Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
International Journal of Advanced Research in Biological Sciences
ISSN : 2348-8069
www.ijarbs.com
Research Article
Isolation and characterization of pathogenic bacteria from Indian Sand Lobster
(Thenus Orientalis) Larval rearing system
M.Rubiya shahana and P.Mahalakshmi
Department of Biochemistry, T.S.Narayanaswami College of Arts and Science, Navalur, Chennai- 600 130,
Tamil Nadu, India
*Corresponding author: maha_lecturer@yahoo.co.in
Abstract
Vibrio fischeri and related bacteria are important pathogens responsible for severe economic losses in the aquaculture industry
worldwide. The Vibrio fischeri is a bioluminescent symbiont that colonizes the light-emitting organs of certain marine animals,
including lobster larval rearing systems. Various studies of the Euprymna scolopes - Vibrio fischeri symbiosis have demonstrated
that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria a mass in dense
aggregations outside the sites of infection. In this study, experiments with green fluorescent protein-labeled symbiotic and
nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates. When hatchling
animals were exposed to 103 to 106 V. fischeri cells/ml added to natural seawater, which contains a mix of approximately 106
nonspecific bacterial cells/ml, V. fischeri cells were the principal bacterial cells present in the aggregations. Furthermore, when
animals were exposed to equal cell numbers of V. fischeri (either a motile or a nonmotile strain) and either Vibrio
parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host’s
habitat, the symbiont cells were dominant in the aggregations. The presence of V. fischeri did not compromise the viability of
these other species in the aggregations, and no significant growth of V. fischeri cells was detected. These findings suggested that
dominance results from the ability of V. fischeri either to accumulate or to be retained more effectively within the mucus.
Viability of the V. fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus. Neither
of the V. fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small
signal molecules are not critical to V. fischeri dominance. Taken together, these data provide evidence that the specificity of the
squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ. The
primary aim of this study was to rear T. orientalis larvae from egg stage to juvenile and to study the growth performance of
laboratory-raised juveniles to establish a basis for exploring the possibilities of aquaculture of this species in India. Due to
pathogenic infection the second day of larvae stage will have high mortality rate so to prevent the high mortality rate, our present
investigation to isolate the particular pathogenic bacteria responsible for pathogenicity from larvae of Thenus Orientalis. The
samples (Infected larvae) were collected from Madras central fisheries centre, Chennai for the study. The samples were collected
and it was used to isolate and rapid identification of pathogenic bacteria from larval rearing. Both biochemical characterization
and molecular conformation (PCR) was done to confirm the pathogenic bacteria.i.e.,Vibrio fischeri. Earlier detection and
conformation of V.fischeri is one of the important tool to prevent the mortality rate.
Keywords: Vibrio fischeri, Euprymna scolopes , squid-vibrio symbiosis.
Introduction
aetiology can be kept in limits of by proper
management and husbandry techniques (Leslie V.A.,
Margaret Muthu Rathinam A.,et al., 2012).
Prevention of diseases and early diagnosis are very
important in the cost effective management of
aquaculture systems. Most of the disease situation,
both non-infectious and infectious disease of unknown
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
organ development (McFall-Ngai, M. J.,et al., 2000,
Visick, K. L., et al., 2000), suggesting that signaling is
occurring between the bacteria and their host.
Marine micro organisms
In contrast to land living organisms, marine organisms
are surrounded by an ambient environment rich in
bacteria and other micro-organisms. Seawater
functions both as a transport and growth medium in
contrast to air, which has been thought only to
function as a transport medium for micro-organisms.
The majority of bacteria causing disease in marine fish
are opportunistic pathogens that are present as part of
the normal seawater microflora. Thus, marine
organisms share an ecosystem with the microorganisms responsible for their disease. Members of
the genus Vibrio are natural marine inhabitants,
playing important roles in nutrient cycling and
forming associations with zooplankton (Thompson
F.L., et al., 2004). Accordingly, many
The mechanisms by which V. fischeri cells attach to
and colonize the light organ tissues of juvenile hosts
are just beginning to be described in detail. For
example, aggregation of V. fischeri cells in a hostderived mucus-like matrix is an early event that is
required for these cells to find and enter the pores that
lead to the nascent light organ crypts (Nyholm, S. V.,
et al., 2000). In addition, evidence exists that mannose
residues present on the cells lining the crypts may
function as receptors for the colonizing bacteria
(McFall-Ngai, M. J., et al., 1998) and that bacterial
fimbriae are involved in this process (Feliciano, B.
and Ruby E.G., 1999).
Vibrio species are pathogenic to cultured crustacean
zooplanktonic larval forms, including the three closely
related species Vibrio harveyi (Prayitno S.B.,
Latchford J.W., 1995, Robertson PAW, et al., 1998),
V.campbellii (Hameed ASS et al., 1996, SotoRodriguez SA et al., 2006), and the recently described
V. owensii (Cano-Gómez A, et al 2010).
Lobsters
Lobsters are among the most priced seafood delicacies
enjoying a special demand in international markets.
Scyllarid lobsters contribute to about 8 % of the
world's lobster production. The genus Thenus acquires
significance in the Indo - west Pacific (from the east
coast of Africa through the Red Sea and India, up to
Japan and the northern coast of Australia). While
India's lobster production averaging about 2000 metric
ton (MT) annually has been on the decline. India is
one of the leading producers of the flat-head lobster,
Thenus orientalis, which has been a relatively late
introduction in Indian seafood exports. However the
annual landing of this resource has fallen drastically
from about 600 MT to about 130 MT over a span of a
decade (1991 -2001). In 2001 the export of sand
lobster tails from India was about 70 MT, which is less
than half the quantity exported in 1991. There is an
urgent need to evolve strategies for decreasing the gap
between supply and demand and to strike a balance
between the fished and the fishable quantity of
lobsters in Indian waters.(Joe K. Kilzhakudan).
Vibrio Fisheri
The marine bacterium Vibrio fischeri is the specific
symbiont of the light-emitting organ of the sepiolid
squid Euprymna scolopes (McFall-Ngai, M. J. 1999).
