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Journal of Biology and Medical Sciences - JBMS

An Open Access International Journal published by University of Babylon, Iraq
Vol.1 2013

Isolation and Identification of Streptomyces from Different

Sample of Soils
Ali. Al-Saadi, NooraMajidHameed, Eman Mohammad Jaralla
University of Babylon, College of Science
[email protected]

Abstract
A total of 36 actinomycetes were isolated and purified from soil samples collected from agricultural soils in Hilla.
The isolates were morphologically distinct on the basis of spore mass color, reverse slide color, aerial and
substrate mycelia formation and production of diffusible pigment. Only two isolates which were S.A.2 and
S.S.10 was selected for further investigation due to its strong antibacterial activity against six pathogenic bacteria
which were (Staphylococcus aureus, Escherichia coli, Pseudomonas aeroginosa,Serratiamarcescens, Klebsiella
pneumonia, Aeromonashydrophila).These two isolates was identified asStreptomycesorientalisand Streptomyces
humidus respectively based on its morphological, cultural, physiological, microscopic features, utilization of
carbon sources, biochemical characteristics and molecular analysis of the 16S rRNA gene primers.
Keywords :actinomycetes, spore mass color, 16S rRNA gene primers.

Introduction
Actinomycetes are a group of prokaryotic organisms phylogenetically grouped as gram-positive bacteria with
high guanine + cytosine in their DNA. Most of them are in subclass Actinobacteridae, order actinomycetales
comprising of 14 suborders, 49 families, and over 140 genera (Adegboye and Babalola, 2012).They are
filamentous bacteria which produce two kinds of branching mycelium, aerial mycelium and substrate mycelium.
Actinomycetes constitute a significant component of the microbial population in most soils and Streptomyces a
count for 90% of the total Actinomycetes population (Poopal and Laxman, 2009). They produce a wide range of
secondary metabolites and more than 70% of the naturally derived antibiotics that are currently in clinical use
are derived from soil actinomycetes (Elardoet al., 2009).In particular, the genus Streptomyces accounts for
about 80% of the actinomycete natural products reported to date (Bull and Stach, 2007). The genus
Streptomyces was proposed by Waksman &Henrici for aerobic and spore formingActinomycetes (Williams et al.
1989).They are well known by a linear chromosome, complex morphological differentiation(El-Gendyet al.,
2008b). The most interesting property of Streptomyces is the ability to produce bioactive secondary metabolites
such as antibacterials, antifungals, antivirals, antitumoral, anti-hypertensives and mainly antibiotics and
immunosuppressives (Patzer and Volkmar, 2010). Many species belonging to the genus Streptomyces are well
known as biocontrol agents that inhibit or lyse several soil borne and air borne plant pathogenic fungi (Sousa et
al., 2008).

Materials and methods

Samples collection
Agricultural soil samples was collected from diffirent sites in Hilla at various depth of surface, ranging from
layers of 15 to 20 cm depth. The samples were collected in sterile small plastic containers by using a trowel and
properly labeled indicating the date of collection and the depth and transferred to the laboratory for the study.
Isolation of Streptomyces from soil
Soil samples were mixed thoroughly and passed through 2 mm sieve filter to remove gravel and debris. The
samples were kept at 55 °C for 5 min, for pre - treatment. In conventional dilution plate technique, 1 gm of
soil sample was suspended in 9 ml of sterile water and successive dilution was made upto 10-4. An aliquot (0.5
ml) of suspension from the last dilution test tube was spread on yeast-malt extract agar medium (ISP-2 according
to (Pridhamet al., 1957)and incubated for 7-9 days at 28-30°C. After incubation period, the plates were
examined for typical colonies of Streptomyces. The typical round, small, opaque, compact, frequently pigmented
colonies were examined under a light microscope (100X). The colonies that bear typical Streptomyces
morphology were purified and sub-cultured on Yeast extract-Malt extract agar plates and stored for further assay
(Bernard, 2007).
Initial screening of the pure isolates
Preliminary screening for inhibitory metaboliteproducing ability of the isolate was tested by Cross streakmethod
against one Gram positive bacteria(Staphylococcus aureus), five Gram negative bacteria (Escherichia coli,
Pseudomonas aeroginosa, Serratiamarcescens, Klebsiellapneumoniae, andAeromonashydrophila). The isolate
was inoculated as a single streak in the centre of the petridish containing Mueller Hinton medium and incubated
at 28°C for 3-4 days to permit growth and antibiotic production. Later the test bacteria were inoculated by
streaking perpendicular to the growth of isolate. The plates were incubated for 24-48 hours at 37°C. After

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Journal of Biology and Medical Sciences - JBMS
An Open Access International Journal published by University of Babylon, Iraq
Vol.1 2013

incubation, inhibition of test bacteria around the growth of isolate was taken as positive for inhibitory activity
(Nanjwadeet al., 2010 ).

