- ข้อความ
- ประวัติศาสตร์
INTRODUCTION‘Chilika’ is the larges
INTRODUCTION
‘Chilika’ is the largest lagoon in the subcontinent which is
situated along the east coast of India between latitude 19o
28’ and 19o 54’ N, and longitude 85o 05’ and 85o 38’E.
This is a shallow, brackish-water lake formed due to the
silting action of the Mahanadi River, which drains into the
northern end of the lake, and the northerly currents in the
Bay of Bengal, which have formed a sandbar along the
eastern shore (Arya and Lakhotia, 2006). It is the largest
brackish water wetland complex in Asia, declared as a
Ramsar site under the convention on “Wetlands of
International Importance”. This lagoon is presently under
threat from both natural and anthropogenic pressures
(Nayak et al., 2004). The marine realm constitutes the
major habitat of the biosphere covering 73 % of the earth
surface which provides the largest inhabitable space for
living organisms, particularly microbes. Marine microbes
flourish not only in the surface water of the sea, but also in
the lower and abyssal depths from coastal to the offshore
regions and from the general oceanic to the specialized
niches like blue waters of coral reefs to black smokers of
hot thermal vents at the sea floor.
As the water being served as major solvent, it plays an
important role in the pharmaceutical industries.
It is
considered as the primary source of contamination for
pharmaceutical products (Tamara et al., 1998). Microbes
present in the marine waters represent a potential source
for commercially important bioactive compounds and their
bioremediation capabilities are also remarkable. There is
an increasing demand of biodiversity from natural
resources for development of therapeutic drugs.
Antibiotics are broadly used as chemotherapeutic agents
which, albeit in a very low quantity, can inhibit the
pathogenic activity of microorganisms. The potential
contribution of marine organisms to the discovery of new
bioactive molecules is increasingly challenging (Sponga et
al., 1999; Skulberg, 2000). The microorganisms have
become a significant attraction as natural source of
bioactive molecules with a broad range of biological
activities, such as antibiotics, antivirals, antitumorals,
antioxidant and anti-inflammatory (Okami, 1982; Kamei et
al., 1987; Nunez et al., 2006; Uzair et al., 2009; Shankar
et al., 2010). Evidence of phycochemical and
pharmacological studies on microbes is available in the
literature (Zeeshan et al., 2010; Odeyemi et al., 2010;
Bragadeeswaran et al., 2010).
Previously, there were a number of researchers that had
worked on ‘Chilika’ regarding hydrological
characterization, water quality variation, antimicrobial
activity and physiochemical variation (Rao et al., 1981;
Nayak et al., 2004; Patra et al., 2009, Patra et al., 2010).
Unfortunately progress made on pharmaceutical studies
of marine microorganism from ‘Chilika’ is inadequate. This
underpinned the present study with a view to investigating
the physiological, biochemical, and serological
characterization of bacteria isolated from marine waters of
‘Chilika’ aiming at their exploitation for pharmaceutical
benefits.
MATERIALS AND METHODS
Isolation and characterization of bacterial strains
The total study area is comprised of 18 sampling stations
covering three different sectors of the ‘Chilika’ lagoon i.e.
Central, Southern and Outer Channel (Figure 1A, 1B)
during the period from January to March 2008. The exact
sampling locations were fixed by using Global Positioning
System (GPS). All the water samples were collected in the
morning hours between 7:00 A.M-11:00 A.M. During
sample collection necessary precautions had been taken
to collect undisturbed water samples in the lake. Samples
were collected in pre-sterilized polypropylene bottles (500
mL). Physical parameters like temperature and pH of the
samples were determined on the spot of collection. Then
the water samples were kept in the icebox and transferred
to laboratory for further analysis and the samples were
preserved at 4ฐC before isolation and identification in the
laboratory. The experiments were carried out within 4
months of collection. Chemicals used for preparation of
reagents in the present investigation were of analytical
reagent grade and for preparation of solutions doubledistilled
water was used. One milliliter of water sample
was subjected through serial dilution to obtain 10-8
dilution. After that 1mL of 10-8 diluted sample was mixed
with 10 mL of sterilized Nutrient broth medium and then it
was incubated at 37 ฐC in a BOD incubator for 24 h. After
incubation a loop-full of microbial culture were picked up
from different colonies and streaked separately on the
different agar-gelled sterilized media and the plates were
incubated at 37 ฐC for 24 h for isolation of pure culture.
The colony characteristics such as colour, appearance,
and shape of the isolate were recorded. Further,
morphological, and biochemical tests were carried out in
the laboratory by following standard microbiological
methods described by Cappuccino and Sherman (2002)
for identification and characterization of isolated
microorganism. The isolated strains were also
characterized through different physiological factors such
as pH, temperature and salinity to observe their tolerance
capacity in the stressed environmental conditions.
Collection and preservation of pre-immunized serum
For production of antisera 3-4 month-old white rabbit (2.5 -
3 kg) were selected and kept in separate cage and
labeled properly. The pre-immunized sera of the rabbit
used for the test were collected before 7 days of
immunization with different a ntigens to confirm that the
rabbit did not contain any antisera. The sterilized tube
containing fresh drawn blood was kept at room
temperature for 1-2 h for clot formation. The clots was
stirred with glass rod and was kept overnight in
refrigerator to allow the clot contraction. The plasma were
centrifuged (3000 rpm, 15 min). The straw-coloured serum
was withdrawn into a sterilized centrifuge tube which was
labeled properly. For preservation of serum, it was added
with 0.02 % sodium azide (NaNH2) to give a final
concentration of 1:5000 and kept in a deep freeze at -20
ฐC for further use.
Serological test
Preparation of whole cell antigen: The antigen was
prepared from the freshly grown bacterial cultures. The
bacteria were cultured on NSA medium in test tube at 27ฐC for 24 h. The test tube was filled with PBS or saline water.
The culture was scraped with sterilized inoculation
needle. The scraped bacteria with saline water were
transferred to the centrifuge tube. The bacterial growth
was mixed thoroughly with the help of a vortex mixture to
make it completely homogenized. The whole cell of
bacteria was centrifuged (3000 rpm, 30 min, room
temperature). The supernatant was discarded and the
pellet was re-suspended with saline water and
centrifuged. The process was repeated three times to
make the bacteria free from any foreign material. After the
last centrifugation bacteria were re-suspended in normal
saline/butler saline, collected in sterilized specimen tube,
labeled properly, preserved and stored in deep freeze at -
20 ฐC for further use.
Preparation of antigen for agglutination test: The
antigens were prepared by suspending live bacteria in
buffer saline as described earlier except that the antigen
prepared was more concentrated having an optical
density of 0.5 using a Gallen Kamp colorimeter at 520
mm.
Immunization of rabbits for antisera production and
harvesting of immunized serum: The rabbits were
immunized by intra-muscular and intravenous injections.
Both right and left legs were used for intra-muscular
injection. In case of intravenous injection, marginal vein of
ear was used. Three doses of antigen (0.5 mL, 1.0 mL,
and 1.5 mL) were injected intra-muscularly at four days
interval. Acetone was used to dilate the vein. The
designed quantity was injected slowly into the blood
system. A booster injection was given 4 days after the last
injection to accelerate antibody production. Five or seven
days after the booster injection, the rabbits were held by
cardiac puncture for collection of antisera. The procedure
of collection of blood separation and preservation of
antiserum was the same as described earlier.
Antigen-antibody reaction tests: Mixture of antigen and
antibody showing positive reaction resulted in the
formation of precipitation due to binding of antibodies with
their respective antigens. Two different serological tests
were conducted to record the precipitations or positive
reaction of antigen and antibodies. The gel diffusion test
was performed following the method as originally
described by Ouchterlony (1958, 1962) while the tube
agglutination test was carried out for homologous and
heterologous antigens using a national standard method
(NHS, 2010).
Antibiotic sensitivity test
Antibiotic sensitivity test of the identified bacteria were
repeated 3 times for each strain using 10 different
antibiotics namely norfloxacin, tetracycline, ciprofloxacin,
neomycin, nalidixic acid, ofloxacin, chloramphenicol,
nitrofurantoin, streptomycin and amoxicillin (Hi- Media,
Mumbai, India) by disc diffusion method (Bauer et al.,1966). Antibiotic activity was measured in terms of zoneinhibition (mm diameter).