The nascent light organ of a newly hatched E.
scolopes juvenile is axenic, but cells of V. fischeri
present in the surrounding seawater serve as an
inoculum that passes through pores on the surface of
the organ and proliferates within epithelium-lined
internal crypt spaces (Nyholm, S.V., et al., 2000). The
colonization process requires that the bacteria migrate
past several different host cell types on their way into
the organ (Nyholm, S.V.,et al., 1998) and then become
securely associated with the microvillar surface of the
crypt cells (Lamarcq, L. H., et al., 1998). Periodic
expulsion of over 95% of the symbiotic bacterial
population every morning (Ruby, E. G., et al., 1993)
may further select for closely adhering V. fischeri
cells, which become increasingly invested in the
microvilli during the first few days after colonization
(Lamarcq, L. H., et al., 1998). In addition, the
presence of the bacteria induces both reversible and
irreversible stages in the programme of normal light
Scyllarid lobsters, encompassing over 70 species, have
been given little importance. Biological research has
been limited and in most instances has dealt with
species of some economic importance (Ben-Tuvia,
1968). Thenus is the only genus in 7 scyllarid genera
that is economically significant (Jones, 1990). Species
of this genus contribute to many of the demersal trawl
fisheries which operate along the tropical coasts of the
Indian Ocean and the Western Pacific region (Ivanov
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
and Krylov, 1980) and are becoming commercially
exploited species (Department of Fisheries 1997;
FAO, 2010). The species in India was described
earlier as Thenus orientalis (Chhapgar and
Deshmukh, 1964). It is commonly known as sand
lobster, slipper lobster or shovel-nosed lobster. Sand
lobsters are bottom dwellers and prefer sandy and
muddy habitats of 10-50 m depth (Uraiwan, 1977;
Jones, 2007; FAO, 2010).
Australia, it is more widely known as the Moreton
Bay bug after Moreton Bay, a location in Queensland.
In Singapore, both the flathead lobster and true
crayfish are confusingly called crayfish. They are
popularly used in many Singaporean dishes. T.
orientalis has a strongly depressed body, and grows to
a maximum body length of 25 centimetres (9.8 in), or
a carapace length of 8 cm (3.1 in).
Thenus orientalis has an Indo-West Pacific
distribution, ranging from the east coast of Africa
(southern Red Sea to Natal) to China, southern Japan,
the Philippines and along the northern coast of
Australia from Western Australia to Queensland. They
are also caught on a small scale off the shores of
Malaysia and Singapore.
Thenus Orientalis
T. orientalis is known by a number of common names.
The United Nations Food and Agriculture
Organization prefers the name flathead lobster, while
the official Australian name is Bay lobster. In
The scyllarid lobsters, commonly called sand lobsters,
slipper lobsters or squat lobsters, constitute one of the
important crustacean resources in the Indo-Pacific
region. These lobsters grow to a moderate size and
support fisheries of localised importance. In India,
only one species of sand lobster, Thenus orientalis
(Lund) occurring along both west and east coasts,
forms a resource of commercial importance in Gujarat,
Maharashtra and Tamil Nadu.(Deshmukh V.D.,
2001).
Australia. The larval cycle is completed in 26-30 days
and juveniles attain a size of 150 g (the minimum legal
size for export) in about 300 days. The only shorter
duration of the larval phase is an advantage in captive
rearing of the sand lobster as compared to the spiny
lobsters.
Lobsters are among the most priced seafood delicacies
enjoying a special demand in international markets. As
against a world average annual production of 2.1 lakh
tonnes, India's average annual lobster production is
about 2000 tonnes. With the distinction of being
perhaps, the only seafood resource in India's trade
economy, which remains relatively low down the
ladder in terms of quantity of production but brings in
maximum foreign exchange, lobsters have been the
subject of study for more than two decades now.
The sand lobster Thenus orientalis ranks next to spiny
lobsters and tiger shrimp in export value. It is one of
the most promising candidates for lobster aquaculture
in India. Complete larval development of T. orientalis
was achieved for the first time in India at the Kovalam
Field Laboratory of CMFRI (Joe.K.Kizhakudan et al.,
2004, 2005). There has been only one other earlier
report of a similar achievement in T. orientalis from
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
The lobster fishery in India is supported
by two groups of lobsters - the spiny
lobsters
(Palinurus
homarus,
P.
polyphagus, P. ornatus and P. versicolor)
and the scyllarid lobster (Thenus
orientalis). Scyllarid lobsters contribute to
about 8% of the world's lobster
production. The genus Thenus acquires
significance in the Indo-west Pacific (from
the east coast of Africa through the Red
Sea and India, up to Japan and the
northern coast of Australia), The sand
lobsters are represented by a single species
in India's lobster hery - Thenus orientalis
(Joe K. Kizhakudan)
.
Lobster in World Production
Lobsters are considered as highly priced
delicacies and command high prices in the
domestic and export markets. Lobster
fishery which remained as subsistence
fishery till the sixties bas now flourished
into a commercial) activity earning
valuable foreign exchange for the country.
Of the 25 species reported from the Indian
coast, 6 are known to tha recommercial
importance and include Panulirus
homarus, P. polyphagus, P. ornatus,
Puerulus sewelli and Thenus orientalis.(
K.K. Vijayan,N.K. Sanil et al 2010).
\
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
World production of lobsters average
about 2.1 lakh tones per annum. Annual
lobster production of India averaging
about 2000 tonnes has been steadily
declining over the year.
and abiotic factors. ( K.K. Vijayan, N.K.
Sanil and Krupesh Sharma 2010).
Larval rearing system
Lobsters have a complex and prolonged
life cycle , which often involves several
planktonic ("free floating") larval stages.
Larvae (phyllosoma) were reared in
treated seawater of salinity 37-39 ppt and
pH 8-8.2 and fed on a combination of
fresh clam meat and live zooplankton. The
larval cycle is completed in 26 days (Joe
K. Kizhaludan 2005).
meat starts. Hatching occurs in batches
over a period of 36 – 42 hours, mostly
during the night hours. The larvae that
hatch out on the second night are usually
found to be more active and healthy and
are better for rearing experiments.
Complete larval rearing was achieved with
the help of wild zooplankton and fresh
clam meat.
