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Sclerotinia sclrotiorum
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CONTINUE
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()7
[1] Curtis F. Gerald & Patric O. Wheatley, Applied Numerical

Analysis, fifth edition, 1994, America, Addison Wesley Publishing
Company.
[2] Richard L. Burden & J. Douglas Faires, Numerical Analysis,
fifth edition, 1993, America, Prindle, Weber & Shmidt.
Garlic
Escherichia coli

, , ,
:

,
, E. coli

%80
,
E. coli
. Ceftriaxone
:(Garlic) Allium sativum

Liliaceae

], [7

],[6
Allins Alkyl
cystinesulfoxides
Allicine diallal-
disylphid-mone oxide

[11] E. coli
Klebsilla, E.coli, , Bacillus sp
Enterobacter sp, staphylococcus sp,Salmonella sp,
,pseudomonds sp, streptococcus sp ,vibrio sp, [12].
Escherichia coli
Enterobacteriacea
( )Flagella
,

Antigens P- fimbriae
( )Antigen K

],[13
) Urinary Tract infection (UTI

.[8] E. coli

57

E. coli
E. coli

Eosin mehtylene blue Agar Macconkey
AgarIndol test
-:
10
25

,

24 60

Mueller Hinton Agar E. coli
24
.
-:
20 200
CH3OH %75
24

.E . coli
:
. %75
: Mueller Hinton Agar

40C
15-10 .

(.)12mg\ml 8mg\ml 4mg\ml
: 24
60
Mueller Hinton agar
24-18
.
24
10 1
25

( )1 E. coli
:
] [5 5-4
10

( .)Turbidity Macfarland Standerd
Mueller Hinton Agar

24-18

AMC
) %65( 37
) %35( 20
TE
) %40( 23
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CRO
)% 82( 47
)%2( 1
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CXM
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) %4( 2
) %19( 11
57 E. coli
( )
8 ( ) %14
49 ( ) % 84 ()1
30

20
20
15
15
)Intermed( I )Sensitive( S ) Resistant( R

E.coli ,
E .coli
( )4mg/ml %68
( )8mg/ml %81 E.coli
( )12mg/ml
%88 (. )2
( )2 E. coli
10
1
2 3
0
60 - 75 46 - 59 31 - 45 16 - 30 0 - 15

8mg/ml

12mg/ml

E .coli
S
)%68( 39
)%81( 46
)%88( 50
) %14( 8
)%11( 6
)%7( 4
)%18( 10
)%9( 5
)%5(3
(: )1 ( )
( =.)57
E. coli
. ,

E .coli
Eosin methylene blue agar
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E .coli
.

E. coli
, %82 Ceftriaxone
Cefuroxime %77
)F( Furadentin
, %75 Amoxycillin Clavinic acid
E.coli %65
Tetracycline ,%40 (.)1

mg/ml
4
( ) 3 E .coli

E.coli

4mg/ml

8mg/ml

12mg/ml
) %79( 45
)%9( 5
)%18( 10
)%14( 8
)%91( 52
)%82( 47
) %7( 4
E .coli
( )12mg/ml , %79 ( )4mg/ml
( )8mg/ml ,E .coli

E.coli .
( 4mg/ml )8mg/ml
( )12mg/ml ,% 33E. coli
(. )4
- - 2015
26
( ) 4 E .coli

E .coli

4mg/ml

8mg/ml

12 mg/ml
I
S
R
0
0
)%100( 57
0
)%9( 5
)%91( 52
)%33(19
)%14( 8
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E.coli
E .coli .
E.coli

( )30 _16

].[10

E.coli ] [1

26mm
][4
E.coli
,
, E.coli
%88 12mg/ml
Ceftriaxone
E.coli %82
Cefotaxime
%77
E. coli 12mg/ml %79
] [2
Cefotaxime
, E.coli
Amoxycillin clavulanic acid
%65
4mg/ml %68 Tetracycline
%40
%33 ,12mg/ml
] [3 E.coli
Tetracycline
. E.coli
] , [9
E.coli

.200mg/ml
Allium sativum
Allicine
Scordinin A
A Phytonicidine 57 E.
coli

E .coli
,
E. coli )(CRO
Ceftriaxone )CXM(Cefotaxime
), Amoxycilin and calvonic Acid (AMC

E. coli

AMC , CXM
,
, )TE(Tetracyclinr

E.coli

-1 ( )2011 ,
37 . 2
-2 ( )2011

( )3 ( )2 (. )161-151
-3 ( )2008

.

4- Abubakar,E.M. (2009): Efficacy of crude extracts of garlic
(Allium sativum linn) against nosocomial Eschrichia coli
.Journal of medicinal plants Research vol. 3(4) PP 179-185.
5- Bauer, A.w.;Kirby,W.M. N.; Sherries, J.C.; and
Turck,M. (1966): Antibiotic susceptiobility testing by
standredizd single disk method American.Journal Of clinical
pathology., 45:493-496.
6- Block, E. (1985): The chemistry of garlic and onion. Sci.
Am, 254:114-119.
7- Dausch, J. C. and Diw-Mixon(1990): Allium sativumprew.
Meel., 19:346-361.
8- De Mouy, D.; Armeng, M.; Lefevre, M. and Discamps,
G.(1989) :Recovery rates of urinary tract microorganism in
office practice susceptibility to seven antimicrobial drugs in
cluding the amoxicillin- clavulanic acid combination pathol
.Bial.,37(5):402-405.
9-Garba,I.; Umar,A,I.; Abdulrahman, A.B.; Giyyani, M.
B.; Aliyu, M. S.;Zango, V.V and Muhammad, A. (2013):
pHytochemical AND ANTIBACTERIAL PROPERTIES of
GARLIC EXTRACTS. University Zaria, Nigeria. Bayero.
Journal of pure and Applied sciences., 6 (2) 45-48 (2013).
10-Lucas, M.J. &Cunningam F.G. (1993): Urinary infections
in pregnancy .Clin .Obstet Gynecol.36:855.
11-Rees, L.P.; Minney, N.T.; plammer,J.H. and
skyrme,D.A.(1993):Aguantitative assessment of the
antimicrobial activity of garlic Biotechnol., 9:303-307 .
12-Reuter,H.p.; kock and Lawson D.L.(1996):Therapeutic
effects and applications in: garlic : The science and theThe
rapeutic Applications of Allium sativum L. and related
species. 2nd Ed. Pp.,135-212.
13-Stenquist, K.;Sanberg, G.& Lidinjanson F.(1987):
Virulence factors of E.coli in urinary isolates from pregnant
women J.infect .Dis .156:870-877.
Sclerotinia sclrotiorum
, , ,

Sclerotia

2 10
] [1

] [2

.
: S. sclerotiorum
.

.

.

S. sclerotiorum
24

.
S. sclerotiorum

.

% 7.5 . :

: Sclerotinia
.
sclrotiorum
,S. sclerotiorum : , , .
, ,
Fruits vegetable used in

:this study

] [1
20 .%100 S.
sclerotiorum, S. minor, S.trifolium, S. asari, S. nivalis
] [ 18 S. sclerotiorum

].[18 S. sclerotiorum
$200
1999 $100 ].[ 7
Boland and Hall [8] 1994 S. sclerotiorum
75 294 ( 278 )26% 4045
( )6.9% 408
S. sclerotiorum

.

- / /
- / /
- / /
- / /
19

()Cucurbita pepo ( Cucumis
)astivus ( )Solanumme longena ( )Daucus carota
( )Brassica oleracea ( )Pisum sativum
.
Medicinal plants used in the study:
()1
( )1
S. sclrotiorum
Origanum
spp.
Lamiaceae
Mentha spp.
Lamiaceae
20
:
:Pathogenicity experiments
:Diameter of infected zone
S. sclerotiorum
S. sclerotiorum :
( )Cucurbita pepo( )Daucus carota
( (Brassica oleracea
()Solanum melongena
( )Pisum sativum ( )Cucums sativus
) )cork porer 10

5 7
and
] [9,4
( )
20 1 .
:
:

( %3 )Sodium Hypochlorite

25
( 7 10 )
. (
)Hypocotyl El-Samre at al.
] [11 ] [12 S. sclerotiorum
10 PD 20
5 ( 2010)

) (Cotyledons 20
22 10 .

.
.S. sclerotiorum
.The effect inhibitory of plant extracts on S. sclerotiorum
] [16
10
500 200
Magnetic Stirrer 15

Centerfuge 15
, ( )100
.
Cellulose acetate filter 0.2
.m
S.sclerotoirum (5.02.5
) %7.5 PDA ,
8
20 4
( ) .

. :
A : B
.
Results
Diameter of infected

( )1 S. sclerotiorum

(.)1
( )2
200
126157179

64 94

.
( ) 1 ( )2
S. sclerotiorum
3

2 1

%900
%580
%280

%341
%164
.%133
( )2

S. sclerotiorum
(
) (.)2
33

2
6.035
................... - 2015-
21
4.414
6.920
.

%241

%164
10.52.6 8.93.1
%133 ( )3

7.53.1 6.32.5

5.82.5
.

)
( 3
4.72.2.
24

) .)A-3 48
.

( .)B-3 72

(.) C-3
96

(.) D-3
( )1 S.sclerotiorum 7
( )2 S.sclerotiorum 7

( )2
S.sclerotiorum 7
Histopatholgical Study
S. sclerotiorum
Hypocotyl

Microtome
96 72 4824
.
). (3
()
( )3 S. sclerotiorum (-)A -24

( )4 S. sclerotiorum ( -24

()D -96( -)C -72( -)B -48
)D -96( -)C -72( -)B -48( -)A .
( )2 S. sclerotiorum

( )2 ,

L.S.D.
33
23
9
2
6
30
17.166
2.19

/
()g
6.47
1.46
0.41
0.13
0.29
6.92
2.596
0.017

/
()mm
()mm
8.93.1
5.56
7.53.0
5.05
5.82.5
3.83
4.72.2
3.52
6.32.5
4.73
10.52.6
9.65
5.390
1.02
S.sclerotiorum

( )% 7.5 % 5 % 2.5% 0.0
S. sclerotiorum Sclerotia

% 7.5
%56.92 %74.07
( )% 0.0 ( )3 ( )4 (.)5
% 5.0
%39.66 %62.22
. %2.5
% 48.88 ,

.
( )5( )4 ( )3

22

.
( ) 3
S. sclerotoirum
LSD
0.0
2.5
5.0
7.5
0.0
2.5
5.0
7.5
LSD

()%
66.66
61.31
100
100
66.66
45.75
75.17
100
4.04

()%
43.33
54.96
73.63
79.99
43.33
36.43
49.97
71.91
4.07

()%
16
51.33
69.37
76.12
16
22.97
45.94
29.35
3.87

()%
0
48.88
62.22
74.07
0
0
38.51
56.96
4.41

(%5.0 % 2.5
) % 7.5 S.
.sclerotiorum
S. sclerotiorum

S. sclerotiorum 7.5
% % 5.0 7.5
% % 2.5 5.0
% % 2.5

S. sclerotiorum

] [6;19
] [13
F. oxysporum

Menthol Menthon
] [15; 14

( )Limonene
( )Phellendrene ( )Pinene
( )Terpinol
( )Geraniol ( )Eugernol ( [5] )Linalnol

.

: :
( )4 S.
.sclerotoirum
.1997
-1
, - -

-.
( .)1985
-2
.
995
( .)1981
-3
.395
-4 ( .)2000
- -. 72
: :
( ) 5 S.
sclerotoirum
5- Awadalla, O. (2008). Antifungal activity of some medicinal

plant extracts on different phyto-pathologenic Fusarium spp.
with special reference to controlling wilt disease of tomato
plants, in: N. Egypt. J. Microbiol., 20:190-201.
6- Alyet, A.; Abdel-Kader, D.; Hilal, A.; Atia, M.; and Nada,
M. (2003). Performance of some medicinal and aromatic
plant products, especially water extracts, in controlling
powdery mildew disease of squash caused by
Sphaerothecafuliginea, in: 10th congress of Phytopathology,
Giza, Egypt, p.; 195-217.
7- Boltion, M.D.; Thomma, B.P.H. J.; Nelson, B.D.
(2006).Sclerotiniasclerotiorum (Lib.)deBary: Biology and
molecular traits of cosmopolitan pathogen. Molecularplant
pathology.7.(1):1-16.
8- Bolland, G.J.; Hall, R. (1994). Index of plant hosts of
Sclerotiniasclerotiorum. Canad J Plant Pathol 16:93-108.
____________________________________________
,1 ,
,2 ,
2015- - ...................
23
9- Budage , S.P.; Mcquilken, M.P.; Fenlon, J.S. and Whipps,

J.M. (1995). Use of Coni othyriumminitans and Gliocladium
Virens For Bioligical Control of Sclerotinia sclerotiorum.
10-Chiba, S. and Uozumi, T. (1974). Studies on factors affecting
the occurrence of barn rots caused by Erwiniaaroideae,
Sclerotiniasclerotiorum, Botrytissp. and Botryosporium sp.,
and the preventive affect by transfer of infected tobacco
leaves to unfavourable environment on development of barn
rots.Bulletin of the Morioka Tobacco Experiment Station 10.
141- 166.
11- El-Samra, I.A .; El-Faham, Y.M . and Kamara, A.M.
(1981). Selective induction of infection cushions by
Rhizoctoniasolani in relation to host reponses. Phytopathol. Z,
102:122-126.
12- El-Faham, Y.M. and Aboshosha , S.S. (1987). Formation of
infection chushion by Rhizoctoniasolani Kuhn in relation to
host isolate compatibility. Com. In. Sci. and Dev. Res., 19:
225- 245.
13- El-Shetehy, M.H. (2009). Control of damping off disease of
control. M.sc. Thesis, Faculty of Sci., Tanta Uni., 279 pp.
14- Edinger, P. (1973). How to Grow Herbs, Lane books
Menlopark, California, U. S. A., 64-65. pp
15- Jamil, R.M; Abu-Al-Futuh, I.M; Hasan, M. A.; Al-Essa,
L.Y. and Sheikh I.A. (2001). Antibacterial effects of extracts
from Menthalongifolia, Jordan J. Appl. Sci., 1999-2001: 13:2.
16- Ladd, H.L.; Jacobson, M. and Buriffim, C. (1987). Beetles
extract prom Neem tree as feeding Deternts. J Econ. Entomol
.71:803-810.
17- Mazen,
M.M.
(1995).
Pathological
studies
on
SclerotiniasclerotiorumAffecting some legume crops. MSc.
Thesis, Fac. Of Agric., Cairo University , 112pp.
18- Purdy, L.H. 1979. Sclerotinia sclerotiorum: history, diseases
and symptomatology, host range, geographic distribution, and
impact. Phytopathology 69:875-880.
19- Seyed, M.R.;Gholamreza, Z. and Ghade, M. (2012). The
Evaluation of some Biological Activity of MenthaLongifolia
(L) Huds Growing wild in Iran. Pharmacologia. 3:535-538.
:

,
.
2.2 :Even Function

)(
:
: - - -
- - - -
.
;)() = (

1807 ,

.