Plasmid DNA isolation and agarose gel
electrophoresis
The alkaline-lysis mini-prep method (Wizard Plus
Minipreps DNA Purification Systems, Promega, USA) was
used for plasmid extraction from all the four bacterial
isolates. Overnight-grown bacterial cultures (3-5 mL) were
pelleted by centrifugation (10,000 x g, 10 min),
supernatant decanted and pellet re-suspended in 300 OL
of Cell Re-suspension Solution (50 mM Tris, pH 7.5; 10
mM EDTA; 100 Og/mL RNase A). The re-suspended cells
were transferred to 1.5 mL micro-centrifuged tube to
which 300 OL of Cell Lysis Solution (0.2 M NaOH, 1 %
SDS) was added and samples thoroughly mixed. To the
cleared lysate 300 OL of Neutralization Solution (1.32 M
potassium
‘Chilika’ is the largest lagoon in the subcontinent which is
situated along the east coast of India between latitude 19o
28’ and 19o 54’ N, and longitude 85o 05’ and 85o 38’E.
This is a shallow, brackish-water lake formed due to the
silting action of the Mahanadi River, which drains into the
northern end of the lake, and the northerly currents in the
Bay of Bengal, which have formed a sandbar along the
eastern shore (Arya and Lakhotia, 2006). It is the largest
brackish water wetland complex in Asia, declared as a
Ramsar site under the convention on “Wetlands of
International Importance”. This lagoon is presently under
threat from both natural and anthropogenic pressures
(Nayak et al., 2004). The marine realm constitutes the
major habitat of the biosphere covering 73 % of the earth
surface which provides the largest inhabitable space for
living organisms, particularly microbes. Marine microbes
flourish not only in the surface water of the sea, but also in
the lower and abyssal depths from coastal to the offshore
regions and from the general oceanic to the specialized
niches like blue waters of coral reefs to black smokers of
hot thermal vents at the sea floor.
As the water being served as major solvent, it plays an
important role in the pharmaceutical industries.
It is
considered as the primary source of contamination for
pharmaceutical products (Tamara et al., 1998). Microbes
present in the marine waters represent a potential source
for commercially important bioactive compounds and their
bioremediation capabilities are also remarkable. There is
an increasing demand of biodiversity from natural
resources for development of therapeutic drugs.
Antibiotics are broadly used as chemotherapeutic agents
which, albeit in a very low quantity, can inhibit the
pathogenic activity of microorganisms. The potential
contribution of marine organisms to the discovery of new
bioactive molecules is increasingly challenging (Sponga et
al., 1999; Skulberg, 2000). The microorganisms have
become a significant attraction as natural source of
bioactive molecules with a broad range of biological
activities, such as antibiotics, antivirals, antitumorals,
antioxidant and anti-inflammatory (Okami, 1982; Kamei et
al., 1987; Nunez et al., 2006; Uzair et al., 2009; Shankar
et al., 2010). Evidence of phycochemical and
pharmacological studies on microbes is available in the
literature (Zeeshan et al., 2010; Odeyemi et al., 2010;
Bragadeeswaran et al., 2010).
Previously, there were a number of researchers that had
worked on ‘Chilika’ regarding hydrological
characterization, water quality variation, antimicrobial
activity and physiochemical variation (Rao et al., 1981;
Nayak et al., 2004; Patra et al., 2009, Patra et al., 2010).
Unfortunately progress made on pharmaceutical studies
of marine microorganism from ‘Chilika’ is inadequate. This
underpinned the present study with a view to investigating
the physiological, biochemical, and serological
characterization of bacteria isolated from marine waters of
‘Chilika’ aiming at their exploitation for pharmaceutical
benefits.
MATERIALS AND METHODS
Isolation and characterization of bacterial strains
The total study area is comprised of 18 sampling stations
covering three different sectors of the ‘Chilika’ lagoon i.e.
Central, Southern and Outer Channel (Figure 1A, 1B)
during the period from January to March 2008. The exact
sampling locations were fixed by using Global Positioning
System (GPS). All the water samples were collected in the
morning hours between 7:00 A.M-11:00 A.M. During
sample collection necessary precautions had been taken
to collect undisturbed water samples in the lake. Samples
were collected in pre-sterilized polypropylene bottles (500
mL). Physical parameters like temperature and pH of the
samples were determined on the spot of collection. Then
the water samples were kept in the icebox and transferred
to laboratory for further analysis and the samples were
preserved at 4ฐC before isolation and identification in the
laboratory. The experiments were carried out within 4
months of collection. Chemicals used for preparation of
reagents in the present investigation were of analytical
reagent grade and for preparation of solutions doubledistilled
water was used. One milliliter of water sample
was subjected through serial dilution to obtain 10-8
dilution. After that 1mL of 10-8 diluted sample was mixed
with 10 mL of sterilized Nutrient broth medium and then it
was incubated at 37 ฐC in a BOD incubator for 24 h. After
incubation a loop-full of microbial culture were picked up
from different colonies and streaked separately on the
different agar-gelled sterilized media and the plates were
incubated at 37 ฐC for 24 h for isolation of pure culture.
The colony characteristics such as colour, appearance,
and shape of the isolate were recorded. Further,
morphological, and biochemical tests were carried out in
the laboratory by following standard microbiological
methods described by Cappuccino and Sherman (2002)
for identification and characterization of isolated
microorganism. The isolated strains were also
characterized through different physiological factors such
as pH, temperature and salinity to observe their tolerance
capacity in the stressed environmental conditions.
Collection and preservation of pre-immunized serum
For production of antisera 3-4 month-old white rabbit (2.5 -
3 kg) were selected and kept in separate cage and
labeled properly. The pre-immunized sera of the rabbit
used for the test were collected before 7 days of
immunization with different a ntigens to confirm that the
rabbit did not contain any antisera. The sterilized tube
containing fresh drawn blood was kept at room
temperature for 1-2 h for clot formation. The clots was
stirred with glass rod and was kept overnight in
refrigerator to allow the clot contraction. The plasma were
centrifuged (3000 rpm, 15 min). The straw-coloured serum
was withdrawn into a sterilized centrifuge tube which was
labeled properly. For preservation of serum, it was added
with 0.02 % sodium azide (NaNH2) to give a final
concentration of 1:5000 and kept in a deep freeze at -20
ฐC for further use.
Serological test
Preparation of whole cell antigen: The antigen was
prepared from the freshly grown bacterial cultures. The
bacteria were cultured on NSA medium in test tube at 27ฐC for 24 h. The test tube was filled with PBS or saline water.
The culture was scraped with sterilized inoculation
needle. The scraped bacteria with saline water were
transferred to the centrifuge tube. The bacterial growth
was mixed thoroughly with the help of a vortex mixture to
make it completely homogenized. The whole cell of
bacteria was centrifuged (3000 rpm, 30 min, room
temperature). The supernatant was discarded and the
pellet was re-suspended with saline water and
centrifuged. The process was repeated three times to
make the bacteria free from any foreign material. After the
last centrifugation bacteria were re-suspended in normal
saline/butler saline, collected in sterilized specimen tube,
labeled properly, preserved and stored in deep freeze at -
20 ฐC for further use.
Preparation of antigen for agglutination test: The
antigens were prepared by suspending live bacteria in
buffer saline as described earlier except that the antigen
prepared was more concentrated having an optical
density of 0.5 using a Gallen Kamp colorimeter at 520
mm.
Immunization of rabbits for antisera production and
harvesting of immunized serum: The rabbits were
immunized by intra-muscular and intravenous injections.
Both right and left legs were used for intra-muscular
injection. In case of intravenous injection, marginal vein of
ear was used. Three doses of antigen (0.5 mL, 1.0 mL,
and 1.5 mL) were injected intra-muscularly at four days
interval. Acetone was used to dilate the vein. The
designed quantity was injected slowly into the blood
system. A booster injection was given 4 days after the last
injection to accelerate antibody production. Five or seven
days after the booster injection, the rabbits were held by
cardiac puncture for collection of antisera. The procedure
of collection of blood separation and preservation of
antiserum was the same as described earlier.
Antigen-antibody reaction tests: Mixture of antigen and
antibody showing positive reaction resulted in the
formation of precipitation due to binding of antibodies with
their respective antigens. Two different serological tests
were conducted to record the precipitations or positive
reaction of antigen and antibodies. The gel diffusion test
was performed following the method as originally
described by Ouchterlony (1958, 1962) while the tube
agglutination test was carried out for homologous and
heterologous antigens using a national standard method
(NHS, 2010).
Antibiotic sensitivity test
Antibiotic sensitivity test of the identified bacteria were
repeated 3 times for each strain using 10 different
antibiotics namely norfloxacin, tetracycline, ciprofloxacin,
neomycin, nalidixic acid, ofloxacin, chloramphenicol,
nitrofurantoin, streptomycin and amoxicillin (Hi- Media,
Mumbai, India) by disc diffusion method (Bauer et al.,1966). Antibiotic activity was measured in terms of zoneinhibition (mm diameter).