CMFRI has successfully demonstrated the
breeding of spiny lobsters and sand
lobsters, and further R&D may eventually
lead to the hatchery production of baby
lobsters and mariculture of lobsters.
Lobster rearing systems, viz breeding
larval and fattening are always prone to
disease occurrence due to various biotic
Larval rearing of lobsters in captive
conditions has always posed a problem
owing to the complexity of their life cycle
with delicate larval stages. The key
bottleneck for lobster aquaculture is the
hatchery-nursery phase. Like the spiny
lobster, the sand lobster, too has a
complex and prolonged life cycle, though
not as prolonged as in the case of the
former. There are four larval (phyllosoma)
stages which metamorphose and settle
finally as the post-larval nisto stage in
about 26 – 30 days. Moulting occurred in
the late evening or night hours. The
number of days taken for phyllosoma fed
on clam meat and live ctenophores
collected from the sea, to settle as nisto
was 26 – 30 days. The average lengths of
the intermoult period for each stage of
larval rearing were:
Phyllosoma I
Phyllosoma II
Phyllosoma III
Phyllosoma IV
The
phyllosoma
larva
is
characteristically flattened, leaf-like and
transparent. The cephalic shield is much
broader the thorax. The abdomen is very
short and narrow. The pereiopods arise
from the thoracic region. The nisto is a
non-feeding stage. It resembles the adult
lobster but has a transparent exoskeleton.
Moulting to nisto marks the end of the
planktonic phase of the animal’s life and
the nisto settles to the substratum stage. It
does not swim actively unless disturbed
and prepares for the next moult in another
2-3 days, following which feeding on clam
The hatchery was divided into three
rearing sections. The rearing system in
each section was modified to suit the
habitat requirement at different stages of
larval metamorphosis. Phyllosoma I (Plate
1a) were stocked in glass beakers at 5/litre
of seawater. Feed was given twice daily.
Mortality and moulting were recorded
daily. Upon reaching the Phyllosoma III
stage (Plate 1b), the larvae were
transferred to floating plastic basins with
perforated bottom kept in mildly aerated
7-9recirculating
days
seawater, in 1 tonne tanks
5-6run
days
with biofilters (Closed Recirculatory
7 days
System). When the larvae began
7 days
exhibiting morphological changes and
stopped feeding, indicating a readiness to
moult into the nisto stage, they were
transferred to bigger tanks provided with
sand bottom and different substrates to
enable larval attachment before moulting
into the nisto. Minimum light exposure
was given to the larvae during the entire
experiment. The nistos were maintained in
the same tanks. Feeding was stopped till
the formation of the first seed.
Salient Observations in Juvenile rearing
of T. orientalis
The animals show a preference for soft
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substrate during the day. They respond
promptly to the introduction of feed into
the tank. Clam meat induces good
chemoreception even during day hours.
Molt death syndrome is frequently
observed with difficulties in shedding and
hardening of the soft body post molt – this
can be possibly overcome with feed
supplements which can provide a wellbalanced nutrition.
at the National lnstitute of Water and
Atmosphere Research Ltd (NIWA)
(Wellington and Auckland) from J.
edwardsli to J. verrauxl. larval rearing is
carried out mostly along the lines
described by Illingworth et at. (1997),
employing a recirculating upwelling
system with no biofilter.
Although reports of research conducted in
different parts of the world indicate the
amenability of lobsters to being cultured in
closed systems (Kittaka and Booth, 1980).
Lobster aquaculture is still a virgin arena
in India. The primary aim of this study
was to rear T. orientalis larvae from egg
stage to juvenile and to study the growth
performance of laboratory-raised juveniles
to establish a basis for exploring the
possibilities of aquaculture of this species
in India.
Males approach maturity faster and at a
relatively smaller size; the activity perhaps
leads to damages/erosion of uropod, and
their survival rates show a sudden decline.
Cannibalism is not exhibited by this
species.
Amenable to captive rearing in high
density systems.
Amenable to polyculture with the Indian
white shrimp, F. indicus.
Disease
Lobster rearing in other parts of the
world
In India lobster farming is in its infancy
and hence limited information is available
on the diseases and pathogens encountered
in lobsters in our country. Other than
bacterial shell diseases, the only major
disease reported in lobster from India is a
case of suspected gaffekemia. The major
diseases encountered in lobsters are:
Japan has been leading the world's nations
in initiating research on lobster
aquaculture. Complete larval rearing has
been successfully achieved in different
parts of the world in panulirid lobsters Jasus lalandi (Kittaka, 1988), Palinurus
elephas (Kittaka & Ikegami, 1988),
Panulirus japonlcus (Kittaka & Kimura,
1989) and P. interruptus (Johnson, 1956)
and in scyllarid lobsters - S. americanus
(Robertson, 1968), Ibacus ciliatus and
I.novemdentatus (Takahashi and Saisho,
1978), I. alticrenatus (Atkinson and
Boustead,1982), Scyllarus demani (Ito
and lucas,1990) and I. peronii (Marinovic
et al.,1994) Kittaka (1997) has obtained
the highest survival of phyllosoma for J.
verreauxl and has reared several hundred
pueruli (Robertson 1968) has described
the larval life span of scyllarid lobsters to
last from 30 days to nine months,
depending on the species and influential
factors. Highly promising results achieved
by the Japanese with the larval culture of
J. verrauxl (the eastern rock lobster) has
shifted the focus of larval culture research
Ciliate disease of lobsters
Caused
by
a
holotrich
ciliate,
Anophryoides, an opportunistic parasite,
which attaches to and destroys
haemocytes, sometimes causing mortality.
PARAMOEBIASIS
Caused by parasites of the genus
Paramoeba; under stressful environmental
conditions can lead to high mortalities.
MICROSPORIDOSIS
Caused by various microsporidian
parasites. Spores occur in the muscle
fibres causing the muscle to appear
"milky" or "cooked" in living specimens.
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DINOFLAGELLATE BLOOD
DISEASE
STUDY
Caused by Hematodinium - like parasitic
dinoflagellates in the haemolymph. Most
major organs and tissues appear to be
invaded before the parasite enters the
haemolymph in high numbers leading to
death.
Mortalities due to stress and diseases in
lobster holding facilities are frequently
noted, causing serious setbacks systems.