.

_
.
+
)
( () = +
=1
, ,

.

)( -:
x
2x
x
2x
+a 2 cos
++b1 sin b2 sin
L
L
L
L
A+a 1 cos
2.3 : Odd Function

)(
)(
; )(() =
2.4 : Periodic Function
)(
; )( = ) ( +
> 0
)()( period
)(
.
( + ) = () ,
( ) Piecewise Continuous
[1],[3] :Function
] : [, ] [,
:
- )( ] [,
1 , 2 , , < < < < 1 < 2

). . . (1
+
)
( +
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) (, 1 ), (1 , 2 ), , ( ,
, , ()1
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-2 [1],[2],[3] :
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2.1 Trigonometric Fouries

:Series
)( ] [ ,
)( )( ] .[ ,
27
28
([1],[2],[3] -: )1
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)( )( () = 2 0
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2
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= 0
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n=1,2,..
2
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=
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)( = )(
=1
1
1
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2
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n
-
n
n
= )(
(1)2
)(
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.
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: () =

= )() = (
(=
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sin nx dx a n = a 0 = 0
ex -e-x
2
, bn = 0 sin hx sin(nx) dx = 0
1
] )( = [ () =
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0
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=1
1
1
1

] = 2 [ 2 + 3 4 +
+1
(1)+1
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)(
)(1
2
3
4
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=
. 2
(2 + 1
+1
)( ) (, .2
:
28
29
.................... - 2012
.
] [, + 2
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2
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(1)+1
=
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+1
=1
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1
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0
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) (,
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: 3
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{ = )(
< < 1 ; 2
1 +2
() () ,

;n
-2(-1)k-1
;n=2k-1 , k=1,2,
(2k-1)
+2
n=1,2,
] [ , +
:
+
1
(),
1 +
() (),

+
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0
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7
1
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+
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][3, 3
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2
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2
2
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sin nx |

n
2
2
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= )(
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2
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=1
30
+2
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0
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2
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])
2
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1
)( )(
=1
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2
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(),
1
1
n
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sin
n
2
( )( =
)
c+2
1
1
bn= f(x) sin(nx) dx = [ 2 sin (nx) dx + sin(nx) dx - sin(nx) dx
3
)2(1
( ) +
= )(
:
2
=
)(
2
) (4 2 + 2 2
:
4
])([1
) + 2 2
(42 2
)(
)sin h(km
1
2nx
=)f(x
( +2mk sin h(km) n=1 2 2 2 2 cos
) +
m
- 6 ) [3]: (, +
4n +m k
2
(
])
(42 2 + 2 2
mk
[ ])(4[1
(-: )3
)(
-7 [1],[3] :convergence of Fourier series

0
2
2
= )(
( [ +
( ) +
])
2
=1
)(
)( ] [-L,L
) 0 (,:
0 )(
)( . 0
0 )(
)( :
2
() ,
=1
= 0
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= ) (0
2
2
2
( )( =
) , = 1,2, ,
2
2
( )(
) , = 1,2,
= ; =
:
)( () + +
= )() = (
2
.
)( () = :5
) (0,

)( )(
30
)(
31
.................... - 2012
:
2
( +
( ) +
)
2
= )(
1
nx
1
nx
-1
n
(a n = f(x)cos
()dx= f(x)cos
=)dx
(sin
),
L
L
2
2
n
2
-2
=1
-L
1
nx
1
nx
1
)( = ) ( = n
(bn = f(x)sin
()dx= f(x)sin
=)dx
][ cos ( ) -2 cos(n) +1
:
L
L
2
2
n
2
-2
0
)(
)(
( [ +
( ) +
])
2
)( :
= )(
=1
( )
a0
= +
])n=1 [a n cos(n)-bn sin(n
) +
=1
)
+
)=1 (1
0
2
-L
( ) (
() =
=1
( 2 () + 1
= :
: )( )(2,2
0
)(
)(
( [ +
( ) +
])
2
)( = 1; = 0 :
= )(
() = 1 () = 0
=1
0+
a0
= +
])n=1 [a n cos(n)+bn sin(n
2
+
)=1 (1
1+ () = 2 1 () = 1
0
2
)( " "
:
; = =
:
+
)=1 (1
0
2
}) = {(2,0) (0,1) (1,2
= )() = (
: )( :
: )( ][2,2
0; 2 < 0
() = { 1; 0 1
2; 1 < 2
: = 1; = 0
= 0 :
: )( ,2L=4 .L=2
+ () = 1; () = 0
0
)( :
:
= )(
( +
( ) +
)
2
11
= 0,
=1
( +
( ) +
)
2
2
2
2
1
1
3
= )( = )(
2
2
= +
)( (0 + ) +
0
(0) = +
0
=1
00
=0
= +
0
)( (0 + )
0
(0) = +
0
) (0 ) (0 )(
; = 0 = 0
:
= 0
32
+ () + () 1 + 0 1
0
(0) = 0
=
=
2
2
2
; = 1:
1- Donal (1990 ) Applied Partial Differential Equations:

PWS. D.W. TRIM, The University Of Manitoba
() = 2; () = 1
1+

22
= +
11
= +
)f(1+x)- lim+ f(x

x1
2- Mathematical Methods For Physicists, George Arfken,

Miami University, Oxford, Ohio. 3rd ed,1985, Academic
press
(1) = +
0
3- Modern Mathematical Analysis Prother. Morry H.

Protter _ University of California _Berkeley, California and
Charles 4- B. Morrey, JR_ University of California
_Berkeley, California,1964,Addison-Wesley.
= 0,
)( (1 + )
1
(1) = +
=0
) (1 ) (1 )(
; = 1
= 1 :
0
+ () + () 2 + 1 3
1
(1) = 1
=
=
2
2
2
=
1; = 0
3 1
, .
2 2
:
= 2; = 2
() = 0; () = 2
2+
:
0-0
=0,
x
= lim+
2-2
=0
-x
)f(-2+x)- lim+ f(x
x0
= lim+
x0
x2
x
)f(2-x)- lim- f(x
x2
-x
= 0
2
2
= 1 1
. = 2; = 2
fR' (-2)= lim+

x0
fL' (2)= lim+

x0
) (1 ) (1 )(
; = 2
= 2; = 2 :
+ () + () 0 + 2
2
(2) = (2) = 2
=
=1
2
2

}) {(2,0) (0,1) (1,2
32
, -
5
,1 ,2 3, ,4
: :
( ,)Cr ( ,)Mn ( ,)Fe ( ,)Co ( ,)Ni
( ,)Cu ( ,)Zn ( )Cd ( )Pb
.

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atomic
plasma
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Microwave
.)spectrometer
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.
4-1 imsa242171@gmail.com
5 s_elw1970@yahoo.com
(.)2(,)1
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:
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33
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.
( :)1

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Periforms
Mugiliod
Mugilidae
:

. 100
,
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1.0
1.4
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0.8
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1.2
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0.8
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1.5
( )2 ( ) Muscles
( )6
250 3
(Glass
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. 3
.
250 .
( Microwave Plasma,
)Atomic Emission Spectrometer, Agrilent 4100
:
( ,)Cr ( ,)Mn
( ,)Fe ( ,)Co ( ,)Ni ( ,)Cu
( ,)Zn ( )Cd ()Pb (
) .

( )W.H.O, 1985 IAEA -
) 407 (2OO3 (.)3
( )4 ( )5
( .g/g ) ( )4 ( )5

( )5 ( )6 .
0.5
100.0
-
0.1
0.6-0.5
-
3.0
75-10
-
IAE
A407
(2OO
)3
2.0
WH
O
(1985
)
1-0.5
( )4 ( ) Liver
C
r
Mn
Fe
C
o
Ni
C
u
Z
n
C
d
P
b
( )3 ( ) Gills
()3 g/g
2.0
34
- - 2015
35
( )4
160
140
120
100
80
60
40
20
Fe
Ni
Zn
Cd
48.23 0.53 14.33 0.55
148.62 0.23 19.58 0.33

35.08 0.07 8.66 0.5
( )Mean STD
g/g
Cr
1.63 1.14
0.33 0. 41
1.58 0.31
Mn
5.01 0.36
1.97 0.36
3.4 0.84
Co
0.94 0.64
0.56 0.17
0.14 0.16
Cu
3.59 0.56
8.66 6.00
2.88 0.45
Pb
17.08 8.29
6.19 1.65
17.57 3.40
( )6
( )5 ( )6

( )
(.) 8
20
10
Cr
Mn
Co
Cu
Pb
1.63 5.01 0.94 3.59 17.08
0.33 1.97 0.56 8.66 6.19
0.14 2.88 17.57
3.4
,

,
.
1.58
( )5
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, ,

( )Bioindicator
. ( )5
,

] [ 7
.
.
( )5
Fe Pb Zn Mn Cu Cr Co Cd Ni
Fe Zn Cu Pb Mn Co :
Cr Cd Ni Fe Pb :
Zn Mn Cu Cr Cd Co Ni
.

(.)11()9()5
SPSS
. ( )6 ()9( )8( )7

( )6

Cr-Co, CrNi
( )Mean STD g/g

Cr-Mn, Cr-ZnCrCd
Mn-Zn
Mn-Co, Mn-Cu
Mn-Pb, Mn-Fe
Mn-Ni, Mn-Cd
Fe
48.23
16.63
148.62
30.86
35.08 9.47
Co-Zn
Ni
0.53 0.40
0.23 0.27
0.07 0.14
Co-Cu,
Co-Pb CoFe, CoCd
Zn
14.33
4.64
19.58
4.206
8.66 1.128
Cd
0.55 0.05
0.410.33
0.50 0.09
35
Cu-Ni
Cu-Zn, Cu-Cd
Pb-Ni
Fe-Ni
Ni-Zn, NiCd
Pb-Zn, Pb-Cd
Fe-Zn, Fe-Cd
Zn-Cd
Cr-Cu,
Cr-Fe
Cr-Pb
Co-Ni
Cu-Pb,
Cu-Fe
Pb-Fe
(9) Namin J.I., Mohammadi M., Heydari S., and

Rad M., (2011). Heavy Metals Cu, Zn, Cd and
Pb in Tissue, Liver of Esox Lucius and Sediment
from the Anzali International Lagoon- Iran,
Caspian J. Env. Sci., Vol. 9 No.1 pp. 1-8
(10) Al-Weher M. S., (2008). Levels of Heavy
Metal Cd, Cu and Zn in Three Fish Species
Collected from the Northern Jordan Valley, Jordan,
Jordan Journal of Biological Sciences, Vol.1, No. 1,
pp. 41 46.
(11) Safahieh A., Taghi M. R., Monikh F.A.,
Savari A., and Doraghi A., (2011). Heavy metal
Concentration in Belanger's Croaker Fish,
Johniusbelangerii from Petrochemical Waste
Receiving Estuary in the Persian Gulf, Iran, 2 nd
International Conference on Environmental
Engineering and Applications, Singapore.
250
200
150
100
50
0
g/g
Cr
Mn
Co
Cu
Pb
Fe
Ni
Zn
Cd
)1) VandenBroek, J. I.; Gledhill, K. S.And Morgan, D.

G. (2002). Heavy Metal Concentration in the Mosquito
Fish Gambusiaholbrooki in the Manly Lagoon
Gatchment. In: UTS , Freshwater Ecology Report 2002
Department of Environmental science, University of
Technology , Sydney.
(2) Gulfraz, M.; Ahmad , T. And Afzal, H. (2001).
Concentration Levels of Heavy and Trace Metals in the
Fish and Relevent Water from Rawal and Mangla Lakes.
Online Journal of Biological Science, Vol1 , No,5, pp
414- 416.
. ,) 3(
.)2000( . .
, ) 4(
.)2007(
Heckel Aspiusvorax Heckel Barbusluteus
Heckel Barbusgrybus
Hypophthalmicthyesmolotrix Richardson
, ,
.
(5) Aktaruzzaman, M. ,Chowdhury M., Fardous, Z. ,
Alam, M. , Hossain, S. and Fakhruddin, M.
Ecological Risk Posed by Heavy Metals Contamination
of Ship Breaking Yards in Bangladesh.,Int. J. Environ.
Res., Vol, 8,No2,pp 469-478.
(6) Denton, G.R.W. and C. Burdon-Jones (1982).
Environmental Effects on Toxicity of Heavy Metals to
Two Species of Tropical Marine Fish from Northern
Australia. Chemistry in Ecology,Vol, 2,pp 233-249.
)7) Erdem, C., 1990. Cadmium accumulation in liver,
spleen, gill and muscle tissues of Tilapia nilotica (L.).
Turk. J. Biochem., Vol. 15,pp. 13-22.
(8) Nicolas R. Bury, Paul A. Walker and Chris N. Glover,
(2002). Nutritive Metal Uptake in Teleost Fish, The
Journal of Experimental Biology Vol.206,pp. 11-23.
36
) 7(
g/g
250
200
150
100
50
0
Cr
Co
Cu Fe
Ni
) 8(
g/g
250
200
150
100
50
0
Cr
Cu
Pb
Fe
) 9(

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350
300
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150
5
6
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362.7 5.21
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1.200 13.33
303.9 5.14
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0.933 13.33
387.3 5.29
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0.670 12.86
382.3 5.29
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1.170 13.86
313.7 5.14
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0.047
0.700 12.62
352.9 5.29
0.415
0.062
0.733 12.86
40
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-1 , ( )2014
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-16 , , .
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Journal of
Science
Biannual Refereed Academic Journal
Issued by
The Faculty of Science
(Vol. 2)
(Issue 2)
( August 2015)
Journal of Science
Vol. (2) Issue (2) August-2015
Correspondences:
Journal of Science,
Faculty of Science,
Misurata University,
PO Box 2478,
Misurata, Libya.
Email:
journalofscience2013@gmail.com
Journal of Science
Contents
No.
1
Topic
Comparison of Microscopy and Culture Methods for Detection of