Plasmid DNA isolation and agarose gel
electrophoresis
The alkaline-lysis mini-prep method (Wizard Plus
Minipreps DNA Purification Systems, Promega, USA) was
used for plasmid extraction from all the four bacterial
isolates. Overnight-grown bacterial cultures (3-5 mL) were
pelleted by centrifugation (10,000 x g, 10 min),
supernatant decanted and pellet re-suspended in 300 OL
of Cell Re-suspension Solution (50 mM Tris, pH 7.5; 10
mM EDTA; 100 Og/mL RNase A). The re-suspended cells
were transferred to 1.5 mL micro-centrifuged tube to
which 300 OL of Cell Lysis Solution (0.2 M NaOH, 1 %
SDS) was added and samples thoroughly mixed. To the
cleared lysate 300 OL of Neutralization Solution (1.32 M
potassium
0/5000
INTRODUCTION‘Chilika’ is the largest lagoon in the subcontinent which issituated along the east coast of India between latitude 19o28’ and 19o 54’ N, and longitude 85o 05’ and 85o 38’E.This is a shallow, brackish-water lake formed due to thesilting action of the Mahanadi River, which drains into thenorthern end of the lake, and the northerly currents in theBay of Bengal, which have formed a sandbar along theeastern shore (Arya and Lakhotia, 2006). It is the largestbrackish water wetland complex in Asia, declared as aRamsar site under the convention on “Wetlands ofInternational Importance”. This lagoon is presently underthreat from both natural and anthropogenic pressures(Nayak et al., 2004). The marine realm constitutes themajor habitat of the biosphere covering 73 % of the earthsurface which provides the largest inhabitable space forliving organisms, particularly microbes. Marine microbesflourish not only in the surface water of the sea, but also inthe lower and abyssal depths from coastal to the offshoreregions and from the general oceanic to the specializedniches like blue waters of coral reefs to black smokers ofhot thermal vents at the sea floor.As the water being served as major solvent, it plays animportant role in the pharmaceutical industries.It isconsidered as the primary source of contamination forpharmaceutical products (Tamara et al., 1998). Microbespresent in the marine waters represent a potential sourcefor commercially important bioactive compounds and theirbioremediation capabilities are also remarkable. There isan increasing demand of biodiversity from naturalresources for development of therapeutic drugs.Antibiotics are broadly used as chemotherapeutic agentswhich, albeit in a very low quantity, can inhibit thepathogenic activity of microorganisms. The potentialcontribution of marine organisms to the discovery of newbioactive molecules is increasingly challenging (Sponga etal., 1999; Skulberg, 2000). The microorganisms havebecome a significant attraction as natural source ofbioactive molecules with a broad range of biologicalactivities, such as antibiotics, antivirals, antitumorals,antioxidant and anti-inflammatory (Okami, 1982; Kamei etal., 1987; Nunez et al., 2006; Uzair et al., 2009; Shankaret al., 2010). Evidence of phycochemical andpharmacological studies on microbes is available in theliterature (Zeeshan et al., 2010; Odeyemi et al., 2010;Bragadeeswaran et al., 2010).Previously, there were a number of researchers that hadworked on ‘Chilika’ regarding hydrologicalcharacterization, water quality variation, antimicrobialactivity and physiochemical variation (Rao et al., 1981;Nayak et al., 2004; Patra et al., 2009, Patra et al., 2010).Unfortunately progress made on pharmaceutical studiesof marine microorganism from ‘Chilika’ is inadequate. Thisunderpinned the present study with a view to investigatingthe physiological, biochemical, and serologicalcharacterization of bacteria isolated from marine waters of‘Chilika’ aiming at their exploitation for pharmaceuticalbenefits.MATERIALS AND METHODSIsolation and characterization of bacterial strainsThe total study area is comprised of 18 sampling stationscovering three different sectors of the ‘Chilika’ lagoon i.e.Central, Southern and Outer Channel (Figure 1A, 1B)during the period from January to March 2008. The exactsampling locations were fixed by using Global PositioningSystem (GPS). All the water samples were collected in themorning hours between 7:00 A.M-11:00 A.M. Duringsample collection necessary precautions had been takento collect undisturbed water samples in the lake. Sampleswere collected in pre-sterilized polypropylene bottles (500mL). Physical parameters like temperature and pH of thesamples were determined on the spot of collection. Thenthe water samples were kept in the icebox and transferredto laboratory for further analysis and the samples werepreserved at 4ฐC before isolation and identification in thelaboratory. The experiments were carried out within 4months of collection. Chemicals used for preparation ofreagents in the present investigation were of analyticalreagent grade and for preparation of solutions doubledistilledwater was used. One milliliter of water samplewas subjected through serial dilution to obtain 10-8dilution. After that 1mL of 10-8 diluted sample was mixedwith 10 mL of sterilized Nutrient broth medium and then itwas incubated at 37 ฐC in a BOD incubator for 24 h. Afterincubation a loop-full of microbial culture were picked upfrom different colonies and streaked separately on thedifferent agar-gelled sterilized media and the plates wereincubated at 37 ฐC for 24 h for isolation of pure culture.The colony characteristics such as colour, appearance,and shape of the isolate were recorded. Further,morphological, and biochemical tests were carried out inthe laboratory by following standard microbiologicalmethods described by Cappuccino and Sherman (2002)for identification and characterization of isolatedmicroorganism. The isolated strains were alsocharacterized through different physiological factors suchas pH, temperature and salinity to observe their tolerancecapacity in the stressed environmental conditions.Collection and preservation of pre-immunized serumFor production of antisera 3-4 month-old white rabbit (2.5 -3 kg) were selected and kept in separate cage andlabeled properly. The pre-immunized sera of the rabbitused for the test were collected before 7 days ofimmunization with different a ntigens to confirm that therabbit did not contain any antisera. The sterilized tubecontaining fresh drawn blood was kept at roomtemperature for 1-2 h for clot formation. The clots wasstirred with glass rod and was kept overnight inrefrigerator to allow the clot contraction. The plasma werecentrifuged (3000 rpm, 15 min). The straw-coloured serumwas withdrawn into a sterilized centrifuge tube which waslabeled properly. For preservation of serum, it was addedwith 0.02 % sodium azide (NaNH2) to give a finalconcentration of 1:5000 and kept in a deep freeze at -20ฐC for further use.Serological testPreparation of whole cell antigen: The antigen wasprepared from the freshly grown bacterial cultures. Thebacteria were cultured on NSA medium in test tube at 27ฐC for 24 h. The test tube was filled with PBS or saline water.The culture was scraped with sterilized inoculationneedle. The scraped bacteria with saline water weretransferred to the centrifuge tube. The bacterial growthwas mixed thoroughly with the help of a vortex mixture tomake it completely homogenized. The whole cell ofbacteria was centrifuged (3000 rpm, 30 min, roomtemperature). The supernatant was discarded and thepellet was re-suspended with saline water andcentrifuged. The process was repeated three times tomake the bacteria free from any foreign material. After thelast centrifugation bacteria were re-suspended in normalsaline/butler saline, collected in sterilized specimen tube,labeled properly, preserved and stored in deep freeze at -20 ฐC for further use.Preparation of antigen for agglutination test: Theantigens were prepared by suspending live bacteria in
buffer saline as described earlier except that the antigen
prepared was more concentrated having an optical
density of 0.5 using a Gallen Kamp colorimeter at 520
mm.
Immunization of rabbits for antisera production and
harvesting of immunized serum: The rabbits were
immunized by intra-muscular and intravenous injections.
Both right and left legs were used for intra-muscular
injection. In case of intravenous injection, marginal vein of
ear was used. Three doses of antigen (0.5 mL, 1.0 mL,
and 1.5 mL) were injected intra-muscularly at four days
interval. Acetone was used to dilate the vein. The
designed quantity was injected slowly into the blood
system. A booster injection was given 4 days after the last
injection to accelerate antibody production. Five or seven
days after the booster injection, the rabbits were held by
cardiac puncture for collection of antisera. The procedure
of collection of blood separation and preservation of
antiserum was the same as described earlier.
Antigen-antibody reaction tests: Mixture of antigen and
antibody showing positive reaction resulted in the
formation of precipitation due to binding of antibodies with
their respective antigens. Two different serological tests
were conducted to record the precipitations or positive
reaction of antigen and antibodies. The gel diffusion test
was performed following the method as originally
described by Ouchterlony (1958, 1962) while the tube
agglutination test was carried out for homologous and
heterologous antigens using a national standard method
(NHS, 2010).
