyish P +ve

10 S3 Discrete Greyish white P +ve

11 S3 Rugose Greyish P +ve

12 S4 Raised, radiating Whitish red P +ve

13 S4 Discrete Whitish brown P +ve

14 S5 Powdery Blackish white P +ve

15 S5 Powdery, radiating Whitish black P +ve

P= presence of mycelium, +ve = shows positive test

Nature, appearance of colony, presence of short mycelium and positive Gram’s staining
test resembles the characteristics of Actinomycetes.

16
Table 3: Biochemical characteristics of selected Actinomycetes isolates.

Isolates Catalase Oxidase Urease Citrate MR VP

01 +ve -ve -ve +ve +ve -ve

02 +ve -ve -ve +ve +ve -ve

03 +ve -ve -ve +ve +ve +ve

04 +ve -ve -ve -ve -ve -ve

05 +ve -ve -ve +ve +ve -ve

06 +ve -ve -ve +ve +ve -ve

07 +ve -ve -ve +ve +ve -ve

08 +ve -ve -ve +ve +ve -ve

09 +ve -ve -ve +ve -ve +ve

10 +ve -ve +ve +ve +ve -ve

11 +ve -ve -ve +ve +ve -ve

12 +ve -ve -ve +ve +ve -ve

13 +ve -ve -ve +ve +ve -ve

14 +ve -ve -ve -ve -ve -ve

15 +ve -ve -ve +ve +ve -ve

17
 BIOCHEMICAL TEST
16 15 15 15

14 13 13
12
12
Number of isolates

10

4 3
2 2
2
0 0 0
0
catalase Oxidase Urease Citrate MR VP
Biochemical test

positive negative

Figure 2: Biochemical test of isolated Actinomycetes.

Table 4: Carbohydrate fermentation by selected Actinomycetes isolates

Isolates Glucose Fructose Maltose Mannitol Lactose

01 + + + - -

02 + - - + +

03 + + + + +

04 - - + - -

05 + + + + +

06 + - - + +

18
07 + + + + +

08 + + + + +

09 + + + + +

10 + + + + -

11 + - - + -

12 + - + + +

13 + + + + +

14 + - + + +

15 + + - - -

Carbohydrate fermentation test

16
14
14
12
12
Number of isolates

11
10
10 9
8
6
6 5
4
4 3
2 1
0
Glucose Fructose Maltose Mannitol Lactose
carbohydrates

positive negative

Figure 3: Carbohydrate fermentation test of isolated Actinomycetes.

19
PRIMARY SCREENING TEST FOR ANTIBACTERIAL ACTIVITY
OF SELECTED Actinomycetes

None of the isolated Actinomycetes species gave +ve screening test against

1. Escherichia coli
2. Pseudomonas
3. Klebsiella
4. Staphyllococcus

EXTRACT OBTAINED FROM SUBMERGED

FERMENTATION USED IN WELL AGAR DIFFUSSION
METHOD
Although primary screening test result were not as expected and positive, for final
conformation fermentation was carried out and extract obtained were tested by well agar
diffusion method.

By this method also none of the isolated Actinomycetes colonies were found to be
antibiotics producing for the test bacteria.

20
Picture 2: Submerged fermentation and extract obtained after centrifugation

Picture 3: Well agar diffusion test with E. coli.

21
 DISCUSSION

Soil is inhabited by a large number of organisms. Many of soil microbes are useful as they
produce a number of bioactive metabolites, including clinically important antibiotics.
Actinomycetes are abundant in soil, species of Streptomyces in particular. Streptomycetes
represent the dominant Actinomycetes genera in soil and play an important role in recycling
of materials and production of bioactive compounds. A variety of media such as Glycerol-
Yeast extract agar, Starch casein agar, inorganic salts starch agar, Humic acid Vitamin
agar, Kuster’s agar, Yeast extract-malt extract agar, Oat meal agar, Actinomycetes isolation
agar and Glycerol aspargine agar are used for isolation of Actinomycetes. SCA agar is one
of the most commonly used media for isolation of Actinomycetes from a variety of samples
(Xu et al., 1996; Moncheva et al., 2000-2002).

The present study concentrated on isolating Actinomycetes from different places of

Biratnagar. A total of 5 soil samples were collected. A total of 15 Actinomycetes isolates
were recovered on SCA plate. The primary screening for antagonistic efficacy of isolated
Actinomycetes was done by using cross streak and dual inoculation technique. The primary
screening as well as well agar diffusion method revealed no antimicrobial activity of the
isolates. This might be because of the following reasons:

1. The Actinomycetes species isolated from soil sample of Biratnagar may not have
ability to produce antibiotic substances.
2. The amount of antibiotics product produced might be in less concentration to
inhibit the growth of the test organism.
3. The test organism may be resistant strains for the produced antibiotics extract.