Blastocystis hominis in stool samples
Mona Radwan Aboalgasm1, Mohamed Chibani Mohamed1, Abdul
Hafeez
2 and Salem Ramadan
Study of ColorKhan
Change
in Softening Plant Distribution
Network at
Libyan Iron and Steel Company
K.A.B.Najm,1* M.O.Al-Qubbi2, K.A.K.Abo-Zakia3
The Effect of Rheb (Ras) Homolog Enriched in Brain
Expression in Claw Muscle During the Molt Cycle in the
Land Crab, Gecarcinus Lateralis
Ali Moftah Abuhagr 1*Kyle S. MacLea 2Donald L. Mykles 3
Evaluation Dimension of Permanent Teeth Size in MisurataLibya
Adnan M . Hmeida * . Abushahma,S.M ** and Ahmed k. Ben
Omron ***.
Antifungal Potential in Crude Extracts of Five Selected Brown
Seaweeds Collected From the Western Libyan Coast
1Daghman, I. M.;2Khallil, A. M. and 1Fady, A. A.
Highly Production of Omega -3 Polyunsaturated Fatty Acid by
Developed Fermenter Strategy and Design
Thureia. A. El-Sharif
Page
5
11
13
18
22
27
Journal Of Science
Issued by the Faculty of Science
Misurata University
Comparison of Microscopy and Culture Methods for Detection of Blastocystis hominis in stool samples
Mona Radwan Aboalgasm1, Mohamed Chibani Mohamed1, Abdul Hafeez Khan2 and Salem Ramadan
3
Abstract A Sariti
total of 360 stool samples were randomly collected
form patients, presenting different sexes and ages (121 males and
239 females), residing in Wadi Al-shati province. All specimens were
examined by direct smear microscopy, concentration method and two
xenic culture systems (Diphasic Boeck and Drbohlav's and
Monophasic Jone's medium) to detect Blastocystis hominis. The
results shows the low sensitivity of direct smear microscopy (11.29%)
compared to concentration method (15.5%) and in-vitro culture
methods (22.60%). There was significant difference (p<0.05)
between direct smear preparations and the two xenic culture systems
for the detection of B. hominis. There was an almost equal numbers
of positive samples in both culture techniques (85 samples in diphasic
medium and 78 in monophasic medium). Diphasic
Boeck
and
Drbohlav's medium produced highest numbers (7.6006.379) of B.
hominis cells compared to Monophasic Jone's medium (5.0514.938)
after every passage cultures and only the vacuolar morphologic type
of this organism was found in both culture systems. Moreover, a
larger size of vacuolar stage of B. hominis detected in Diphasic
Boeck and Drbohlav's medium, than in Monophasic Jone's medium.
In vitro cultivation does seem worthwhile in the detection of B.
hominis in diagnostic laboratories. Of all the diagnostic techniques
used, diphasic Boeck and Drbohlav's medium was the most sensitive
method for detecting B. hominis in stool specimens.
diagnostic techniques direct smears microscopy, formalinether concentration, and two xenic culture systems (diphasic
Boeck and Drbohlav's medium, and monophasic Jone's
medium) for the detection of Blastocystis hominis in stool
samples. The two xenic media (diphasic and monophasic)
were used to investigate, which culture medium could be more
efficient and suitable for diagnosis of B. hominis in laboratory.
Materials and Methods
During the period of October 2010 to end of June
2011, a total of 360 stool specimens, presenting different sexes
and ages (121 males and 239 females, aged from less than one
year to 90 years) were freshly collected from patients, who
routinely submitted their stool samples for parasitological
analysis to Brack Hospital and Medical Technology
Department, Faculty of Engineering and Technology, Brack.
Each sample was processed and examined
immediately after collection using direct faecal smears
(Normal saline, Iodine, Eosin) [5]. Soon after direct smear
microscopy, the samples were concentrated by formalin-ether
sedimentation technique as described by [5 and 6]. All the
collected faecal specimens were inoculated and cultured into
two culture systems upon arrival at the laboratory, at the same
time after the routine (direct smear examination) in
Monophasic Jone's Medium and Diphasic Xenic System
Boeck and Drbohlav's inspissated egg medium (B-D) LockeEgg Serum (LES) Medium as described by [11] and [37]
respectively. The results for positive samples of B. hominis
of the detection techniques were expressed as percentages, and
statistical analysis was carried out by using chi square test. A
probability (p) value of less than 0.05 was considered as
significant whenever appropriate.
Key words: Prevalence, Blastocystis hominis and Detection methods.
Introduction
Blastocystis hominis is a singlecelled enteric protozoan that
has a world-wide distribution [27]. The wide range in
prevalence of B. hominis seen between countries can be
attributed to several factors such as socioeconomic conditions,
also to the different diagnostic methods used for the detection
([21]. The most common diagnostic technique used worldwide
for identification of Blastocystis is the permanent stain. The
use of xenic cultures, in which Blastocystis is grown in-vitro
with non-specific microorganisms, has been shown to be more
sensitive in detecting this organism, but it is not commonly
used in the diagnostic laboratories [13, 29 and 34]. In Generd,
the diagnosis of human infections with B. hominis is usually
based on detection of its typical vacuolar form in a light
microscopy of faecal samples, directly, either as a
simplesmear or after some form of concentration. Since, this
organism is the most frequent isolate in human stools in Libya
[2; 3 and 8]) and so far only two studies have been carried out
to compare sensitivity of direct smear, concentration and xenic
culture for the detection of B. hominis in stool specimens [8
and 22] , therefore this study was aimed to compare four
1-Medical Department, Faculty of Engineering and Technology,

2-Faculty of Medicine, Sebha University, 3-Libyan Academy,
Misurata
To whom reprint requests should be addressed. e-mail:
salemsrs@yahoo.com.
5
Results
The results of comparison of diagnostic techniques
showed 11.29 , 15.5 , 21.6 and 23.61% positive rates for
B.hominis in stools by directed smear microscopy,
concentration, Jone's medium and Boeck and Drbohlav's
medium respectively. All the stool specimens found positive
in direct smears, were also found positive in concentration,
and both culture methods (40 of 360). Eighty-five (23.61%)
samples were positive in one or more of the
diagnostictechniques. Direct smear microscopy, and
concentration showed significantly low sensitivity (p<0.05)
compared to both culture techniques for identifying B.
Journal of Science
hominis, meanwhile there was significant difference (p<0.05)

between direct smear preparations and the two xenic culture
systems for the detection of B. hominis. No significant

difference (p>0.05) was found between monophasic Jone's
medium and diphasic Boeck and Drbohlav's medium (Table
1).
Table 1: Comparison of methods for the detection of B. hominis in stools.

Samples
examined
Number and percentage of positive samples by different methods

Direct smear microscopy 122 (11.29%)
Concentration
Culture
Normal saline
Iodine
Eosin
Formalin-ether
sedimentation
Monophasic
Jone's Medium
Diphasic Boeck and

Drbohlav's Medium
34 (9.44)
45 (12.5)
43 )11.94)
56 )15.5(
78 )21.6)
85 (23.61)
360
Figures in parentheses indicate percentages.Of all the

diagnostic techniques used, cultivation of stool samples was
the most sensitive method for the detection of B. hominis.
There were 275 stool samples found to be negative
(represented 76.39% of the total samples) and 40 of 360
(11.11%) were found to be positive by all used methods
(direct smear microscopy, concentration and culture
techniques. 14 (3.8%) samples gave negative results in direct
smear microscopy only but were found positive in both
formalin-ether concentration, and culture methods. 22(6.1%)

stool samples found positive in culture techniques only, but
were negative in both direct smear microscopy, and
concentration method. Nine stools samples (2.5%) were
negative in concentration only, but were found positive in both
direct smear microscopy, and culture techniques. There was
no stool sample, which showed negative results in culture
methods, but was found positive in both direct smear
microscopy and concentration (Table 2).
Direct smear
(+Pos)
Concentration
(-Neg)
Culture (+Pos)
Direct smear
(+Pos)
Concentration
(+Pos)
Culture (-Neg)
275
Direct smear (Neg)

Concentration
(-Neg)
Culture (+Pos)
Number of samples
Detection efficiency (%)
Direct smear
(-Neg)
Concentration
(+Pos)
Culture (+Pos)
Methods
Direct smear
(-Neg)
Concentration
(-Neg)Culture
(-Neg)
Status
Direct smear
(+Pos)
Concentration
(+Pos)Culture
(+Pos)
Table 2 : Comparison of the Detection efficiency (%) of direct smear microscopy, formalinether Concentration
and culture methods for the detection of B. hominis.
40
11.11
14
3.80
22
6.10
9
2.50
0
0.00
(+Pos) = Positive, and (- Neg) = Negative

Growth profiles of B. hominis in monophasic and diphasic
cells compared to monophasic Jone's medium ( 5.05 4.94)
but this difference was not statistically significant (p>0.05).
medium are shown in Table 3. Diphasic Boeck and

Drbohlav's medium produced higher numbers (7.60 6.38)of
Table 3 : Growth profile of B. hominis in Monophasic Jone,s Medium and Diphasic Boeek Drbohlav,s Medium
Culture Methods
Number of B. hominis cells

P. value
Range
Mean SD
Monophasic Jone's Medium
78 )21.6)
2-20
5.054.94
Diphasic Boeck and Drbohlav's Medium
85 (23.61)
3-25
7.606.38
Figures in parentheses indicate percentages

.
Number and Percentage of

B. hominis cases
>0.05
Comparison of Microscopy and Culture Methods
Discussion
Blastocystis hominis is a common human intestinal protozoan,
reported in children and adults in developing countries [19].
Diagnosis of B. hominis public health centers, and clinical
laboratories is mostly made by the demonstration of typical
vacuolar form (approximately 10 to 15m in diameter), with a
large central vacuole and 1 to 4 nuclei in the peripheral
cytoplasm. The small forms of B. hominis like multivacuolar,
avacuolar, and cysts are not used for detection in the routine
microscopy, which are also present in the stool samples and
usually are missed during laboratory examinations in direct
smear microscopy. Moreover, asymptomatic infections are

also common in the communities and no doubt these cases are
frequently undetected or underestimated [13]. Missed
diagnosed patients or shedding of B. hominis from
asymptomatic cases may be a vast potential source of infection
humans in the region.
So far only two studies have been carried out in Libya to
compare sensitivity of direct smear, concentration and xenic
culture for the detection of B. hominis in stool specimens
(Table. 4).
Table. 4: Comparison of the prevalence and diagnostic methods used for the detection of Blastocystis hominis in Libya
Reference
Locality/Category
Detection Methods (Prevalence %)

Direct
smear
Concentration
18.55
[24]
Patients attending Central

Laboratory in Sebha.
Libyan patients in Sirte
[23]
[2]
[22]
[25]
[12]
[3]
[9]
[8]
Present study
Culture Techniques
ND
Jone's
Medium
ND
Boeck and Drbohlav's

Medium
ND
26.21
34.1
ND
42.31
29.6
ND
ND
ND
School children in Derna
6.7
ND
ND
ND

Children and neonates admitted
to Ibne-Sina Hospital in Sirte.
Community population in Wadi
Al-Shati.
Raudam Poplation of Rural ares
of Wadi Al-Shati.
Food handlers in Sirte
22.69
ND
ND
ND
12.57
ND
ND
ND
18.3
21.2
ND
ND
20.21
ND
ND
ND
14
21
ND
35.5
Patients attending Brack

Hospital and Medical
Laboratory Department, Brack,
Wadi Al-Shati.
11.26
15.55
21.66
23.61
ND = Not Determined
A prevalence of 26.21%, 34.10% and 42.31% by
using
direct smear, concentration and Boeck and
Drbohlav's Medium respectively were reported from
patients attending Central Laboratory in Sebha [22],
however prevalence of 14.0%, 21.0 % and 35.5% by
direct smear, concentration and Boeck and Drbohlav's
Medium respectively were reported from food handlers in
Sirte [8]. A prevalence of 18.30% and 21.20% in
community population in Wadi Al-Shati were reported by
using direct smear and concentration respectively [3]. AlFellani et al. [2] reported prevalence of 18.55% in patients
attending Central Laboratory in Sebha, Salem et al. [24]

reported prevalence 29.6% in Libyan patients in Sirte,
Sadaga and Kassem [23] reported prevalence 6.7% School
children in Derna , Saleh [25] reported prevalence 22.69%
in patients attending Central Laboratory in Sebha, Kassem
et a.l [12] reported prevalence 12.57% in Children and
neonates admitted to Ibne-Sina Hospital in Sirte , Gelani et
al. [9] reported prevalence 20.21% in patients attending
Brack Hospital and Brack Medical Laboratory by using
direct smear method only (Table. 4).
Journal of Science
In the present study, the culture techniques