Antibiotic sensitivity test
Antibiotic sensitivity test of the identified bacteria were
repeated 3 times for each strain using 10 different
antibiotics namely norfloxacin, tetracycline, ciprofloxacin,
neomycin, nalidixic acid, ofloxacin, chloramphenicol,
nitrofurantoin, streptomycin and amoxicillin (Hi- Media,
Mumbai, India) by disc diffusion method (Bauer et al.,1966). Antibiotic activity was measured in terms of zoneinhibition (mm diameter).
Plasmid DNA isolation and agarose gel
electrophoresis
The alkaline-lysis mini-prep method (Wizard Plus
Minipreps DNA Purification Systems, Promega, USA) was
used for plasmid extraction from all the four bacterial
isolates. Overnight-grown bacterial cultures (3-5 mL) were
pelleted by centrifugation (10,000 x g, 10 min),
supernatant decanted and pellet re-suspended in 300 OL
of Cell Re-suspension Solution (50 mM Tris, pH 7.5; 10
mM EDTA; 100 Og/mL RNase A). The re-suspended cells
were transferred to 1.5 mL micro-centrifuged tube to
which 300 OL of Cell Lysis Solution (0.2 M NaOH, 1 %
SDS) was added and samples thoroughly mixed. To the
cleared lysate 300 OL of Neutralization Solution (1.32 M
potassium
buffer saline as described earlier except that the antigen
prepared was more concentrated having an optical
density of 0.5 using a Gallen Kamp colorimeter at 520
mm.
Immunization of rabbits for antisera production and
harvesting of immunized serum: The rabbits were
immunized by intra-muscular and intravenous injections.
Both right and left legs were used for intra-muscular
injection. In case of intravenous injection, marginal vein of
ear was used. Three doses of antigen (0.5 mL, 1.0 mL,
and 1.5 mL) were injected intra-muscularly at four days
interval. Acetone was used to dilate the vein. The
designed quantity was injected slowly into the blood
system. A booster injection was given 4 days after the last
injection to accelerate antibody production. Five or seven
days after the booster injection, the rabbits were held by
cardiac puncture for collection of antisera. The procedure
of collection of blood separation and preservation of
antiserum was the same as described earlier.
Antigen-antibody reaction tests: Mixture of antigen and
antibody showing positive reaction resulted in the
formation of precipitation due to binding of antibodies with
their respective antigens. Two different serological tests
were conducted to record the precipitations or positive
reaction of antigen and antibodies. The gel diffusion test
was performed following the method as originally
described by Ouchterlony (1958, 1962) while the tube
agglutination test was carried out for homologous and
heterologous antigens using a national standard method
(NHS, 2010).
Antibiotic sensitivity test
Antibiotic sensitivity test of the identified bacteria were
repeated 3 times for each strain using 10 different
antibiotics namely norfloxacin, tetracycline, ciprofloxacin,
neomycin, nalidixic acid, ofloxacin, chloramphenicol,
nitrofurantoin, streptomycin and amoxicillin (Hi- Media,
Mumbai, India) by disc diffusion method (Bauer et al.,1966). Antibiotic activity was measured in terms of zoneinhibition (mm diameter).
Plasmid DNA isolation and agarose gel
electrophoresis
The alkaline-lysis mini-prep method (Wizard Plus
Minipreps DNA Purification Systems, Promega, USA) was
used for plasmid extraction from all the four bacterial
isolates. Overnight-grown bacterial cultures (3-5 mL) were
pelleted by centrifugation (10,000 x g, 10 min),
supernatant decanted and pellet re-suspended in 300 OL
of Cell Re-suspension Solution (50 mM Tris, pH 7.5; 10
mM EDTA; 100 Og/mL RNase A). The re-suspended cells
were transferred to 1.5 mL micro-centrifuged tube to
which 300 OL of Cell Lysis Solution (0.2 M NaOH, 1 %
SDS) was added and samples thoroughly mixed. To the
cleared lysate 300 OL of Neutralization Solution (1.32 M
potassium
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INTRODUCTION
‘Chilika’ is the largest lagoon in the subcontinent which is
situated along the east coast of India between latitude 19o
28’ and 19o 54’ N, and longitude 85o 05’ and 85o 38’E.
This is a shallow, brackish-water lake formed due to the
silting action of the Mahanadi River, which drains into the
northern end of the lake, and the northerly currents in the
Bay of Bengal, which have formed a sandbar along the
eastern shore (Arya and Lakhotia, 2006). It is the largest
brackish water wetland complex in Asia, declared as a
Ramsar site under the convention on “Wetlands of
International Importance”. This lagoon is presently under
threat from both natural and anthropogenic pressures
(Nayak et al., 2004). The marine realm constitutes the
major habitat of the biosphere covering 73 % of the earth
surface which provides the largest inhabitable space for
living organisms, particularly microbes. Marine microbes
flourish not only in the surface water of the sea, but also in
the lower and abyssal depths from coastal to the offshore
regions and from the general oceanic to the specialized
niches like blue waters of coral reefs to black smokers of
hot thermal vents at the sea floor.
As the water being served as major solvent, it plays an
important role in the pharmaceutical industries.
It is
considered as the primary source of contamination for
pharmaceutical products (Tamara et al., 1998). Microbes
present in the marine waters represent a potential source
for commercially important bioactive compounds and their
bioremediation capabilities are also remarkable. There is
an increasing demand of biodiversity from natural
resources for development of therapeutic drugs.
Antibiotics are broadly used as chemotherapeutic agents
which, albeit in a very low quantity, can inhibit the
pathogenic activity of microorganisms. The potential
contribution of marine organisms to the discovery of new
bioactive molecules is increasingly challenging (Sponga et
al., 1999; Skulberg, 2000). The microorganisms have
become a significant attraction as natural source of
bioactive molecules with a broad range of biological
activities, such as antibiotics, antivirals, antitumorals,
antioxidant and anti-inflammatory (Okami, 1982; Kamei et
al., 1987; Nunez et al., 2006; Uzair et al., 2009; Shankar
et al., 2010). Evidence of phycochemical and
pharmacological studies on microbes is available in the
literature (Zeeshan et al., 2010; Odeyemi et al., 2010;
Bragadeeswaran et al., 2010).
Previously, there were a number of researchers that had
worked on ‘Chilika’ regarding hydrological
characterization, water quality variation, antimicrobial
activity and physiochemical variation (Rao et al., 1981;
Nayak et al., 2004; Patra et al., 2009, Patra et al., 2010).
Unfortunately progress made on pharmaceutical studies
of marine microorganism from ‘Chilika’ is inadequate. This
underpinned the present study with a view to investigating
the physiological, biochemical, and serological
characterization of bacteria isolated from marine waters of
‘Chilika’ aiming at their exploitation for pharmaceutical
benefits.
MATERIALS AND METHODS
Isolation and characterization of bacterial strains
The total study area is comprised of 18 sampling stations
covering three different sectors of the ‘Chilika’ lagoon i.e.
Central, Southern and Outer Channel (Figure 1A, 1B)
during the period from January to March 2008. The exact
sampling locations were fixed by using Global Positioning
System (GPS). All the water samples were collected in the
morning hours between 7:00 A.M-11:00 A.M. During
sample collection necessary precautions had been taken
to collect undisturbed water samples in the lake. Samples
were collected in pre-sterilized polypropylene bottles (500
mL). Physical parameters like temperature and pH of the
samples were determined on the spot of collection. Then
the water samples were kept in the icebox and transferred
to laboratory for further analysis and the samples were
preserved at 4ฐC before isolation and identification in the
laboratory. The experiments were carried out within 4
months of collection. Chemicals used for preparation of
reagents in the present investigation were of analytical
reagent grade and for preparation of solutions doubledistilled
water was used. One milliliter of water sample
was subjected through serial dilution to obtain 10-8
dilution. After that 1mL of 10-8 diluted sample was mixed
with 10 mL of sterilized Nutrient broth medium and then it
was incubated at 37 ฐC in a BOD incubator for 24 h. After
incubation a loop-full of microbial culture were picked up
from different colonies and streaked separately on the
different agar-gelled sterilized media and the plates were
incubated at 37 ฐC for 24 h for isolation of pure culture.
The colony characteristics such as colour, appearance,
and shape of the isolate were recorded. Further,
morphological, and biochemical tests were carried out in
the laboratory by following standard microbiological
methods described by Cappuccino and Sherman (2002)
for identification and characterization of isolated
microorganism. The isolated strains were also
characterized through different physiological factors such
as pH, temperature and salinity to observe their tolerance
capacity in the stressed environmental conditions.
