FARHANNA MOHAMMED

870131565028

IDENTIFICATION AND ISOLATION OF PROBIOTIC MICROORGANISM IN EEL

Introduction

True eels (Anguilliformes) are an order of fish, which consists of four suborders, 19 families,
110 genera and approximately 600 species. Most eels are predators. The term "eel" is also
used for some other similarly shaped fish, such as electric eels and spiny eels, but these are
not members of the Anguilliformes order.

True eels are elongated fishes, ranging in length from 5 centimetres (2.0 in) in the one-jawed
eel (Monognathus ahlstromi) to 3.75 metres (12.3 ft) in the giant moray. They have no pelvic
fins, and many species also lack pectoral fins. The dorsal and anal fins are fused with the
caudal or tail fin, to form a single ribbon running along much of the length of the animal.

Most true eels prefer to dwell in shallow waters or hide at the bottom layer of the ocean,
sometimes in holes. These holes are called eel pits. Only the Anguillidae family regularly
lives in fresh water, and returning to the sea to breed. Some eels dwell in water as deep as
4,000 metres (13,000 ft), or are active swimmers (the family Nemichthyidae — to a depth of
500 metres (1,600 ft).

Eels possess a flat and transparent larva, called a leptocephalus. These drift in the surface
waters of the sea feeding on dissolved nutrients, before developing into a young eel, referred
to as an elver, and seeking out the adult habitat.

Probiotics are dietary supplements of live bacteria or yeasts thought to be healthy for the host
organism. According to the currently adopted definition by FAO/WHO, probiotics are: ‘Live
microorganisms which when administered in adequate amounts confer a health benefit on the
host. Probiotic bacterial cultures are intended to assist the body's naturally occurring gut
flora, an ecology of microbes, to re-establish themselves.

Combining both term, the research’s goal is to identify the supplement of live bacteria
thought to be healthy for the host in eel fish in term of assisted the eel fish’s normal flora to
develop immunocompetent in the eel fish.

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Statement of Problem

Delimitation: The limitation of this research is “probiotic microorganism” and “eel fish”. It
will included the morphological and biochemical properties of probiotic bacteria obtain from
the eel fish while for the eel itself some benefits and physical properties will be discussed, no
further research will be made others than mentioned terms.

Limitation: To isolate the unknown probiotic bacteria from eel fish, the culture medium and
suitable biochemical test method must first be known. This will encounter some difficulty
because to get the correct media and compatible method, we need to exactly know the strain
species. In this case, several species of probiotic bacteria which is used from previous study
to get the media and method will be the reference.

Hypothesis: The expected result from this research is the presence of probiotic bacteria such
as Lactobacillus sp. and Bacillus sp. from the eel fish. The research will face any risk of not
getting any sample of pro biotic bacteria due to habitat of eel fish and its source of food.

Objectives

The purpose of this research proposal is to identify and isolate the probiotic microorganism in
eel. The research is also to determine the beneficial and compatibility of pro biotic for future
used.

Statement of Significant

Significant of this research is to obtain the microorganism or the bacteria which are believe to
be probiotics source in eel fish. The complete analysis including morphological, chemical
properties and PCR analysis will be the final result of all. From the result, we may have some
idea on feeding the eel fish in industry on more suitable probiotics for the sake of mortality
rate and economics benefits.

Literature Review
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Eel common name for any fish of the 10 families constituting the order Anguilliformes, and
characterized by a long snakelike body covered with minute scales embedded in the skin.
Eels lack the hind pair of fins, adapting them for wriggling in the mud and through the
crevices of reefs and rocky shores. Most species are marine; the largest and most diverse
group are the morays, family Muraenidae, sharp-toothed and vicious.(Schweld, R. 2002)

Japanese eel (Anguilla japonica) is one of 18 species of genus Anguilla that distributes widely
in Taiwan, mainland China, Japan, and Korea (Ege 1939; Jespersen 1942). Japanese eel
Anguilla japonica is known to be a typical fatty fish (Ohshima, 1985; Ozaki, Koga,
Takahashi, Adachi, & Yamauchi, 2008). Japanese eel is one of the most valuable cultured
species in Japan. Some 100,000 and 300 tons of cultured and wild eels, respectively, are
supplied to the food market of Japan a year (Tachiki, 1996).

