BIOCHEMICAL TESTS FOR MICROBIOLOGY

Various biochemical test are used for the identification of bacteria based on the character of a
particular bacteria to give its positive and negative result for a particular biochemical test.
1. The Indole Test
The test organism is inoculated into tryptone broth, a rich source of the amino acid
tryptophan. Indole positive bacteria such as Escherichia coli produce tryptophanase, an
enzyme that cleaves tryptophan, producing indole and other products. When Kovac's
reagent (p-dimethylaminobenzaldehyde) is added to a broth with indole in it, a dark pink
color develops. The indole test must be read by 48 hours of incubation because the indole
can be further degraded if prolonged incubation occurs. The acidic pH produced by
Escherichia coli limits its growth.
2. The Methyl Red and Voges-Proskauer Tests
The methyl red (MR) and Voges-Proskauer (VP) tests are read from a single inoculated
tube of MR-VP broth. After 24-48 hours of incubation the MR-VP broth is split into two
tubes. One tube is used for the MR test; the other is used for the VP test. Media contains
glucose and peptone. All enterics oxidize glucose for energy; however the end products
vary depending on bacterial enzymes. Both the MR and VP tests are used to determine
what end products result when the test organism degrades glucose. E. coli is one of the
bacteria that produces acids, causing the pH to drop below 4.4. When the pH indicator
methyl red is added to this acidic broth it will be cherry red (a positive MR test).
Klebsiella and Enterobacter produce more neutral products from glucose (e.g. ethyl
alcohol, acetyl methyl carbinol). In this neutral pH the growth of the bacteria is not
inhibited. The bacteria thus begin to attack the peptone in the broth, causing the pH to
rise above 6.2. At this pH, methyl red indicator is a yellow color (a negative MR test).
The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B
(potassium hydroxide). When these reagents are added to a broth in which acetyl methyl
carbinol is present, they turn a pink-burgundy color (a positive VP test). This color may
take 20 to 30 minutes to develop.
3. Catalase Test
Catalase is the enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2.
Hydrogen peroxide is often used as a topical disinfectant in wounds, and the bubbling
that is seen is due to the evolution of O2 gas. H2O2 is a potent oxidizing agent that can

wreak havoc in a cell; because of this, any cell that uses O2 or can live in the presence of
O2 must have a way to get rid of the peroxide. One of those ways is to make catalase.
4. The Citrate Test
The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow
utilizing citrate as its sole carbon and energy source. Simmon's media contains
bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at
acidic pH's (around 6), and gradually changes to blue at more alkaline pH's (around 7.6).
Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color.
Growth of bacteria in the media leads to development of a Prussian blue color (positive
citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative.
5. Oxidase Test
The oxidase test identifies organisms that produce the enzyme cytochrome oxidase.
Cytochrome oxidase participates in the electron transport chain by transferring electrons
from a donor molecule to oxygen. The oxidase reagent contains a chromogenic reducing
agent, which is a compound that changes color when it becomes oxidized. If the test
organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple
within 15 seconds.
6. Nitrate Reduction Test
Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to
nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to
perform nitrification on nitrate and nitrite to produce molecular nitrogen. Nitrate broth
contains nutrients and potassium nitrate as a source of nitrate. After incubating the nitrate
broth, add a dropperful of sulfanilic acid and a-naphthylamine. If the organism has
reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid. When
sulfanilic acid is added, it will react with the nitrous acid to produce diazotized sulfanilic
acid. This reacts with the a-naphthylamine to form a red-colored compound. Therefore, if
the medium turns red after the addition of the nitrate reagents, it is considered a positive
result for nitrate reduction.If the medium does not turn red after the addition of the
reagents, it can mean that the organism was unable to reduce the nitrate, or it could mean
that the organism was able to denitrify the nitrate or nitrite to produce ammonia or
molecular nitrogen. Therefore, another step is needed in the test. If the medium does not
turn red after the addition of the nitrate reagents, add a small amount of powdered zinc.
Be careful, as powdered zinc is hazardous! If the tube turns red after the addition of the
zinc, it means that unreduced nitrate was present. Therefore, a red color on the second
step is a negative result. The addition of the zinc reduced the nitrate to nitrite, and the