Collection and preservation of pre-immunized serum
For production of antisera 3-4 month-old white rabbit (2.5 -
3 kg) were selected and kept in separate cage and
labeled properly. The pre-immunized sera of the rabbit
used for the test were collected before 7 days of
immunization with different a ntigens to confirm that the
rabbit did not contain any antisera. The sterilized tube
containing fresh drawn blood was kept at room
temperature for 1-2 h for clot formation. The clots was
stirred with glass rod and was kept overnight in
refrigerator to allow the clot contraction. The plasma were
centrifuged (3000 rpm, 15 min). The straw-coloured serum
was withdrawn into a sterilized centrifuge tube which was
labeled properly. For preservation of serum, it was added
with 0.02 % sodium azide (NaNH2) to give a final
concentration of 1:5000 and kept in a deep freeze at -20
ฐC for further use.
Serological test
Preparation of whole cell antigen: The antigen was
prepared from the freshly grown bacterial cultures. The
bacteria were cultured on NSA medium in test tube at 27ฐC for 24 h. The test tube was filled with PBS or saline water.
The culture was scraped with sterilized inoculation
needle. The scraped bacteria with saline water were
transferred to the centrifuge tube. The bacterial growth
was mixed thoroughly with the help of a vortex mixture to
make it completely homogenized. The whole cell of
bacteria was centrifuged (3000 rpm, 30 min, room
temperature). The supernatant was discarded and the
pellet was re-suspended with saline water and
centrifuged. The process was repeated three times to
make the bacteria free from any foreign material. After the
last centrifugation bacteria were re-suspended in normal
saline/butler saline, collected in sterilized specimen tube,
labeled properly, preserved and stored in deep freeze at -
20 ฐC for further use.
Preparation of antigen for agglutination test: The
antigens were prepared by suspending live bacteria in
buffer saline as described earlier except that the antigen
prepared was more concentrated having an optical
density of 0.5 using a Gallen Kamp colorimeter at 520
mm.
Immunization of rabbits for antisera production and
harvesting of immunized serum: The rabbits were
immunized by intra-muscular and intravenous injections.
Both right and left legs were used for intra-muscular
injection. In case of intravenous injection, marginal vein of
ear was used. Three doses of antigen (0.5 mL, 1.0 mL,
and 1.5 mL) were injected intra-muscularly at four days
interval. Acetone was used to dilate the vein. The
designed quantity was injected slowly into the blood
system. A booster injection was given 4 days after the last
injection to accelerate antibody production. Five or seven
days after the booster injection, the rabbits were held by
cardiac puncture for collection of antisera. The procedure
of collection of blood separation and preservation of
antiserum was the same as described earlier.
Antigen-antibody reaction tests: Mixture of antigen and
antibody showing positive reaction resulted in the
formation of precipitation due to binding of antibodies with
their respective antigens. Two different serological tests
were conducted to record the precipitations or positive
reaction of antigen and antibodies. The gel diffusion test
was performed following the method as originally
described by Ouchterlony (1958, 1962) while the tube
agglutination test was carried out for homologous and
heterologous antigens using a national standard method
(NHS, 2010).
Antibiotic sensitivity test
Antibiotic sensitivity test of the identified bacteria were
repeated 3 times for each strain using 10 different
antibiotics namely norfloxacin, tetracycline, ciprofloxacin,
neomycin, nalidixic acid, ofloxacin, chloramphenicol,
nitrofurantoin, streptomycin and amoxicillin (Hi- Media,
Mumbai, India) by disc diffusion method (Bauer et al.,1966). Antibiotic activity was measured in terms of zoneinhibition (mm diameter).
Plasmid DNA isolation and agarose gel
electrophoresis
The alkaline-lysis mini-prep method (Wizard Plus
Minipreps DNA Purification Systems, Promega, USA) was
used for plasmid extraction from all the four bacterial
isolates. Overnight-grown bacterial cultures (3-5 mL) were
pelleted by centrifugation (10,000 x g, 10 min),
supernatant decanted and pellet re-suspended in 300 OL
of Cell Re-suspension Solution (50 mM Tris, pH 7.5; 10
mM EDTA; 100 Og/mL RNase A). The re-suspended cells
were transferred to 1.5 mL micro-centrifuged tube to
which 300 OL of Cell Lysis Solution (0.2 M NaOH, 1 %
SDS) was added and samples thoroughly mixed. To the
cleared lysate 300 OL of Neutralization Solution (1.32 M
potassium
‘Chilika’ is the largest lagoon in the subcontinent which is
situated along the east coast of India between latitude 19o
28’ and 19o 54’ N, and longitude 85o 05’ and 85o 38’E.
This is a shallow, brackish-water lake formed due to the
silting action of the Mahanadi River, which drains into the
northern end of the lake, and the northerly currents in the
Bay of Bengal, which have formed a sandbar along the
eastern shore (Arya and Lakhotia, 2006). It is the largest
brackish water wetland complex in Asia, declared as a
Ramsar site under the convention on “Wetlands of
International Importance”. This lagoon is presently under
threat from both natural and anthropogenic pressures
(Nayak et al., 2004). The marine realm constitutes the
major habitat of the biosphere covering 73 % of the earth
surface which provides the largest inhabitable space for
living organisms, particularly microbes. Marine microbes
flourish not only in the surface water of the sea, but also in
the lower and abyssal depths from coastal to the offshore
regions and from the general oceanic to the specialized
niches like blue waters of coral reefs to black smokers of
hot thermal vents at the sea floor.
As the water being served as major solvent, it plays an
important role in the pharmaceutical industries.
It is
considered as the primary source of contamination for
pharmaceutical products (Tamara et al., 1998). Microbes
present in the marine waters represent a potential source
for commercially important bioactive compounds and their
bioremediation capabilities are also remarkable. There is
an increasing demand of biodiversity from natural
resources for development of therapeutic drugs.
Antibiotics are broadly used as chemotherapeutic agents
which, albeit in a very low quantity, can inhibit the
pathogenic activity of microorganisms. The potential
contribution of marine organisms to the discovery of new
bioactive molecules is increasingly challenging (Sponga et
al., 1999; Skulberg, 2000). The microorganisms have
become a significant attraction as natural source of
bioactive molecules with a broad range of biological
activities, such as antibiotics, antivirals, antitumorals,
antioxidant and anti-inflammatory (Okami, 1982; Kamei et
al., 1987; Nunez et al., 2006; Uzair et al., 2009; Shankar
et al., 2010). Evidence of phycochemical and
pharmacological studies on microbes is available in the
literature (Zeeshan et al., 2010; Odeyemi et al., 2010;
Bragadeeswaran et al., 2010).
Previously, there were a number of researchers that had
worked on ‘Chilika’ regarding hydrological
characterization, water quality variation, antimicrobial
activity and physiochemical variation (Rao et al., 1981;
Nayak et al., 2004; Patra et al., 2009, Patra et al., 2010).
Unfortunately progress made on pharmaceutical studies
of marine microorganism from ‘Chilika’ is inadequate. This
underpinned the present study with a view to investigating
the physiological, biochemical, and serological
characterization of bacteria isolated from marine waters of
‘Chilika’ aiming at their exploitation for pharmaceutical
benefits.
MATERIALS AND METHODS
Isolation and characterization of bacterial strains
The total study area is comprised of 18 sampling stations
covering three different sectors of the ‘Chilika’ lagoon i.e.
Central, Southern and Outer Channel (Figure 1A, 1B)
during the period from January to March 2008. The exact
sampling locations were fixed by using Global Positioning
System (GPS). All the water samples were collected in the
morning hours between 7:00 A.M-11:00 A.M. During
sample collection necessary precautions had been taken
to collect undisturbed water samples in the lake. Samples
were collected in pre-sterilized polypropylene bottles (500
mL). Physical parameters like temperature and pH of the
samples were determined on the spot of collection. Then
the water samples were kept in the icebox and transferred
to laboratory for further analysis and the samples were
preserved at 4ฐC before isolation and identification in the
laboratory. The experiments were carried out within 4
months of collection. Chemicals used for preparation of
reagents in the present investigation were of analytical
reagent grade and for preparation of solutions doubledistilled
water was used. One milliliter of water sample
was subjected through serial dilution to obtain 10-8
dilution. After that 1mL of 10-8 diluted sample was mixed
with 10 mL of sterilized Nutrient broth medium and then it
was incubated at 37 ฐC in a BOD incubator for 24 h. After
incubation a loop-full of microbial culture were picked up
from different colonies and streaked separately on the
different agar-gelled sterilized media and the plates were
incubated at 37 ฐC for 24 h for isolation of pure culture.
