International Journal of Chemical Studies 2019; 7(3): 3282-3288
P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2019; 7(3): 3282-3288
© 2019 IJCS
Received: 15-03-2019
Accepted: 20-04-2019
Yog Raj
1. Department of Microbiology
Himachal Institute of Life
Sciences, Paonta Sahib,
Himachal Pradesh, India
2. Agrotechnology of Medicinal,
Aromatic and Commercially
Important Plants Division,
CSIR-IHBT, Palampur,
Himachal Pradesh, India
V Singh
Department of Microbiology
Himachal Institute of Life
Sciences, Paonta Sahib,
Himachal Pradesh, India
Arti Chauhan
Department of Microbiology
Himachal Institute of Life
Sciences, Paonta Sahib,
Himachal Pradesh, India
Correspondence
Yog Raj
1. Department of Microbiology
Himachal Institute of Life
Sciences, Paonta Sahib,
Himachal Pradesh, India
2. Agrotechnology of Medicinal,
Aromatic and Commercially
Important Plants Division,
CSIR-IHBT, Palampur,
Himachal Pradesh, India
Isolation, characterization and screening of novel
antibiotic producing bacteria from natural
habitats of Western Himalayas and industrial
waste soil samples
Yog Raj, V Singh and Arti Chauhan
Abstract
Antibiotics, potential against multi-drug resistant (MDR) pathogenic bacteria is currently become a vital
research area. The present research approach is focused on the isolation of antibiotic producing bacteria
from the soil samples collected from several diverse habitats viz. preserved areas of Himalayas, natural
water resources, rhizosphere of medicinal plants, agricultural soil, forest soils, industrial waste soil from
district Kullu and Poanta Sahib (HP). The bacterial isolates were characterized on the basis of their
morphology, further confirmed by biochemical and haemolytic activity tests. Antibiotic producing ability
of isolates was confirmed by zone of inhibition around bacterial colonies. A total of 30 isolates belonging
to six different genera i.e. Micrococcus, Bacillus, Lactococcus, Pseudomonas, E.coli and Proteus,
antibiotic producing bacteria were found in different soil samples. Out of these, 12 potential antibiotic
producing strains showed their inhibitory response against pathogenic microorganisms. These results
suggested that after proper standardization and processing, soil isolates having antibiotic producing
capability can be used commercially.
Keywords: Antibiotics, MDR, haemolytic activity, rhizosphere, zone of inhibition
Introduction
The word “antibiotic” is derived from Greek term antibiosis which means “against life”.
Antibiotics are medicine that kill or prevent the growth of bacteria, used to cure diseases. More
than 4000 antibiotics have been isolated, but only 50 have achieved wide usage while rest of
the other antibiotic compounds being toxic to humans and animals, high production costs
failed to attain commercial importance [1]. Antibiotics are natural drugs produced by several
fungi or bacteria unlike chemotherapeutic drugs which are manmade substances. Antibiotics
have long been considered the “magic bullet” that would end infectious disease.
Antimicrobial drugs are generally classified into two categories; one includes the synthetic
drugs, such as sulfonamides and quinolones, the second antibiotics synthesized by
microorganisms. Antibiotics, potential against multi-drug resistant (MDR) pathogenic bacteria
is currently become a vital research area. Natural bioresources having novel structures have
been observed to retain valuable biological activities. Producers of antibiotic can be found in
rivers, lakes, decaying plants and animal remains etc. but majority of microorganisms that
produce antibiotic inhabits soil. Many species of Bacillus are of considerable importance due
to antibiotic producing abilities [2]. Bacillus sp. produces diverse range of antibiotics such as
bacitracin and gramicidin which is produce by Bacillus licheniformis and Bacillus brevis
respectively. These antibiotics are active against many gram positive organisms such as
Stapylococci, Streptococci, anaerobic cocci, Cornyebacter and Clostridia but not against most
other gram negative bacteria [3]. Nowadays, much attention has been given to the increase in