nitrite in the medium formed nitrous acid, which reacted with sulfanilic acid. The
diazotized sulfanilic acid that was thereby produced reacted with the a-naphthylamine to
create the red complex. If the medium does not turn red after the addition of the zinc
powder, then the result is called a positive complete. If no red color forms, there was no
nitrate to reduce. Since there was no nitrite present in the medium, either, that means that
denitrification took place and ammonia or molecular nitrogen were formed.
7. Urease Test
Urease broth is a differential medium that tests the ability of an organism to produce an
exoenzyme, called urease that hydrolyzes urea to ammonia and carbon dioxide. The
broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and
the pH indicator phenol red. Phenol red turns yellow in an acidic environment and
fuchsia in an alkaline environment. If the urea in the broth is degraded and ammonia is
produced, an alkaline environment is created, and the media turns pink. Many enterics
can hydrolyze urea; however, only a few can degrade urea rapidly. These are known as
"rapid urease-positive" organisms. Members of the genus Proteus are included among
these organisms. Urea broth is formulated to test for rapid urease-positive organisms.
The restrictive amount of nutrients coupled with the use of pH buffers prevent all but
rapid urease-positive organisms from producing enough ammonia to turn the phenol red
pink.
8. Phenol Red Broth
Phenol Red Broth is a general-purpose differential test medium typically used to
differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH
indicator), a Durham tube, and one carbohydrate. We use three different kinds of phenol
red broths. One contains glucose; one contains lactose, and the last contains sucrose. The
objective of the exercise is to determine which organisms can utilize each sugar. Phenol
red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4.
If the organism is able to utilize the carbohydrate, an acid by-product is created, which
turns the media yellow. If the organism is unable to utilize the carbohydrate but does use
the peptone, the by-product is ammonia, which raises the pH of the media and turns it
fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be
produced. If it is, an air bubble will be trapped inside the Durham tube. If the organism is
unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be
formed.
9. Casease Test

Skim milk agar is a differential medium that tests the ability of an organism to produce an
exoenzyme, called casease, that hydrolyzes casein. Casein forms an opaque suspension in
milk that makes the milk appear white. Casease allows the organisms that produce it to
break down casein into smaller polypeptides, peptides, and amino acids that can cross
the cell membrane and be utilized by the organism. When casein is broken down into
these component molecules, it is no longer white. If an organism can break down casein,
a clear halo will appear around the areas where the organism has grown.
10. Gelatinase Test
Nutrient gelatin is a differential medium that tests the ability of an organism to produce
an exoenzyme, called gelatinase, that hydrolyzes gelatin. Gelatin is commonly known as
a component of gelled salads and some desserts, but it's actually a protein derived from
connective tissue. When gelatin is at a temperature below 32C (or within a few degrees
thereof), it is a semisolid material. At temperatures above 32C, it is a viscous liquid.
Gelatinase allows the organisms that produce it to break down gelatin into smaller
polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized
by the organism. When gelatin is broken down, it can no longer solidify. If an organism
can break down gelatin, the areas where the organism has grown will remain liquid even
if the gelatin is refrigerated.
11. Lipase Test
Tributyrin agar is a differential medium that tests the ability of an organism to produce an
exoenzyme, called lipase, that hydrolyzes tributyrin oil. Lipases break down lipids (fats).
Tributyrin oil is a type of lipid called a triglyceride. Other lipase tests use different fat
sources such as corn oil, olive oil, peanut oil, egg yolk, and soybean oil. Lipase allows
the organisms that produce it to break down lipids into smaller fragments. Triglycerides
are composed of glycerol and three fatty acids. These get broken apart and may be
converted into a variety of end-products that can be used by the cell in energy production
or other processes. Tributyrin oil forms an opaque suspension in the agar. When an
organism produces lipase and breaks down the tributyrin, a clear halo surrounds the areas
where the lipase-producing organism has grown.
12. Starch Hydrolysis
Starch agar is a differential medium that tests the ability of an organism to produce
certain exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze
starch. Starch molecules are too large to enter the bacterial cell, so some bacteria secrete
exoenzymes to degrade starch into subunits that can then be utilized by the organism.
Starch agar is a simple nutritive medium with starch added. Since no color change occurs