Secondary screening
The two active isolates were inoculated on fermentation broth medium according to (Ahmed, 2007), after
incubation for 10-15 days at 28°C, the cultures were filtered by Waksman No.1 filter and the antimicrobial agent
extracted using organic solvent Ethyl acetate (V:V), then tested for its inhibitory activity by agar well diffusion
method (Atta, 2010).

Identification of S.A.2 and S.S.10 isolates:

The isolates S.A.2 and S.S.10 was further characterized based on morphological, biochemical, cultural,
physiological features and microscopic characterization. Cultural characteristics were tested in yeast extract-malt
extract agar (ISP-2), oatmeal agar (ISP-3according to (Kuster, 1959a), inorganic salt-starch agar (ISP-4
according to (Kuster, 1959a.), glycerol-asparagine agar (ISP-5according to(Pridham and Lyons, 1961), peptone-
yeast-extractiron agar (ISP-6 according to (Tresner and Danga, 1958), tyrosine agar (ISP-7 according to
(Shinobu, 1958) and carbone utilization medium (ISP-9 according to (Modified from Pridham and Gottlieb,
1948).Biochemical tests including (starch and gelatin hydrolysis, voges-proskauer, citrate utilization, Indole,
methyl red, oxidase,H2S production and blood hemolysis according to (MacFaddin, 2000), lecithinase
production according to (Janda and Bottone, 1981), catalase according to (Colleeet al., 1996).. In addition to
genomic DNA extraction according to method recommended by SolGnet kit for DNA extraction and molecular
amplification of 16S rRNA gene primersF, (5'ACGTGTGCAGCCCAAGACA3) and R,(5'-
ACAAGCCCTGGAAACGGGGT-3) (Atta et al., 2011).

Results and Discussion

Antimicrobial activity assay
The two active isolates showed inhibitory activity against six test pathogenic bacteria as shown in table (1 and 2)
and which were chosen for further investigation. S.A.2 isolate showed inhibitory activity against
(Staphylococcus aureus, Escherichia coli, Pseudomonas aeroginosae and Klepsiellapneumoniae) but negative
for (Serratiamarcescens and Aeromonashydrophila), while S.S.10 isolate showed inhibitory activity against all
test bacteria but with less ability to inhibit growth of test pathogenic bacteria than S.A.2 isolate.

Table (1) Primary screening of Streptomyces isolates for antimicrobial activity

Test bacteria Results
S.A.2 S.S.10
Staph. aureus + +
E. coli ++ +
Pseudo.aeroginosae + +
Serr. marcescens - +
Kleb. pneumonia + +
Aero. hydrophila - +

Table (2) Inhibition zone of culturefiltrate extract of the two isolatesagainst test organisms on
MuellerHinton agar medium measured bymm., (R: Resistant).
Test bacteria Results
S.A.2 S.S.10
Staph. aureus 18 9
E. coli 20 9
Pseud. aeroginosae 15 7
Serr. marcsesenc R 13
Kle. pneumonia 8 14
Aer. hydrophila R 11

Identification of Actinomycete isolates

Morphology, as previously mentioned, has alwaysbeen an important characteristic used to identifyactinomycete
strains, and, in fact, it was theonly characteristic used in many early descriptions,particularly of Streptomyces
species in the first feweditions of Bergey's manual. Morphological observationsare best made on a variety of
standard cultivationmedia. Several of the media suggested forthe International Streptomyces Project(Shirlingand
Gottlieb, 1966) and by(Pridhamet al., 1956) have proven to be useful in ourhands for the characterization of

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Journal of Biology and Medical Sciences - JBMS
An Open Access International Journal published by University of Babylon, Iraq
Vol.1 2013

strains accessionedinto the Actinomycetales Culture Collection.Most clinical microbiologylaboratories offer

presumptive identification of Streptomycesspecies, ie, long filamentous gram-positive bacteria thatgrow
aerobically and are negative for partial acid-fast stain.These features distinguish Streptomyces species from
othermorphologically similar genera within Actinomycetales, ie,anaerobic Actinomycetes, and aerobic
NocardiaandRhodococcusthat are usually partially acid-fast. The actinomycete isolates was identified on the
basis of microscopic, cultural, biochemical and molecular characteristics.Scanning electron microscopy can
providefar more detailed information concerning the sporulationmicromorphology of actinomycetes,
particularlythose whose spore structures are associatedwith the vegetative mycelium. Both of two isolates spore
chain arrangement was examined under light microscope and have spore chains fexibiles and smooth spore
surface under scanning electron microscope.