antibiotic resistance as several microbial species and strains become resistant, many diseases
have become difficult to treat. Research in finding newer antibiotics and enhancing
productivity of such agents has become an essential action [4, 5]. The vast majority of novel
antibiotics have been detected by screening of “wild isolates” acquired from soil and other
natural habitats.
The present study directed the isolation and screening of potential antibiotic producing bacteria
from soil, optimization and characterisation of antibiotic producing strain, production of
antibiotics by recovered strain, determining the prevalence of recovered isolates and to check
~ 3282 ~
International Journal of Chemical Studies
antimicrobial activity of recovered isolates against some
pathogenic bacteria viz. Pseudomonas (2488), Proteus (425),
Streptococcus (655), Bacillus cereus (1305) and
Staphylococcus (96).
Material and Methods
The present study was carried out in the Department of
Microbiology, Himachal Institute of Life Sciences, Poanta
Sahib (HP).
Collection of Soil Sample
In systematic screening for isolation of antibiotic producing
bacteria a total 22 soil samples were collected from several
diverse habitats viz.preserved areas of Himalayas, Natural
water resources, rhizosphere of medicinal plants, agricultural
soil, forest soils,industrial waste soil from district Kullu and
Poanta Sahib (HP). The samples were taken after removing
approximately 3 cm of the soil surface, deep up to 20 cm as
most of the bacterial population is concentrated on upper
layer of the soil. Soil samples were collected with sterile
spatula in clean dry and sterile polythene bags, transferred to
the Department of Microbiology, HILS at Poanta Sahib and
kept at 4 0C for further processing.
Processing of Samples
Soil samples (50g each) were dissolved in 100 ml of sterile
distilled water to make soil suspensions and after that with the
help of Whatman filter paper obtained almost clear solution
devoid of soil, stones and debris. Then filtrated solution was
serially diluted to ten folds.
Isolation and Sub Culturing of Bacteria
The nutrient agar medium was used for the isolation of
bacteria; 28g of nutrient agar powder dissolved in 1000 ml of
distilled water, sterilized in autoclave for 15 min. at 121 0C
after cooling poured in Petri dishes and allowed to solidify.
Portions of the soil suspension (100 μl) inoculated on the
nutrient agar media by spread plate method were incubated at
370C for 24h. Bacterial colonies with clear zone were
observed and further sub cultured into nutrient agar slants
using a sterile wire loop, incubated at 37 0C for 24h for
preservation and purify the isolates. These slant bottles
containing the bacterial isolates were kept in refrigerator at 4
0
C for short time storage prior biochemical tests for
identification.
Identification of Bacterial Strains
The selected strains were identified by means of physical,
biochemical and haemolytic characterisation.
Physical Characterization
Colony Morphology
Shape, size, colour, elevation, margin of colony and
appearance were observed in overnight plate culture and
noted down.
Gram Staining
Thin smear of bacterial culture was prepared and then heat
fixed. Few drops of Crystal Violet were added on the smear
for 60 sec. and removed with distilled water. The smear was
then covered with Gram’s iodine solution for 60 sec. and then
the smear was treated with alcohol followed by counterstained
with safranin dye to the smears for 30 sec. Slide was then
washed with distilled water and let it air dry, observed under
~ 3283 ~
light microscope under 100x oil emersion objective to observe
the colour, shape, size and arrangement of bacterial cells.
Biochemical Characterization
The various biochemical tests viz., Indole, Methyle Red,
Voges Proskaur, Citrate utilization testes were carried out for
identification of isolates. Cappuccino Sherman’s Manual of
Microbiology, 7th Ed [6].
IMVIC Test
Indole Test
Bacterial colony was inoculated in 1% tryptophan broth in a
test tube. After incubation period of 48 h at 37 0C, one
millilitre (1ml) of chloroform was added to the broth. The test
tube was shaken gently, then 2.1 ml of Kovac’s reagent were
