Nativa, Sinop, v. 11, n. 2, p. 161-165, 2023.
Pesquisas Agrárias e Ambientais
DOI: https://doi.org/10.31413/nativa.v11i2.15434
ISSN: 2318-7670
Isolation and identification normal flora bacteria from different areas of
Ninava governorate
Ahmed Mohamed TAHER1, Ibrahim Omar SAEED 1*
1 Department of Biology, Collage of Science, University of Tikrit, Salah Al-Din, Iraq.
*E-mail: dr.ibrahim1977@tu.edu.iq
Submission: 05/07/2023; Accepted on 06/09/2023; Published on 06/12/2023.
ABSTRACT: In this study, different types of bacteria were isolated and diagnosed from soils collected from
different areas of Ninava governorate, new strain of bacteria was discovered, based on biochemical tests and
genetic examination (16S rRNA) and the strain was recorded under accession number (MW690182) in the
Gene Bank (NCBI). The isolated bacteria were Bacillus Subtilis strain that given new name (AHM1976) it was
positive for gram stain, motility, hemolytic, beta type.
Keywords: isolation; identification; normal flora bacteria.
Isolamento e identificação de bactérias da flora normal de diferentes áreas da
província de Ninava, Iraque
RESUMO: Neste estudo, diferentes tipos de bactérias foram isolados e diagnosticados em solos coletados em
diferentes áreas da Província de Ninava, Iraque. Foi descoberta uma nova cepa de bactéria, com base em testes
bioquímicos e exame genético (16S rRNA), sendo essa cepa registrada sob o número de acesso (MW690182)
no Gene Bank (NCBI). A bactéria isolada foi a cepa Bacillus Subtilis, que recebeu novo nome (AHM1976),
testando de forma positiva para coloração de gram, motilidade, hemolítica, tipo beta.
Palavras-chave: isolamento; identificação; bactérias da flora normal.
1. INTRODUCTION
Bacteria are among the most widespread forms of life on
Earth. One gram of soil contains more than 40 million
bacterial cells and nearly a million bacterial cells per
millimeters of fresh water (WHITMAN, 1998; RAMBO et
al., 2019). Bacteria make up the biomass that exceeds the
biomass of plants and animals (KUNDAVVARAM, 2015).
Because of the large number of bacterial species that live
on Earth, they have not been fully identified and identified.
Therefore, many technological and biological techniques
have been developed to identify these different types, such as
the VITEK2 device, DNA hybridization, PCR techniques,
which are the most popular at the present time, and
biochemical tests.
Among these bacteria spread in the soil are Gram-
positive Bacillus.subtilis bacteria, which is one of the best
types of bacteria that are used for basic research on bacteria
because of its unique characteristics such as its response to
inappropriate environmental conditions such as lack of food
sources, temperatures, pH, osmotic pressure, and the
formation of a rich (Biofilm) layer It contains sugar, protein,
fatty compounds and nucleic acids (MIROUZE; DUBNAU,
2016). Therefore, bacteria B. subtilis were used in laboratory
studies aimed at discovering the basic characteristics of gram-
positive bacteria and the formation of spores (EARL et al.,
2008).
2. MATERIALS AND METHODS
2.1. Collection of samples
Soil samples were randomly collected from different
areas of Nineveh governorates. Soil samples were taken from
five areas. Samples are put in sterile polyethylene bags and
the required information (sample number, date, weight) was
recorded on them and transferred to the laboratory and kept
in the refrigerator at a temperature of 4 C° until use.
2.2. Isolation of bacteria
The collected soil samples were mixed homogeneously
and passed from a 2 mm sieve to get rid of the unwanted
gravel and impurities. The dilution and plate method was
used to isolate the bacteria by adding 1 g of soil samples to 9
ml of distilled water (DW) and after homogenization, the
dilution series was prepared from 10-1 to 10-4, after which the
decanting method was used, where 1 ml of each dilution was
transferred to a sterile Petri dish and added to it the nutrient
medium, MacConkey agar medium and blood agar medium,
with three repetitions for each dilution, and the dishes were
incubated at a temperature of 37 C° for 24 hours. 48 hours.