Cross streak method is one of the widely used preliminarily screening methods to detect
the possible antagonistic potential of Actinomycetes isolates. This technique forms the basis
to delineate potent isolates for secondary screening procedures. The absence of growth or
a less dense growth of test organisms near the Actinomycetes isolates was considered
positive for production and secretion of antimicrobial metabolite by the isolates (Sahin and
Ugur, 2003).
22
As all the isolates did not performed well in primary screening for antibacterial activity,
for its conformation further the process was carried out performing submerged
fermentation and well agar diffusion method where also the positive result could not be
obtained.

In this study, all the isolates taken were characterized on the basis of cultural, microscopic,
staining, biochemical and carbohydrate fermentation characteristics. The colonies of
isolates exhibited appearance such as raised, powdery, rugose, radiating, compact and
discrete being characteristic of Actinomycetes. Although the colors of mycelia could not be
distinguished but presence could be easily observed. Most of the isolates belonged to grey
series. All isolates were Gram positive and nonacid-fast. The isolates were biochemically
versatile in terms of utilization of various sources. On the basis of cultural characteristics,
microscopic features together with biochemical characteristics, all 15 isolates were found
to resemble species of the genus Streptomyces. Results of Gram’s staining, biochemical
test and carbohydrate test are in agreement with other investigators (Kekuda et al. 2015;
Rai et.al 2016).

Morphology plays an important role in distinguishing Streptomyces from other sporing

Actinomycetes and in the characterization of Streptomycete species. Information on cultural
features and characteristic spore arrangement together with biochemical properties assists
classification of Actinomycetes as members of the genus Streptomyces (Cao et al., 2004;
Oskay et al., 2004).

In the present study, all the isolates were grown in YEMA broth and the culture filtrate was
extracted using solvents chilled acetone. The solvent extracts were screened for
antimicrobial activity by agar well diffusion assay. Solvents of different polarity such as
butanol, ethyl acetate, acetone, hexane, ethanol and methanol have been shown to extract
metabolites efficiently (Sahin and Ugur, 2003).

The distribution of Actinomycetes varied with the texture and cultivation status of the soil,
with the highest number found in sandy soil along the river banks (Rai et al., 2016) where
as our finding varies from this point as alluvial soil harbors highest number.

23
 CHAPTER-7

CONCLUSION

In the present study, the isolated Actinomycetes from different soil sample of Biratnagar
resembles Streptomyces species. As the isolates did not show antibacterial efficacies
against four of the tested organism. The Biratnagar Actinomycetes are not considered to be
promising resources for the development of therapeutic agent. Moreover alluvial soil
harbors more number of Actinomycetes in Biratnagar soil sample.

Further studies on isolation of Actinomycetes from soil sample of Biratnagar and their
antibacterial activities determination are to be carried out.

24
 REFERENCES

1. Oskay M, Tamer AU and Azeri C. 2004. Antibacterial activity of some

Actinomycetes isolated from farming soils of Turkey. African J Biotechnol.
3(9):441-446.
2. Kumar N, Singh RK and Mishra SK. 2010. Isolation and screening of soil
Actinomycetes as sources of antibiotics active against bacteria. International
Journal of Microbiology Research. 2:12-16.
3. Jeffrey LS. 2008. Isolation, characterization and identification of Actinomycetes
from agriculture soils at Semongok, Sarawak. African Journal of Biotechnology.
7:3697-3702.
4. Arifuzzaman M, Khatun MR and Rahman H. 2010. Isolation and screening of
Actinomycetes from sundarbans soil for antibacterial activity. African Journal of
Biotechnology. 9:4615-4619.
5. Alanis AJ. 2005. Resistance to antibiotics: are we in the post-antibiotic era?
Archives Med Res. 36:697-705.
6. Enright MC. 2003. The evolution of a resistant pathogen--the case of MRSA. Curr
Opinion Pharmacol. 3(5): 474-479.
7. Agadagba SK. 2014. Isolation of Actinomycetes from Soil. Journal of
Microbiology Research. 4(3):136-140.
8. Gebreyohannes G, Moges F, Sahile S and Raja N. 2013. Isolation and
Characterization of Potential Antibiotic Producing Actinomycetes from Water and
Sediments of Lake Tana, Ethiopia. Asian pacific Journal of Tropical Biomedicine.
3(6):426-435.
9. Rai M, Bhattarai N, Dhungel N and Mandal PK. 2016. Isolation of antibiotic
producing Actinomycetes from soil of Kathmandu valley and assessment of their
antimicrobial activities. International Journal of Microbiology and Allied Sciences.
2(4):22-26.
10. Manandhar S, and Sharma S. 2013. Practical approach to Microbiology (2nd ed.).
Kathmandu, Nepal: National book centre.