added and this was also shaken gently and allowed to stand
for 20 min. The formation of red colouration at the top layer
indicated positive and yellow colouration indicates negative.
Methyl Red
The isolates were inoculated in MR-VP medium and
incubated at 37 0C for 24 h. Few drops of methyl red reagent
were added after incubation of 24 h. The presence of red
colour indicated positive test.
Voges Proskauer Test
The isolates were inoculated in MR-VP medium and
incubated at 370C for 24 h. After 24 h, 3 drops of 40% KOH
and 9 drops of alpha-naphthol reagent were added.
Appearance of red colour showed positive test.
Citrate Utilization Test
Isolates were streaked on Simmon citrate slants and incubated
at 37 0C for 24 h, appearance of blue colour indicated for
positive test.
Haemolytic Activity/ Blood Agar Culture
For the identification of pathogenic and non-pathogenic
isolates, growth of bacteria was studied on blood agar by
preparing sterile plates of blood agar media, adding 10% of
blood in media. Subsequently streak isolated strains on the
plates. Incubate the plates for overnight at 370C and study the
growth of bacteria on plates.
Screening for Anti-Microbial Activity
In present investigation of antibiotic production was studied
under different time periods i.e., 1 to 20 days of incubation.
Screening are based on the using the broth cultures of
antibiotic producing strains in agar well diffusion method and
their supernatants separately in case of disc diffusion method.
Agar Well Diffusion Method
Prepare 5 test tubes, each containing 5 ml of MHB (Muller
Hinton Broth). After autoclaving, pathogenic strains were
inoculated and kept in incubator at 370C for 24 h.
Subsequently the recovered isolates were inoculated in NB
(Nutrient Broth) after autoclaving. It was kept on shaker for
48 h. Spread100l of pathogenic strain on MHA (Muller
Hinton Agar) plates with the help of sterile glass spreader.
Then bore wells of 6 mm diameter on the MHA plates, pour
the isolates in to the wells with the help of micropipette. The
plates were kept for 48 h incubation period after that
inhibition zones were observed.
International Journal of Chemical Studies
Disc Diffusion Method
Recovered isolates were inoculated in 50 ml nutrient broth
and was kept in a rotatory shaker at 37 0C for 48 h. After 48 h
decant the 5 ml of broth culture into eppendorf test tubes and
centrifuged at 5000 rpm for 10 min. at 4 0C.The 5 pathogenic
strains were grown in MHA having 100μl of inoculum. Small
discs were punched out from filter paper and were spreaded
on the MHA plates. The filter discs were dipped in the
centrifuged broth culture in eppendorf tube for 10 to 15
minutes prior to the placement of discs on MHA plates. The
plates were kept for incubation of 48 h. Zone of inhibitions
were observed around the filter paper.
Result and Discussion
Isolated Microorganism from Soil Sample
In the course of screening of novel antibiotic producing
bacteria from soil samples, many antibiotic producing cultures
were recovered from soil sample taken from Poanta sahib and
district Kullu (HP). In the present study, total 30
phenotypically different strainspossessing capability to
producing antibiotics were isolated on NA medium (Nutrient
Agar)from 22 soil samples, showingcolonies with clear zone
after incubationperiod of 24 hat 37 0C. Colonies which showed
clear zone were preserved on NA slants for further use. In a
study [7] a total 56 isolates having potential to produce
antibiotic were recovered from 25 soil samples collected from
different areas of Lahore. In earlier study [8] reported 10
bacterial isolated from different location within Birnin Kebbi
metropolis from which only 5 (50%) of isolates were active.
Characterization of Selected Isolates
Recovered 30 isolates were characterized and identified by
their physical characterization, possessing white, yellow and
mucoid colony having rod and cocci shape (Table1),
morphological examinations of isolates are much similar to
earlier study [9]. Gram staining of recovered isolates showed
12 strains gram negative for reaction showing pink colour and
rest 18were found gram positiveshowing blue or purple colour