2.3. Purification
Depending on the shape, texture and color of the
bacterial colonies growing on the petri dishes, the bacterial
isolates were purified and with the help of the differential and
selective media (MacConkey and Mannitol) as well as the
nutrient medium so that this technique was repeated to
obtain pure isolates, after obtaining the pure isolates, they are
stored formally. Sterilized and safe at a temperature of 4 C°.
2.4. Identify and diagnose bacteria
2.4.1. Phenotypic diagnosis
The phenotypic characteristics of the developing colonies
were relied on the nutrient agar media in terms of texture,
shape, size, height, color and odor in the diagnosis of
bacterial isolates (LOGAN, 2009).
Isolation and identification normal flora bacteria from different areas of Ninava governorate
Nativa, Sinop, v. 11, n. 2, p. 161-165, 2023.
162
2.4.2. Microscopic diagnosis
A Loop campaign was taken from the developing
colonies and spread them in a circular motion on a glass slide
after being heat-fixed and stained with a gram stain
(COLLEE et al, 1996), depending on the chemical and
physical properties of the cell wall and using an oily lens of
100x strength, the shapes of bacteria were identified (SONI,
2013).
2.4.3. Biochemical Indemnification Test
1. The Oxidase Test
A drop of oxidase reagent is placed on a filter paper, after
which a portion of the bacterial culture is transferred by a
sterile wood stick. We wait about 10 seconds. The positive
result is the appearance of a blue color (FORBES et al.,
2007).
2. The catalase test
We transfer a Loop campaign from the bacterial colony to
a glass slide and sprinkle it with a few drops of catalase
reagent (3% hydrogen peroxide). The appearance of bubbles
is evidence of a positive result (FORBES et al., 2007).
3. Voges – Proskauer test
After preparing the MR-VP medium in laboratory tubes,
these tubes were inoculated with bacterial growth and
immunized at 37 C° for 24 hours. After the incubation
period, 0.5 ml of VP1, VP2 solution was added, waiting for
about 30 minutes, the appearance of the red color evidence
of a positive result (ABEDIN, 2015).
4. Motility
The movement medium was inoculated by the stabbing
method using the needle with bacterial growth and incubated
for a period of 24 hours at a temperature of 37 C°. The
appearance of a cloudy area in the movement medium is
evidence of the positive of the examination as for the growth
of the stabbing area, only evidence of the negative result
(FORBES et al., 2007).
5. Citrate consumption test
The medium of consumption of citrate was inoculated
with the bacterial inoculum and immunized for 24 hours at
37 C°. The transformation of the medium from green to blue
is evidence of positive testing (CAPPUCCINO; SHERMAN,
2010).
6. Mannitol test
The bacterial isolates were cultivated on a medium of
mannitol for a period of 24 hours at a temperature of 37 C°.
The color of the medium changed from red to yellow,
indicating a positive result (fermentation of mannitol sugar),
while the remaining red color was considered a negative
result (non-fermentation of mannitol sugar) (MUDAWI et
al., 2007).
7. Test of indole production
The tubes containing the indole medium were inoculated
with bacterial growth and immunized for 24 hours at a
temperature of 37 C°, after which 5-6 drops of the Kovac’s
reagent were added, the appearance of the red ring
representing the positive result and its absence representing
a negative result (MACFADDEN; JEAN, 2013).
8. The urease test
A slant tube containing urea medium was inoculated with
bacterial growth by the planning method and immunized for
48 hours at a temperature of 37 C°. The color of the medium
changed from yellow (acid) to pink (alkaline) representing the
positive result, and the reason is due to the ability of the
bacteria to obtain the production of the enzyme urea, which
works on the analysis of urea and the production of
ammonia, which increases the alkalinity of the medium and
there is evidence in the medium that turns pink in the alkaline
conditions (COLLEE et al., 1996).
9. Triple iron sugar test
An oblique tube containing TSI-iron agar medium was
inoculated with bacterial growth by planning method and
pricking to the depth of the medium and incubated 48 hours
at 37 C°. After the end of the incubation period, the color of
the medium, gas formation, and hydrogen sulfide production
were observed (ATLAS, 1995; COLLEE, 1996).
2.5. Molecular Biology Identification
2.5.1.DNA Extraction
The analysis kit provided by Geneaid was used to extract
DNA from bacterial samples.