Most of the disease situation, both noninfectious and infectious disease of
unknown aetiology can be kept in limits of
by proper management and husbandry
techniques. Infectious diseases and
adverse environmental conditions might
produce similar clinical symptoms in
lobsters making the exact diagnosis
difficult. However the primary cause of
infections (by virus, bacteria or fungus) or
infestations by algae, parasites might be
the deteriorating environmental stock.
Failure to adjust and adapt to
environmental stresses like crowding, poor
nutrition, low levels of dissolved oxygen
and sudden changes in salinity and
temperature
weakens
the
innate
immunological
resistance
and
the
opportunistic pathogens make progression
to cause lobsters.
JUVENILE LOBSTER VIBRIOSIS
Caused by Vibrio anguillarum and other
Vibrio sp. which are ubiquitous in stressful
culture conditions and are often lethal.
LAGENIDIUM DISEASE
Caused by the fungus Lagenidium sp.,
penetrates and fills larvae with mycelia
giving a white, opaque appearance and is
usually lethal. Usually considered related
to poor husbandry and can be prevented
by better sanitation. Burn spot disease of
juvenile lobsters Caused by Fusarium sp.,
resulting in black spots on exoskeleton and
brownish discoloration of the gills in
larvae/juveniles and is due to poor
husbandry practices.
The samples (Infected larvae) were
collected from Madras central fisheries
centre, Chennai for the study. The samples
were collected and it was used to isolate
and rapid identification of pathogenic
bacteria from larval rearing.
HALIPHTHOROS FUNGUS DISEASE
Infiltration of the exoskeleton of post
larvae by mycelia causing extensive
damage and melanisation, causing
mortality.
Hence the present study was design with
the objective to identity the pathogenic
organisms responsible for the increased
mortality rate in Indian sand lobster larval
rearing system.
EPIBIONT FOULING
Persistent low level mortalities of juvenile
lobsters have been reported in rearing
systems associated with moderate to heavy
growth of epibionts and Leucothrix-like
bacteria.
The results was interpreted in consultation
with
the
senior
scientist
Dr.Joe.K.Kilzhakudan. Its is humbly
hoped, that study may be helpful in future
to detect earlier, we can prevent mortality
rate of larval rearing practices.
Infections with the larval forms of
nematodes,
trematodes
and
acanthocephalans and Nemertean worms
feeding on lobster eggs are also reported
(K.K. Vijayan, N.K. Sanil and Krupesh
Sharma 2010).
MATERIALS AND METHODS
COLLECTION OF SAMPLES
The present study is to isolate the pathogenic
bacteria from larvae of Indian sand lobster.
The samples (Infected larvae) were collected
SCOPE AND OBJECTIVE OF THE
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
from Madras central fisheries centre,
Chennai for the study. The samples were
collected and it was used to isolate and
rapid identification of pathogenic bacteria
from larval rearing.
MICROBIAL
ISOLATION
CHARACTERIZATION
clean cover slip was taken. The inoculum
loop was flame sterilized and allowed to
cool fall in 2 seconds. The loop was
inserted into the over night broth culture
of the test organisms. A single drop of the
culture ws placed on to cover slip. On the
four sides of the cover slip vaseline was
applied. The cavity glass slide was placed
on to the cover slip. On lifting the slide a
drop can be observed. This preparation
was observed under 40 Hetz microscope
and the motility was recorded.
AND
The sample obtained were divided into
two batches as larvae sample (A) and
water sample (B) by centrifugation of the
sample at 1500 rpm for 15 minutes. The
larvae pellet (A) was separated from the
supernatant (B). Both A and B were used
as the samples for the microbial isolation
and characterization.
INDOLE TEST
Inoculate the tubes of tryptone broth with
the organisms, and incubate for24-48 hours
at 37 C. Add 0.2 ml of kovac’s reagent and
shake. Allow to stand for few minutes and
read the result.
GRAM’S STAINING
Procedure
Prepare a smear following the instructions
given for simple stain. Add reagent 1
crystal violet so that it cover the whole
smear. Allow to act for one minute. Rinse
with tap water. Add few drops of gram’s
iodine to cover the smear. Allow to react
for 30 seconds to one minute. Rinse with
tap water. Decolorize with 95% ethanol.
As this step is crucial for gram’s staining
procedure, do as follows. Hold the slide in
a slanting position. Add the alcohol drop
by drop with a help of a droping bottle on
the top of the slide so that the alcohol runs
over the smear and decolorizes it. The
drop of alcohol that falls down the slide is
coloured. Continuously add the alcohol
drop by drop, and at one stage the falling
drop will be colourless. Stop adding
alcohol and immediately wash it under a
running tap water. This decolourisation
may take 30 seconds to one minute
depending upon the density of the smear.
Cover the smear with safranine and allow
the stain to act for 1 minute. Rinse with
water. Blot dry, and examine under oil
immersion objective.
METHYL RED (MR) AND
VOGESPROSKAUER (VP) TEST
Inoculate the organisms into MR/VP broth,
incubate at 37 C for atleast 48 hours. Divide
the broth into two equal halfs and to one add
0.5 ml of MR reagent. To the other half add
0.2 ml VP reagent A an 0.2 ml of VP reagent
B. gently mix and allow it to stand for 15
min
OXIDASE
A
solution
of
1
%
pphenylenedihydrochloride is prepared in
distilled water ( 5 mg in 5 ml). With the
pencil make the mark on the strip as T,
C+, C-. Soak the paper with few drops of
the reagent and keep it on a slide or
petridish. With the help of a clean glass
rods/plastic loop or platinum wire pick a
colony from 24 hours growth of the
organisms and controls and rub over the
filter paper. Use different loops for each
organism. Observe the colour change to
blue or purple within 10 seconds. Filter
paper strips are soaked in 1 % pphenylenediaminedihydrochloride solution
in distilled water and dried at 37 C
overnight in the dark. Keeping paper well
separated. The dried papers are stored in
MOTILITY TEST
A clean cavity glass slide was taken and a
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
brown bottles at C. After soaking the dried
paper with few drops of distilled water.
18-24 hours.
Add 0.5 ml reagent A and 0.5 ml reagent
B in that order and read the results.