New Zealand has two main species of fresh-water eel, the shortfin eel (Anguilla australis),
which is also found in southeastern Australia and at some Pacific islands, and the endemic
longfin eel (A. dieffenbachii) (McDowall, 1990).

Transferring the eels, Anguilla japonica or A. anguilla, from fresh water to sea water results
in an increase in plasma electrolytes and a decrease in body weight; maximal changes are
observed 2 days after transfer (Oide & Utida, 1968; Mayer & Nibelle, 1970; Kirsch, 1972;
Kirsch & Mayer-Gostan, 1973).

The European eel (Anguilla anguilla L.) spawns in the Sargasso Sea and the resulting larvae
(Leptocephali) migrate northeast to coastal areas whilst exhibiting a diel vertical migration.
After metamorphosis on the continental shelf, glass eels enter estuaries mostly from October
to April /May and migrate up estuaries for a phase of growing into the yellow eel stage
(Tesch, 1977).

The long distance oceanic migrations of thousands of kilometers that are made by the
freshwater eels of the genus Anguilla have fascinated biologists for more than a century
(Tsukamoto et al. 2002), but the unique morphology and biology of their leptocephali still
remains mysterious. These virtually transparent larvae have laterally compressed bodies that
reach large sizes compared to most Wsh larvae. Their bodies are Wlled with a gelatinous
material that provides structural support and increased buoyancy (Pfeiler, 1999) as they

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migrate in the surface layer of the ocean from their oVshore spawning areas to their terrestrial
freshwater habitats using ocean currents.

Commercial eel fishers supply a monthly catch and effort return.These data are cross-checked
against monthly returns from eel processors and any significant discrepancies between the
two datasets are investigated. Reported commercial fishery catches show the rapid increase in
catches in the late 1960s to a peak of 2077 t in 1972, 25 years of markedly fluctuating
catches, and a general decline since 1994/ 1995 to currently a little over 700 t (Sullivan et al.,
2005).

Apart from small quantities of glass eels that can be caught for research purposes, it is not
legal in New Zealand to catch or export glass eels. High overseas prices for glass eels in the
1970s stimulated experimental capture of this life stage in the Waikato River, the river with
New Zealand’s largest recruitment. As much as 6 t was caught in a single year (Jellyman,
1979). The population of the European eel Anguilla anguilla is in decline (Dekker, 2004).

Japanese eels (Anguilla japonica Temminck & Schlegel) taken from a commercial source
were decapitated and the gut excised immediately and stored at –40 °C. A boiled-water
extract of eel gut (361 g wet mass) was prepared following a method previously described
(Uesaka et al., 1994a; Uesaka et al., 1994b).

Probiotics can be defined as a food (feed) or drug containing live microbes that, when
ingested, is expected to give beneficial physiologic effects to the host animal through
microbial actions (Ishibashi and Yamazaki, 2001).

According to Yasuds and Taga (1980) probiotic bacteria would be found to be useful not only
as food but also as biological controllers of fish disease and activators of nutrient
regeneration.

Consideration of the safety of probiotics is therefore of extreme importance. The safety of the
microbes that have been used traditionally in probiotics can be conformed through a long
period of experience (Mayra-Makien and Bigert, 1993).

Kennedy et al. (1998) used probiotic bacteria in the culture of marine fish larvae. They
identified and used probionts for the culture of common snook, red drum, spotted sea trout

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and striped mullet. They then observed that the application of probiotic bacteria to larval fish
tanks (from egg through transformation) increased survival, size uniformity, and growth rate.

Bacillus sp. is often antagonistic against other fresh water fish pathogenic bacteria
(Gatesoupe, 1999; Rengipipat et al., 2000). The results obtained in many countries have
indicated that some of the bacteria used in probiotics (Lactobacilli) are capable of stimulating
the immune system (Fuller, 1992).

Carnevali et al. (2004) isolated Lactobacillus fructivorans (AS17B) from sea bream (Sparus
aurata) gut, and then administered it during sea bream development using Brachinons
plicatilis and/or Artemia salina and dry feed as vectors.

Previously, Gildberg et al. (1997) had analysed the effect of a probiotic of lactic acid bacteria
in the feed of Atlantic cod fry (Gadus morha) on growth and survival rates.