1. The precipitate of bacteria that were grown in the liquid
medium is taken into a 1.5ml Eppendorf tube
2. Add 200 μl to an Eppendorf tube with Lysozyme at a
concentration of 0.8 mg / 200 ml with mixing with Vortex.
3. The tube was incubated at 37 C° for 30 minutes and
the tube was turned over every 3 minutes during the
incubation period.
4. Add 20 μl of Proteinase K and mix Vortex
5. The mixture was incubated at 60 C° for 10 minutes.
6. Add 200 μl of GB buffer, mix with Vortex and incubate
at 70 C°.
7. 200 μl of absolute ethanol are added and mixed
manually, then the mixture is transferred to the GD tube
installed in the collection tube and a (16000g) centrifugation
is performed for 30 seconds and the filtrate is removed, then
600 μl of the washing solution are added and centrifuged at
the same speed and time before the sediment is removed. Re-
centrifuge for 3 minutes to get rid of all washing solution
residues.
8. The GD column is transferred to a new 1.5 ml tube
and 100 μl of dissolving solution are added, then left for 3
minutes. After that, centrifugation at 16000g for 30 seconds
is carried out, then the DNA is kept at -20 ° C until use.
9. Quanti Fluor® (Promega, USA) was used to measure
DNA concentration. 199µl quantus stain was mixed with 1µl
of DNA sample and left in a dark place for 5 minutes after
which the DNA concentration was measured by a quantus
™ fluorometer.
2.5.2. DNA in Gel Electrophoresis
Electrophoresis was performed to separate the extracted
DNA mixture and test it according to the following steps:
(MISHERA et al., 2009; SAMBROOK; RUEESL, 2006).
1. To prepare the 1% agarose gel, 1 g of agarose powder
was dissolved in 100 ml of TBE 1x buffer and 3 μl of red safe
dye was added by using a heat source with constant stirring
until boiling. Then it was left to cool down to a temperature
of (60-50 C°).
Taher & Saeed
Nativa, Sinop, v. 11, n. 2, p. 161-165, 2023.
2. The tray was prepared for pouring gel, and the two
edges of the template were surrounded by adhesive tape and
the comb was fixed to create the wells at a distance of 1 cm
from the edge of the template. After that, the gel was poured
into the template, which is placed completely horizontally,
taking into account that the pouring was quietly in order to
avoid the formation of bubbles and leave for a period 30
minutes to harden, after which the comb and tape gently
lifted from the template and placed in the electrophoresis
bath that contains buffer1x TBE so that the agarose gel
covered approximately 1 mm because the large amount
causes the DNA bundles not to appear in the horseshoe
shape or straight and evenly.
3. Migration samples were prepared by mixing 5 µl of
DNA sample with 3 µl of loading solution
4. Using a (2000bp) ladder by taking 6 µl of it and mixing
it with 2 µl of bromophenol dye solution and taking out 5 µl.
5. The electric migrate was Done under a voltage of 100V
for a period of 60 minutes.
6. The gel was examined by UV Trans illumination and
photographed by a camera.
2.5.3. Polymerase chain reaction
The DNA concentration in all study samples is adjusted
by dilution with TBE buffer solution to obtain the required
concentration for PCR reactions and it was (50)
nanogram(ng) / µl per sample. As the Master Reaction
mixture was prepared for each PCR reaction by mixing the
DNA sample and the initiator of each gene with the master-
mix components inside a 0.2 ml Eppendorf tube supplied by
the English company Biolabs, the reaction volume was fixed
to 20 μl with distilled water, the mixture was in the Microfuge
device for a period between (3-5) seconds to ensure that the
components of the reaction were mixed, then the reaction
tubes were inserted into the Thermocycler device for the
purpose of performing the amplify reaction using the special
program for each reaction, after which the sample was loaded
into the wells of pre-prepared agarose gel at a concentration
of 1% with the addition of the volumetric guide Ladder DNA
prepared by Biolabs in one of the wells, then the samples are
migrated by running the Electrophoresis device for a period
of (60-70) minutes, after which the gel is photographed using
the UV Trans illumination device.
The PCR polymerase technique was used to amplify the
DNA encoded for the 16S rRNA gene and using the prefixes
shown in Table (1) with the DNA extracted in paragraph
(2.5.1), 4 μl (100 ng) of DNA template were added with 1 μl
(10 picomole) of each Gene-specific prefix to Master Mix
contents.