in the medium when organisms hydrolyze starch, we add iodine to the plate after
incubation. Iodine turns blue, purple, or black (depending on the concentration of iodine)
in the presence of starch. A clearing around the bacterial growth indicates that the
organism has hydrolyzed starch.
13. Triple Sugar Iron Agar
Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a
small amount of glucose (dextrose), ferrous sulfate, and the pH indicator phenol red. It is
used to differentiate enterics based on the ability to reduce sulfur and ferment
carbohydrates. As with the phenol red fermentation broths, if an organism can ferment
any of the three sugars present in the medium, the medium will turn yellow. If an
organism can only ferment dextrose, the small amount of dextrose in the medium is used
by the organism within the first ten hours of incubation. After that time, the reaction that
produced acid reverts in the aerobic areas of the slant, and the medium in those areas
turns red, indicating alkaline conditions. The anaerobic areas of the slant, such as the butt,
will not revert to an alkaline state, and they will remain yellow. This happens with
Salmonella and Shigella
14. Decarboxylation Test
Decarboxylase broth tests for the production of the enzyme decarboxylase, which
removes the carboxyl group from an amino acid. Decarboxylase broth contains nutrients,
dextrose (a fermentable carbohydrate), pyridoxal (an enzyme cofactor for decarboxylase),
and the pH indicators bromcresol purple and cresol red. Bromcresol purple turns purple at
an alkaline pH and turns yellow at an acidic pH. We also add a single amino acid to each
batch of decarboxylase broth. The three amino acids we test in our decarboxylase media
are arginine, lysine, and ornithine. The decarboxylase test is useful for differentiating the
Enterobacteriaceae.Each decarboxylase enzyme produced by an organism is specific to
the amino acid on which it acts. Therefore, we test the ability of organisms to produce
arginine decarboxylase, lysine decarboxylase, and ornithine decarboxylase using three
different but very similar media. If the organism is unable to ferment dextrose, there will
be no color change in the medium. If an organism is able to ferment the dextrose, acidic
byproducts are formed, and the media turns yellow. As the organisms ferment the
dextrose, the media initially turns yellow, even when it has been inoculated with a
decarboxylase-positive organism. The low pH and the presence of the amino acid will
cause the organism to begin decarboxylation. If an organism is able to decarboxylate the
amino acid present in the medium, alkaline byproducts are then produced. Arginine is
hydrolyzed to ornithine and is then decarboxylated. Ornithine decarboxylation yields
putrescine. Lysine decarboxylation results in cadaverine. These byproducts are sufficient
to raise the pH of the media so that the broth turns purple. If the inoculated medium is

yellow, or if there is no color change, the organism is decarboxylase-negative for that

amino acid. If the medium turns purple, the organism is decarboxylase-positive for that
amino acid.
15. Coagulase Test
The coagulase test identifies whether an organism produces the exoenzyme coagulase,
which causes the fibrin of blood plasma to clot. Organisms that produce catalase can form
protective barriers of fibrin around themselves, making themselves highly resistant to
phagocytosis, other immune responses, and some other antimicrobial agents. The
coagulase slide test is used to identify the presence of bound coagulase or clumping
factor, which is attached to the cell walls of the bacteria. Bound coagulase reacts with the
fibrinogen in plasma, causing the fibrinogen to precipitate. This causes the cells to
agglutinate, or clump together, which creates the "lumpy" look of a positive coagulase
slide test. You may need to place the slide over a light box to observe the clumping of
cells in the plasma.The coagulase tube test has been set up as a demo for you to observe
in class. This version of the coagulase test is used to identify the presence of either bound
coagulase or free coagulase, which is an extracellular enzyme. Free coagulase reacts with
a component of plasma called coagulase-reacting factor. The result is to cause the plasma
to coagulate. In the demo, the coagulase plasma has been inoculated with
Staphylococcus aureus and Staphylococcus epidermidis and allowed to incubate at 37C
for 24 hours. Staphylococcus aureus produces free coagulase; Staphylococcus
epidermidis does not. The coagulase test is useful for differentiating potentially
pathogenic Staphylococci such as Staphylococcus aureus from other Gram positive,
catalase-positive cocci.