These are classified in the group of Gram-positive bacteria. They usually have no mobility
and are nonsporulating bacteria that produce lactic acid. Some members of this group contain
both rods (lactobacilli and carnobacteria) and cocci (streptococci). Different species of lactic
acid bacteria (such as Streptococcus, Leuconostoc, Pediococcus, Aerococcus, Enterococcus,
Vagococcus, Lactobacillus, Carnobacterium) have adapted to grow under widely different
environmental conditions. They are found in the gastrointestinal tract of various endothermic
animals, in milk and dairy products, seafood products, and on some plant surfaces (Ring &
Gatesoupe, 1998).

Methodology

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Lactic acid producing bacteria (probiotic bacteria) isolated from eel digestive tract:

1) The eel fish was collected from fresh water river. The eel fish were washed with
sterile distilled to remove the unwanted particles.
2) Then the animals were dissected to remove the digestive tracts by the sterilization
condition. The digestive tracts were homogenized in the same sterile distilled water
for centrifugation.
3) After centrifugation the supernatant was taken and serially diluted in sterile distilled
water in the test tubes to 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8 dilution and were pour
plated on nutrient agar plate and incubated for 24 h at room temperature.
4) Individual colonies were taken and inoculated in McConkey broth. Then the broths
were incubated for 18-24 h at 37°C.
5) One hundred microliter of culture was inoculated in Bacillus selective medium and
Lactobacillus bulgaricus agar plate and different colonies were selected and subjected
to confirmatory tests.

Biochemical Test
Grams staining: Heat-fixed smears should be stained for 1 min with crystal violet or
methylene blue dye, washed in tap water, covered with Grams iodine for 1 min re-washed,
decolorized by a few seconds in acetone-alcohol and counterstained for 30 sec in safranin.
The smears are washed thoroughly and gently air-dried and observed under the oil immersion
objective.

Methyl Red test (MR): The test is used to identify bacteria producing stable acids by
mechanisms of mixed acid fermentation of glucose. Inoculate the isolated bacteria with
buffered glucose broth and incubate at 37°C for 48 h. After incubation add a few drops of
methyl red solution to the culture, read immediately. A red color represents a positive test.

Catalase test: This test is can be used to detect the enzyme catalase. This enzyme is
responsible for protecting bacteria from hydrogen peroxide (H2O2) accumulation, which can
occur during aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to the
organism. Catalase breaks H2O2 down into water and O2. To perform the catalase test simply
smear a small amount of the test organism onto the lid of a Petri plate/culture dish. Then add
a drop of hydrogen peroxide to the smear. If bubbles become visible (these would be the O2
bubbling up) then the test is positive and you can conclude that the organism makes catalase.
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A lack of bubbles indicates the absence of catalase. A loopful of the culture was placed on a
slide and few drops of 10% hydrogen peroxide were added. The slides were observed for
effervescence.

PCR identification:

DNA extraction of the bacteria

A singe colony grown overnight was transferred into a new 1.5ml eppendorf tube. Cell was
pelleted by microcentrifuge at 13000rpm for 1 minute. Cell pellet obtained was resuspended
in 700μl of TE buffer (pH 8) and 25% SDS and the mixture was vortexed. The samples were
incubated in water bath at 60°C for 15 minutes until the solution become clear. The samples
were inverted every 5 minutes during the incubation. Then, 800μl of chloroform
isoamylalcohol was added and mixed gently. The resultant cell lysate solution was
centrifuged at 13000rpm for 1 minute. 200μl of upper aqueous layer was carefully transferred
into a new sterile 1.5ml eppendorf tube. An equal volume of 3M sodium acetate was added
and mixed gently. A double volume of cold isopropanol was added and centrifuged at
13000rpm for 10 minutes. The resultant supernatant was discarded. The pellet was washed
twice with 500μl of 70% cold ethanol and spun at 12000rpm for 5 minutes. The ethanol
solution was discarded. The pellet was dried at room temperature and resuspended in 50μl of
sterile distilled water. The DNA solution was stored at -4°C until further use.