under light microscopy (Fig.1 and Table 2). Biochemical
characterisation coupled with Bergery’s Manual of
Determinative Bacteriology [10] conferred the presence of
bacteria probably to the genera Micrococcus, Bacillus,
Lactococcus, Pseudomonas, E.coli and Proteus (Fig. 2a-c and
Table 3).
Haemolytic test showed that out of 30 isolates, 12 were non-
pathogenic i.e. they showed  haemolysis, while the other 18
were pathogenic i.e. they showed  and  haemolysis (Fig.3).
Screening for Antibacterial Activity
Results are based on the screening of antimicrobial activity
using the cultures of antibiotic producing strains in agar well
diffusion method and their supernatants separately in case of
disc diffusion method. It was found that maximum zone of
inhibition showed by recovered isolatesafter an incubation
period of 7 days.
Agar Well Diffusion Method
Antibiotics produced by the isolates showed variable zones of
Table 1: Morphological Characterization of Recovered Isolates
Sr. No. Characters
1 Colour White, Yellow, creamish and mucoid with clear zone
2 Colony size
3 Shape
~ 3284 ~
inhibition against five bacterial pathogens after 48 h of
incubation period. Out of 30 strains 5 potential antibiotic
producing strains (APS-3, 9, 12, 15, 17) showed zone of
inhibition 14, 20, 16, 29 and 10 mm respectively against
pathogenic strain Pseudomonas (2488). In case of
Streptoococcus (655),7 strains (APS-1, 3, 4, 6, 7, 12, 17)
showed clear zone of inhibition of 16, 21, 17, 12, 17, 14, 10
mm for Bacillus and Staphylococcus 6 strains (APS-1, 3, 4, 7,
15, 17) and only 3 strains (APS-3, 4, 6) showed clear zone of
inhibition of 18, 12, 12, 10, 11, 15 mm and 20, 10, 22 mm
respectively while there were no zone of inhibition observed
against Proteus (425) (Fig. 4a-f and Table 4). In previous
report [11] discovered the production of bacitracin and subtillin
by Bacillus sp. Similarly [12] investigated that bacitracin
produced by Bacillus sp. inhibits Escherichia coli and
Staphylococcus aureus. Whereas [8] reported isolates Bacillus
alvei, Enterobacter aerogene, Micrococcus roseus showed
inhibitory response towards S. aureus, Pseudomonas sp. and
Shigella sp. with clear zone of inhibition 15, 17 and 12 mm
respectively.
Disc Diffusion Method
In this experiment, small disc were punched out from a sterile
filter paper and dipped in the centrifuged broth culture of
recovered isolates in 5ml eppendorf test tube for 10 to 15 min.
then placed on MHA plates for observation of clear zone after
48 h of incubation. Result revealed that APS-12, 17 showed
inhibitory activity againstpathogenic strain Pseudomonas
(2488) having zone 10 and12 mm. APS-1, 3, 12 showed
inhibition zone of 14, 15, 10 mm against Streptococcus (655).
For Bacillus and Staphylococcus APS-5 and APS-3, 6 showed
zone of 14 mm and 10, 17 mm respectively (Fig. 5 and Table
5). In other study [13] investigated, two isolates 5 and 6
inhibited the growth of S. aureus and P. vulgris with zone
having diameter of 11 and 12 mm respectively.
Conclusion
The soil microbial isolate were showing antibiotic activity
under normal growth conditions was found inhibiting
pathogenic strains of the gram positive bacilli and cocci. The
present data focused on obtaining local microbial isolates
which have the ability to produce antimicrobial agent.
Exposing potential antibiotic producing strains with
competing organisms, its production can be enhanced. Based
on the above experimental study, we reached to the following
conclusions that the strain APS-15 was found to be having
better antimicrobial activity against Pseudomonas in
comparison with other soil isolates, which have been
investigated. APS-12 and APS-17 showed minimum zone of
inhibition against Staphylococcus. Proteus showed resistance
to all isolates. It is concluded that Bacillus species and
Micrococcus sp. isolated during the course of this research
from soil samples have potential of produce antibiotics. These
results suggested that after proper standardization and
processing, soil isolates having antibiotic producing capability
can be used commercially.
Morphology
Medium
Rod and cocci shape
International Journal of Chemical Studies