Immunological Diagnosis
Antigen Antibody reactions are highly specific and sensitive. This forms the basis of
immunodiagnostics. These tools are used for the qualitative and quantitative estimation of the
pathogens and/or the protective antibody. These tests can be used at the farm level without the
aid of the instruments. It is apparent that conventional diagnostic methods rely solely on the
microscopic examination and its visual recognition, which requires a great deal of experience
with the organisms that often change so dramatically in their morphology during the course of
their development. Therefore, in the evolution of disease diagnostic procedures in aquaculture,
antibody-based (protein-based) immunodiagnosis plays a crucial role. This method has the
advantage over other traditional method in that it can detect sub-clinical/latent/carrier sate of
infection and can also discriminate the antigenic differences. This technique is relatively rapid
and more specific and sensitive. Further refinement of conventional immunodiagnostic
techniques has resulted in the development of monoclonal antibody-based techniques and this
has increased the accuracy of detection and has allowed studying the pathogenesis of diseases.

Nevertheless, the specificity of antibodies also limits their usefulness because major antigens are
not conserved among life stages of certain pathogens (Bartholomew et al., 1995). Although there
is an array of polyclonal and monoclonal antibody-based diagnostics available for various
aquatic animal pathogens.
1. Agar Gel Precipitation Test,
2. Agglutination Test,
3. ELISA,
4. Dot ELISA,
5. Latex Agglutination Test and
6. Fluorescent Antibody Test.
1. Agar Gel Precipitation Test
In this test, antibody and possible antigens are placed in wells in agar plates and allowed to
diffuse toward one another. The antibody is placed in a center well and antigens (specific or
nonspecific) are placed in surrounding wells. When an antibody and its specific antigen meet
one another and are at the proper concentrations, the precipitate will form a visible white line
between the two wells. This line is called a precipitin line.
2. Agglutination Test
This test is used to identify unknown antigens; blood with the unknown antigen is mixed
with a known antibody and whether or not agglutination occurs helps to identify the antigen;
used in tissue matching and blood grouping and diagnose. In the direct agglutination test,
serum is added to a suspension of cells that have the surface self-Ag to be tested. If the
individual's serum contains the specific auto-Abs, Ig will bind and, at the appropriate Ab
concentration, the cells will become cross linked. This will cause agglutination, and the cells
will form a mat at the bottom of the test well. Auto-Abs attached to a patient's cells can be
detected by the addition of a second Ab and observed for agglutination. Selective soluble
self-Ags can also be used to assay auto-Abs by attaching them to the surface of red blood
cells. This latter type of agglutination test is called passive or indirect hemagglutination.
3. ELISA
The purpose of an ELISA is to determine if a particular protein is present in a sample and if
so, how much. There are two main variations on this method: you can determine how much

antibody is in a sample, or you can determine how much protein is bound by an antibody.
The distinction is whether you are trying to quantify an antibody or some other protein. In
this example, we will use an ELISA to determine how much of a particular antibody is
present in an individual's blood. ELISAs are performed in 96-well plates which permits high
throughput results. The bottom of each well is coated with a protein to which will bind the
antibody you want to measure. Whole blood is allowed to clot and the cells are centrifuged
out to obtain the clear serum with antibodies (called primary antibodies). The serum is
incubated in a well, and each well contains a different serum. A positive control serum and a
negative control serum would be included among the 96 samples being tested. After some
time, the serum is removed and weakly adherent antibodies are washed off with a series of
buffer rinses. To detect the bound antibodies, a secondary antibody is added to each well. The
secondary antibody would bind to all human antibodies and is typically produced in a rodent.
Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase.
These enzymes can metabolize colorless substrates (sometimes called chromagens) into
colored products. After an incubation period, the secondary antibody solution is removed and
loosely adherent ones are washed off as before. The final step is the addition the enzyme
substrate and the production of colored product in wells with secondary antibodies
bound.When the enzyme reaction is complete, the entire plate is placed into a plate reader
and the optical density (i.e. the amount of colored product) is determined for each well. The
amount of color produced is proportional to the amount of primary antibody bound to the
proteins on the bottom of the wells.
4. DOT ELISA
Dot-ELISA (Enzyme Linked Immunosorbent Assay) is an extensively used immunological
tool in research as well as analytical/diagnostic laboratories. In sandwich Dot-ELISA, the
antigen is sandwiched directly between two antibodies which react with two different
epitopes on the same antigen. Here one of the antibodies is immobilized onto a solid support
and the second antibody is linked to an enzyme. Antigen in the test sample first reacts with
the immobilized antibody and then with the second enzyme-linked antibody. The amount of
enzyme linked antibody bound is assayed by incubating the strip with an appropriate
chromogenic substrate, which is converted to a coloured, insoluble product. The latter
precipitates onto the strip in the area of enzyme activity, hence the name Dot-ELISA. The
enzyme activity is indicated by intensity of the spot, which is directly proportional to the
antigen concentration.
5. Latex Agglutination Test
The latex agglutination test is a laboratory method to check for certain antibodies or antigens
in a variety of bodily fluids including saliva, urine, cerebrospinal fluid or blood. In latex
agglutination, an antibody (or antigen) is coated on the surface of latex particles which is