PCR method

PCR master mix:

Component Stock Final 5 rxn

dH2O 195.25 μL
Taq buffer 10x 1x 25 μL
MgCl2 50 mM 1.5 mM 7.5 μL
dNTP’s 10 mM 0.25 mM 6.25 μL
Forward 20 μM 0.4 μM 5 μL

primer
Reverse 20 μM 0.4 μM 5 μL

primer
Taq 5 U/μL 0.02 U/μL 1 μL

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polymerase

First, the extracted DNA is move into the special PCR tube. Next, the first primer is added
into the tube followed by the second primer. The nucleotides that make up the DNA code
(dNTP’s) are then added into the same tube. Lastly, all the reaction mix which are DNA
polymerase, reverse transcriptase, MgCl2, buffer and distilled water are added up in the tube.
The tube is inserted into thermal cycler for the reaction to begin and amplify the DNA. The
resulted product is then ready to be loaded into the agarose gel for electrophoresis.

Gel Electrophoresis method:

Pouring an agarose gel

The melted agarose is pour into the gel space until the gel is about 5 mm deep. The agarose
will be leave harden, which should take 5-10 minutes.

Electrophoresis of PCR reactions

Using the micropipet with a clean tip, 5 µl gel loading dye is pipet into the PCR reaction
tube. Load both the PCR reactions and standard DNA markers sample into the gel. Load
samples into the wells.Pour TAE buffer carefully so it fills the electrophoresis apparatus and
just covers the gel. Run that gel. Plug the electrodes into the electrophoresis apparatus. Plug
the power source into an outlet and set the voltage to about 100 V (max = 120 V). Let the gel
run until the dye migrates about 5-6 cm from the wells (about 20-25 minutes). Turn off the
power supply, disconnect the electrodes, and remove the top of the electrophoresis apparatus.
Carefully remove the gel. The gel can be wrapped in plastic wrap and stored in the
refrigerator or placed it in the staining tray for DNA staining.

Staining gels to examine PCR reactions

Place gel in staining tray. Using gloves, remove the plastic from the ethidium bromide sheet
and place the ethidium bromide paper on the gel. Gently rub the paper with fingers to make
sure it is contacting the gel all over. Stain for about 10 minutes. Put the gel on the UV light
box and, with the UV shield down, view the gel. Take a polaroid picture of the gel.

Stain gels and enter digital images into BioNumerics database. The source of the bacteria
isolated from eel may now be identified by comparison of these fingerprint images to the
fingerprints from known sources in the database. The same database contains isolates from
both known and unknown sources, so that comparisons can be easily made.

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Chapter outline

Chapter 1- Plan of the study

1.1- Objective of the study

1.2- Introduction
1.3- Statement of problem (hypothesis)
1.4- Statement of significant
1.5- Methodology

Chapter 2- Review of related research and literature

2.1- Literature review

Chapter 3- Analysis of observation

3.1- Result and observation (picture, graph, table,)

3.2- Discussion

Chapter4- summary, finding, conclusion and recommendation

4.1- Conclusion

Data Collection

Data will be collected from February 2009 until November 2009. Qualitative data will be
collected via journal and internet research or any other media. The qualitative data is more on
focusing into literature review and previous research on the same study.

For the quantitative data, they will be collected from the lab experiment which will be
conducted during the research. It will include morphological analysis, biochemical analysis
and Polymerase Chain reaction (PCR) analysis. The data will be collected from at least 20
samples to get the exact result.

Costing and Expenses

Item Cost

Live eel RM20.00/kg

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Medium (Mc Conkey) RM 250.00

Other necessary chemical and apparatus RM 150.00

which may not available in lab

Grand Total RM470.00

Time Frame

Data will be collected from February 2009 until November 2009. The lab research will take
about 10 months to complete.

ITEM FEB MAR APR MAY JUNE JULY AUG SEP OCT NOV
LITERATURE
REVIEW
DATA
COLLECTION

DATA
ANALYSIS
THESIS
WRITING
THESIS
SUBMITTION

Conclusion

The result from this research is the presence of probiotic bacteria such as Lactobacillus sp.
and Bacillus sp. from the eel fish. This research will reveal particular probiotic
microorganism in eel fish and their benefits to the species will be highly and clearly
understood. This research also will determine what kind of probiotic bacteria that can inhibit
other pathogenic microorganisms in eel fish.

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