(diphasic Boeck and Drbohlav's medium and monophasic
Jone's medium) detected significantly (23%) infection of B.
hominis than direct smear microscopy (11.27%) (Table 1).
This finding is similar to results of [8; 17; 35 ; 37], who
reported that culture method (Boeck and Drbohlav's
medium) was more sensitive than microscopy direct smear,
and /or permanent stained smears of stool specimens. This
observation is also similar to [34, 32; 28, 33], who found
monophasic Jone's medium most effective than direct
smear microscopy, and / or concentration technique.
Similarly, Dogruman et al. [7] compared direct smear
microscopy in iodine stain, permanent stained smears in
trichrome, immunofluoresence assay and monophasic
Ringer's culture medium for the detection of B. hominis in
stool samples. Moreover, Roberts et al. [21] compared the
sensitivity of diphasic Boeck and Drbohlav's and
monophaisc tryptone, yeast extract, glucose methionine9
medium with permanently stained smears using a modified
ironhematotoxylin stain. Results showed low sensitivity of
permanent stained smears compared to two culture systems
(Table 1).
The results demonstrate that both culture methods
(diphasic Boeck and Drbohlav's and monophasic Jone's
medium) detected almost equal number of positive samples
for B. hominis (Table 1). The results are in accord with
[21], who reported that both diphasic Boeck and
Drbohlav's medium and monophasic tryptone, yeast extract,
glucose, methionine-9 medium) showed equal effectiveness
for the detection of B. hominis in clinical stool samples.
The results from this study also found higher growth profile
of
B. hominis in diphasic medium compared to
monophasic medium (Table 3). Similar observation has
been reported by Roberts et al. [21], who observed high
numbers of B. hominis cells growth in diphasic medium
than monophasic medium. The increased in the numbers of
positive samples of B. hominis using stool culture methods
may be attributed to the organism needing more time to
grow and replicate and appeared to be due to change of cyst
and multivacuolar forms into vacuolar forms during
cultivation of stools (as mostly vacuolar stages were seen in
both culture systems). Moe et al. [35] also observed that
cyst forms of B. hominis isolated from human faeces,
developed into a large number of vacuolar forms in short
term in- vitro culture.
Increased in numbers and size of this organism was also
observed during the culture of stool samples (Table 3).
These results were the same as those [4; 13;16; 26 and 35].
Although
the examination of direct wet smears is
convenient and inexpensive, but it frequently leads to a
false-negative results. The main problem with simple direct
smear is that small numbers of B. hominis are present in
the stool samples may go undetected or at least
unrecognized.
The better sensitivity, and higher yield growth profile of B.
hominis in diphasic Boeck and Drbohlav's medium may
be due to that of egg slant of this medium, providing more
surface area for the growth of this organism. The results
showed that concentration method was found relatively
more sensitive (15.5%) than the average of direct smear

microscopy (11.27%) (Table 1).This increased in the
detection efficiency of B. hominis in concentration method,
compared with direct smear microscopy is probably due to
presence of small numbers of B. hominis cells in the faecal
specimens, which are missed during routine direct smear
microscopy. The results are in accordance with [1; 10; 14;
20; and 30], who reported that concentration methods are
beneficial, compared to direct smear microscopy. However,
others have reported that concentration methods have no
advantages over direct smear microscopy for the detection
of B. hominis in the stool specimens [13; 15;18; 29 and 31].
They assumed that low detection efficiency appears due to
necessary steps of shaking and centrifugation in formalin
ether technique that lead to rupture of the vacuolar,
multivacuolar and granular forms of B. hominis during the
procedure.
In the present study, there is also increased in the positive
samples of B. hominis in concentration negative stool
samples in both xenic culture methods. This may be due to
presence of smaller forms (other than vacuolar) of B.
hominis in the faecal materials, which successfully grow,
and multiply in diphasic and monophasic medium.
Similarly, [13], [29] and [31] also reported that stool
specimens found negative in formalinether concentration
technique were found positive for B. hominis in stool
culture.
Conclusion
This study demonstrated that B. hominis detection
efficiency was found to be more in culture methods,
followed by faecal concentration in formalinether
sedimentation and direct smear microscopy. Similar
observation have been reported by others[13; 22; 31 and
33]. These workers have reported that in-vitro cultivation of
faecal material is effective and had advantages over direct
smear microscopy, and concentration method.
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Thathaisong. U. and Murgthin, M. (2002): In-vitro
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Journal Of Science
Misurata University
Study of Color Change in Softening Plant Distribution Network at
Libyan Iron and Steel Company
K.A.B.Najm,1* M.O.Al-Qubbi2, K.A.K.Abo-Zakia3
AbstractThe effect of color change in drinking water was studied
through the measurments of of some physicochemical paramters

,such as color, turbidity and iron. The results of analysis appeared
that the measured parameters in some samples were excceded the
acceptable standard limits. This indicated the cause of color change
in drinking water.
Water samples collection was processed according to APHA

standard[5]. In this study 24 samples of drinking water were
taken from four different sites in water distribution network,
one in each site for 6 months from January to June.
Some physicochemical parameters such as, color, turbidity
and Fe were determined according to procedures in APHA
standard[5]. Turbidity measured by turbid-meter model
2100A.Colorand Fe measured by colorimeter DR 890.
Keywords-Color change , Drinking water, Iron , Softening Plant,

Libyan Ironand Steel Company.
Introduction
Results and Discussion
Degradation of drinking water quality in distribution networks

represents a problem forwater supply in urban area.
Color in drinking-water is usually due to the presence of
colored organic matter (primarily humic and fulvic acids)
related to the humus fraction of soil or the presence of iron
and other metals, either as natural impurities or as corrosion
products[1].
Turbidity in drinking-water may be due to the
presence of inorganic particulate matter in some groundwater
or sloughing of biofilm within the distribution system. High
turbidity value can protect microorganisms from the effects of
disinfection thereby can stimulate bacterial growth[1].
The residual iron in drinking water can react with
disinfectants (neutralizing them)and the presence of residual
iron in drinking water can also cause proliferation ferruginous
bacteria sources of soft deposit. Growth of ferruginous
bacteria can reduce the diameter of the pipe or cause corrosion
[2]. Ochre deposition can cause a seriously draulic clogging
problem in drinking water network. The problem increases
when the iron in the pipeline may be incrystalline form or
amorphous sform. Dehydration of amorphous formas soluble
Fe(II), is oxidized to insoluble Fe(III) either biotically via
bacteria, or abiotically [3]. Oxyhydroxides of iron formed
insoluble Fe(III) either biotically via bacteria, or abiotically.
Oxyhydroxides of iron formed gives crystalline form. The
crystallization is a complex electrochemical and
physicochemical phenomenon between the network surface
and water composition.
In oxygenated medium, the divalent iron (Fe2+) and in
slightly alkaline medium the trivalent iron (Fe3+) take the form
of ferric oxide Fe2O3 with rust-colored or ferric hydroxide
Fe(OH)3. These forms are insoluble. Ferric oxide crystallizes
as Fe2O32H2O,or likeFe2O3H2O. The increase in the iron and
turbidity in drinking water would be attributable to the release
of soft deposits into bulk water [4].
1-K.A.B.Najm, Collage of Education - Al-Asmarya Islamic

University Zletin, PH-00218925504107. E-mail: :kabnch@hotmail.com
1- Collage of Education - Al-Asmarya Islamic University Zletin
2-Collage of Education - University of Misurata-Misurata
3-Libyan Iron& Steel Company-Misurata
The results of measurements are shown in Table(1)
.Color value of 8.6(Pt-unit) was highest at Mechanical

Building with bright brown, while the lowest value of 0.5 (Ptunit) was recorded at water exit of Store Containers with no
color and transparent. The average value ranged from 5.6 (Ptunit) in Mechanical Building and 0.8 (Pt-unit) for Store
Containers. The values at Central Laboratories and
Maintenance Building exceeded the permissible limit of 0-5
(Pt-unit) with change the color of water samples to bright
brown.
The values of turbidity of all measured samples were
below the permissible limit of 0-5 NTU, except at
Maintenance Building of 7.6 NTU and Central Laboratories of
6.0 NTU . The high value of turbidity higher than 5.0 NTU
can protect microorganism from effects of disinfection and
can stimulate bacterial growth.
Iron values of all samples in studied sites were below
the permissible limits of 0-0.3 ppm, except the Mechanical
10
Study of Color Change in Softening Plant

Building of 0.46 ppm. The increase Iron in few samples
,revealed that Iron corrosion in a form of soluble molecules or
suspension in drinking water causing the red color .In this case
the Iron precipitated with time in the form of red hydroxide
inside water taps. Increasing Iron over 0.3 ppm become
unplearsentand cause water hardness, which causes what is
known as Haemo Chromatists, leading in this case destruction
of tissues due to accumulation of Iron[6].Also ,color can be
strongly influenced by presence of Iron due to corrosion.
References
1- World Health Organisation. " Drinking Water Standards and
Health Advisories Office of Water U.S. Environmental Protection
Agency Washington", 2011.
2- Desjardins, C. B. Koudjonou and R. Desjardins"Laboratory
study of ballasted flocculation," Water Research, 36, 2002, pp. 744754.
3- Kuntze,H. "Iron clogging in soils and pipes". Analysis and
treatment. Bulletin No. 10 of
4-The German Association for Water Resorurces and Land
Improvement (ed.Kuntze.H)Verlag Paul Parey, Hamburg, 1982, pp.
1-22.
5- M.T. Lehtola, T.K. Nissinen, T.I. Miettinen, P.J. Martikainen
and T, Vartiainen"Removal of 6- soft deposits from the
distribution system improves the drinking water quality,"
WaterResearch, 38, 2004, pp. 601-610.
7- APHA (2005)."Standard methods for examination of water and
wastewater", 21st.ed., American Public Health Association
Publication, Washington DC, USA,
8- A.Jacobs, )1997): Iron overload-Clinical and Pathological"
Seminaryin Hematology,pp14-89.
11
Journal Of Science
Misurata University
The Effect of Rheb (Ras) Homolog Enriched in Brain Expression in Claw Muscle During the Molt Cycle
in the Land Crab, Gecarcinus Lateralis
Ali Moftah Abuhagr 1*Kyle S. MacLea 2Donald L. Mykles 3
Abstract: Muscle atrophy in crustaceans is necessary for the

progression of molting and coordinated by the action of ecdysteroid
hormones. The insulin/metazoan target of rapamycin (mTOR) pathway,
previously shown to be important for growth, development,
reproduction, and metamorphosis in insects, is a likely target of
ecdysteroid regulation. As complete cDNAs for Rheb the activator of
mTOR, have been cloned from Gecarcinus lateralis (Gl-Rheb). Levels
of Rheb increase dramatically during molting induced by either eyestalk
ablation (ESA) or multiple limb autotomy (MLA) and showed a
correlation with levels of ecdysteroid in the claw muscle, in both
molting paradigms. These data indicate that Gl-Rheb is an important
activator of crustacean mTOR signaling in the molt cycle in response to
ecdysteroids.
atrophy occurs coincident with extensive remodeling

of the contractile apparatus [9], increased protein synthesis
[10], and increased protein degradation [7], [8]. The
induction of atrophy is dependent on increased ecdysteroid
levels in circulating hemolymph [3], [4], [11].
One of the potential effectors of the ecdysteroid
changes seen in molting is the mTOR (metazoan target of
rapamycin) signaling pathway.
mTOR complex 1
(mTORC1) is now recognized as the pathway involved in
inhibition of protein synthesis in mammalian skeletal muscle
[12-], [13], [14]. TORC1 is a part of the insulin/insulin-like
growth factor (IGF) pathway that is important for controlling
growth in eukaryotic cells and is a serine/theonine protein
kinase that regulates protein synthesis at the translational
level by phosphorylating the S6 kinase (s6k) and 4E-binding
protein 1 (4E-BP1) [15], [16], [17]. The mTOR pathway has
been studied before in insects, and was shown to be
important for nutrient-dependent growth [18]. The single
study of this pathway in crustaceans, which examined p70S6
kinase in the brine shrimp Artemia franciscana, showed that
mTOR activity is present, and increases, in early
preemergence development after quiescence, when protein
synthesis is restored [19]. Based on the previous
observations that elevated ecdysteroids have only a minor
effect on gene expression of myofibrillar proteins [20], that
increased premolt protein synthesis is correlated with
increased ribosomal activity [10], and that premolt protein
synthesis is inhibited by rapamycin, we hypothesize that this
effect on mTOR is a direct effect of ecdysteroid.
The insulin/mTOR pathway is highly conserved among
all metazoans and is a critical nutrient sensor for growth
[17]. In insects, it has been extensively studied, especially in
its role in growth, development, reproduction, and
metamorphosis [18]. Of particular interest given the results
of this study is the role of the protein Rheb (ras homolog
enriched in brain) in the insulin/mTOR signaling pathway.
The Rheb family of GTP-binding proteins is a unique group
within the larger Ras superfamily of G-proteins [21].
Originally identified in rat brain, two paralogs of Rheb are
found in mammals while single copies have been found in
arthropods [22], [23]. All Rheb proteins have guanine
nucleotide binding functions, located within a series of G
boxes [24], and have intrinsic GTPase activity in all species
examined, including D. melanogaster. Rhebs primary
function is as the critical upstream activator of mTOR, likely
through its interaction with FKBP38 [25], [26].
To assess the role of Rheb in claw muscle during the
molt cycle in the land crab, Gecarcinus lateralis, the
complete cDNAs for Rheb was cloned from G. lateralis (Gl-
Key words: Claw Muscle, Eyestalk ablation, Rheb, Crustacea,

Ecdysteroid, Molting, mRNA, Muscle atrophy, Multiple limb autotomy.
Introduction
In mammals as well as in invertebrates, skeletal
muscle is a plastic tissue that changes greatly in response to
physiological cues. Although there are many differences
between these divergent animals in terms of their skeletal
muscle physiology, some important components have been
shown to be important in both types of organisms. In
particular, there are important similarities in the processes of
atrophy and hypertrophy in both types of organism.
Mammalian muscles have been shown to be altered under
conditions such as altered nervous innervation, hormonal
manipulation, stretching, disuse atrophy, and disease wasting,
etc.[1], [2]. A simpler model of these processes is found in the
decapod crustaceans.
In decapods, we see a regular pattern of atrophy and
hypertrophy in skeletal muscles [3], [4]. Given the necessity,
among organisms with a rigid exoskeleton, of molting in
order to grow larger, withdrawal of muscles from the old
exoskeleton is a necessary precursor. In particular, the basiischial joint at the base of the chelipeds is a constriction
point for withdrawal of the large closer muscle of the
propodus [5], [6], and so atrophy of claw muscles is a
requirement for successful molting. Afterwards, expansion
of the new exoskeleton and subsequent hypertrophy of the
skeletal muscles completes the growth process for that molt
cycle.
This extensive atrophy in crustacean claw muscles is
seen in muscles of the chelipeds only, and not in thoracic or
walking leg muscles, which do not undergo appreciable
atrophy during the molt process [7], [8]. Claw muscle
*Lecturer ,Dental Sciences Department, College of Medical Technology,

Misurata-Libya
**Lecturer ,Dental Sciences Department, College of Medical Technology,
Misurata-Libya
***Consultant orthodontics, Altager Dental Center , , Misurata-Libya
13
The effect of Rheb (Ras) homolog enriched in brain expression