Table 2: Gram Staining Characterization of Recovered Isolates Table 4: Antimicrobial Activity of Isolates by Agar Well Diffusion
Method
Strain Colour and Morphology Type and Bacteria
APS-1 Pink and Micrococci Gram Negative Zone of inhibition in mm
APS-2 Blue and Micrococci Gram Positive Bacillus
Isolates Pseudomonas Proteus Streptococcus Staphylococcus
cereus
APS-3 Pink and Bacilli Gram Negative (2488) (425) (655) (96)
(1305)
APS-4 Blue and Micrococci Gram Positive APS-1 N.Z N.Z 16 18 N.Z
APS-5 Pink and Bacilli Gram Negative APS-2 N.Z N.Z 6 N.Z N.Z
APS-6 Pink and Bacilli Gram Negative APS-3 14 N.Z 21 12 20
APS-7 Blue and staphylococci Gram Positive APS-4 N.Z N.Z 17 12 10
APS-8 Blue and staphylococci Gram Positive APS-5 N.Z N.Z N.Z 8 8
APS-9 Blue and Micrococci Gram Positive APS-6 N.Z N.Z 12 9 22
APS-10 Blue and staphylococci Gram Positive APS-7 N.Z N.Z 17 10 N.Z
APS-8 N.Z N.Z 9 N.Z N.Z
APS-11 Blue and staphylococci Gram Positive
APS-9 20 N.Z N.Z 5 N.Z
APS-12 Blue and staphylococci Gram Positive APS-10 N.Z N.Z N.Z N.Z N.Z
APS-13 Pink and staphylococci Gram Negative APS-11 N.Z N.Z N.Z N.Z N.Z
APS-14 Blue and Bacilli Gram Positive APS-12 16 N.Z 14 N.Z 3
APS-15 Blue and staphylococci Gram Positive APS-13 N.Z N.Z N.Z N.Z N.Z
APS-16 Blue and Bacilli Gram Positive APS-14 N.Z N.Z N.Z N.Z N.Z
APS-17 Blue and staphylococci Gram Positive APS-15 29 N.Z 9 11 N.Z
APS-18 Blue and Micrococci Gram Positive APS-16 N.Z N.Z N.Z N.Z N.Z
APS-19 Blue and Micrococci Gram Positive APS-17 10 N.Z 10 15 3
APS-18 N.Z N.Z N.Z N.Z N.Z
APS-20 Pink and staphylococci Gram Negative
APS-19 N.Z N.Z N.Z N.Z N.Z
APS-21 Blue and Bacilli Gram Positive APS-20 N.Z N.Z N.Z N.Z N.Z
APS-22 Blue and Bacilli Gram Positive APS-21 N.Z N.Z N.Z N.Z N.Z
APS-23 Pink and Micrococci Gram Negative APS-22 N.Z N.Z N.Z N.Z N.Z
APS-24 Pink and staphylobacilli Gram Negative APS-23 N.Z N.Z N.Z N.Z N.Z
APS-25 Blue and staphylobacilli Gram Positive APS-24 N.Z N.Z N.Z N.Z N.Z
APS-26 Pink and Micrococci Gram Negative APS-25 N.Z N.Z N.Z N.Z N.Z
APS-27 Pink and staphylobacilli Gram Negative APS-26 N.Z N.Z N.Z N.Z N.Z
APS-27 N.Z N.Z N.Z N.Z N.Z
APS-28 Blue and Micrococci Gram Positive
APS-28 N.Z N.Z N.Z N.Z N.Z
APS-29 Pink and staphylobacilli Gram Negative APS-29 N.Z N.Z N.Z N.Z N.Z
APS-30 Pink and Bacilli Gram Negative APS-30 N.Z N.Z N.Z N.Z N.Z