known as sensitized latex. When a sample containing the specific antigen (or antibody) is
mixed with the milky-appearing sensitized latex, it causes visible agglutination
6. Fluorescent Antibody Test
It is a laboratory test that uses antibodies tagged with fluorescent dye that can be used to
detect the presence of microorganisms. This method offers straight-forward detection of
antigens using fluorescently labeled antigen-specific antibodies. Because detection of the
antigen in a substrate of patient sample (cellular smear, fluid or patient- inoculated culture
medium) is the goal, DFA is seldom quantitative.

Molecular diagnosis
Molecular techniques are potentially faster and more sensitive than culture, serology, and
histology methods that are traditionally used to identify fish pathogens. During the last 15 years
or so, molecular techniques have been increasingly employed to diagnose fish diseases. These
techniques include polymerase chain reaction (PCR), restriction enzyme digestion, probe
hybridization, in situ hybridization, and microarray. Pathogens can be detected from
asymptomatic fish by molecular diagnostic techniques so disease outbreak could be prevented.
Thus antibiotic treatment can be reduced so that creation of antibiotic resistant bacteria may be
eliminated. In this paper molecular techniques for detection of fish pathogens are reviewed and
the potential for their application are discussed. The application of new techniques as a routine
tool in a diagnostic laboratory is an area where relevant literature is scarce and this may
contribute to the reticence of some to adopt these methods.
1. Polymerase Chain Reaction
Polymerase chain reaction is a technique for amplifying a specific region of DNA,
defined by a set of two "primers" at which DNA synthesis is initiated by a thermostable
DNA polymerase. Usually, at least a million-fold increase of a specific section of a DNA
molecule can be realized and the PCR product can be detected by gel electrophoresis. The
regions amplified are usually between 150-3,000 base pairs (bp) in length. Primer design
is important to obtain greatest possible sensitivity and specificity. Therefore, the primers
should be sufficiently long to allow a high annealing temperature and reduce the
opportunity for nonspecific primer annealing, but primers that are too long may facilitate
nonspecific annealing even to regions of DNA that are not perfectly complementary to
the primer sequence. The reaction includes template DNA that may be in various forms,
from a simple tissue lysate to purified DNA, primers, polymerase enzyme to catalyze
creation of new copies of DNA, and nucleotides to form the new copies. During each
round of the thermocycling reaction, the template DNA is denatured, primers anneal to
their complementary regions and polymerase enzyme catalyses the addition of

nucleotides to the end of each primer, thus creating new copies of the target region in
each round. Theoretically, the increase in amount of product after each round will be
geometric. Reverse transcriptase polymerase chain reaction (RT-PCR) is used to detect
specific mRNA and determine levels of gene expression. Compared to the two other
commonly used techniques for quantifying mRNA levels, Northern blot analysis and
RNase protection assay, RT-PCR can be used to quantify mRNA levels from much
smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA
from a single cell. Over the last several years, the development of novel chemistries and
instrumentation platforms enabling detection of PCR products on a real-time basis has led
to widespread adoption of real-time RTPCR as the method of choice for quantitative
changes in gene expression. Furthermore, real-time RT-PCR has become preferred
method for validating results obtained from array analyses and other techniques that
evaluate gene expression changes on a global scale. The sensitivity and specificity
achieved in a well-designed RT-PCR make it an ideal tool for use in the surveillance and
monitoring of covert infections. As in the eukaryotes, the prokaryotic rRNA genes
contain highly conserved sequences. The potential utility of conserved regions to identify
or amplify the rRNA genes, followed by exploitation of more variable regions of the
genes or spacers to detect or identify bacteria that may be difficult or even impossible to
culture has long been recognized.
2. Multiplex PCR
New developments such as design of PCR conditions that can detect several pathogens at
one time in a multiplex reaction will improve time and cost-efficiency of this
methodology, countering one of the major arguments against the adoption of these
techniques as routine. In multiplex PCR more than one target sequence can be amplified
by including more than one pair of primers in the reaction. Multiplex PCR has the
potential to produce considerable savings of time and effort within the laboratory without
compromising test utility. Since its introduction,multiplex PCR has been successfully
applied in many areas of nucleic acid diagnostics, including gene deletion analysis,
quantitative and RNA detection. In the field of infectious diseases, the technique has been
shown to be a valuable method for identification of viruses, bacteria, fungi and parasites.
3. Labeling and detection of nucleic acids
A variety of labeling and detection systems exist for nucleic acid probes. Radioisotopes
were once the norm but, in the interests of researcher safety, other methods are becoming
increasingly popular. The variety of labels and detection methods now available can
provide a system suitable for any application, from dot blots to in situ hybridization.
These include labeling with a variety of haptens such as biotin or digoxygenin and