Rheb) [27]. After we designed qPCR primers, we
analyzed the cDNAs synthesized for Rheb expression (GlRheb).
Meterials And Methods
Animals
Adult Gecarcinus lateralis were maintained in the lab at 27
C in 75-90% relative humidity with intermolt individuals
kept in communal plastic cages lined with aspen bedding
wetted with 5 p.p.t. Instant Ocean (Aquarium Systems,
Mentor, OH). Crabs that have autotomized 5-8 walking
limbs (multiple limb autotomy, MLA) were kept in the same
environmental conditions in quart size (~11) plastic cages in
playground sand wetted with 10 p.p.t. Instant Ocean. MLA
crab sand cages were shielded from room illumination
behind a cloth curtain, mimicking the constant darkness
conditions previously shown to shorten the premolt period
[28].
Precocious molting is induced by two main
methods: multiple limb autotomy (MLA) and eyestalk
ablation (ESA, removal of eyestalks). Progression of
animals through the premolt period for both methods was
monitored by measurement of the length of limb regenerates
called the regeneration or R index (=length of regenerate
100 / carapace width). The R index value varies between 0
and ~24 [29]. For MLA experiments, all animals had
autotomized 8 walking legs. ESA experiments were done as
previously and monitored on the basis of time elapsed since
treatment administration.
RNA purification and cDNA synthesis
Total RNA was isolated from land crab tissues (claw
muscle) using TRIzol reagent (Life Technologies, Carlsbad,
CA) as described previously [10]. Briefly, tissues (50-200
mg) were homogenized in 1-2 ml TRIzol and centrifuged at
12,000 g for 15 min at 4 C. Supernatants were phenolchloroform extracted and RNA in the aqueous phase was
precipitated using isopropanol (0.75 ml per 1 ml TRIzol
reagent). RNA was treated with DNase I (Life
Technologies), extracted twice with phenol: chloroform:
isoamyl alcohol (25:24:1), precipitated with isopropanol,
washed twice with 75% ethanol in DEPC water, and
resuspended in nuclease-free water. First-strand cDNA was
synthesized using 1 g total RNA in a 20 l total reaction
with SuperScript III reverse transcriptase (Life
Technologies) and oligo-dT(20)VN primer (50 mol/l; IDT,
Coralville, IA). RNA was treated with RNase H (Fisher
Scientific, Pittsburgh, PA) and stored at -80 C.
A LightCycler 480 thermal cycler (Roche Applied
Science, Indianapolis, IN) was used to quantify levels of GlRheb. Reactions consisted of 1 l first strand cDNA or
standard, 5 l 2 SYBR Green I Master mix (Roche Applied
Science), 0.5 l each of 10 mM forward and reverse primers
and 3 l nuclease-free water. PCR conditions were as
follows: an initial denaturation at 95 C for 5 min, followed
by 45 cycles of denaturation at 95 C for 10 s, annealing at
62 C for 20 s, and extensions at 72 C for 20 s, followed by
melting curve analysis of the PCR product. Transcript
concentrations were determined by use of the LightCycler
480 software (Roche, version 1.5) using a series of dsDNA
14
gene standards produced by serial dilutions of PCR product

for each gene (10 ag/l to 10 ng/l). The absolute amounts
of transcript in copy numbers per g of total RNA in the
cDNA synthesis reaction were calculated based on the
standard curve and the calculated molecular weight of
dsDNA products.
Statistical analyses and software
Statistical analysis was performed using JMP 5.1.2,
6.0.0, or 8.0.2 (SAS Institute, Cary, NC). Group variances
were analyzed using a Brown-Forsythe test and found to be
equal (P < 0.05). Means for different developmental stages
were compared using analysis of variance (ANOVA). All
data not plotted as individual points are represented as mean
1 S.E. and the level of significance was set at = 0.05.
Linear regression analysis of log-transformed data was
performed using Excel 2010 (Microsoft, Redmond, WA) and
JMP.
The Rheb gene (ras homolog enriched in brain), an
activator of the metazoan target of rapamycin (mTOR) were
obtained from RT-PCR and RACE [27]. cDNAs containing
the complete ORF of Gl-Rheb. Three variables are used to
describe the data presented for qRT-PCR: time, R-index,
and molt stage. Events post-ESA or post-ecdysis were
measured in time since that event. R-index was utilized to
allow comparison between ESA and MLA methods of molt
induction. The molt stage classifications were modified for
G. lateralis by Skinner [30] with both methods. The highly
conserved insulin/IGF/mTOR signaling pathway is found in
all metazoans and has an important role as a nutrient sensor
[25], critical for growth and development in insects and
other invertebrates [26], [31], [32]. It was clear the
components of this pathway would be regulated during the
molt cycle of crustaceans [27].
Quantitative reverse transcriptase-polymerase chain
reaction (qPCR) was used to analyze levels of land crab GlRheb in cDNA from claw muscle (CM), as animals
progressed through the molt cycle, and compared with the
corresponding hemolymph ecdysteroid hormone levels.
Induction of molting used one of two classical methods
MLA and ESA. cDNA samples were analyzed for Rheb
expression over the course of the molt cycle.
The
hemolymph ecdysteroid levels increased in response to both
methods of molt induction (Fig. 1A and Fig. 2A). As noted,
ESA induced a rapid 5-fold increase that was then followed
by a slower, but prolonged increase, topping out at 12-fold
higher than the intact levels (Fig. 1A). MLA induced
changes in hemolymph ecdysteroid levels were slower than
the changes seen in ESA, and only a 7-fold increase was
observed in mid-premolt (Fig. 2A) in those animals.
However, late in premolt there was an even greater peak in
ecdysteroids, reaching an average of 28-fold higher than
intact animals. Although postmolt hemolymph levels are not
possible to measure in ESA animals due to mortality, the
levels observed in MLA animals in the 10 days immediately
following molt were very low.
Journal of Science
The eyestalk ablation (ESA) method used to

examine the Rheb transcriptional response of animals in the
intermolt phase of the molt cycle. ESA had a strong effect
on expression of Gl-Rheb, the important regulator of mTOR
activity, in muscle (Fig. 1B). Claw muscle shows significant
increases in Gl-Rheb expression by 1 day post-ESA,
however, the expression increased rapidly between 3 and 7
days post-ESA, peaking at 3.9-fold above the intact level
also at 14 days post-ESA. This change in expression in CM
is sustained, with no significant drop observed after the 7
day post-ESA level. In this experiment, we observed a
substantial increase (3.9-fold) in claw muscle Gl-Rheb
following ESA (Fig. 1B) that was sustained between through
the end of the experiment. The largest changes are seen in
Rheb, consistent with its role as the key activator of mTOR
[33], [34].
B.
8
60
A.
40
6
20
0
5
Intact 1 day 3 day 7 day 14 day
Day Post-ESA
B.
100
Intact 1 day 3 day 7 day 14 day
Day Post-ESA
Figure 1. Effects of eyestalk ablation (ESA) on hemolymph

ecdysteroid titer (A) and claw muscle expression of Gl-Rheb (B)
in G. lateralis. Animals were ES-ablated at Day 0, hemolymph and
claw muscle tissues were collected from intact (Day 0) and at 1 day
and at 3days and at 7 days and 14 days post-ESA. The Gl-Rheb
mRNA levels were quantified by quantitative reverse transcriptasepolymerase chain reaction (qPCR). Means of ESA animals that
were significantly different shared same letters indicate significant
difference between molt stages. All data are presented as means
s.e.m. Sample sizes varied with molt stage (Intact Day 0, n = 12;
Days 1, 3, and 7, n = 10; Day 14, n = 8).
Induction of molting by the more natural process of

multiple limb autotomy method showed a more gradual
increase in hemolymph ecdysteroids (Fig. 2A) than that seen
for ESA (Fig. 1A) consistent with a more gradual entrance
into premolt. Animals in the multiple limb autotomy
experiment showed changes over the molt cycle for Rheb
expression (Fig. 2A). In MLA experiment, intact intermolt
animals were compared with animals that had begun
progressing through premolt as measured by their R-index
value. Since MLA is a natural process in land crabs, animals
survive to undergo a normal ecdysis, and it is therefore
possible to monitor expression after defined time periods
after ecdysis.
Expression of Gl-Rheb in land crabs was greatly
affected in claw muscle over the molt cycle of the MLA
experiment (Fig. 2B). Claw muscle transcript abundance of
Gl-Rheb increased steadily from intact levels over the course
80
Log Rheb
80
of premolt, reaching significant levels by mid-premolt

(R=15) and peaking in late premolt (R=22) at 3.4-fold higher
expression than intact levels (Fig. 2B). Following ecdysis,
claw muscle copy numbers of Gl-Rheb declined rapidly to
return to intact levels. In this experiment, expression in claw
muscle Gl-Rheb following MLA increased slowly at first
(R=10) and then more quickly (R=15-22), peaking with a
large increase (3.4-fold) over the intact levels, followed by a
return to intact levels in postmolt. This result is very similar
to what was observed in the ESA experiment (above).
The abundance of Gl-Rheb transcripts was
generally associated with higher levels of circulating
ecdysteroids (Fig. 2B) in claw muscles. This was found in
both MLA (Fig. 2B) and ESA (Fig. 1B) but the association
was stronger in ESA animals. This positive correlation
between the claw muscle expression of Gl-Rheb with
increasing hemolymph ecdysteroid levels is especially
evident in the log-transformed data for MLA and ESA. The
linear regressions in the log-transformed data show a clear,
modest positive association in claw muscle that is significant
(P<0.05) in both molt induction paradigms.
Ecdysteroid level (pg/l)
100
Log Rheb
Ecydysteroid level (pg/l)
15
60
40
6
20
0
5
10-R
15-R
22-R 2 day 10 day

R-Rvalue
Intact 10-R 15-R 22-R 2 day 10 day

R-Rvalue
Figure 2. Effects of multiple leg autotomy (MLA) on hemolyph

ecdysteroid titers (A) and claw muscle expression of Gl-Rheb
(B) in G. lateralis. Hemolymph ecdysteroid levels were quantified
by ELISA. Gl-Rheb mRNA levels at early premolt (10-R), mid
premolt (15-R), late premolt (22-R), 2 days postmolt, and 10 days
postmolt were quantified by quantitative reverse transcriptasepolymerase chain reaction (qPCR). All data are presented as mean
1 S.E. Sample sizes varied with molt stage (Intact, N=15; R-10,
N=10; R-15, N=11; R-22, N=10; 2 and 10 days post-molt, N=13).
Means that are significantly different from each other shared same
letter.
Taken together, the largest transcriptional effects on

the mTOR pathway seen under both molt induction methods
were for claw muscle Gl-Rheb expression (Fig. 1B, 2B).
Since Rheb is a GTPase and active as an effector of mTOR
in its GTP-bound state [33], [34], [35], [36], we would
expect cellular processes to turn greater transcription of
Rheb into a greater pool of active Rheb, capable of
activating mTOR. This is what was seen with exogenous
expression of Rheb via transfection of human cellsgreater
expression of Rheb even in the presence of inhibitors greatly
increased all read-outs of mTOR activity [36], [37] Because
of this, we believe that Gl-Rheb transcriptional changes in
The effect of Rheb (Ras) homolog enriched in brain expression

the molt process are indications of upregulation of mTOR
(kinase) activity, particularly in claw muscle tissues. It has
long been appreciated that claw muscle atrophy in premolt is
occurring along with increased protein synthesis [10]. This
observed upregulation of mTOR activity and its well
understood downstream effectors of p70S6kinase and 4EBP1, important for increasing translation of mRNA into
protein [17].
This increase in protein synthesis necessary for
atrophy of claw muscles is dependent on increased
ecdysteroid levels in circulating hemolymph [3], [4]. How
exactly ecdysteroid titers are mediating this process is a key
area of investigation. It is clear from the analysis that Rheb
transcript levels increase with increasing ecdysteroid levels
in the hemolymph in both MLA and ESA.This increase in
transcripts is only seen in claw muscle tissue which needs to
undergo atrophy, and not in thoracic muscle, which does not.
Conclusion
In summary, Gl-Rheb is regulated in different manners in
atrophy processes in skeletal muscle. The increase noted in
Gl-Rheb transcript levels in premolt claw muscle is
correlated with elevated hemolymph ecdysteroid titers. This
supports our hypothesis that ecdysteroids are causing an
increase in mTOR signaling components, and particularly
the mTOR activator Rheb, during premolt, probably through
interaction with nuclear receptors and increased transcription
it is clear that the cellular machinery of mTOR component
regulation of growth/muscle remodeling in the context of
claw muscle atrophy, while capable of being regulated by
ecdysteroid.
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17
Journal Of Science
Misurata University
Evaluation Dimension of Permanent Teeth Size in Misurata-Libya

Adnan M . Hmeida * . Abushahma,S.M ** and Ahmed k. Ben Omron ***.
satisfactory substitutes for this term.

Although there is a general consensus with regard to the
use of landmarks for this dimension (mesiodistal tooth
diameter or width), investigators have used different
landmarks for the purpose. The anatomic contact points
between individual teeth are agreed upon in defining
mesiodistal limits of a tooth. It should be borne in mind that
the distance between the contact points may not be the
widest dimension of a tooth crown. The latter, very often, is
notaccessible for measurement on dental casts and is more
suitable for extracted teeth or teeth ondried skulls. However
some idea of the maximum tooth width may be obtained by
measuring the width of their labial or buccal surfaces from
interproximal aspects.
Some anthropologists have used the marginalridges for
measurement of mesiodistal tooth width.[18] These ridges
have also been recommended by the Federation Dentaire
International (FDI). McCanne [19] considered this method a
suitable approach for determining tooth width in the casts of
repaired cleft lip and palate patients. However, most clinical
studies, particularly those investigating crowding or dental
irregularities, have used contact points to define mesiodistal
tooth width.
Recent studies had used two demission(2-D) and there
dimension (3-D), to investigate the limitations of existing
methodologies and to develop and validate new methods for
obtaining true 3-D measurements, including curvatures and
volumes, in order to enhance discrimination to allow
increased differentiation in studies of dental morphology and
development. The validity of a new methodology for the 3-D
measurement of teeth is compared against an established 2D system. The intra- and inter-observer reliability of some
additional measurements, made possible with a 3-D
approach, are also tested [34].
The intent of this study was to establish the maximum
mesiodistal tooth width in a sample from Libya , and also
to be able to compare tooth widths for Libyans with other
racial groups. The establishment of tooth width for Libyan
population will enable further studies to help in predicting
crowding in mixed dentition analyses in future.
Abstract: The purpose of this study was to establish the mesiodistal

tooth width of permanent teeth in a Libyan population sample. The
measurements were obtained from 30 pairs of randomly selected
dental casts. The subjects age ranged from 13 to 20 years. An
electronic digital caliper was used for the measurements.
Descriptive statistics was used for the analysis of the data. The
results of this study presents the mean values of the mesiodistal
tooth width of permanent teeth in both arches, the error of the
method, and the coefficient of variation were calculated . The error
of the method in the present study ranged from 0.02 mm to 0.30
mm, with the first molars exhibiting the least coefficient of
variation while the central and the lateral incisors showed the most.
Hence, the results obtained could be of help in clinical orthodontics
for space assessment. Results are also of importance to
anthropologists and may be used as a basis for future studies.
Key words:
dimension.
mesiodistal width, orthodontics, proximal ,
Introduction
Reliable measurements of the human dentition are
needed in many disciplines of dentistry. Such measurements
are generally made from dental casts or directly from the
teeth in the oral cavity. These measurements are
predominantly used for research and clinical purposes,
particularly in orthodontics. Application of such data in the
day to day clinical practice has, however, remained limited.
In the past, researchers have employed the contact method
using simple instruments such as a pair of dividers with a
millimeter ruler [1,2,3] or sliding calibrated calipers for
dental cast measurements.[4,5] Other researchers have used
the non-contact methods, which include standard
photographs[6] photocopies,[7] sophisticated occlusograms
[8] and laser holograms of the occlusal aspects of the
teeth.[9] Computerized methods for collecting information
from photographs and photocopies have also been described,
saving considerable time and effort.[10,11] Various terms
have been used to define the tooth width, and there is a
difference of opinion as to what constitutes the tooth width
and how best to measure it. The mesiodistal dimension of a
tooth, i.e. the distance between its mesial and distal surfaces,
which is the commonly used measure of the occlusal size of
the tooth,[12] has been variously defined as diameter,[13,14]
breadth[15,16] and width.[17] According to Moorrees[14]
the term crown length used by some investigators, as
synonymous with mesiodistal teeth diameter, is not
appropriate. Neither length nor breadth is completely