N.Z-No Zone
Table 3: Biochemical Characterization of Isolates
Table 5: Antimicrobial Activity of Isolates by Disc Diffusion
Isolates Indole MR VP Citrate Utilization Probable identified Species
Method
APS-1 - + - + Micrococcus roseus
APS-2 - - - - Bacillus/ Micrococcus luteus Zone of inhibition in mm
APS-3 - + - - Lactococcus lactis Pseudomonas Proteus Streptococcus Bacillus cereus Staphylococcus
Isolates
APS-4 - - - - Bacillus/ Micrococcus luteus (2488) (425) (655) (1305) (96)
APS-1 N.Z N.Z 14 8 N.Z
APS-5 - + - - Lactococcus lactis
APS-2 N.Z N.Z 6 N.Z N.Z
APS-6 - + - + Micrococcus roseus APS-3 N.Z N.Z 15 N.Z 10
APS-7 - - - - Bacillus/ Micrococcus luteus APS-4 N.Z N.Z N.Z N.Z N.Z
APS-8 - + - - Bacillus/ Micrococcus luteus APS-5 N.Z N.Z N.Z 14 N.Z
APS-9 - - - - Bacillus/ Micrococcus luteus APS-6 N.Z N.Z N.Z N.Z 17
APS-10 - + - - Lactococcus lactis APS-7 N.Z N.Z 6 9 N.Z
APS-11 - - - - Bacillus/ Micrococcus luteus APS-8 N.Z N.Z 6 N.Z N.Z
APS-12 - + - + Micrococcus roseus APS-9 N.Z N.Z N.Z N.Z N.Z
APS-13 - - - + Pseudomonas aeruginosa APS-10 N.Z N.Z N.Z N.Z N.Z
APS-11 N.Z N.Z N.Z N.Z N.Z
APS-14 - - - + Pseudomonas aeruginosa
APS-12 10 N.Z 10 8 N.Z
APS-15 - - - - Bacillus/ Micrococcus luteus APS-13 N.Z N.Z N.Z N.Z N.Z
APS-16 + + - - E.coli/Proteus vulgaris APS-14 N.Z N.Z N.Z N.Z N.Z
APS-17 - + - - Lactococcus lactis APS-15 5 N.Z 8 N.Z N.Z
APS-18 - - - + Pseudomonas aeruginosa APS-16 N.Z N.Z N.Z N.Z N.Z
APS-19 - + - - Lactococcus lactis APS-17 12 N.Z N.Z N.Z N.Z
APS-20 - - - - Bacillus/ Micrococcus luteus APS-18 N.Z N.Z N.Z N.Z N.Z
APS-21 - - - - Bacillus/ Micrococcus luteus APS-19 N.Z N.Z N.Z N.Z N.Z
APS-20 N.Z N.Z N.Z N.Z N.Z
APS-22 - - - - Bacillus/ Micrococcus luteus
APS-21 N.Z N.Z N.Z N.Z N.Z
APS-23 - - - - Bacillus/ Micrococcus luteus APS-22 N.Z N.Z N.Z N.Z N.Z
APS-24 - + - + Micrococcus roseus APS-23 N.Z N.Z N.Z N.Z N.Z
APS-25 - + - - Lactococcus lactis APS-24 N.Z N.Z N.Z N.Z N.Z
APS-26 - + - - Lactococcus lactis APS-25 N.Z N.Z N.Z N.Z N.Z
APS-27 - - - - Bacillus/ Micrococcus luteus APS-26 N.Z N.Z N.Z N.Z N.Z
APS-28 - - - - Bacillus/ Micrococcus luteus APS-27 N.Z N.Z N.Z N.Z N.Z
APS-29 - - - - Bacillus/ Micrococcus luteus APS-28 N.Z N.Z N.Z N.Z N.Z
APS-30 - - - + Pseudomonas aeruginosa APS-29 N.Z N.Z N.Z N.Z N.Z
APS-30 N.Z N.Z N.Z N.Z N.Z
APS-Antibiotic producing strain