Table 1. Universal primers.
Tabela 1. Primários universais.
Primer
Sequence
Bp size
Ref.
Forward
Reverse
CACAGACACACGCTG
ACGCTGACCA
1200 Kumar et
al. (2016)
The reaction was carried out according to the leaflet
supplied with the kit supplied by Geneaid with a volume of
20 μl according to the Table 2. The program shown in Table
3 was followed to amplify the DNA samples with PCR
technology.
Table 2. Components and volumes of the polymerization reaction
mixture.
Tabela 2. Componentes e volumes da mistura de reação de
polimerização.
Volume
Reaction components
5 µl
Master MIX
1 µl
Primer Forward
1 µl
Primer Reverse
5 µl
DNA
8 µl
dd.H2O
20 µl
Total
Table 3. DNA amplification program.
Tabela 3. Programa de amplificação de DNA.
No. Stage
Temperature
(°C) Time
Cycle
number
1. Initial denaturation
95 6 min. 1
2. denaturation 95 45 sec.
35
3. Annealing 56 1 min.
4. Extension 72 1 min.
5. Final extension 72 5 min. 1
2.5.4. Electrophoresis of samples
The same method was used for DNA detection, where a
solution (1500bp) ladder was placed in the first hole of the
gel in order to locate the 16S rRNA Gene in the carried
DNA. After the migration process was completed, the
samples were examined by a (UV Trans illumination) device
to view the orange beams that were photographed by the
camera.
2.5.5. DNA extraction from agarose gel
The bands resulting from the PCR reaction were extracted
from the gel to be purified and sent for a nucleotide sequence
sequencing test, based on the analysis kit supplied by
Geneaid, according to the steps:
1. The bands are cut from the agarose gel using a sterile
scalpel with the removal of the largest amount of gel
surrounding the bands.
2. Transfer approximately 300 mg of gel piece to a 1.5 mL
Eppendorf tube and add 500 μl of buffer solution DF and
mix using a Vortex device.
3. The tube is incubated at a temperature of 55-60 C° for
15 minutes to ensure a complete piece of gel with inverting
the tube every 3 minutes during the incubation period. The
tube is left to cool down at room temperature.
4. Transfer 800 μl of the sample mixture to a DF column
installed in the collection tube, then perform a centrifugation
with a force of 16000g for 30 seconds and the filtrate is
removed.
5. Re-install the DF column again to the collection tube,
add 600 μl of washing solution and leave for one minute.
6. Conduct a centrifuge with the same force of 16000g
for 30 seconds and get rid of the filtrate.
7. The previous process is repeated.
8. Conduct a 3-minute centrifugation to ensure that the
DF column is dry.
9. The collection tube is discarded and the DF column is
transferred to a new 1.5 mL tube.
10- (20-50 μl) of Ellution buffer solution are added to
the center of the column.
11- Leave for two minutes to ensure absorption of the
dissolving solution.
Isolation and identification normal flora bacteria from different areas of Ninava governorate
Nativa, Sinop, v. 11, n. 2, p. 161-165, 2023.
164
12- Two-minute centrifugation of 16000g to obtain the
dissolved DNA.
2.5.6. Detection of genetic sequences
After the completion of the DNA extraction process and
the PCR process, the samples were sent to Macrogen (South
Korea), where 20 μl of each sample was placed in an
Eppendorf tube and 50 µm of the primers were placed in
other Eppendorf tubes to determine the genetic sequence of
the bacterial isolates, depending on the Genetic Analyzer
3130 device. Equipped from Japanese Hitachi company.
The gene sequences were matched with data available at
the National Center Biotechnology Information (NCBI).
3. RESULTS
3.1. Isolation and identification of bacteria:
The results of Table 4 showed the bacterial growth that
was cultured on nutrient agar medium, the growth of
bacterial isolate from soil samples, which were collected from
different areas of Nineveh governorates, at dilutions from 10-
1 to 10-4, Studies differ in the possibility of isolating different
types of bacteria, as Azeez; Shareef (2012) managed to obtain
six bacterial isolates from different regions of Nineveh
Governorate and its dependent regions, while (JASIM, 2016).