The colony characteristics such as colour, appearance,
and shape of the isolate were recorded. Further,
morphological, and biochemical tests were carried out in
the laboratory by following standard microbiological
methods described by Cappuccino and Sherman (2002)
for identification and characterization of isolated
microorganism. The isolated strains were also
characterized through different physiological factors such
as pH, temperature and salinity to observe their tolerance
capacity in the stressed environmental conditions.
Collection and preservation of pre-immunized serum
For production of antisera 3-4 month-old white rabbit (2.5 -
3 kg) were selected and kept in separate cage and
labeled properly. The pre-immunized sera of the rabbit
used for the test were collected before 7 days of
immunization with different a ntigens to confirm that the
rabbit did not contain any antisera. The sterilized tube
containing fresh drawn blood was kept at room
temperature for 1-2 h for clot formation. The clots was
stirred with glass rod and was kept overnight in
refrigerator to allow the clot contraction. The plasma were
centrifuged (3000 rpm, 15 min). The straw-coloured serum
was withdrawn into a sterilized centrifuge tube which was
labeled properly. For preservation of serum, it was added
with 0.02 % sodium azide (NaNH2) to give a final
concentration of 1:5000 and kept in a deep freeze at -20
ฐC for further use.
Serological test
Preparation of whole cell antigen: The antigen was
prepared from the freshly grown bacterial cultures. The
bacteria were cultured on NSA medium in test tube at 27ฐC for 24 h. The test tube was filled with PBS or saline water.
The culture was scraped with sterilized inoculation
needle. The scraped bacteria with saline water were
transferred to the centrifuge tube. The bacterial growth
was mixed thoroughly with the help of a vortex mixture to
make it completely homogenized. The whole cell of
bacteria was centrifuged (3000 rpm, 30 min, room
temperature). The supernatant was discarded and the
pellet was re-suspended with saline water and
centrifuged. The process was repeated three times to
make the bacteria free from any foreign material. After the
last centrifugation bacteria were re-suspended in normal
saline/butler saline, collected in sterilized specimen tube,
labeled properly, preserved and stored in deep freeze at -
20 ฐC for further use.
Preparation of antigen for agglutination test: The
antigens were prepared by suspending live bacteria in
buffer saline as described earlier except that the antigen
prepared was more concentrated having an optical
density of 0.5 using a Gallen Kamp colorimeter at 520
mm.
Immunization of rabbits for antisera production and
harvesting of immunized serum: The rabbits were
immunized by intra-muscular and intravenous injections.
Both right and left legs were used for intra-muscular
injection. In case of intravenous injection, marginal vein of
ear was used. Three doses of antigen (0.5 mL, 1.0 mL,
and 1.5 mL) were injected intra-muscularly at four days
interval. Acetone was used to dilate the vein. The
designed quantity was injected slowly into the blood
system. A booster injection was given 4 days after the last
injection to accelerate antibody production. Five or seven
days after the booster injection, the rabbits were held by
cardiac puncture for collection of antisera. The procedure
of collection of blood separation and preservation of
antiserum was the same as described earlier.
Antigen-antibody reaction tests: Mixture of antigen and
antibody showing positive reaction resulted in the
formation of precipitation due to binding of antibodies with
their respective antigens. Two different serological tests
were conducted to record the precipitations or positive
reaction of antigen and antibodies. The gel diffusion test
was performed following the method as originally
described by Ouchterlony (1958, 1962) while the tube
agglutination test was carried out for homologous and
heterologous antigens using a national standard method
(NHS, 2010).
Antibiotic sensitivity test
Antibiotic sensitivity test of the identified bacteria were
repeated 3 times for each strain using 10 different
antibiotics namely norfloxacin, tetracycline, ciprofloxacin,
neomycin, nalidixic acid, ofloxacin, chloramphenicol,
nitrofurantoin, streptomycin and amoxicillin (Hi- Media,
Mumbai, India) by disc diffusion method (Bauer et al.,1966). Antibiotic activity was measured in terms of zoneinhibition (mm diameter).
Plasmid DNA isolation and agarose gel
electrophoresis
The alkaline-lysis mini-prep method (Wizard Plus
Minipreps DNA Purification Systems, Promega, USA) was
used for plasmid extraction from all the four bacterial
isolates. Overnight-grown bacterial cultures (3-5 mL) were
pelleted by centrifugation (10,000 x g, 10 min),
supernatant decanted and pellet re-suspended in 300 OL
of Cell Re-suspension Solution (50 mM Tris, pH 7.5; 10
mM EDTA; 100 Og/mL RNase A). The re-suspended cells
were transferred to 1.5 mL micro-centrifuged tube to
which 300 OL of Cell Lysis Solution (0.2 M NaOH, 1 %
SDS) was added and samples thoroughly mixed. To the
cleared lysate 300 OL of Neutralization Solution (1.32 M
potassium
การแปล กรุณารอสักครู่..
บทนำ
'chilika ' เป็นทะเลสาบที่ใหญ่ที่สุดในทวีปซึ่ง
ตั้งอยู่ตามชายฝั่งตะวันออกของอินเดีย ระหว่างละติจูด 19o
28 ' และ 19o 54 ' N และลองจิจูด 85o 05 ' และ 85o 38'e.
นี่คือตื้น ทะเลสาบน้ำกร่อยที่เกิดขึ้นเนื่องจากการกระทำของ
silting waters world - class . kgm แม่น้ำซึ่งไหลใน
ตอนเหนือสุดของทะเลสาบ และเหนือกระแสใน
อ่าวเบงกอลซึ่งมีรูปแบบที่สันทรายตามชายฝั่งตะวันออก ( อารียา และ lakhotia
, 2006 ) มันเป็นที่ใหญ่ที่สุด
น้ำกร่อยพื้นที่ชุ่มน้ำซับซ้อนในเอเชีย ประกาศเป็น
Ramsar เว็บไซต์ภายใต้อนุสัญญาว่าด้วย " ชายเลน
ความสำคัญระหว่างประเทศ " ทะเลสาบนี้เป็นปัจจุบันภายใต้การคุกคามจาก
แรงกดดันทั้งจากธรรมชาติและมนุษย์
( นาแยค et al . , 2004 ) อาณาจักรทะเลถือเป็น
ที่อยู่อาศัยหลักของชีวมณฑลครอบคลุมร้อยละ 73 ของโลก
พื้นผิวซึ่งมีพื้นที่ใหญ่ที่สุดสำหรับ inhabitable
สิ่งมีชีวิต โดยเฉพาะจุลินทรีย์ จุลินทรีย์ทะเล
เจริญรุ่งเรืองไม่เพียง แต่ในน้ำในทะเล แต่ยังลดลงและระดับความลึกจากก้นสมุทร
นอกชายฝั่งในภูมิภาคและจากมหาสมุทรทั่วไปกับเฉพาะ
niches เหมือนน้ำทะเลสีฟ้าของปะการังสีดำระบายความร้อนร้อนสูบบุหรี่ของ
อย่างที่พื้น ทะเล น้ำที่ถูกเสิร์ฟเป็นตัวทำละลายที่สำคัญ มันเล่นเป็นบทบาทสำคัญในอุตสาหกรรมเภสัชกรรม
ถือว่าเป็นหลักเป็นแหล่งที่มาของการปนเปื้อนสำหรับ
ผลิตภัณฑ์เภสัชกรรม ( Tamara et al . , 1998 ) . จุลินทรีย์ที่มีอยู่ในน้ำทะเล
แหล่งที่มีศักยภาพแทนสำหรับสารออกฤทธิ์ทางชีวภาพที่สำคัญในเชิงพาณิชย์และความสามารถ
ค่าของพวกเขาจะยังโดดเด่น มีอุปสงค์เพิ่มขึ้นของความหลากหลายทางชีวภาพ
จากทรัพยากรธรรมชาติเพื่อการพัฒนายารักษาโรค .