Figure (1 ) Mycelium morphology of (A) S.A.2 isolate and (B) S.S.10 isolate

Figure (2 ) Spores morphology for (A) S.A.2 isolate and (B) S.S.10 isolate
The colonies was white and Grey on ISP-3 medium respectively, with no melanin production and no diffusible
pigments in ISP-6 and ISP-7. Both of isolates could utilize Arabinose, Fructose, Xylose and Mannitole but
negative forRaffinose, Sucrose and cellulose, In case of S.A.2 could utilize Inositol while S.S.10 negative for
Inositol.

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Journal of Biology and Medical Sciences - JBMS
An Open Access International Journal published by University of Babylon, Iraq
Vol.1 2013

Table (3) Morphological and physiological characteristics of Streptomyces isolates on ISP media
ISP media S.A.2 S.S.10
Aerial Substrate Aerial Substrate
mycelium mycelium mycelium mycelium
ISP - 2 / Yeast – Malt extract agar White Yellow White Yellow
ISP - 3 / Oatmel agar White White Gray Yellow
ISP - 4 / Inorganic salts agar Gray Gray Gray yellow
ISP - 5 / Glycerol Asparagine agar Yellow Yellow Yellow Yellow
ISP - 6 / Peptone-yeast extract iron agar - -
(melanin production)
ISP – 7/ Tyrosine agar (melanin White / - White / - White / - White / -
production)
ISP – 9/ Utilization of Carbone sources
Positive control (D-Glucose) + +
Negative control (no Carbone source) - -
Arabinose + +
Raffinose - -
Fructose + +
Sucrose - -
Xylose + +
Mannitole + +
Cellulose - -
Inositole + -
Diffusible pigments - -
The two isolates was positive for Voges-proskauer, citrate, H2S and starch hydrolysis but negative for methyl
red, indole and catalase, first isolate was positive for Gelatine liquefaction and oxidase while the second isolate
was negative, blood hemolysis test was positive in second isolate but negative in first isolate. Theseresults
emphasized that the actinomycetes isolate relatedto a group of Streptomyces (Williams, 1989 and Hensyl,1994).
In view of all the previously recorded data of the two isolates, it could be stated that the isolates (S.A.2 and
S.S.10) belonging to Streptomycesorientalisthat agreed with(Antonova et al., 2005)andStreptomyces
humidusrespectively (Kuster, 1972). Regarding the spore chain type of Streptomyces humidus, (Pridham and
Tresner, 1975) apportioned itto the Spiralesbut later, in the description only referred to it as atypical Retinaculum
Apertum. (Pridham and Tresner, 1975) also apportioned the spore mass of this actinomycete to the Grey colour
series. (Hnamuraet al.,1956 ; Waksman, 1961 and Gauze et al.,1983) regarded S. humidusto be closely related
toS. hygroscopicusand couldProducedihydrostreptomycin, humidin and cobalamines (Hnamuraet al. 1956 and
Pridham and Tresner, 1975).

Table (4) Biochemical tests for Streptomyces isolates

Test S.A.2 S.S.10

Gram stain + +
Indole - -
Methyl red - -
VogesProskauer + +
Citrate Utilization + +
Lecithinase + -
Starch Hydrolysis + +
Gelatineliquifaction + -
Catalase - -
Blood Hemolysis - +Alpha
H2S Production Acidic – Acidic No gas No H2S Acidic – acidic
No gas No H2S
Oxidase + -

This result was supported by molecular amplification of 16S rRNA gene primers thatshowed 1.5kbp fragments
for both isolates which indicates that the two isolates belonged to the genus Streptomyces.

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Journal of Biology and Medical Sciences - JBMS
An Open Access International Journal published by University of Babylon, Iraq
Vol.1 2013

DNA ladder S.A.2 S.S.10 S.A.2 S.S.10

Figure (3) Amplification of 16S rRNA gene primers for the two isolates

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