CATALASE
Slide Method
TSI MEDIUM
Transfer pure growth of the organism from
the agar to a clean slide with a loop or
glass rod. Immediately add a drop of 3%
hydrogen peroxide to the growth. Observe
the release of the bubbles.
Dissolve the ingredients, check the pH,
dissolve the agar by boiling. Check the pH
again and then distribute in 3-4 ml
quantities in 12 ×100 mm test tubes.
Autoclave at 121 C for 15 min and allow it
to set in such a way that about 1 inch butt
and a slope is obtained. Pick
up
the
organisms from the top of a single colony
from primary isolation plate or from pure
growth with a straight wire and inoculate
by stabbing down the center of agar butt
carefully. Withdraw the inoculating wire
carefully and then streak the surface of the
slant. Incubate at 37 C. Read the result only
after 18 -24 hours incubation
ONPG TEST
Aseptically mix 25 ml of ONPG solution
to 75 ml peptone water and distribute in
0.5 ml amounts in 10 × 100 mm tubes, cap
and store at -20 C. solution should not be
yellow.Inoculate the tube of ONPG broth
with heavy suspension of the organisms,
incubate at 37 C for 1 -24 hours and read
the results.
CITRATE UTILIZATION TEST
GELATIN LIQUIFICATION TEST
Melt the agar, distribute in 1-2 ml
quantities in 12 × 100 mm test tubes.
Autoclave at 121 C for 15 minutes and
allow it to solidify in a slanting position.
Inoculate a drop of 4 -6 hour old culture in
to the medium and incubate for 18 – 24
hours or longer and read the result.
50 ml of gelatin medium was prepared and
5 ml each was distributed into 9 clean 10
ml test tubes and plugged with nonabsorbant cotton. These tubes were
marked as H1,H2.H3,H4,H5,W1,W2,W3.
And were subjected to autoclave (121°C
for 15 minutes). After cooling of tubes,
one inoculums loop of the test microbes
were stab into the respective culture tubes
and incubated at room temperature for 48
hrs. After the incubation period the tubes
were
transferred
to
refrigeration
temperature for 4 hrs. After the incubation
period the tubes were thawed to room
temperature and the gelatin liquefication
was observed by tilling the tubes. Positive
culture tubes the liquid can be observed at
the region of stabbing and the nature of the
medium can be observed at the stab of the
medium.
UREASE TEST
Prepare the base, sterilize by autoclaving
at 121 C for 15 min. cool to 50 C in water
bath and then add 5 ml of filter sterilized
40 % urea solution. Mix, distribute in 2-4
ml amounts in 12 ×100 mm test tubes.
Allow the medium to solidify in a slanting
position in such a way to get half inch but
and one inch slant. Inoculate the slant with
a drop of 4-6 hour growth of bacterium in
broth and incubate at 30/37 C for 18-24
hours or longer.
BLOOD AGAR
NITRATE REDUCTION TEST
Blood: collect sheep blood from the
jugular vein in sterile 3.80% trisodiun
citrate (1 ml citrate solution for 10 ml
Inoculate the nitrate broth with organisms
from pure culture and incubate at 37 C for
138
Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
blood) prepare the basal medium nutrient
agar , sterilise and cool to 50 - 55 C in a
water bath. Add aseptically, with a sterile
pipette, 10 ml of blood to 90 ml of the
base. Mix and pour the plates. Care must
be taken to avoid air bubbles in the
medium. Allow it to set at ambient
temperature. Incubate the plates at 37°C to
check for sterility.
Incubate the two tubes with colonies from
pure culture. Overlay each tube with 4 -5
mm sterile mineral oil. Incubate for 4 days
and read the results.
CARBOHYDRATE UTILIZATION
TEST
Dissolve the ingredients, melt the agar,
check the pH and distribute in 100 ml
amounts in small bottles/flasks and
sterilize at 121°C for 15 min. Dissolve
each 1 gram of maltose, mannose,
mannitol, sucrose, lactose, glucose,
cellibiose in 10 ml distilled water and
sterilize by filteration ( 0.2 micron filter )
and store in refrigerator. Cool O-F
medium base to 55°C add 10 ml sugar
solution to 100 ml of the medium.
Dispense of 5 ml quantities in 12 × 100
mm test tubes and allow to set in an
upright position to get the solid butt.
Incubate 2 tubes of O-F glucose (and other
sugars) medium with the organism by
picking up the colonies with straight wire
and stabbing the butt thrice. Overlay one
of the two tubes with sterile (neutral)
mineral oil up to 1 cm or melted paraffin
and petroleum gelly. The other tube is not
overlaid. (a third tube un inoculated with
organism may be overlaid with mineral oil
and incubated to check the acidity of the
oil). The tubes are incubated at 35 – 37°C
for atleast 4 days and examined daily for
production of acid.
MEC CONKEY AGAR
Weight the ingredients. Dissolve by
boiling in a water bath and then autoclave
at 15 lbs 15 minutes. Cool to 55°C and
pour the plates. Allow them to set at abient
temperature. Allow the surface to dry in an
incubator and inoculate the above
organisms mentioned. Note the growth
and colony characteristics.
EFFECT OF SALT
CONCENTRATION ON GROWTH
Prepare 150 ml of glucose broth in 250 ml
flask, adjust the pH to 7.2. Take 8 test
tubes, mark 1 – 8. To tube no 4 and 8 add
10 ml of GB mark as control. To tube no 1
and 5 put 0.05 gram of NaCl and add 10
ml GB (0.5%). To tube no 2 and 6 put 0.1
gram of NaCl and add 10 ml GB (1%). To
tube no 3 and 7 put 0.2 gram of NaCl and
add 10 ml of GB (1.5%). Sterilize all the
tubes at 121°C for 15 min. in an autoclave.
Inoculate tubes 1 – 4 with E.coli and 5 – 8
with candida albicans. Inoculate at 37°C
for 48 hours and observe the growth and
record.