~ 3285 ~
International Journal of Chemical Studies

Fig 1: Gram staining of recovered isolates

A. Control B. Positive result C. Negative result

Fig 2: Biochemical characterization of isolates

Fig 3: Haemolytic activities of recovered isolates

Fig 4b: Antibiotic producing strains (APS-1, 3, 4, 6, and 7) showing

zone of inhibition against pathogenic strain Streptococcus (655)

Fig 4a: Antibiotic producing strains (APS-3, 9, 12, 15, and 17) Fig 4c: Antibiotic producing strains (APS-1, 3, 4, 15, and 17)
showing zone of inhibition against pathogenic strain Pseudomonas showing zone of inhibition against pathogenic strain Bacillus cereus
(2488) (1305)

~ 3286 ~
International Journal of Chemical Studies

Fig 4d: Antibiotic producing strains (APS-3, 4,5, 6 and 12) showing
zone of inhibition against pathogenic strain Staphylococcus (96) Fig 4e: Control plate

Fig 4: Isolates showing antimicrobial activity againstpathogenic test organism in agar well diffusion method

Fig 5a: Antibiotic producing strain (APS-5) showing zone of

Fig 5c: Antibiotic producing strains (APS-3, 6) showing zone of
inhibition against pathogenic strain Bacillus cereus (1305)
inhibition against pathogenic strain Staphylococcus (96)

Fig 5b: Antibiotic producing strains (APS-1, 3, 12) showing zone of Fig 5d: Antibiotic producing strains (APS- 12, 17) showing zone of
inhibition against pathogenic strain Streptococcus (655) inhibition against pathogenic strain Pseudomonas (2488)

Fig 5: Isolates showing antimicrobial activity against pathogenic test organism in disc diffusion method

Acknowledgement and support throughout my project work. I am obliged for his

With abundant pleasure and deep sense of appreciation, I persistent, selfless devotion towards the progress of my
express my heartiest thanks to my guide Dr Virender Singh, project work and his immense endurance to listing my
Head, Department of Microbiology, Himachal Institute of problems and provide an appropriate solution for them.
Life Sciences Paonta Sahib (HP) for his exceptional guidance

~ 3287 ~
International Journal of Chemical Studies

References
1. Smith JE. Biotechnology. 3rd ed. Cambridge university
press, United Kingdom, 1996, 125-137.
2. Waites MJ, Morgan NL, Rockey JS, Higton M. Industrial
Microbiology an Introduction. Blackwell science Ltd,
London, 2008, 288.
3. Evoy Mc, G. Ahes drug information Amr. Soc. Hospital
Pharm. USA, 1993.
4. Sundaramoorthi C, Vengadesh PK, Gupta S, Karthick K,
Tamilselvi N. Production and characterization of
antibiotics from soil-isolated actinomycetes. Int. Res. J
Pharm. 2011; 2(4):114-118.
5. Retinowati W. Identification of Streptomyces sp-MWS1
producing antibacterial compounds. Indonesian J. Trop.
Infect. Dis. 2010; 1(2):82-85.
6. Cappuccino, James G, Natalie Sherman. Microbiology: a
laboratory manual. 1996, 115-118.
7. Yunus FN, Kanwal F, Rashid F, Ashraf A, Iqbal MN,
Xiao S. A Comparative Study on Isolation and
Identification of Bacillus thuringiensis from Different
Localities of Gujranwala City. PSM. Biol. Res. 2016;
1(1):34- 38.
8. Abdulkadir M, Waliyu S. Screening and Isolation of the
Soil Bacteria for Ability to Produce Antibiotics. Eur. J
Appl. Sci. 2012; 4(5):211-215.
9. Singh AP, Singh RK, Mishra S. Studies on Isolation and
Characterization of Antibiotic Producing Microorganisms
from Industrial Waste Soil Sample. Open Nutraceuticals
J. 2012; 5:169-173.
10. Holt JG, Krieg NR, Sneath PHA, Stately JT, Williams St.
Bergey’s Manual of Determinative Bacteriology. 9 th ed,
Baltimore, Williams and Wilkins. 1994, 787.
11. Al-Ajlani MM, Hasnain S. Bacteria Exhibiting
Antimicrobial Activities; Screening for Antibiotics and
the Associated Genetic Studies. Open Conf. Proc. J 2010;
1:230-238.
12. Prescott ML, Harley PJ, Klein AD. Microbiology. 7th ed.
Publishing Group, 2008, 42-51, 232-233, 762-764.
13. Kaur S, Kaur J, Pankaj P. Isolation and characterization
of antibiotic producing microorganisms from soil
samples of certain area of Punjab region of India. Int. J
Pharm. Clin. Res. 2014; 6(4):312-315.

~ 3288 ~