25
11. Waksman S.A, Schatz A. and Rynolds D.M. 2010. Production of antibiotic
substances by Actinomycetes. Annals of the new Yorkacademy of sciences
websites: https://doi.org/10.1111/j.1749-6632.2010.05861.
12. Galatenko, OA, Terekhova LP. Isolation of antibiotic-producing Actinomycetes
from soil samples exposed to UV light. 1990 Nov:35(11):6-8.
13. Hotam S. Chaudhary, JayprakashYadav,Anju R. Shrivastava,Smriti Singh, Anil K.
Singh, and NatrajanGopalan. Antibacterial activity of Actinomycetes isolated from
different soil samples of Sheopur .J Adv Pharm Technol Res. 2013 Apr-Jun; 4(2):
118–123. doi: 10.4103/2231-4040.111528
14. Grasso L.L, Martino D.C. and Alduina R. “Production of actibacterial compounds
from Actinomycetes” .Antibacteria – Basics and biotechnological applications,
Italy. Page 178- 192.
15. Kekuda, P.T.R., Onkarappa, R., Gautham, S.A., Shobha,K.S., and Raghavendra,
H.L. (2015). Antimicrobial, antioxidant and cytotoxic activityof Streptomyces
species from western ghat soils of Karnataka, India. Science, Technology and Art
Research Journal 4(2): 164-180.
16. Xu, L.H., Li, Q.R., Jiang, C.L. (1996). Diversity of Soil Actinomycetes in Yunnan,
China. Applied and Environmental Microbiology 62(1): 244-248
17. Moncheva, P., Tishkov, S., Dimitrova, N., Chipeva, V., Nikolova, S.A.,
Bogatzevska, N. (2000-2002). Characteristics of soil Actinomycetes from
Antarctica. Journal of Culture Collections 3: 3-14.
18. Sahin, N., Ugur, A. (2003). Investigation of the antimicrobial activity of some
Streptomyces isolates. Turkish Journal of Biology 27: 79-84.

26
Limitation of study

27
 Appendix 1

List of materials

A. Equipments

1. Autoclave
2. Electric heater
3. Laminar air flow
4. Incubator
5. Hot air oven
6. Microscope
7. pH meter
8. Refrigerator
9. Micropipette
10. Centrifuge

B. Glassware

1. Beaker
2. Conical flak
3. Glass rod
4. Glass slides
5. Petri plates
6. Pipette
7. Reagent bottles
8. Test tubes
9. A measuring cylinder

C. Miscellaneous

1. Inoculating loop
2. Inoculating needle

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 3. Immersion oil
4. Plastic bag
5. Plastic shield tube

D. Chemical and reagents

1. Ethanol
2. Crystal violet
3. Hydrogen peroxide
4. Methyl red
5. Saffranin
6. Gram’s iodine
7. Chilled acetone
8. Tris-phosphate buffer
9. Gram’s decolouriser

E. Microbiological media

1. Starch casein agar

2. Muller hinton agar
3. Nutrient agar
4. Yeast malt extract broth
5. Nutrirent broth
6. Centrimide agar
7. MSA media

F. Biochemical media

1. MR-VP Broth
2. Simmon’s Citrate Agar
3. Urease
4. SIM

29
 G. Others

1. Soil sample
2. Distilled water
3. Membrane filter of 90 mm diameter

Appendix-2

Composition of media

1. Nutrient agar

 Peptone (5gm)
 Sodium chloride (5gm)
 Meat extract B (1.5gm)
 Yeast extract ?(1.5gm)
 Agar (15gm)

2. Nutrient broth

 Peptic digest of animal tissue (5gm)

 Sodium chloride (5gm)
 Beef extract (1.5gm)
 Yeast extract (1.5gm)

3. Muller Hinton Agar

 Casein acid hydrolysate (17.5gm)

 Beef extract powder (2gm)
 Starch (1.5gm)
 Agar (17gm)

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4. Starch Casein Agar

 Soluble starch (10gm)

 Casein (0.3gm)
 KNO3 (2gm)
 NaCl (2gm)
 K2HPO4 (2gm)
 MgSO4.7H2O (0.05gm)
 FeSO4.7H2O (0.01gm)
 CaCO3 (0.02gm)
 Agar (18gm)
 Distilled water (1000ml)
 pH (7.0±0.1) at 25ºC

5. Phenol red carbohydrate broth

 Trypticase or proteose peptone No.3 (10 gm)

 Sodium Chloride (5 gm)
 Beef extract (1 gm)
 Phenol red (7.2 ml of 0.25% phenol red solution) (0.018 gm)

6. Centrimide agar

 Gelatin (20 gm)

 Potassium sulfate (10 gm)
 Magnesium chloride (1.4 gm)
 Centrimide base (0.3 gm)
 Glycerine (10 ml)
 Agar (13.6 gm )
 Distilled water (1000 ml)

31
Cultural characteristics of selected actinomycetes

32