Isolation of 44 bacterial isolates from soils for different
regions of Sulaymaniyah Governorate.
Table 4. Phenotypic characteristics of bacterial isolate.
Tabela 4. Características fenotípicas do isolado bacteriano.
Isolate
Bacillus subtilis
strain
(AHM1976)
Nature of
colonies on
solid nutrient
medium
Shape
Circular
Colour
Light pink
Texture
Creamy
Appearance
Dark
Hight
Flat
Edge
Regular
Gram stain
+
Cell pool
Single and binary
Cell shape
Rod
3.2. Biochemical tests
Biochemical tests, which included the tests shown in
Table 5 based on (Collee et al., 1996; Macfadden; Jean, 2013),
where the test for catalase, oxidase, hemolysis, and culture
was performed on McConkey's medium, mannitol medium,
motility test, IMVIC group of tests, as well as TSI test (Triple
Sugar Iron) and urease test, the growing isolates were
diagnosed based on colony shape, color, edge shape,
interaction with gram stain and biochemical assays (Brookse
et al., 2013) as shown in Tables 4 and 5 and Figures 1 and 2.
3.3. Molecular Diagnostics
The final diagnosis of bacterial isolates was made using
DNA Sequencing technology due to the ability of this
technique to diagnose genus and species with high accuracy
and in less time than traditional methods of diagnosis, where
special primers are used to amplify this gene, which contains
specialized regions that allow the diagnosis of genus and
species. This gene consists of many nucleotides (JENKIS et
al., 2012).
16S rRNA technology is considered one of the basic
criteria for classifying and identifying bacteria due to the
difficulty of genetic mutations in them, and because this gene
contains high change regions between bacterial species and
provides an independent sequence for each bacterial species.
(ABDULLAH; MAHDI,2016).
Table 5. Biochemistry tests for bacterial isolate.
Tabela 5. Testes bioquímicos para isolado bacteriano.
N°
Tests
Bacillus subtilis
strain (AHM1976)
1
Motility
+
2
Indole
-
3
Methyl red
+
4
Voges
-
Proskaoer
-
5
TSI
A
\
A
6
Oxidase
+
7
Catalase
+
8
Urease
-
9
Simmon
Citrate
-
10
MacConkey agar
-
11
Mannitol salt agar
+/Non
-
F
12
Blood hemomysis
β
Figure 1. Bacterial cell growth on nutrient agar and it,s Shaps under
microscope.
Figura 1. Crescimento de células bacterianas em ágar nutriente e
suas formas ao microscópio.
Figure 2. Bacterial cell growth on Blood and MacConkey agar.
Figura 2. Crescimento de células bacterianas em ágar Blood and
MacConkey.
3.4. Polymerase chain reaction PCR
The results of electrophoresis on an agarose gel of DNA
samples extracted from bacterial isolates using the 16S rRNA
gene primer showed that the DNA bundles were about
1200bp as shown in Figure 3.
3.5. Detection of a nucleotide sequence
The results of samples sent to Macrogen Company in
North Korea, and in comparison, with the database of the
National Center for Biotechnology NCBI, showed the
discovery new strain of bacteria, and it is registered on the
NCBI website for the first time with new names and given a
special number called an Accession number (MW690182).
Taher & Saeed
Nativa, Sinop, v. 11, n. 2, p. 161-165, 2023.
Figure 3. Agarose gel electrophoresis showing the analysis of the
PCR product for the 16S rRNA gene of bacterial isolate.
Figura 3. Eletroforese em gel de agarose mostrando a análise do
produto de PCR para o gene 16S rRNA do isolado bacteriano.
4. DISCUSSION
Through this research, it became clear to me that the
bacteria present in different soils of Nineveh Governorate
are exposed to mutations. The reason for this may be due to
the excretion of factory waste and war remnants in different
environments. It is also clear that the effect of fertilizers and
hydrocarbons on the sequence of genes in the natural
bacteria of the soil is obvious.
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Author Contributions: The two authors participated in all stages
of the article, read and agreed to the published version of the
manuscript.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Study data/imagens can be obtained
by request to the corresponding author or the second author, via e-
mail.
Conflicts of Interest: The authors declare no conflict of interest.
Supporting entities had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the
manuscript, or in the decision to publish the results.