Thirty pairs of pre-treatment orthodontic study casts
with equal distribution of the two sexes were selected
randomly from the clinical material in the Division of
Orthodontics at Altager Dental Center , Misurata-Libya
. The criteria for selection of the subjects were:
1. Age ranged from 13 to 20 years.
2. Presence and complete eruption of all permanent teeth,
excluding third molars.
3. No conservative treatment other than Class I occlusal
restorations.
*Lecturer ,Dental Sciences Department, College of Medical Technology,

Misurata-Libya
**Lecturer ,Dental Sciences Department, College of Medical Technology,
Misurata-Libya
***Consultant orthodontics, Altager Dental Center , , Misurata-Libya
18
Journal of Science
19
4. No evidence of air blows or fractured teeth.

identification. Measurement of mesiodistal width was
obtained from each dental cast using the electronic digital
caliper*calibrated to the nearest 0.01 mm
[Fig. 1]. The
measurements were made as carefully as possible to avoid 5.
No history of previous measurement.
The sample was found to exhibit different types of
malocclusion with varying degrees of arch crowding and
spacing. The study casts were numbered for ease of any
damage on beaks contact. The caliper beaks were sharpened
on their outer surfaces to improve the access interproximaly.
The maximum mesiodistal width was measured. The caliper
beaks were inserted and held occlusally parallel to the long
axis of the tooth. The beaks were then closed until gentle
contact with the tooth was felt. The measurements included
all permanent teeth from central incisors through first molars
in all four quadrants. All measurements were taken under
natural and neon light.
To study the error of the method between a first and a
second measurement, five pairs of dental casts were selected
randomly to study the differences. All measurements were
performed by one author. Each tooth was measured by using
the electronic digital caliper. This procedure was repeated
after one month. The error of the methodwas calculated by
means of double determination (Table1). The reproducibility
of the caliper measurements was done by measuring a
known length of stainless steel rod twice a day for 30 days
and was found reproducible to 0.065 mm.
Table 2. Means, standard deviations, standard errors of the mean,

minimum and maximum values of the individual tooth widths for
bothsexes (values in millimeters).
Upper Arch
n
mean
s.d.
s.e.m
min.
max.
Tooth
11
30
8.66
0.57
0.10
7.59
9.58
12
30
6.68
0.54
0.10
5.77
8.29
RC
30
7.61
0.48
0.09
6.68
8.68
PM1
30
6.95
0.44
0.08
6.17
8.21
PM2
30
6.62
0.36
0.07
6.14
7.38
M1
30
10.59
0.53
0.10
9.56
11.34
1l
30
8.61
0.57
0.10
7.79
9.97
12
30
6.68
0.51
0.09
5.95
8.37
LC
30
7.58
0.52
0.10
6.67
8.79
PM1
30
6.92
0.45
0.08
6.12
8.11
PM2
30
6.59
0.38
0.07
5.82
7.30
M1
30
10.53
0.51
0.09
9.55
11.70
Lower Arch
l1
30
l2
30
RC
30
PM1
30
PM2
30
M1
30
l1
30
l2
30
LC
30
PM1
30
PM2
30
M1
30
5.45
5.96
6.67
6.86
7.07
10.90
5.45
6.00
6.68
6.94
7.08
10.95
0.64
0.44
0.48
0.51
0.45
0.53
0.63
0.51
0.45
0.53
0.40
0.62
0.11
0.08
0.09
0.09
0.82
0.10
0.11
0.09
0.08
0.10
0.07
0.11
4.80
5.05
5.81
5.85
6.00
9.77
4.82
5.01
5.83
5.92
6.17
9.41
8.46
6.75
7.60
8.42
8.34
11.74
8.38
6.81
7.43
8.51
8.20
12.03
Table 3. Coefficient of variation of individual tooth width
Figure 1. The electronic digital caliper with sharpened beaks

to improve access interproximally.Electronic Digital
Caliper, Mitutoyo, Japan
Table 1. Error of the method by double determination.
Upper arch
Tooth
Central incisor
Lateral incisor
Canine
First premolar
Second premolar
First molar
(11)
(12)
(C)
(PM1)
(PM2)
(M)
right
left
0.12
0.15
0.25
0.12
0.14
0.23
0.09
0.12
0.08
0.08
0.08
0.18
Lower arch
righ
t
0.02
0.10
0.14
0.10
0.30
0.16
left
0.05
0.10
0.07
0.24
0.10
0.09
Tooth
Right
upper arch
Lower arch
l1
l2
C
PM1
PM2
M1
Left
l1
l2
C
PM1
PM2
M1
30
30
30
30
30
30
6.60
8.02
6.35
6.32
5.46
4.96
11.68
7.34
7.15
7.42
6.42
4.88
30
30
30
30
30
30
6.56
7.56
6.84
6.52
5.71
4.83
11.53
8.46
6.72
7.66
5.68
5.62
Results
Table 1 demonstrates the error of the method by double
determination. It was found that the lower right central
incisor exhibited the lowest error measurement (0.02) while
the lower right secondpremolar exhibited the highest (0.30).
Table 2 shows the mean mesiodistal tooth width values for
all teeth in both upper and lower arches for both sexes. The
Evaluation Dimension of Permanent

result showed that the values in the right side in the upper
arch were relatively greater than those in the left side. This
was not true in the lower arch.
Table 3 exhibits the tooth variability in upper and lower
arches. It was observed that the first molars in both arches
showed the least tooth variability while the central and
lateral incisors showed the most.
Discussion
Of all the measurements considered, the estimation of
individual mesiodistal tooth width poses the most
methodological problem. These tooth widths are generally
obtained from contact points which may not coincide with
the points of maximum mesiodistal convexity. Further, these
contact points are not always accessible even with calipers,
and are frequently obscured on an occlusal view. However,
most investigators used plaster casts of dentition for tooth
measurements while few of them did measurements on
natural teeth. This could give rise to errors due to distortion
in the impression material during making of the impression,
due to dimensional changes in the impression material
during setting, and due to changes during setting of the cast
material.
Because of the variability in tooth morphology, the
measurement errors of different teeth, are not the same.
Hunter and Priest[4] found that measurements on casts were
on an average of 0.1mm larger than those of the actual teeth.
They explained that due to the difficulty encountered in
establishing the greatest mesiodistal diameter, particularly in
the maxilla, they did not mention whether this difference is
significant or not. However, Lundstrom[20] recorded
measurements of six anterior teeth by a direct method. He
claimed that no significant differences were observed
between the direct and indirect methods. This could be
explained by the fact that Lundstrom[20] did not measure
the posterior teeth which are difficult to measure due to
inaccessibility.
There was some indication that the order of errors for
different teeth varied between the different methods. For
example, by the direct method (Caliper, Divider) the molars,
particularly the upper, were the least accurately measured
followed by the upper laterals and then the lower second
premolar.[20] By the photographic method the upper lateral
incisors were the least well measured teeth.[20] In this study
the order of errors for different teeth showed that the lower
second premolar
was the least accurately measured
followed by the upper right canine and lower left first
premolar.
Errors of the individual tooth width have been reported
in other studies. The errors found by Lundstrom[20] are in
the range of 0.06 mm 0.25mm and those by Murshid[21]
are 0.06 mm - 0.21 mm while in this study they varied from
0.02 mm to 0.30 mm. This disagreement could be due to
methodological problems. Moorrees and Reed [23] have
provided the average error of only 0.09 mmin all teeth
combined. Lysell[24] found the average error to be 0.13
mm. It appears that the error of the present study (0.13 mm)
20
is similar to what Lysell[24] found and greater than

Moorrees and Reed.[23]
Consideration of the errors of the individual tooth
measurements obtained by other investigators revealed that
some errors are greater (0.38 Robinson[25], 0.51 Miethke
and Menthel[26] )while others are smaller 0.05 - 0.11 (Sanin
&Savara,[27] 0.09 -0.18, Townsend & Brown[28]) than that
of the present study.
Variability in the size the teeth was studied by means of
coefficient of variation. The coefficient of variation in this
study ranged from 4.83 for theme an mesiodistal tooth width
of maxillary first molar to 11.68 for mean mesiodistal tooth
width of the mandible central incisor. The maxillary right
lateral incisor and the mandible right central incisor showed
the greatest coefficient of variation while the first molar in
both jaws showed the lowest. This result is in agreement
with there sults obtained by other investigators.
Lundstrom[29] noticed the greatest coefficient of variation
in the maxillary lateral incisor. Lunt[30] stated that in
general the first molars of both jaws were the teeth in
which the lowest coefficients of variation were most
frequently to be found. Healso stated that the third molar and
lateral incisor of both jaws showed the greatest degree of
variability in size. Barrett et al [31] observed that the third
molar and the lateral incisor in Australian aborigines showed
the greatest variability, while the first molar gave the lowest
values. Axelsson and Kirveskari[32] noticed that the
lateralincisor showed the greatest variability in the maxilla,
the central incisor in the mandible, while the first molars
showed greatest stability in crown form. In the present study,
the third molars were not included. On the other hand,
Hunter and Priest[4] observed in their measurements that
the molars and the lower second premolar showed the most
variability, and the upper canines, and the upper and lower
incisors showed the least. This contradicts with the results of
the current study where by samples of the types of
malocclusion were not separated. However, a study carried
out by Crosby and Alexander[33] showed that there was no
significant statistical difference between the different
malocclusion classes.
The results of the mesiodistal tooth width obtained could
be of help to the clinical orthodontist for space assessment
and of importance to anthropologists. Further, the present
study may be used as a basis for future studies where a
normal occlusion sample is considered
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15. Lundstrom A. Inter-maxilla tooth width ratio
andtooth
alignment
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occlusion
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Master of Science Thesis, University of London.
20. Lundstrom A. (1967). Genetic aspects of variations in
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Hereditas;57:403.
21. Murshid ZA. (1991) Computer aided measurement
ofdental casts with special reference to crowding and arch
form. M.Phil Thesis, University of London.
22.
Lundstrom
A.
(1943)
Intermaxillare
tandbreddsforhallenden Och tandstallningen. Svensk
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crowdingand spacing of the mandible. Am J
PhyAnthropol12:77-88.
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incisorcrowding. Master of Science Thesis,University of
London.
26.Miethke RR, Behm-Menthel A. (1988) Correlation
betweenlower incisor crowding and lower incisor
positionand lateral craniofacial morphology. Am J
Orthodont Dentofac Orthopaed . 94:231-9.
27.Sanin C, Savara BS. (1971) An analysis of permanent
mesiodistal crown size. Am J Orthodont. 59:488-500.
28. Townsend GC, Brown T, Inheritance of tooth size in
Australian aborigines. Am J PhyAnthropol1978;48:305-31.
29 . Lundstrom A. (1948). Tooth size and occlusion in
twins. Thesis (Uppsala) Karger, Basle.
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Danes. Acta Odont Scand 27:55.
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Dentalobservations on Australian aborigines: mesiodistal
crown diameters of permanent teeth. Aust Dent J;8;150.
32. Axelsson G, Kirveskari P. (1983) Crown size of
permanent teeth in Icelanders. Acta Odont Scand 41:181-6.
33. Crosby DR, Alexander CG. (1989) The occurrence of
tooth size discrepancies among different malocclusion
groups. Am J OrthodontDentofacOrthopaed. 95:457-61.
34. Smith R1, Zaitoun H, Coxon T, Karmo M, Kaur G,
Townsend G, Harris EF, Brook A. (2009) Defining new
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Journal Of Science

Misurata University
Antifungal Potential in Crude Extracts of Five Selected Brown Seaweeds Collected from the Western
Libyan Coast
1
Daghman, I. M.;2Khallil, A. M. and 1Fady, A. A.
Abstract : In vitro antifungal screening of six organic extracts of
Rajauria and Abu-Ghannam [24] screened antimicrobial

activity and bioactive compounds of Himanthalia
elongate(Brown Seaweed). In France, Hellioet al. [10]
tested antimicrobial potentiality of many seaweed extracts
and observed a conspicuous decrease in the development of
the fungi tested. In China, Yi et al., [30] used ethanol,
acetone and methanol-toluene to extract antibiotics from 23
species of marine algaeand revealed that the strongest
antifungal activities were exhibited by the ethanol extract
and the least by the methanol-toluene extract. In Korea, Lee
et al. [16] investigated the antifungal activities of Dieckol
isolated from the marine brown alga Ecklonia cava against
Trichophyto nrubrum and reported the minimum inhibitory
concentration of dieckol was 200 M. In India, Indira et al.
[11] evaluated the antimicrobial property of seaweed
Halimeda tuna, and recorded the activity was against nine
fungal strains (Aspergillus niger, Aspergillus
flavus,
Alternaria alternata, Candida albicans, Epidermophyton
floccossum, Trichophytonme ntagrophytes, Trichophyto
nrubrum, Pencillium sp. and Rhizopus sp.). It is recorded
that methanolic extracts exhibited a broad spectrum of
antimicrobial activity compared to the ethanolic and
chloroform extracts. In Pakistan, Khanzada et al. [13]
screened various fractions of ethanolic extract of Solieria
robusta (Rhodophyta) for antifungal activity against 5 fruit
spoiling fungi and reported that all fractions were able to
inhibit fungal growth. Aqueous fraction showed maximum
inhibition ratios followed by methanol, ethyl acetate,
chloroform and ethanol. In Algeria, Saidani et al. [26]
explored antifungal activity of four species of marine algae
of Bejaia coast and reported that all the experimented
extracts exhibited antifungal activity and the highest
inhibiting effect was noted for Rhodomela confervoides (red
algae) and Padina pavonica (brown algae) respectively
against Candida albicans and Mucorram aniannus for the
first one and Candida albicans for the second one.
Aspergillus niger showed resistance against majority of
methanolic extracts.
According to the available literatures, the Libyan
marine algae have been neglected with respect to
assessments of their antifungal activity. Thus, the current
investigation represents an attempt to bridge this gap and
was designed to evaluate the antifungal potential of different
extracts of the five commonest seaweeds which were
collected from the western Libyan coast against eight
economically important fungi.
five seaweeds belong to Phaeophyta (Sargassum vulgare,

