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 Callyspongia

  Callyspongia diffusa

À
 

 


 Basic idea of the paper
0 Symbiotic association
^seudomonas aeruginosa
aeruginosa,, Escherichia coli
coli,,
Vibrio cholera
cholera,, Vibrio parahaemolyticus

0 Bioactive proteins
u in methanol and in aqueous extract
u antibacterial property
u hemolytic activity on chicken erythrocytes

0 Protein estimation was carried out.

0 SDS-PAGE was used to know the molecular

SDS-
weight to the proteins extracted
 ÿethods and materials
0 Study area ± khardhanda beach near Juhu,
ÿumbai coast

0 Handpicking of the Callyspongia diffusa during

low tide

0 Preservation at low temperature

0 Boiled with concentrated HNO3 to extrude the

spicules and confirm the identity based on the
characters proposed by Thomas
 |solation of the associated
Freshbacteria
tissue of sponge
Ļ
cut by sterile blade
Ļ
Homogenized using pestle and mortar
Ļ
Serial dilution
Ļ
pour in different sterile media plates
[ÿac Conkey¶s agar, Vibrio agar, Nutrient agar,
Eosin methyl blue agar]
Ļ
Bacterial associates were identified using Bergey¶s
manual
 Extraction of crude toxin
0 ÿ 
 
 0 


 

mbakus and green)
sponge Squeezing the sand free sponge
Ļ specimens in triple distilled water
dried in airm days) Ļ
Ļ Solution obtained is filtered and
10g tissue soaked in 00ml of dialyzed using sigma dialysis
methanolm
hrs) membrane
00 against D D--glucose
Ļ to remove excess water
Removal of solvent by squeezing the Ļ
sponge supernatant obtained is lyophilized
Ļ using the Labcono Freeze Dry
System
filter through Whatman filter no.1
Ļ
Ļ
Stored at 4°
4°C in refrigerator for further
solvent evaporation at low pressure use
using Buchi Rotavopor at 60°
60°C
Ļ
Storage of extract in refrigerator for
further use
Antimicrobial activity
Petri dish with sterile nutrient agar
was inoculated with the isolated
bacteria
Ļ
Sponge extract was sterilized by
passing each through a 0. m
ÿillipore GV filter
Ļ
Round paper discs were dipped into
0.001 ml of each sponge extract
Ļ
Then placed in centre of the
inoculated Petri dish
Ļ
Bacterial colonies were allowed to
grow overnight at 37°
37°C
Ļ
|nhibition zone around the disc was
observed
Partial purification of crude
extract
0 Partial purification
was carried out using
DEAE cellulose anion
exchange
chromatography
according to the
method of Stempion
 Protein Estimation
mby Lowry & Lopez method)
Standard used was Bovine albumin serum at
concentration ranging from 0.1 to 1 mg/ml
Ļ

ml of alkaline copper reagent was added
Ļ
ÿix well and allow it to stand for 10 minutes
Ļ
Add 0.
ml of dilute folin¶s phenol reagent &
mix well
Ļ
|ncubate for 30 minutes
Ļ
Take absorbance at 6
0nm
spectrophotometrically
Ļ
Protein was estimated using standard graph
 Hemolytic activity
Performed in 96 well µv¶ bottom microtitre plates
Ļ
Selection of different rows for chicken blood
Ļ
-fold serial dilution of crude toxin in 100ml of normal saline and repetition of
above steps up to the last well
Ļ
100l RBC was added to all wells
Ļ
Appropriate controls were included
1% RBC suspension & 100l normal saline ĺnegative control
Ļ
Gentle shaking of plates
And allowed to stand for hrs at room temperature
Ļ
Recorded the results muniform red color suspension ĺpositive hemolysis
Button formation at bottom ĺlack of hemolysis)
Ļ
Reciprocal of highest dilution of crude toxin showing pattern was taken as 1
hemolytic unit mHU)
 SDS--PAGE
SDS
0 One dimension Sodium dodecyl sulphate
polyacrylamide gel electrophoresis was carried
out

0 Proteins were electrophoresed on 1 %

separating gel m0.7
mm thickness) overlaid with

% stacking gel.
 Statistical analysis
0 Statistical analysis was carried out using one
way analysis of variance mANOVA) by SPSS
software package, version-
version-13.0 followed by
Duncan¶s multiple range test mDÿRT).
 Result
0 ÿorphological Bacteria Colony characters
and P. aeruginosa Growth on NAÿ light
light--grey
physiological colonies of 1.
mm
diameter
characteristics
of the bacteria E. coli ÿetallic sheen colonies
on EÿB agar
isolated from
the sponge V. Grey colonies on NAÿ,
species are parahaemolytic ÿAC, 1-
1- mm diameter,
given : us swarm across plate
V. cholera Colonies are round
smooth -3mm, greenish
grey on VA
0 ÿethanolic extract m
00g) of sponge yielded
.4 g of crude
extract, whereas aqueous extract yielded
.09g.

0 The protein content from the sponge was found to be 1.6

mg/ml in methanolic extract and 1.43 mg/ml in aqueous
extract.

0 The extract were tested at different concentrationm
,10 and

1
mg/ml) against the isolated bacteria and none of the
extract inhibited the test bacteria except the aqueous extract
did inhibit V. cholera
cholera..

0 The crude methanolic extract induced hemolysis on chicken

erythrocytes and its hemolytic titre was 14 and also its
hemolytic activity was estimated at 8.46 HU/mg of protein
whereas the hemolytic titre of aqueous extract was 10 and its
activity was estimated at 6.99 HU/mg of protein
0 SDS-PAGE ĺ6 bands in aqueous extract and 8
SDS-
bands in methanolic extract ranging from 14.4 to 116
kDa molecular weight.

0 3 well defined bands of 19.
, 39.0, 66. kDa in both

extract
 Discussion
0
00g sponge yielded
.09gmmethanol) and
.4g mwater) of crude
extract
Ļ compare
Richet ĺ
.8g of crude extract from 490g fresh weight of marine
sponge, Guberea preatense

0 Protein contentĺ corresponding data on protein content of

sponge is not available in literature for comparison

0 Hemolytic activity of both extract are in accordance with earlier

studies [Stempeinĺ halitoxin mîaliclona
mîaliclona),
), Fusetanie ĺsterol
derivatives viz., halistanol sulphate and sokotrasterol sulphate
mhalichondriidae sponge), ÿebs ĺhaemagglutination and
hemolytic activity of aqueous extract m48 tropical sponge
species).
 Conclusion
0 Hemolytic activity indicates cytotoxicity of the extractĺ
extractĺ
worthwhile for studies on their antitumour/anti-
antitumour/anti-neopastic
activities ĺdetection of anticancer compounds

0 Antibacterial agents produced by sponge ĺenhancing

efficiency with sponge retains bacterial food.

0 ÿarine sponge have anti microbial activity against marine

bacteria but no activity against bacteria has been observed
thus confirming the isolated bacteria are symbiotic to marine
sponge studied.

0 The 66. kDa band obtained in SDS-

SDS-PAGE analysis ĺ in
present study might be stress induced protein and this type
of stress induced protein has been reported in marine cat-
cat-
fish.
Thank You