MOLECULAR
CHARACTERIZATION
AMINOACID DECARBOXYLASE
TEST
GENOMIC DNA ISOLATION
1.5ml of overnight culture was taken in an
eppendorf tube. It was centrifuged at
12,000 rpm for 2 minutes to collect the
cell pellet. Supernatant was discarded and
to the pellet 467 l of TE buffer, 30 l of
10%SDS and 3 l of Proteinase K was
added. The content was incubated at 37 C for 1
hour. To this equal volume of Phenol:
Chloroform – 24:1 was added. The
eppendorf tube was centrifuged at 12,000 rpm
for 15 minutes at 4 c. Aqueous phase was
Divide the basal medium into two 100 ml
amounts. To one portion add 1 gram of Larginine. To the second portion is left as
such without amino acid to serve as
negative control. Check the pH again if
needed read just with 1 N sodium
hydroxide before sterilization. Autoclave
at 121°C for 10 min and distribute in 2 – 3
ml amount in 12× 100 mm test tubes.
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
transferred to fresh Eppendorf tube. 1/10th
volume of 3M sodium acetate was added.
Twice the volume of 99.9% Ethanol was
added to aqueous phase. Invert mixed
slowly. It was centrifuged at 12,000 rpm
for 15 minutes at 4 C. The pellet was
washed with 70% ethanol. Supernatant
was discarded and the pellet was air dried.
The pellet was suspended in 20 l of 1X
Tris EDTA buffer.
Purity of the DNA
A260 : A280 ratio = A260 / A280
=
1.8: pure DNA
=
1.7 – 1.9; fairly pure DNA
(acceptable ratio for PCR)
=
less than 1.8; presence of proteins.
=
greater than 1.8; presence of
organic solvent.
POLYMERASE CHAIN REACTION
(PCR)
AGAROSE GEL
ELECTROPHORESIS
Agarose was weighed and transferred to a
conical flask. 50 ml of 1X TAE was
added and Agarose was melted to a clear
solution by heating. It was allowed to
cool until it reached bearable temperature.
2.5µl of ethidium bromide stock solution
was added. A gel casting tray was placed
in a leveling table and the melted agarose
was poured. After the gel solidified, the
comb was taken out carefully. The casted
gel was placed in an electrophoresis tank
and 1X TAE buffer was added until the
gel was completely submerged. DNA
sample was mixed with the gel loading
buffer and loaded into the well.
The
samples were then electrophoresed at 50V
until the gel loading buffer reached 2/3rd of
the gel. This gel was then viewed under
UV Transilluminator.
100ng of DNA is used for molecular
Identification of respective sample. PCR
reaction was performed for 16S rRNA
gene.The PCR tubes were placed in
thermocycler and the samples were
amplified in thermocycler. Amplified
samples were then electrophoresed on
1.5% agarose gel.
SEQUENCING
3µl the amplified PCR product was
subjected
to
1.5%
agarose
gel
electrophoresis and the remaining sample
was subjected for sequencing.
Fig.2: Genomic DNA
QUANTIFICATION OF DNA BY
SPECTRO PHOTOMERIC METHOD
The spectrophotometer was calibrated and
the UV lamp was turned on. The
wavelength was set at 260nm and 280nm.
Absorbance at 260 and 280 nm was set at
zero with TE buffer or sterile water as
blank. 3µl of the DNA was taken in a
quartz cuvette and made up to 3ml with
TE buffer or sterile water. Absorbance of
the sample at 260 and 280 nm was noted.
The concentration of DNA was calculated
using the given formula:
1
2
3
4
5
Lane 1: Genomic DNA from sample 1
Lane 2: Genomic DNA from sample 2
Lane 3: Genomic DNA from sample 3
Lane 4: Genomic DNA from sample 4
Lane 5: 1 Kb DNA ladder
Concentration of dsDNA
A260 X 50µg/ml X dilution factor
140
Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
Table 3: Genomic DNA Analysis using UV Spectrophotometer
Sample
Quantity
Purity
Sample 1
1800ng/µl
1.82
Sample 2
1750ng/µl
1.76
Sample 3
1600ng/µl
1.89
Sample 4
1910ng/µl
1.71
Fig.3: Amplification of 16S rRNA gene from bacteria (TA 51 C)
1
2
3
4
5
Lane 1: Amplified product of 16s rRNA gene from sample 1
Lane 2: Amplified product of 16s rRNA gene from sample 2
Lane 3: Amplified product of 16s rRNA gene from sample 3
Lane 4: Amplified product of 16s rRNA gene from sample 4
Lane 5: 1 Kb DNA ladder
Fig.4: Amplification of 16s rRNA gene from bacteria
1
2
3
4
5
Lane 1: Amplified product of 16s rRNA gene from sample 1
Lane 2: Amplified product of 16s rRNA gene from sample 2
Lane 3: Amplified product of 16s rRNA gene from sample 3
Lane 4: Amplified product of 16s rRNA gene from sample 4
Lane 5: 1 Kb DNA ladderx
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
sample was subjected to centrifugation at 800
rpm for 10 minutes to remove all unground
samples. The supernatant was used as the source
of sample for the microbial isolation and
characterization – (A).
The water sample
obtained from the first time centrifugation was
used as the source of sample for the isolation of
microbes – (B). Both the sample ware serially
diluted with 1M PBS (pH-7.2) and the dilution of
10-1 ,10-2, 10-3, 10-4, 10-5 subjected to lawn
culture on Luminescent media (pH-7.6). A- IS1,
IS2, IS3 IS4, IS5 : IS3 maximum number of
fluorescence colonies (pale green). B- IS6, IS7,
IS8, IS9, IS10 : IS8 maximum number of
fluorescence colonies (blue to purple).
RESULTS
BIOCHEMICAL CHARACTERIZATION
In biochemical investigation the sample
obtained were divided into two batches as
larvae sample (A) and water sample (B) by
centrifugation of the sample at 1500 rpm for 15
minutes. The larvae pellet (A) was separated
from the supernatant (B). Both (A) and (B) were
used as the samples for the microbial isolation
and characterization.