Cystoseira barbata, Dictyo pterismem branacea, Dictyota
dichotoma, and Colpomenia sinosa) against eight fungal species
(Alternaria alternata, Cladosporium cladosporioides, Fusarium
oxysporum, Epicoccum nigrum, Aspergillusniger, Aspergillus
ochraceus, Aspergillus flavus, and Penicillium citrinum).
Cyclohexanic extracts were almost the most active exhibiting a
broad spectrum inhibitory action irrespective to the experimented
algal extract or fungal species whereas both acetone and ethyl
acetate extracts exhibited the lowest antifungal activity. Some algal
extracts did not show recognizable inhibitory actions, and some
others enhanced some fungal species. The experimented fungal
species exhibited variable responses to the tested algal extracts
depending upon the experimented fungal and algal species as well
as the applied extract. Interestingly, some algal extracts exerted
higher antifungal potential in comparable with the patented
antifungal medicine (Nystatin and Clotrimazole). Generally,
Alternari alternata was relatively more resistant to most of the
tested seaweed extracts whereas Fusariumoxy sporum was more
sensitive. The present study confirms the potential use of seaweed
extracts as a source of antifungal compound and may constitute a
basis for promising future applied research that could investigate
the use of seaweeds. We conclude that the Libyan coast is a source
of bioactive compounds with potential applications in controlling
undesired microorganisms in the fields of medicine, pharmacy and
agriculture, as well as food additives and food preservation. This
may encourage the use of natural products for substituting
chemical preservations in food systems.
Key words: Seaweeds - Organic extracts Fungi.

Introduction
Seaweeds are one of these marine organisms that has
been consider as a source of major metabolites that possess
bioactive effects [29]. There are numerous reports of
compounds derived from macro-algae with a broad range of
biological activities, such as antifungal and antibacterial [6
and 22]. Several investigations have been carried out
worldwide. In this respect, in USA, Martin [19], tested
extracts from macro-algae by spraying on plants and
reported a pronounced reduction in the disease incidence of
Botrytis cinerea on strawberries, Erysiphe polygonion
turnips, and reported that macro-algae produce various
biologically active compounds. In Brazil, Peres et al. [23],
reported that ten seaweed extracts significantly inhibited the
Colletotrichum lagenarium growth, but not inhibited
significantly the Aspergillus flavus growth. In Ireland,

Study area and algal samples collection: During the period
from April to May 2013 five of the commonest seaweeds
were collected in clean polyethylene bags by hand picking
Microbiology Department, Faculty of Science, Misurata University, Libya.

Botany and Microbiology Department, Faculty of Science, Assiut
University, Egypt
2
22
Journal of Science
23
from the Libyan western coast (Misurata Elkhoms

regions).Immediately transferred to the laboratory for further
processing, identified to species using the standard literature
and taxonomic keys [20; 21,8; 3, 1] and then described.
Preparation of extracts:
The shaded dry seaweeds were cut into small pieces
and powdered in a mixer grinder. Every sample was
preserved in a freezer until compound extraction. Twenty
grams of the powder was charged in the thimble and
extracted successively with 400 ml of different solvent
(Ethanol, Acetone, Chloroform, Ethyl acetate and
Cyclohexane) using a Soxhlet apparatus for 8h. The extracts
were filtered through bacterial filters (Millipore, 0.45m).
The crude extracts were stored at -20C in airtight brown
bottle for further study.
Fig.2 The vegetative structure of Cystoseira barbata
The experimented fungal species:

Antifungal activity assay:
Antifungal potentiality of the algal extracts is performed
using the disk diffusion technique [9]. Two patented
medicine namely Clotrimazole and Nystatin as a reagentgrade powder were used for comparison as positive controls.
The experimented fungi were separately and uniformly
swabbed across a culture [28]. A filter-paper disk of 6 mm in
diameter was prepared from Whatman No. 1, and
impregnated with the extract to be tested, then placed on the
surface of the agar. The plates were incubated at 282C.
The results are expressed as:(0): No antifungal activity.
( 10 mm inhibition zone): Weak antifungal activity.
(10 15 mm inhibition zone ): Moderate or mild antifungal
activity.
(
15 mm inhibition zone): High or strong antifungal
activity.
Macro-algae (Marine Seaweeds)
Five species belonging to Phaeophyta were isolated and
identified as Sargassum vulgare, Cystoseira barbata,
Dictyopterismem branacea,Dictyota dichotoma and
Colpomenia sinosa. (Figs. 1,2,3,4 and5).
Fig.3 The vegetative structure of Dictyopteris membranacea

The experimented fungal species were isolated from
outdoor air of some hospitals at Misurata and identified as
Alternaria alternata, Aspergillus niger, Aspergillus
ochraceous, Aspergillus flavus, Fusarium oxysporum,
Cladosporium cladosporioides, Epicoccum nigrum and
Penicillium citrinum depending upon the macroscopic and
microscopic characteristic features. Reliable references (Raper
and Fennell, 1965 and Ainsworth ,1971) were used for
identification. Potato dextrose agar (PDA), and Sabouraud
dextrose agar (SDA) media were used.
Fig.4 The vegetative structure of Dictyota dichotoma
The experimented fungal species were isolated from
outdoor air of some hospitals at Misurata and identified as
Alternaria alternata, Aspergillus niger, Aspergillus
ochraceous, Aspergillus flavus, Fusarium oxysporum,
Cladosporium cladosporioides, Epicoccum nigrum and
Penicillium citrinum depending upon the macroscopic and
microscopic characteristic features. Reliable references (Raper
and Fennell, 1965 and Ainsworth ,1971) were used for
identification. Potato dextrose agar (PDA), and Sabouraud
dextrose agar (SDA) media were used.
Fig.5 The vegetative structure of Colpomenia sinosa
. Antifungal activity:-
Fig. 1 The vegetative structure of Sargassum vulgare
The obtained results showed that antifungal activities of

tested seaweeds varied depending upon the experimented
fungal species, seaweed species and the employed solvent.
1- Sargassumvulgare:
Cyclohexane, chloroform and ethanol extracts of S.
vulgare displayed higher antifungal activity than acetone
and ethyl acetate extracts. The highest inhibitory actions of
cyclohexanic extract was recorded against, E. nigrum, F.
oxysporum, C. cladosporioides and A. ochraceous. A
moderate inhibitory action was recorded against P. citrinum,
A. niger, A. alternate and A. flavus.
Antifungal Potential in Crude Extracts of Five Selected

Both A. flavus and A. niger were not affected by the
chloroform extract of
S. vulgare whereas other
experimented fungal species were slightly or moderately
affected. Acetone extract did not display inhibitory action
against all tested fungi except E. nigrum which were
slightly affected. Nearly similar inhibitory action was
recorded for ethyl acetate extract against F. oxysporum and
a moderate inhibitory action was recorded against E.
nigrum. A. alternata, A. flavus, E. nigrum and C.
cladosporioides did not show responses against the ethanol
extract of S. vulgare but the other tested fungi were slightly
affected. (Table,1 and Fig 6 and 7).
Table (1): Antifungal activities of different organic extracts of
Sargassum vulgarein
comparable with Clotrimazole (Cl) and
Nystatin (N) against 8 fungal species using disk diffusion technique
and diameter of the inhibition zone (mm).
Fungal
species
Cl
Sargassum vulgare
(IZ)
12
Alternaria
alternata
11
Penicillium
citrinum
9
Aspergillus flavus
14
Aspergillus niger
8
Aspergillus
ochraceous
25
Epicoccum nigrum
10
Fusarium
oxysporum
7
Cladosporium
cladosporioides
Solvents: A: Cyclohexane
acetate E: Ethanol.
22
A
*11
B
7
C
0
D
0
E
0
19
*14
*7
*8
22
10
20
*10
12
*15
0
0
*8
0
0
0
0
0
0
0
*7
*8
16
*21
11
*10
14
10
*20
*8
*7
*8
16
*19
*7
B:
Chloroform
C: Aceton
24
2- Cystoseira barbata:
The obtained results from Table,(2) revealed that antifungal
activity varied according to the employed organic solvent
and tested fungal species. It was observed that cyclohexane
was the best organic solvent for extracting the effective
antifungal material from the applied algal species and
exhibited the highest antifungal potential particularly against
F. oxysporum followed by, A. ochraceous and E. nigrum.
The weakest inhibitory action of cyclohexanic extract was
recorded against A. alternata and A. flavus. A moderate
inhibitory action was recorded against P. citrinum, A. niger,
and C. cladosporiodes. Chloroform and ethanol extracts
followed cyclohexanic extract as antifungal activity
exhibiting low potentiality against most of tested fungi.
There was no activity recorded in chloroform extract against
A. alternata, A. niger and E. nigrum. A slight antifungal
activity of chloroform extract was recorded against F.
oxysporum followed by A. flavus, A. ochraceous, P.
citrinum, and C. cladosporiodes. Five of the experimented
fungi did not negatively affected by ethanol extract whereas
the other three fungal species were slightly inhibited. Both
acetone and ethyl acetate extracts displayed the lowest
antifungal activity since the former did not display
antifungal activity against all experimented fungi. Similarly,
all experimented fungi except F. oxysporium resisted ethyl
acetate extract. (Table. 2. Fig.8).
Cystoseira barbatain comparable with Clotrimazole (Cl) and
and diameter of the inhibition zone (mm).
D: Ethyl
Fungal species
Fig. 6 Effect of Cyclohexan extract of S. vulgare on A. nigeron

P.D.A plate .
S. vulgare
Alternariaalternate
Penicil iumcitrinum
Aspergil usflavus
Aspergil usniger
Aspergil usochraceous
Epicoccumnigrum
Fusariumoxysporum
Cladosporiumcladosporioides
N Cl
12
11
9
14
8
25
10
7
acetate E: Ethanol
A. ochraceous
Fig (7): Effect of Cyclohexan extract of on on P.D.A plate .
22
19
22
10
20
16
10
16
A
*9
*13
*10
*15
16
16
*23
15
C. barbata (IZ)
B C D
0 0 0
*7 0 0
*8 0 0
0 0 0
*8 0 0
0 0 0
9 0 *7
*7 0 0
E
0
*8
0
0
*8
0
8
0
B: Chloroform C: Acetone D: Ethyl
Journal of Science
Cycl.
25
against 8 fungal species using disk diffusion technique and

diameter of the inhibition zone (mm).
Solvents: A: Cyclohexane B: Chloroform C: Acetone D:
Ethyl acetate E: Ethanol
Clot
Nyst.
Fungal species
Fig (8): Effect of Cyclohexane extract of Cystoseira barbata (A)

on Aspergillus niger in comparable with Nystatin and
Clotramizole (B) on P.D.A plate .
3- Dictyopteris memberanacea:
The highest antifungal activity was recorded for
cyclohexanic extract followed by chloroform and ethanol
extracts. The maximum inhibitory action of cyclohexanic
extract was recorded against F. oxysporum, and a moderate
inhibitory action was recorded against P. citrinum, A.
ochraceous, and E. nigrum. A slight inhibitory action was
recorded for A. alternata, C. cladosporiodes and A. niger.
Chloroform extract did not display inhibitory action against
A. alternata and E. nigrum whereas the remaining fungal
species were slightly inhibited. Both acetone and ethyl
acetate extracts presented the weakest inhibitory action since
all the experimented fungi do not inhibited except F.
oxysporum. A similar slight inhibitory action was recorded
for F. oxysporum when treated with ethyl acetate extract. No
inhibitory action was recorded for ethanol extract against A.
alternata, A. flavus, A. niger, E. nigrum, and C.
cladosporiodes whereas a slight inhibitory action was
recorded against the remaining fungal species (Table 3.Fig.
9).
4- Dictyotadichotoma:
n general, extracts of D. dichotoma displayed low antifungal
potential in comparable with the other applied seaweeds.
Cyclohexanic and ethanolic extracts of D. dichotoma
exhibited higher antifungal activity than the other three
applied extracts. Cyclohexanic extract was more effective
than both employed patented medicine reagents. The tested
fungi varied in response to cyclohexanic extract since A.
alternata and A. niger were not retarded. A. flavus, A.
ochraceusand C. cladosporiodes were slightly or
moderately inhibited but P. citrinum, E. nigrum, and F.
oxysporum were strongly inhibited. Chloroform extract did
not display inhibitory against both A. alternata and A. niger
whereas the remaining of experimented fungi were slightly
inhibited. All tested fungi did not affected by acetone extract
of D. dichotoma, except F. oxysporum which was slightly
inhibited. Similarly, no inhibitory action was recorded
against all tested fungi resulting from ethyl acetate
application. Ethanol extract showed different results ranging
from no inhibitory action against A. alternata, A. flavus, E.
nigrum, and C.cladosporiodes to slight inhibitory action
against the remaining three fungal species (Table 4).
Table (4): Antifungal activities of different organic extracts of D.
dichotoma in comparable with Clotrimazole (Cl) and Nystatin (N)
Alternariaalternata
Penicilliumcitrinum
Aspergillusflavus
Aspergillusniger
Aspergillusochraceous
Epicioccumnigrum
Fusariumoxysporum
N
12
11
9
14
8
25
10
7
22
19
22
10
20
16
10
16
Cl
D. dichotoma
0
*15
*10
0
*10
*15
15
*7
0
*7
7
0
*8
*9
*8
*7
0
0
0
0
0
0
*7
0
0
0
0
0
0
0
0
0
0
*7
0
*7
*7
0
9
0
5- Colpomeniasinosa :
Cyclohexanic extract exhibited the highest antifungal
activity in comparable with the other applied extracts. The
strongest inhibitory action was recorded against F.
oxysporum, followed by, C. cladosporiodes. The weakest
inhibitory action was recorded against A. flavus, A. niger,
A. ochraceus, and E. nigrum. A moderate inhibitory action
was monitored against P. citrinum. Chloroform extract did
not inhibit both A. alternata and A. niger but displayed a
slight inhibitory action against the remaining tested fungi.
Acetone extract exhibited no inhibitory action against all
tested fungi except for C. cladosporiodes which was
strongly inhibited and E. nigrum, which were slightly
inhibited. Ethyl acetate extract exerted the weakest
inhibitory action since all the tested fungi did not negatively
affected by its application except for F. oxysporum which
was slightly inhibited. Ethanol extract showed a slight
inhibitory action against all experimented fungi except for
A.alternata, A. flavus and A. niger which exhibited no
inhibitory action (Table 5. Fig.10).
Colpomeniasinosa in comparable with Clotrimazole (Cl) and
and diameter of the inhibition zone (mm)
Fungal species
Cl
C. sinosa (IZ)
A
Alternariaalternata
12
22
*9
Penicilliumcitrinum
11
19
*15
*7
*6
Aspergillusflavus
22
*10
*9
Aspergillusniger
14
10
12
Aspergillusochraceous
20
*12
*7
*7
Epicoccumnigrum
25
16
*12
*9
Fusariumoxysporum
10
10
*23
*7
*0
16
16
*7
16
*7
Ethyl acetate E: Ethanol.
B: Chloroform
C: Acetone D:
Antifungal Potential in Crude Extracts of Five Selected
Clot.
Nyst.
Cyclo.
Fig.(10): Effect of Cyclohexane extract of Colpomenia sinosa on C.

