Materials and Methods…………………………………………….

Chapter Three

Chapter three

1
 Materials and Methods……………………………………………. Chapter Three

3. Materials and methods

3.1. Table (3-1) The laboratory Equipments and Instruments:

NO. Equipment/Instruments Company/ Origin

1. Autoclave Stermite/ Germany
2. Light microscope England
3. Distillation system Daihan LabTech/ Indonesia
4. Laminar flow hood class II Dewar / China
5. Micropipettes Eppendorf / Germany
6. Refrigerator Hitachi/ Japan
7. Screw cap bottles Pyrex/ England
8. Incubator Cypress Diagnostics/ Belgium

3.2. Table (3-2) The chemical and biological materials

NO. Equipment/Instruments Company/ Origin
1 Absolute Ethanol, Crystal violet, Glycerol (C3H8O3) FLUKA / Switzerland

2 Phosphate buffered saline, Ph 7.2 Himedia / Indian

3 Phenol red Himedia / Indian

4 Trypan blue Himedia / Indian

5 Nutrient agar Himedia / Indian

6 Voges –Proskaur Reagent Mast, UK

7 Blood agar base Himedia, India
8 MR- VP broth Himedia, India
9 Simmon Citrate agar Himedia, India

2
 Materials and Methods……………………………………………. Chapter Three

3.3. Isolation of bacterial isolates

Bacterial samples were collected from three different parts of the Al-Najaf
governorate. These included the river sediment, soil, and rhizosphere soil during
October 2023. From each place, the sample was collected from at least 10 cm of depth
with a sterile inoculating loop/spoon. The sample was stored in a pre-sterilized
Eppendorf and transported to the laboratory of the science college University of Kufa.
Then, the samples were air dried by heating at 70 _C for 1 h in a dryer.
3.3.1. Culture conditions
For the isolation of Bacillus sp., a serial dilution technique was used considering
different aqueous dilutions (10_1 to 10_4) using phosphate-buffered saline (PBS, pH
7.2). A sample from each dilution was then streaked on a nutrient agar (NA) plate and
incubated at 37 _C for 24 h.
3.3.1.1. Morphological and cultural characteristics
The shape, size, color, edge, and appearance of bacterial colonies were studied on
nutrient agar plates after 24 hr of incubation according to (Collee et al.,1996).
3.3.1.2. Microscopical examinations
3.3.1.2.1. Gram's Stain
A single colony of each bacterial isolate was transferred to a clean slide and fixed by
flame, the smear was stained with Gram stain to study its Gram reaction and spore
formation under the light compound microscope (Collee et al.,1996).
3.3.1.2.2 Endospore features
Cultures were used for the detection of endospores present, and endospores’ position
within sporangium (Claus and Berkeley, 1986).
3.3.2. Biochemical identification of Bacillus species
The suspected Bacillus colonies were identified based on morphology and Gram
staining. Subsequent identification tests included hemolysis, starch hydrolysis, gelatin
hydrolysis, citrate test, Voges–Proskauer (VP) test, and growth at different pH and
temperature (Wulff et al.,2002).

3
 Materials and Methods……………………………………………. Chapter Three

3.3.2.1. Catalase Test

Put the amount of bacterial culture 24 hr on a clean slide and then add one drop of
H2O2 concentration (3%) to the culture, the formation of air bubbles is evidence of the
positive result (MacFadden, 2000).
3.3.2.2. Oxidase Test
The filter paper was saturated with the substrate (Tetra-Methyl-P-phenylene diamine
- dihydrochloride), and direct bacterial colony was to be tested age 24 hr on the filter
paper with a sterile wooden stick, the color changed to dark purple through (2-10)
seconds indicating a positive examination (Shields and Cathcart, 2010).
3.3.2.3. Citrate Utilization
This test was an indication of the utilization of citrate by bacteria as a sole carbon
source, in which aslant of Simmons citrate was inoculated with young colonies and
incubated at 30ºC for 3 days, the formation of deep blue color indicated on the positive
result (MacFaddin, 2000).
3.3.2.4. Blood hemolysis test
Blood agar medium was inoculated by bacterial culture using streaking methods,
then dishes were incubated at 37 ºC for 24 hours, the appearance of a definite clear
zone (β-hemolysis) around the colonies indicated the concerned Bacillus colonies
were selected (Katz, 2008).
3.3.2.5. Motility Test
Prepared a serial of tubes containing motility test agar and then inoculated with
bacterial culture by the needle which entered and cut through the agar then tubes were
incubated at 37 ºC for 24 hrs If the growth of your organism spreads out in only one
direction, and if there is a sharply differentiated edge between this growth and the agar,
your organism is non-motile, it has spread side to side only because those are places
where the needle contacted the agar, in other words, the organism only grew where it
was inoculated. But if the growth around the stab line has radiated outwards in all

4
 Materials and Methods……………………………………………. Chapter Three

directions (it will often look fuzzy) or if the entire tube has turned pink, your organism
is motile (Murray and Kazmierczak, 2008)
3.3.2.6. Salt Tolerance Test (Nacl)
on each dish of the dishes containing NaCl (0.1) ml from a bacterial culture that grew
on nutrient broth for a period of 24 hr by spreading method, the dishes were incubated
at 37 ºC for 24 hrs, after incubation observed growth of bacteria in all concentrations
and determine the highest concentration bacteria can grow in it (Collee et al., 1996).
3.3.2.7. Collection of Bacterial Sample
Samples were collected from the graduate laboratory in the Department of
Pathological Analysis, College of Science. Bacillus subtilis, against E. coli, Klebsiella
pneumonia, Proteus mirabilis, Salmonella typhi, Pseudomonas aeruginosa, Staph
aureus, Streptococcus pneumonia
3.3.2.8. Extraction of bacterial cell wall fraction
1- the culture of bacteria was centrifuged at 6000 rpm for 20 min.
2- the pelt is weighted.
3- suspended in sterile PBS.
4- The bacterial suspension was lysed via a sonicator for 30 min.
5- centrifuged at 10000 rpm for 10 min.
6- The prepared lysate was filtered by a 0.22 μ sterile filter.
7- supernatant was removed but pelt, as bacterial lysate and cell wall extract were
collected, and stored at −20°C.
3.3.2.9. Agar diffusion method
By agar diffusion method (Balouiri et al, 2016) Petri plates containing Twenty-five
ml of sterile Muller–Hinton agar inoculated along with E. coli. Klebsiella pneumonia,
Proteus mirabilis, Salmonella typhi, Pseudomonas aeruginosa, Bacillus subtilis, Staph
aureus, and Streptococcus pneumonia after adjusting these bacteria comparison with the
0.5 McFarland standards tube. Cut wells have (6mm) in diameter in agar by using a
sterile Pasteur pipette and remove the agar discs with sterile forceps after that fill wells

5
 Materials and Methods……………………………………………. Chapter Three

with 0.1ml of cell wall extracts of bacillus subtilis then incubated in the upright position
to keep the extraction in the wells at 37 o C for 24 hours. Measured inhibition zone
diameter formed around each well assessment the antibacterial activity of cell
membrane and matching up against amoxicillin.