The pellets was initially subjected for
homogenization with 1M PBS (pH-7.2) and the
Table 4: Biochemical Characterization
Procedure/
H1
H2
H3
H4
H5
W1
W2
W3
Gram’s staining
-
-
-
+
+
+
-
-
Motility Test
(Hanging drop)
+
+
+
-
-
+
+
Indole
+
+
+
-
+
+
+
MR test
+
+
+
+
VP test
-
+
-
-
Oxidase
-
+
+
+
-
Catalase
+
+
+
+
+
ONPG test
-
+
+
+
-
Citrate utilization
Test
-
+
-
+
-
Ureases test
-
-
-
+
-
Nitrate reduction
Tests
-
+
+
+
-
TSI
Gelatin
Acid
Slant
& butt,
Gas+
-
Acid
slant,
no
Gas
+
Acid
slant,
no
Gas
+
+
Acid
Slant
& butt,
Gas+
-
Blood agar
-
+
+
+
-
Mac Conkey Ager
LF
+
NLF
LF
Growth in 0.5%
NaCl
+
+
+
NLF with
Translucent
Colony
+
Growth in 1.0%
NaCl
+
+
+
+
+
Growth in 1.5%
NaCl
-
-
+
-
-
Arginine
-
-
-
Isolates
142
+
+
-
Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
Fig.5 : 10-5 Dilution (IS3 Sample) lawn culture on luminescent media-larvae extract
Fig 6 : Indole Test
Fig 7 : MR Test
Fig. 8: Catalase
Fig. 9: ONPG Test
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Fig. 10: Nitrate reduction test
A- Control
B- Test microbe positive for nitrate reduction (after the addition of Zinc dust )
Fig. 11: TSI Media
Fig. 12: Gelatin
Fig. 13: Blood Agar
Fig. 14: Growth on 1.5% NaCl
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Fig 15: Carbohydrate utilization test of medium
A – Control tube no colour change
B – Maltose
C - Mannitol
Fig.16: Confirmatory Medium: Growth on Luminescent Media
Colonies with yellow pigmentation
Colonies with yellow pigmentation
Colonies with yellow pigmentation
Colonies showing distinctive yellow-orange pigment.
Colonies with pale green fluorescence
Colonies with pale green fluorescence
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Fig.17: Isoalted pure colony on luminescent media
(a) Luminescent media agar
(b) Luminescent media broth
Quandrant streaking-
A- Positive for fluorescences
pale green fluorescence
B- Uninoculated Media
Confirmatory test for W2
Confirmatory medium (pseudomonas
fluorescence medium) Yellow colored
fluorescence’s.
Confirmatory Test: For H3
Growth positive on Luminescent media,
distinctive yellow-orange pigment, with pale
green fluorescence
Sugar utilization test
Confirmatory (Sugar Analysis)
Mannitol
-positive (acid production)
-Negative
Mannose
-Positive
Maltose
Maltose
-positive
Thus W2 is confirmed as Pseudomonas
aeuroginosa.
Mannitol
-positive
Sucrose
-positive
Lactose
-positive
Glucose
-positive
Cellibiose
-positive
Thus H3 is confirmed as vibrio fisheri
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Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–
154
MOLECULAR CHARACTERISATION
Fig.18: Genomic DNA Isolation
Lane 1: Genomic DNA of the given sample.
Lane 2: 1Kb Ladder (10,000 bp, 8000 bp,
6000 bp, 5000 bp, 4000 bp, 3000 bp, 2500 bp,
2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp,
250 bp.
DNA QUANTIIFCATION BY
SPECTROPHOTOMETRIC METHOD
Sample
OD at 260nm
OD at 280nm
Blank
0.000
0.000
--
--
1
0.325
0.179
16250
1.81
CONCENTRATION OF DNA
A260 X 50µg/ml X dilution factor
Dilution Factor = 3ml/3µl = 1000
POLYMERASE CHAIN REACTION
Fig.19: Amplification of 16s rRNA gene
(48°C)
1
2
147
Concentration (ng/µl)
Purity
Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–
154
Lane 1 :
PCR Amplicon
Lane 2 : 1Kb Ladder (10,000 bp, 8000 bp,
6000 bp, 5000 bp, 4000 bp, 3000 bp, 2500 bp,
2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp,
250 bp
Fig.20: Amplification of 16s rRNA gene
(50°C)
1
Lane 1 : PCR Amplicon
Lane 2 : 1Kb Ladder (10,000 bp, 8000 bp,
6000 bp, 5000 bp, 4000 bp, 3000 bp, 2500 bp,
2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp,
250 bp
SEQUENCE
148
2
Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–
154
GRAPHICAL REPRESENTATION
149
Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
Vibrio sp. PaH1.31 16S ribosomal RNA gene, partial sequence
Sequence ID: gb|GQ406715.1|
Length: 1411
Number of Matches: 1
Related Information
Range 1: 46 to 1324 GenBank
Graphics
Next Match Previous Match
Score
2362 bits(1279)
Query1
Sbjct 46
Query61
Sbjct 106
Query121
Sbjct 166
Query181
Sbjct 226
Query241
Sbjct 286
Query301
Sbjct 346
Query361
Sbjct 406
Query421
Sbjct 466
Query481
Sbjct 526
Query541
Sbjct 586
Query601
Sbjct 646
Expect
0.0
Identities
1279/1279(100%)
Gaps
0/1279(0%)
TCGAGCGGCGGACGGGTGAGTAATGCCTAGGAAATTGC
CCTGATGTGGGGGATAACCATT
60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TCGAGCGGCGGACGGGTGAGTAATGCCTAGGAAATTGC
CCTGATGTGGGGGATAACCATT
105
GGAAACGATGGCTAATACCGCATAATACCTACGGGTCA
AAGAGGGGGACCTTCGGGCCTC
120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGAAACGATGGCTAATACCGCATAATACCTACGGGTCA
AAGAGGGGGACCTTCGGGCCTC
165
TCGCGTCAGGATATGCCTAGGTGGGATTAGCTAGTTGGT
GAGGTAATGGCTCACCAAGGC
180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TCGCGTCAGGATATGCCTAGGTGGGATTAGCTAGTTGGT
GAGGTAATGGCTCACCAAGGC
225
GACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACAC
TGGAACTGAGACACGGTCCAG
240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACAC
TGGAACTGAGACACGGTCCAG
285
ACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAAT
GGGCGCAAGCCTGATGCAGCCA
300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAAT
GGGCGCAAGCCTGATGCAGCCA
345
TGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCAC
TTTCAGTCGTGAGGAAGGTAG
360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCAC
TTTCAGTCGTGAGGAAGGTAG
405
TGTAGTTAATAGCTGCATTATTTGACGTTAGCGACAGAA
GAAGCACCGGCTAACTCCGTG
420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TGTAGTTAATAGCTGCATTATTTGACGTTAGCGACAGAA
GAAGCACCGGCTAACTCCGTG
465
CCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAAT
CGGAATTACTGGGCGTAAAGCG
480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAAT
CGGAATTACTGGGCGTAAAGCG
525
CATGCAGGTGGTTTGTTAAGTCAGATGTGAAAGCCCGGG
GCTCAACCTCGGAATAGCATT
540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CATGCAGGTGGTTTGTTAAGTCAGATGTGAAAGCCCGGG
GCTCAACCTCGGAATAGCATT
585
TGAAACTGGCAGACTAGAGTACTGTAGAGGGGGGTAGA
ATTTCAGGTGTAGCGGTGAAAT
600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TGAAACTGGCAGACTAGAGTACTGTAGAGGGGGGTAGA
ATTTCAGGTGTAGCGGTGAAAT
645
GCGTAGAGATCTGAAGGAATACCGGTGGCGAAGGCGGC
CCCCTGGACAGATACTGACACT
660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
705
150
Strand
Plus/Plus
Query661
Sbjct 706
Query721
Sbjct 766
Query781
Sbjct 826
Query841
Sbjct 886
Query901
Sbjct 946
Query961
Sbjct 1006
Query1021
Sbjct 1066
Query1081
Sbjct 1126
Query1141
Sbjct 1186
Query1201
Sbjct 1246
Query1261
Sbjct 1306
Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
GCGTAGAGATCTGAAGGAATACCGGTGGCGAAGGCGGC
CCCCTGGACAGATACTGACACT
CAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATAC
CCTGGTAGTCCACGCCGTAAAC
720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATAC
CCTGGTAGTCCACGCCGTAAAC
765
GATGTCTACTTGGAGGTTGTGGCCTTGAGCCGTGGCTTT
CGGAGCTAACGCGTTAAGTAG
780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GATGTCTACTTGGAGGTTGTGGCCTTGAGCCGTGGCTTT
CGGAGCTAACGCGTTAAGTAG
825
ACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAA
TGAATTGACGGGGGCCCGCACA
840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAA
TGAATTGACGGGGGCCCGCACA
885
AGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAA
GAACCTTACCTACTCTTGACAT
900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAA
GAACCTTACCTACTCTTGACAT
945
CCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAA
CTCTGAGACAGGTGCTGCATG
960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAA
1005
CTCTGAGACAGGTGCTGCATG
GCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTC
1020
CCGCAACGAGCGCAACCCTTA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTC
1065
CCGCAACGAGCGCAACCCTTA
TCCTTGTTTGCCAGCACTTCGGGTGGGAACTCCAGGGAG
1080
ACTGCCGGTGATAAACCGGAG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TCCTTGTTTGCCAGCACTTCGGGTGGGAACTCCAGGGAG
1125
ACTGCCGGTGATAAACCGGAG
GAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACG
1140
AGTAGGGCTACACACGTGCTAC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACG
1185
AGTAGGGCTACACACGTGCTAC
AATGGCGCATACAGAGGGCGGCCAACTTGCGAGAGTGA
1200
GCGAATCCCAAAAAGTGCGTCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AATGGCGCATACAGAGGGCGGCCAACTTGCGAGAGTGA
1245
GCGAATCCCAAAAAGTGCGTCG
TAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTC
1260
GGAATCGCTAGTAATCGTGGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTC
1305
GGAATCGCTAGTAATCGTGGA
TCAGAATGCCACG
GTGAAT
1279
|||||||||||||||||||
TCAGAATGCCACG
GTGAAT
1324
151
Int. J. Adv. Res. Biol.Sci. 1(8): (2014): 130–154
the aquaculture industry worldwide. The vibrio
fischeri is a bioluminescent symbiont that colonizes
the light-emitting organs of certain marine animals,
including lobster larval rearing systems.
DISCUSSION
Subsequently a total of 20 pure colonies were
randomly selected on the basis of different
morphologies and phenotypic identification was done
following the scheme described elsewhere (Alsina
and Blanch 1994; Holt et al 1994). Presumptive tests
included gram staining, motility test, indole test, MR
test, VP test, oxidase, catalase, ONPG test, citrate
utilization test, ureases test, nitrate reduction test, TSI,
Gelatin, Blood agar, Macconkey agar, Growth at
0.5%, 1.0%, 1.5% NaCl, arginine and different sugar
utilization test. On the basis of preliminary
biochemical tests finally 8 isolates with different
characteristics were chosen as representative strains of
all isolates and detailed molecular characterizations
was performed for pathogenic bacteria.
The indian sand lobster, Thenus Orientalis, is potential
valuable candidate as an aquaculture species but V.
fischeri related species outbreaks during the extended
larval life cycle are major constraints for the
development of a breeding programme for the
aquaculture of this species at a commercial level.
Bacterial identification methods such as conventional
biochemical tests and universal 16s rRNA gene
sequencing were done to conform the pathogen. V.
fishceri is an important pathogen and is extremely
difficult to identify because it is phenotypically
diverse. Hence PCR technique was employed using
16S rRNA sequences to reduce the duration of
identification of this species, a valuable tool for a
rapid accurate detection and hence earlier treatment
can be administered which may increase the survival
rate from vibriosis.
The PCR technique was found to assist the
confirmation of identity of 8 different isolates of
suspected Vibrio fisheri from infected lobsters larvae.
The time factor indicates that affected lobsters larvae
can be diagnosed in stipulated timings with accurate
results than previously spend on biochemical
morphological testings.
References
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investigations of the Israel south Red Sea
expedition. Rep. No. 33. Bull. Sea Fish. Res. Stn.
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Chhapgar, B. F. and Deshmukh, S. K. 1964. Further
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Cano-Gómez A, Goulden EF, Owens L, Høj L. 2010.
Vibrio owensii sp.nov., isolated from cultured
crustaceans in Australia. FEMS Microbiol. Lett.
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Department of Fisheries 1997. Fish and other aquatic
animals of Thailand,
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