some bacterial species have antagonistic activity against some fungal species like E.coli Vs A. niger

1. RESEARCH PROJECT
RS401
META LAB DIAGNOSTIC LABORATORY
ANTAGONISTIC ACTIVITY OF SOME BACTERIAL SPECIES AGAINST
OTHER MICROORGANISMS ISOLATED FROM THE SOIL
External Supervisor
Dr. Mohamed Hussien
Medical Operation Manager at
Metalab Diagnostic
Laboratories
Internal Supervisor
prof. Salwa Sabet
Professor at Faculty of
Biotechnology MSA University
By/ Marlin Milad
ID/ 171663

2. Table of content
I. Introduction
II. Aim
III. Methodology
IV. Results
V. Conclusion
VI. Acknowledgment
VII.Reference

3. Introduction
The antagonistic activities of soil microorganisms can be used as
antibiotics or antifungals for pathogenic bacterial or fungal species for
plants to minimize any loss of plant yield. The usage of bacteria in
biological control projects is based on many variables like taxonomic
role and physiological characteristics such as species and growth cycle,
geographical condition, and soil biota
(Ar & Kalantari, 2011).

4. Introduction cont…
Therefore, the screening of a significant amount of bacteria in different
geographical areas increasing the chance of discovering novel probiotic
strains of broad-spectrum antifungal efficacy that lead to maximize the
opportunity for insulation of bacteria useful as abundant suppliers of
antifungal bioactive metabolites, and also the long term viability of bio-
control factors to use against fungus. (Ramzan, N., Noreen, N.,
Shahzad, S., and Al, E. T., 2014).
A. niger is one of the most popular pathogenic
fungi that cause mold for a lot of plant crops
during storage or cause diseases while planting
(Pitt and Hocking, 2013).

5. Introduction cont…
Celery is an Umbelliferaceae family member. It is the third vegetable
kind used in salad in the world. It has important
role as medical treatment in which it contain
essential nutrients such as nitrogen, phosphorous,
and potassium. Some bacteria and fungi have
pathogenic effect on celery plant such as
E. Coli O: 157: H7, Staphylococcus aureus,
lactobacillus plantarun, and pseudomonas
acruginosa bacterial species and A. niger,
Geotrichumm,and Rhodotorula fungal species
(Kooti, Wesam, Daraei and Nahid, 2017).

6. Aim
The aim of this study is isolating and
identifying some of bacterial and
fungal species from the peat moss soil
of Celery plant by using some of
biochemical tests such as lysine, urea
base, TSI, and MIO test and
microscopic examinations to examine
the antagonistic activity of them
against each other by using Agar-
diffusion technique.

7. Methodology
Purification of some Bacterial and Fungal colonies
Media preparation
Nutrient
agar
Sabouraud
Dextrose
agar
MacConkey
agar
CLED agar
Mannitol
Salt agar
Bile Esculin
agar
Soil sample collection

8. Examination of Antagonistic Activity of Bacteria against Fungi
Identification of Bacterial colonies
Gram staining Biochemical tests
Identification of fungal colony
1- Lysine Iron test
2- Urea Base test
3- TSI test
4- MIO test
5- Mannitol test

9. Results
Nutrient Agar media
and Bacterial culture
Figure (1): Growth of different
bacterial soil isolates on nutrient agar
medium after an incubation period of
96 hours.
Sabouraud Dextrose Agar
media and fungal culture
Figure (2): Growth of different fungal soil
isolates on Sabouraud Dextrose agar
medium after an incubation period of 96
hours.

10. Results cont…
Purification of the fungal colony
Figure (3): Growth of isolated fungal colony on Sabouraud
Dextrose agar medium after an incubation period of 96
hours.

11. Results cont…
Purification of the Bacterial colonies
Figure (4):Growth of isolated
Bacterial colony on Nutrient
agar medium after an
incubation period of 96 hours.
Figure (5):The culturing of Bacillus subtilis
on (a) MacConkey agar media, (b) CLED
agar media, (c) Bile esculin media, and (d)
Nutrient agar media
Figure (6): The culturing of
Staphylococcus aureus on (a)
MacConkey agar media, (b) CLED
agar media, (c) Nutrient agar media,
and (d) Bile esculin media.

12. Results cont…
Figure (7):Culturing of Escherichia coli on (a) MacConkey, (b)
CLED, (C) Starch agar and (d) bile esculin agar media.

13. Results cont…
Identification of isolated and purified microorganisms
Fungal strain
40 um
Figure (8): Microscopic image of
purified fungal cells section with part of
its agar medium on glass slide.

14. Results cont…
Identification Bacterial strains
Gram Staining
Figure (9): Microscopic image
of the gram stained klebsiella.
Figure (10): Gram staining of (a) Escherichia coli, (b) Staphylococcus
aureus and (c) Bacillus subtilis.

15. Biochemical tests
Identification Bacterial strains
1- Lysine Iron test
Figure (11):C1: the control of slant-shape test tube of Lysine test after 24 hours. A: Lysine test result on slant-shape test tube
after the streaking of purified gram-negative bacteria and incubation for 24 hours. C2: the control of butt-shape test tube of
Lysine test after 24 hours. B: Lysine test result on butt-shape test tube after the injection of purified gram negative bacteria and
incubation for 24 hours.D: Lysine test result on petri-dish after the streaking of purified gram negative bacteria and incubation
for 24 hours.

