Chapter four Results and discussion

Chapter four

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Chapter four Results and discussion

4. Results and discussion

4. 1. Morphological characteristics of the Bacillus species

Through laboratory tests for the growth of bacterial isolates on nutrient agar
colonies, most isolates appeared to be circular, the colony varies in size but more are
relatively large, smooth, and opaque, with a round edge that quickly turns to the edge
of lobed with growth, less whitish or cream colored colony and these features match
what pointed it (Collee et al., 1996), of culture character for colonies of genus B.
subtilis.

4.2. Microscopic characteristics of Bacillus species

Microscopic examination of the results of the bacterial colony isolates installed
on slides and dyed with gram stain showed that Bacillus isolates were gram-positive,
cells rods in shape (figure 4.1). some occur singly, others arranged in pairs or chains,
endospore formation, and spores were ellipsoidal in shape and central in position,
these results were in agreement with (Collee et al.,1996 and MacFadden,2000) on
the microscopic forms of B. subtilis cells and the nature of regularity.

Figure (4.1) Bacillus subtilis cells under an optical microscope magnification X100
strongly after incubation for 24 hours at 37 ºC.

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Chapter four Results and discussion

4. 3. Biochemical Test of Bacillus species

The results of biochemical tests recorded in (table 4.2), were considered

complementary to the initial identification of Bacillus isolates belonging to the
species B. subtilis.
Table (4.2) Results of biochemical tests of Bacillus subtilis

NO. Biochemical Tests Results

1 Catalase +
2 Oxidase ‫ــ‬
3 Haemolysis Beta-hemolysis
4 Motility +
5 Citrate Utilization +
6 NaCL (1% - 8%) +
7 NaCL (9% and 10%) ‫ــ‬

4.4. cell wall extraction of bacillus subtilis as antibacterial

In–vitro Activity of Cell Wall Extraction as Antibacterial The antibacterial

activity assay of cell wall extraction of Bacillus subtilis was done against E. coli,
Klebsiella pneumonia, Proteus mirabilis, Salmonella typhi, Pseudomonas
aeruginosa, Bacillus subtilis, Staph aureus, Streptococcus pneumonia were
estimated by determining the diameters of zones of growth inhibition on bacterial,
and match with the inhibition zone of amoxicillin as control, the result show all
bacteria sensitive to cell wall extraction of bacillus subtilis (100%) and the inhibition
zone ranging from 25 to33mm, compare with amoxicillin that all bacteria resistance
to it (100%) according to( Khochamit., et al 2015) and the results are presented as
shown in table (1) and figures (1)

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Chapter four Results and discussion

Table (1): Concentration of LPS Ug/ml, Size of inhibition zone diameter for
three replicated (mm)

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Chapter four Results and discussion

(Lee et al., 2021) found that The total amount of extract loaded into each well to
perform the agar well diffusion assay was 20 μL. Therefore, we calculated that the
bactericidal activity of 1 μL out of 5.5 mL extract from transgenic B. subtilis C117
and C166 strains against V. parahaemolyticus was approximately equivalent to the
potency of 47.07 and 25.2 ng of Tetracycline, respectively. also (Khochamit et al.,
2015) appeared that the Antibacterial activity of cell-free supernatants containing
crude bacteriocin(s) produced by Bacillus spp. KKU213. Bacillus cereus MC11++,
Listeria monocytogenes DMST23145++

The cell wall extract contains components such as peptidoglycan and

lipoteichoic acid that can physically interact with the cell membranes of other
bacteria. This interaction can disrupt the integrity of the cell membrane, leading to
leakage of cellular contents and eventual cell death (Zhou et al., 2022). The cell wall
extract may contain substances that interfere with the synthesis or assembly of the
bacterial cell wall. By targeting the essential processes involved in cell wall
formation, the extract can inhibit the growth and survival of susceptible bacteria.
The cell wall extraction process may release or concentrate antimicrobial
compounds produced by Bacillus subtilis. These compounds can include
antimicrobial peptides, enzymes, or secondary metabolites that exhibit antibacterial
activity against other bacteria (Zhou et al ., 2022).

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