cladosporioides (A) in comparable with Nystatin and Clotramizole
(B) on P.D.A plate .
26
sensitive for the majority of the tested algae and the strong
inhibitory action was recorded particularly for cyclohexanic
extracts. Cyclohexanic extract of
Cystoseira barbata,
Colpomenia sinosa, and Sargassum vulgare displayed the
highest inhibitory actions against Fusarium oxysporum. No
inhibitory action was recorded against Fusarium oxysporum
with ethyl acetate extract of D. dichotoma, acetone extract of
S. vulgare, C. barbata, Colpomenia sinosa and H.
musciformis. The other extracts of the experimented
seaweeds exhibited slight inhibitory actions against Fusarium
oxysporum. Similarly, Kim and Kim [14] reported that
extract of Nostoc commune displayed significant inhibitory
action against Fusarium oxysporum f. sp. lycopersici. Asnad
and Tanver [4] recorded antifungal activity of the water and
ethanol extracts of eight seaweed species against Fusarium
solani and F. oxysporum isolated from deteriorated fruits and
vegetables. Galal et al.[7] reported that crude ethyl acetate
extract of Padinagymnospora and methanolic extract of
Codium fragile exhibited strong activity against Fusarium
oxysporum. Lavanya and Veerappan [15] reported the
minimum inhibitory action of Sargassumwightii was recorded
in methanol water extracts against Fusarium udum.
Discussion
The present study is an endeavor towards the
screening antifungal activities through crude extracts of five
marine macro-algal species belonging toPhaeophyta against
eight fungal species. The marine seaweeds were extracted
with five different organic solvents.
The present screening revealed that cyclohexanic
extracts were almost the most effective and exhibited a broad
spectrum inhibitory action irrespective to the experimented
algal extract or fungal species. On the contrary, some algal
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OceanolLimnol, 19:327331.
Journal Of Science
Misurata University
Highly Production of Omega-3 Polyunsaturated Fatty Acid by Developed Fermenter Strategy and Design
Thureia. A. El-Sharif
Abstract Nowadays researchers were focused on production of
Fan et al. (2007) are also produced in a batch culture

around 47% of PUFA from S. Mangrovei, while Ren et al in
2009 has been developed enzymatic technique in the batch to
produce 60% of PUFA from Schizochytrium sp. HX-308.
Moreover Hong et al. (2011) carried out the conventional
one-factor-at a-time (OFAT) optimization where final DHA
concentration was successfully increased to 2.8 g/L from the
initial concentration of 62.0 g/L (5 L scale, 72 h). Recently
Armenta et al. (2015) has been reached about 85.9% of
Docosahaexonic acid from Thraustochytrid strain F24-2.
Although by developing batch culture strategy technique
such as fed -batch and continuous culture are used most
frequently in the laboratory and are most useful for industrial
processes [12,25]. Jasuja et al., 2010 reported that in his
review Schizochytrium have used different types of
fermentation parameters viz. production of DHA by glucose
fed batch, acetic acid fed batch, sodium acetate fed batch etc.
Before that fed batch fermentation process developed by
Bailey et al., 2003 to produce high cell density and DHA
production in two stages ; the resulting in biomass densities of
at least 100.0 g dry cell/L in the fermentation broth and at least
20% of their dry cell weight as lipids.
Thus the aim of this study was to developed a technique in
batch culture strategy and design to produce high amounts of
cell biomass and high productivity of PUFA in a short time
with inexpensive requirements.
-3 polyunsaturated fatty acid (PUFA) from microalgae in high

amounts. Some species of microalgae that have been produced of -3
polyunsaturated fatty acid (PUFA), collected from coastal Libyan
seawater, were investigated. In classical batch culture isolates were
grown on optimal conditions to standard medium (1000 ml ) of batch
culture at pH:8, temperature:25 C, agitation: 200 rpm of orbital
shaking, it was cultured for 5 days. The results in classical batch were
yielded about 6.0g 1L of cell biomass, 2.0g / 1Lof total lipid, and
50% of -3 polyunsaturated fatty acid.
Developing the strategy and design of batch culture could increase
the amounts of -3 polyunsaturated fatty acid from these isolates.
The results of the newly invention batch culture reached to 35g / 1L of
cell biomass, and 23 g 1L of total involving about 73% of -3
polyunsaturated fatty acid. Therefore this newly invented of
developing batch design is a very promising in an industrial use for
the production of -3 polyunsaturated fatty acid .
Keywords- -3 polyunsaturated fatty acid (PUFA), Batch culture,

standard medium.
Introduction
nowadays importantly is omega-3 polyunsaturated fatty
acids (-3PUFAs) become one the most medically important
substances nowadays.
These are included Docosahexaenoic acid (DHA),
Eicosapentaenoic Acid (EPA), and Alpha-linolenic acid
(ALA) [17]. It played an importantly in human nutrition, in
nutraceutical and pharmaceutical development and in
preventing human disease such as infertility disease, cancer
disease, diabetic disease, Alzheimer disease, as well as in
preventing of AIDs infections[12]. However the main source
of polyunsaturated fatty acids (PUFAs) is fishes, such sardine
and salmon [14]. Researchers have established production of
-3 poly-unsaturated fatty acid by many species of
heterotrophic* microalgae such a Thraustochytrid sp., and
Schizochytrium sp those are considered as oil sources instead
of fish oil[13]. Schizochytrium. limacinum SR21 is a
promising algae strain for DHA in highyield production and it
has been commercially of large scaling [18]. According to the
previous study that they can be produced -3 polyunsaturated
fatty acid from different species of heterotrophic microalgae in
high potential rates by optimizing media conditions and
compositions [9,11,12, 21,23, 24, 25]. Such further studied
results to developed culture strategy as Chi et al in 2007 could
be obtained 34%of PUFA from S. Limacinium MYA-1381
/SR21
Martial and Methods

Samples were collected from different areas of the coastal
Benghazi seawater by used Pine pollen baiting method [8].
Transferred the Pine pollen grains into BY+ agar medium with
antibiotics mixture for 4 days until the white to orange colored
colonies become raise. Pure colonies that those identified as a
chytrids in an axenic culture of BY+. Agar medium could be
maintained in slant subculture for 4-5 weeks.
Classical Batch Culture
The standard medium used in classical batch culture was broth
medium [21] This medium consisted of glucose, 20 g/L; yeast
extract, 1 g\L; Glutamic acid, 5 g\L. The basal medium
contained NaCl2, 25 g/L; KH2PO4, 1 g/L; KCl2 , 1 g/L; Mg
SO4.7H2O, 5 g/L; CaCl2 , 0.02 g/L; trace elements, 5 ml ;
vitamins, 1ml. The standard medium of 1000 mL was
Botany department , Benghazi University, Libya, Benghazi . E-mail:

horiyaalsharif@gmail.com
Inoculated with 10 mL of a suspension. The concentrated of a

suspension was prepared to achieve cell count of 10 6-108
28
Journal of Science
29
Developed Batch Culture

-*By the same standard medium composition, suspension
and the cultivation conditions of 25C temperature, new
fermenter strategy was developed. Such strategy developed as
harvested culture precipitation in three end cycles to started
new cycle by adding new standard medium reached to
1000mL in the same flask. Furthermore some changed in
design were developed in this experimented by connected
aeration tube 0.1inch in size diameter with the flask on an
angle 45. The end fermentation harvested was taken place 12
days.
Lipid Extraction and Analysis
Dry cell biomass was obtained by heating in a 60C oven for
48hours. Total lipid was extracted in chloroform: methanol,
2:1 [4]. Fatty acid extraction was performed by direct
transesterifiction protocol and then analyzed by gas
chromatography.
Optical Density(660 nm)
cells/mL, and the optical density was 0.5 at 660 nm. Then the
seed culture for 48h are incubated in classical batch
fermentation culture at 25C for 2-5 days with 200 rpm in an
orbital shaker.
12
10
8
6
4
2
0
2
1.5
1
0.5
0
0
24h 48h 72h 96h 120h 144h

Time (h)
Figure 2. Growth and Cell Biomass Curves

for classical batch
OD Y1
In this experiment some required conditions has controlled

such as agitation by tube aeration also the position of
fermenter controlled to be at 45 angle in order to allow the
air motivation increasable and then formation the colonies
participation in the bottom. The colonies formation as
participation in the bottom of fermenter due to ectoplasmic net
elements considered as one of morphological characterization
are used for Light microscope identification.
Result and Discussion

In Classical Batch Culture Results
The isolates were identified and studied in batch culture.
Optical density and cell biomass measurements
by
standardized medium are showed that the exponential phase
extent to about 72h, and the culture entered stationary phase
after 96 hr ( Fig. 1). Moreover dry cell biomass was reached to
maximum about 6.0 g L, while the total lipid of it
measurement have been 2.0 g L, that those involved about
50% of -3poly unsaturated fatty acid (table 1).
TABLE 1
COMPARAISON RESULTS BETWEEN CLASSICAL BATCH AND

DEVELOPED BATCH
media
DCW
(g/L)
Total
OMEGA-3
Lipid(g/L) (%)
Classical
batch
4.5-6.0 1.8-2.0
45-50%
Developed
batch
30-35
69.9-73%
20-23
In developed Batch Culture

In the present study, the modified methods for batch
culture was more successes comparing with classical batch
culture. It was produced amounts of the cell biomass
reached up to (35 g L); total lipid (23 g L) and 73% -3
polyunsaturated fatty acid Fig.(2). it is inexpensive and the
period of cultivation takes 12 days only. In fact, devolving
some strategy such participative the harvested shown more
successful in this technique than in a classical batch culture.
Figure (2): results of developed batch culture (g 1L):

a, harvested by filtration; b, cell biomass after dry in 60 C oven for 48h
In this study , it has been manipulated or optimized of media

requirements and physical conditions e.g., carbon, nitrogen,
temperature, dissolved oxygen, salinity, and pH. Those
factors could affect
culture productivity of -3
polyunsaturated fatty acid in a classical batch [3,6, 11,12, 18].
Study done by Lewis et al.,1999 referred that some
biotechnology they have been done since 1990s and used for
productions of -3 polyunsaturated fatty acid from different
species of heterotrophic microalgae ( which has been used in
this study). In the present study, we suggest developmental
strategy and design as a suitable technique for production of
-3 polyunsaturated fatty acid from these microalgae isolates.
Conclusion
In this study, the modified fermenter strategy and design
promote production of -3 polyunsaturated fatty in high
amount, reached up to 73%, therefore the developmental
Highly Production of Omega-3 Polyunsaturated Fatty Acid

strategy suggested as a new invention technology suitable for
polyunsaturated fatty acid production from these isolates. also
we recommended to apply this technique in up scaling more
than 100L as well as the modifications of standard medium by
alternatively of local medium sourcing.
Acknowledgment
I wish to thankful of Botany department, Faculty Science, ,
in Benghazi University also introduce too thank to any helping
and supporting from plant department, Faculty of science in
Musrata University.
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( )Mean STD g/g

(9) Namin J.I., Mohammadi M., Heydari S., and

)1) VandenBroek, J. I.; Gledhill, K. S.And Morgan, D.

Spectrophotometer Flame HITACHI 180-30, Equip

( )3 Ca- Cu- Zn Pb Fe-

Penecilliumchrysogenium - Aspergillus. sp4

24- Das, D., (2002). Health Hazard in Stone Quarries and

Vol. (2) Issue (2) August-2015

Vol. (2) Issue (2) August-2015

Comparison of Microscopy and Culture Methods for Detection of

Key words: Prevalence, Blastocystis hominis and Detection methods.

1-Medical Department, Faculty of Engineering and Technology,

Vol. (2) Issue (2) August-2015

hominis, meanwhile there was significant difference (p<0.05)

systems for the detection of B. hominis. No significant

Table 1: Comparison of methods for the detection of B. hominis in stools.

Number and percentage of positive samples by different methods

Diphasic Boeck and

Figures in parentheses indicate percentages.Of all the

formalin-ether concentration, and culture methods. 22(6.1%)

Direct smear (Neg)

(+Pos) = Positive, and (- Neg) = Negative

medium are shown in Table 3. Diphasic Boeck and

Number of B. hominis cells

Monophasic Jone's Medium

Diphasic Boeck and Drbohlav's Medium

Figures in parentheses indicate percentages

Number and Percentage of

Comparison of Microscopy and Culture Methods

smear microscopy. Moreover, asymptomatic infections are

Detection Methods (Prevalence %)

Patients attending Central

Boeck and Drbohlav's

School children in Derna

Patients attending Central

Patients attending Brack

attending Central Laboratory in Sebha, Salem et al. [24]

Vol. (2) Issue (2) August-2015

In the present study, the culture techniques