16. 2- Urea Base test
Figure (12): C1: the control of Butt-shape test tube of Urea base test after 24 hours. A: Urea base test result on butt-
shape test tube after the injection of purified gram negative bacteria and incubation for 24 hours. C2: the control of
slant-shape test tube of urea base test after 24 hours. B: Urea base test result on slant-shape test tube after the
streaking of purified gram negative bacteria and incubation for 24 hours.C: the control of petri-dish of urea base test
after 24 hours.S: Urea base test result on petri-dish after the streaking of purified gram-negative bacteria and
incubation for 24 hours.

17. 3- TSI test
Figure (13): Triple Sugar Iron test results. Where (C) is the control tube, (K) is the klebsiella, (E) is the
Escherichia coli, (B) is the Bacillus subtilis and (S) is the Staphylococcus aureus strain.

18. 4- MIO test
Figure (14): C: The control of butt-shape
test tube of MIO test after 24 hours. K:
MIO test result on butt-shape test tube
after the injection of purified gram
negative bacteria and incubation for 24
hours.
Figure (15):shows the MIO test results. Where (S) is the
Staphylococcus aureus, (B) is the Bacillus subtilis, (E) is the
Escherichia coli, and (C) is the control tube.
C K

19. 5- Mannitol test
Figure (16):Mannitol test results for the bacterial strains. Where (C) is the control tube, (E) is the
Escherichia coli, (B) is the Bacillus subtilis and (S) is the Staphylococcus aureus strain.

20. Results cont…
Antagonistic Activity of Klebsiella against Fungi on Sabouraud
Dextrose agar media
Figure (17): A: Result of antagonistic activity examination of klebsiella bacterial species as antibiotic disk against streaked
Aspergillus niger pathogenic fungi on Sabouraud Dextrose agar media. B: Result of antagonistic activity examination of
streaked klebsiella bacterial species against streaked Aspergillus niger pathogenic fungi by 50/50 on Sabouraud Dextrose agar
media

21. Antagonistic Activity of E. coli and Bacillus spp against Fungi on
Sabouraud Dextrose agar media
Figure (18): A: Reduction in fungal mycelial growth by Escherichia coli
B: Reduction in fungal mycelial growth by Bacillus spp.

22. 0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
klebsiella Staphylococcus Bacillus E. coli
growth of Asp.
Niger
Figure (19): Antagonistic activity levels of klebsiella, staphylococcus, bacillus, E. against
A. niger and growth rate of A. niger in the presence of each species as agonist

23. Conclusion
Soil microorganisms were reported that they may be used as biological
control factors for plant pathogens. Celery plant was chosen to isolate
some bacterial and fungal species from its soil according to the
importance of it for the community, and examine the antagonistic
activity of each species against each other. E. coli, Bacillus spp.,
Staphylococcus spp., and Klebsiella spp. bacterial species and
Aspergillus niger were isolated and identified. The higher antagonistic
activity levels against A. niger were shown by E. coli and Bacillus spp.
which were 90% and 64% respectively, while the lower antagonistic
activity levels were shown by Staphylococcus and Klebsiella.

24. Bacterial species that may be used as antifungals have the ability to
produce chitinase enzyme and more lysis components such as
siderophores and HNC fungus killer that call cause breakdown for
fungal cell wall and cell death. E. coli, Bacillus spp., and Staphylococcus
might produce one or more of fungicidal metabolites or the fast growth
of each species might help them to overcome the growth of fungi. But
about Klebsiella, it might be 74 needed another agar media to show
different antagonistic activity level. We cannot generalized the
antagonistic activity results of this study as this study was done by
using one agar media type to examine each bacterial species against A.
niger and was done for one time and not repeated again.

25. But in general there are a lot of studies approved that E. coli and
Bacillus had antagonistic activity against pathogenic A.niger and other
pathogenic fungi. The researchers should care and study more in this
topic to detect and exclude more metabolites can be extracted from
bacterial species present in the soil and used as antifungals to limit the
crops mold and the food are able to be stored for long time.

26. Acknowledgment
I would like to thank Prof. Ayman Diab for giving me this great
opportunity to carry out my research project in such great
laboratories. Also, I would especially like to thank Dr. Gehan Safwat
for her continuous support and advice which were priceless. I would
like to offer my appreciation and thanks to my external supervisor: Dr.
Mohamed Hussein and my internal supervisor Prof. Salwa Sabet for
their efforts and continuous support. I offer my Acknowledgment and
my great feelings for Dr. Mariam Zakaria, Dr. Aya Samir and Dr. Samah
Mohamed for their help in the practical of this research that I have
learned. I would like also to thank Dr. Mohamed Galal for his help.
A special thanks to my family, friends, and colleagues who supported
me, and incented me to strive towards my goal

27. Reference
Ar, R., & Kalantari, S. (2011). Molecular identification of
antagonistic bacteria from Tehran soils and evaluation of their
inhibitory activities toward pathogenic fungi, 3(3), 140–146.
Kooti, W., & Daraei, N. (2017). A Review of the Antioxidant Activity
of Celery ( Apium graveolens L ), 22(4), 1029–1034.
https://doi.org/10.1177/2156587217717415
Pitt and Hocking. (2013). Fungi and Food Spoilage.
Ramzan, N., Noreen, N., Shahzad, S., & Al, E. T. (2014). INHIBITION
OF IN VITRO GROWTH OF SOIL-BORNE PATHOGENS BY COMPOST-
INHABITING INDIGENOUS BACTERIA AND FUNGI, 46(3), 1093–
1099.