ยาปฏิชีวนะวงกว้างใช้เป็นชนิดตัวแทน
ซึ่งแม้ว่าในปริมาณที่น้อยมากสามารถยับยั้งเชื้อโรค
กิจกรรมของจุลินทรีย์ ศักยภาพ
ผลงานของสิ่งมีชีวิตในทะเลเพื่อการค้นพบโมเลกุลของสารถูกท้าทายมากขึ้น ( sponga ใหม่
อัล et . , 1999 ; skulberg , 2000 ) จุลินทรีย์ได้กลายเป็นแหล่งท่องเที่ยวที่สำคัญเป็นแหล่ง
ธรรมชาติของโมเลกุลทางชีวภาพที่มีช่วงกว้างของกิจกรรมทางชีวภาพ
, เช่น ยาปฏิชีวนะ ยาต้าน antitumorals
, , สารต้านอนุมูลอิสระและต้านการอักเสบ ( หญิง , 1982 ;
คาเมอิ et al . ,1987 ; นูนเยซ et al . , 2006 ; uzair et al . , 2009 ; Shankar
et al . , 2010 ) หลักฐานการศึกษาทางเภสัชวิทยาใน phycochemical และ
จุลินทรีย์ที่มีอยู่ในวรรณคดี ( zeeshan et al . , 2010 ; odeyemi et al . , 2010 ;
bragadeeswaran et al . , 2010 ) .
ก่อนหน้านี้มีจำนวนของนักวิจัยที่ได้ทำงานใน ' '
chilika เกี่ยวกับลักษณะทางอุทกวิทยา
, การเปลี่ยนแปลงคุณภาพน้ำ ,ฤทธิ์ต้านจุลชีพและการเปลี่ยนแปลง (
physiochemical Rao et al . , 1981 ;
นาแยค et al . , 2004 ; ภัทร et al . , 2009 , ภัทร et al . , 2010 ) .
แต่ความก้าวหน้าในการศึกษาเภสัชกรรม
ทะเลจุลินทรีย์จาก ' chilika ' ไม่ นี้
สนับสนุนการศึกษาที่มีมุมมองที่จะสืบสวน
ทางสรีรวิทยา ชีวเคมี และการศึกษาคุณสมบัติของแบคทีเรียที่แยกได้จากปลาทะเลน้ำ
'chilika ' เป้าหมายในการแสวงหาประโยชน์ตนเพื่อประโยชน์ทางเภสัชกรรม
.
วัสดุและวิธีการ การแยกและการศึกษาคุณสมบัติของแบคทีเรียสายพันธุ์
พื้นที่ศึกษาทั้งหมดประกอบด้วย 18 ตัวอย่างสถานี
ครอบคลุมสามภาคต่าง ๆ ของ ' ' (
chilika ลากูน ภาคกลาง ภาคใต้ และภายนอกช่อง ( รูปที่ 1A 1B )
,ในช่วงเดือนมกราคม - มีนาคม 2551 แน่นอน
ตัวอย่างสถานที่ถูกกำหนดโดยการใช้ระบบตำแหน่งทั่วโลก ( GPS )
. ตัวอย่างน้ำทั้งหมดถูกเก็บในชั่วโมงเช้าระหว่าง 7 : 00 น.
a.m-11:00 ในระหว่างการเก็บตัวอย่างเป็นมาตรการป้องกันที่ได้ถูกถ่าย
เก็บตัวอย่างน้ำมองเห็นทิวทัศน์ในทะเลสาบ ตัวอย่าง
เก็บตัวก่อนฆ่าเชื้อ ขวดโพรพิลีน ( 500
มิลลิลิตร )พารามิเตอร์ทางกายภาพ เช่น อุณหภูมิ และ pH ของ
ตัวอย่างในจุดของคอลเลกชัน งั้น
ตัวอย่างน้ำถูกเก็บไว้ในช่องแช่แข็ง และโอน
เพื่อปฏิบัติการสำหรับการวิเคราะห์ต่อไปและจำนวน
เก็บไว้ที่ 4 องศาเซลเซียสก่อนการแยกและระบุใน
ห้องปฏิบัติการ ทำการทดลองภายใน 4
เดือนของคอลเลกชัน สารเคมีที่ใช้สำหรับการเตรียม
สารเคมีในการตรวจสอบปัจจุบันมีสารเคมีเกรดวิเคราะห์
และการเตรียมการแก้ปัญหา doubledistilled
น้ำที่ใช้ หนึ่งมิลลิลิตรของน้ำตัวอย่าง
ถูกกระทำผ่านอุทิศถวายเพื่อให้ได้ 10-8
เจือจาง หลังจากนั้น 1ml ของ 10-8 ตัวอย่างเจือจางผสม
10 มิลลิลิตรน้ำสารอาหารและฆ่าเชื้อขนาดกลางแล้ว
ถูกบ่มที่ 37 องศาเซลเซียสใน BOD Incubator เป็นเวลา 24 ชั่วโมงหลังจาก
บ่มเพาะห่วงเต็มของวัฒนธรรมจุลินทรีย์ถูกหยิบขึ้นมา
จากอาณานิคมที่แตกต่างกันและลายต่างหาก
วุ้นเจลฆ่าเชื้อต่าง ๆสื่อและแผ่นถูก
บ่มที่ 37 องศาเซลเซียสเป็นเวลา 24 ชั่วโมงในการแยกเชื้อบริสุทธิ์
โคโลนีลักษณะเช่นสี , ลักษณะ ,
และรูปร่างของแยกถูกบันทึกไว้ เพิ่มเติม เกี่ยวกับ การทดสอบทางชีวเคมี
,
ทดลองใน
'chilika ' เป็นทะเลสาบที่ใหญ่ที่สุดในทวีปซึ่ง
ตั้งอยู่ตามชายฝั่งตะวันออกของอินเดีย ระหว่างละติจูด 19o
28 ' และ 19o 54 ' N และลองจิจูด 85o 05 ' และ 85o 38'e.
นี่คือตื้น ทะเลสาบน้ำกร่อยที่เกิดขึ้นเนื่องจากการกระทำของ
silting waters world - class . kgm แม่น้ำซึ่งไหลใน
ตอนเหนือสุดของทะเลสาบ และเหนือกระแสใน
อ่าวเบงกอลซึ่งมีรูปแบบที่สันทรายตามชายฝั่งตะวันออก ( อารียา และ lakhotia
, 2006 ) มันเป็นที่ใหญ่ที่สุด
น้ำกร่อยพื้นที่ชุ่มน้ำซับซ้อนในเอเชีย ประกาศเป็น
Ramsar เว็บไซต์ภายใต้อนุสัญญาว่าด้วย " ชายเลน
ความสำคัญระหว่างประเทศ " ทะเลสาบนี้เป็นปัจจุบันภายใต้การคุกคามจาก
แรงกดดันทั้งจากธรรมชาติและมนุษย์
( นาแยค et al . , 2004 ) อาณาจักรทะเลถือเป็น
ที่อยู่อาศัยหลักของชีวมณฑลครอบคลุมร้อยละ 73 ของโลก
พื้นผิวซึ่งมีพื้นที่ใหญ่ที่สุดสำหรับ inhabitable
สิ่งมีชีวิต โดยเฉพาะจุลินทรีย์ จุลินทรีย์ทะเล
เจริญรุ่งเรืองไม่เพียง แต่ในน้ำในทะเล แต่ยังลดลงและระดับความลึกจากก้นสมุทร
นอกชายฝั่งในภูมิภาคและจากมหาสมุทรทั่วไปกับเฉพาะ
niches เหมือนน้ำทะเลสีฟ้าของปะการังสีดำระบายความร้อนร้อนสูบบุหรี่ของ
อย่างที่พื้น ทะเล น้ำที่ถูกเสิร์ฟเป็นตัวทำละลายที่สำคัญ มันเล่นเป็นบทบาทสำคัญในอุตสาหกรรมเภสัชกรรม
ถือว่าเป็นหลักเป็นแหล่งที่มาของการปนเปื้อนสำหรับ
ผลิตภัณฑ์เภสัชกรรม ( Tamara et al . , 1998 ) . จุลินทรีย์ที่มีอยู่ในน้ำทะเล
แหล่งที่มีศักยภาพแทนสำหรับสารออกฤทธิ์ทางชีวภาพที่สำคัญในเชิงพาณิชย์และความสามารถ
ค่าของพวกเขาจะยังโดดเด่น มีอุปสงค์เพิ่มขึ้นของความหลากหลายทางชีวภาพ
จากทรัพยากรธรรมชาติเพื่อการพัฒนายารักษาโรค .
ยาปฏิชีวนะวงกว้างใช้เป็นชนิดตัวแทน
ซึ่งแม้ว่าในปริมาณที่น้อยมากสามารถยับยั้งเชื้อโรค
กิจกรรมของจุลินทรีย์ ศักยภาพ
ผลงานของสิ่งมีชีวิตในทะเลเพื่อการค้นพบโมเลกุลของสารถูกท้าทายมากขึ้น ( sponga ใหม่
อัล et . , 1999 ; skulberg , 2000 ) จุลินทรีย์ได้กลายเป็นแหล่งท่องเที่ยวที่สำคัญเป็นแหล่ง
ธรรมชาติของโมเลกุลทางชีวภาพที่มีช่วงกว้างของกิจกรรมทางชีวภาพ
, เช่น ยาปฏิชีวนะ ยาต้าน antitumorals
, , สารต้านอนุมูลอิสระและต้านการอักเสบ ( หญิง , 1982 ;
คาเมอิ et al . ,1987 ; นูนเยซ et al . , 2006 ; uzair et al . , 2009 ; Shankar
et al . , 2010 ) หลักฐานการศึกษาทางเภสัชวิทยาใน phycochemical และ
จุลินทรีย์ที่มีอยู่ในวรรณคดี ( zeeshan et al . , 2010 ; odeyemi et al . , 2010 ;
bragadeeswaran et al . , 2010 ) .
ก่อนหน้านี้มีจำนวนของนักวิจัยที่ได้ทำงานใน ' '
chilika เกี่ยวกับลักษณะทางอุทกวิทยา
, การเปลี่ยนแปลงคุณภาพน้ำ ,ฤทธิ์ต้านจุลชีพและการเปลี่ยนแปลง (
physiochemical Rao et al . , 1981 ;
นาแยค et al . , 2004 ; ภัทร et al . , 2009 , ภัทร et al . , 2010 ) .
แต่ความก้าวหน้าในการศึกษาเภสัชกรรม
ทะเลจุลินทรีย์จาก ' chilika ' ไม่ นี้
สนับสนุนการศึกษาที่มีมุมมองที่จะสืบสวน
ทางสรีรวิทยา ชีวเคมี และการศึกษาคุณสมบัติของแบคทีเรียที่แยกได้จากปลาทะเลน้ำ
'chilika ' เป้าหมายในการแสวงหาประโยชน์ตนเพื่อประโยชน์ทางเภสัชกรรม
.
วัสดุและวิธีการ การแยกและการศึกษาคุณสมบัติของแบคทีเรียสายพันธุ์
พื้นที่ศึกษาทั้งหมดประกอบด้วย 18 ตัวอย่างสถานี
ครอบคลุมสามภาคต่าง ๆ ของ ' ' (
chilika ลากูน ภาคกลาง ภาคใต้ และภายนอกช่อง ( รูปที่ 1A 1B )
,ในช่วงเดือนมกราคม - มีนาคม 2551 แน่นอน
ตัวอย่างสถานที่ถูกกำหนดโดยการใช้ระบบตำแหน่งทั่วโลก ( GPS )
. ตัวอย่างน้ำทั้งหมดถูกเก็บในชั่วโมงเช้าระหว่าง 7 : 00 น.
a.m-11:00 ในระหว่างการเก็บตัวอย่างเป็นมาตรการป้องกันที่ได้ถูกถ่าย
เก็บตัวอย่างน้ำมองเห็นทิวทัศน์ในทะเลสาบ ตัวอย่าง
เก็บตัวก่อนฆ่าเชื้อ ขวดโพรพิลีน ( 500
มิลลิลิตร )พารามิเตอร์ทางกายภาพ เช่น อุณหภูมิ และ pH ของ
ตัวอย่างในจุดของคอลเลกชัน งั้น
ตัวอย่างน้ำถูกเก็บไว้ในช่องแช่แข็ง และโอน
เพื่อปฏิบัติการสำหรับการวิเคราะห์ต่อไปและจำนวน
เก็บไว้ที่ 4 องศาเซลเซียสก่อนการแยกและระบุใน
ห้องปฏิบัติการ ทำการทดลองภายใน 4
เดือนของคอลเลกชัน สารเคมีที่ใช้สำหรับการเตรียม
สารเคมีในการตรวจสอบปัจจุบันมีสารเคมีเกรดวิเคราะห์
และการเตรียมการแก้ปัญหา doubledistilled
น้ำที่ใช้ หนึ่งมิลลิลิตรของน้ำตัวอย่าง
ถูกกระทำผ่านอุทิศถวายเพื่อให้ได้ 10-8
เจือจาง หลังจากนั้น 1ml ของ 10-8 ตัวอย่างเจือจางผสม
10 มิลลิลิตรน้ำสารอาหารและฆ่าเชื้อขนาดกลางแล้ว
ถูกบ่มที่ 37 องศาเซลเซียสใน BOD Incubator เป็นเวลา 24 ชั่วโมงหลังจาก
บ่มเพาะห่วงเต็มของวัฒนธรรมจุลินทรีย์ถูกหยิบขึ้นมา
จากอาณานิคมที่แตกต่างกันและลายต่างหาก
วุ้นเจลฆ่าเชื้อต่าง ๆสื่อและแผ่นถูก
บ่มที่ 37 องศาเซลเซียสเป็นเวลา 24 ชั่วโมงในการแยกเชื้อบริสุทธิ์
โคโลนีลักษณะเช่นสี , ลักษณะ ,
และรูปร่างของแยกถูกบันทึกไว้ เพิ่มเติม เกี่ยวกับ การทดสอบทางชีวเคมี
,
ทดลองใน
การแปล กรุณารอสักครู่..
ภาษาอื่น ๆ
การสนับสนุนเครื่องมือแปลภาษา: กรีก, กันนาดา, กาลิเชียน, คลิงออน, คอร์สิกา, คาซัค, คาตาลัน, คินยารวันดา, คีร์กิซ, คุชราต, จอร์เจีย, จีน, จีนดั้งเดิม, ชวา, ชิเชวา, ซามัว, ซีบัวโน, ซุนดา, ซูลู, ญี่ปุ่น, ดัตช์, ตรวจหาภาษา, ตุรกี, ทมิฬ, ทาจิก, ทาทาร์, นอร์เวย์, บอสเนีย, บัลแกเรีย, บาสก์, ปัญจาป, ฝรั่งเศส, พาชตู, ฟริเชียน, ฟินแลนด์, ฟิลิปปินส์, ภาษาอินโดนีเซี, มองโกเลีย, มัลทีส, มาซีโดเนีย, มาราฐี, มาลากาซี, มาลายาลัม, มาเลย์, ม้ง, ยิดดิช, ยูเครน, รัสเซีย, ละติน, ลักเซมเบิร์ก, ลัตเวีย, ลาว, ลิทัวเนีย, สวาฮิลี, สวีเดน, สิงหล, สินธี, สเปน, สโลวัก, สโลวีเนีย, อังกฤษ, อัมฮาริก, อาร์เซอร์ไบจัน, อาร์เมเนีย, อาหรับ, อิกโบ, อิตาลี, อุยกูร์, อุสเบกิสถาน, อูรดู, ฮังการี, ฮัวซา, ฮาวาย, ฮินดี, ฮีบรู, เกลิกสกอต, เกาหลี, เขมร, เคิร์ด, เช็ก, เซอร์เบียน, เซโซโท, เดนมาร์ก, เตลูกู, เติร์กเมน, เนปาล, เบงกอล, เบลารุส, เปอร์เซีย, เมารี, เมียนมา (พม่า), เยอรมัน, เวลส์, เวียดนาม, เอสเปอแรนโต, เอสโทเนีย, เฮติครีโอล, แอฟริกา, แอลเบเนีย, โคซา, โครเอเชีย, โชนา, โซมาลี, โปรตุเกส, โปแลนด์, โยรูบา, โรมาเนีย, โอเดีย (โอริยา), ไทย, ไอซ์แลนด์, ไอร์แลนด์, การแปลภาษา.
- วันนี้เป็นวันลอยกระทง ฉันมีความสุขมาก
- ammonia, coined in 1782 by Swedish chemi
- there has been migration of Buddhist and
- none left...*laugh*
- What about Sunday ?
- yields
- 1. Perceiving the usefulness of theoreti
- Is there a lake in the university?
- ฉันกลัวจะรบกวนคุณเกินไป
- very high vitamin C help treat eye infla
- In 1997 photographs of sheep call Dolly
- You shine It when I'm alone
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- A perk that's big with Umpqua staff is t
- Im final master architecture in iran(teh
- ไฮเทค
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- กองปราบฯ ใช้ช่วงเวลานี้ชำระล้างอิทธิพลเถ
- My sister children
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- Rational choice mounts a challenge to th
- Figure 2.9 show A-Z characters when you
- นอกจากครูจะเป็นผู้สอนคนให้มีความรู้ความส
- นอกจากครูจะเป็นผู้สอนคนให้มีความรู้ความส