detection by antibody binding coupled with fluorescent, chemiluminescent or

colorimetric detection methods
4. Restriction enzyme digestion
Restriction enzymes (or restriction endonucleases) cleave DNA in a very specific fashion.
Type II restriction enzymes, most commonly used for DNA analysis and genetic
engineering, each have a unique nucleotide sequence at which it cuts a DNA molecule. A
particular restriction enzyme will cleave DNA at that only recognition sequence that is
often a six base pair palindromic sequence, but others recognize four or even eight base
pair sequences. A common use for restriction enzymes is to generate a "fingerprint" of a
particular DNA molecule. Because of the sequence specificity of restriction enzymes,
these enzymes can cut DNA into discrete fragments which can be resolved by gel
electrophoresis. This pattern of DNA fragments generates a "DNA fingerprint" and each
DNA molecule has its own fingerprint. Other restriction enzymes can be used to further
characterization of a particular DNA molecule. The location of these restriction enzyme
cleavage sites on the DNA molecule can be compiled to create a restriction enzyme map.
These maps are very useful for identifying and characterizing a particular DNA plasmid
or region. Restriction enzymes recognize specific short sequences of DNA and cleave the
DNA at that site. Single nucleotide changes can result in the gain or loss of a restriction
site, thus altering the number of fragments produced following digestion of DNA. These
RFLP can be visualized following gel electrophoresis of the digested DNA to separate the
fragments according to size. Differences in the RFLP profiles have revolutionized
criminal investigations and have become powerful tools in the identification of
individuals in paternity and maternity cases, population genetics, and in the diagnosis of a
variety of diseases.
5. Restriction Fragment Length Polymorphism (RFLP)
RFLP is a technique in which organisms may be differentiated by analysis of patterns
derived from cleavage of their DNA. If two organisms differ in the distance between sites
of cleavage of a particular restriction endonuclease, the length of the fragments produced
will differ when the DNA is digested with a restriction enzyme. The similarity of the
patterns generated can be used to differentiate species (and even strains) from one
another. Isolation of sufficient DNA for RFLP analysis is time-consuming and labor
intensive. However, PCR can be used to amplify very small amounts of DNA, usually in
2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be
analyzed in a shorter time.
6. Amplified Fragment Length Polymorphism (AFLP)

A rapid PCR-based technique, AFLP can be used for typing prokaryotes and eukaryotes.
The method is based on the selective PCR amplification of genomic restriction fragments
of the whole genome and has been shown to be rapid, reproducible, and highly
discriminatory. Selected markers are amplified in a PCR, which makes AFLP an easy and
fast tool for strain identification in agriculture, botany, microbiology, and animal
breeding. The AFLP method used was essentially that described by Valsangiacomo et al.,
(1995). AFLP analysis belongs to the category of selective restriction fragment
amplification techniques, which are based on the ligation of adapters to genomic
restriction fragments followed by a PCR-based amplification with adapter-specific
primers. For AFLP analysis, only a small amount of purified genomic DNA is needed;
this is digested with two restriction enzymes, one with an average cutting frequency (like
EcoRI) and a second one with a higher cutting frequency (like MseI or TaqI). Doublestranded oligonucleotide adapters are designed in such a way that the initial restriction
site is not restored after ligation, which allows simultaneous restriction and ligation,
while religated fragments are cleaved again. An aliquot is then subjected to two
subsequent PCR amplifications under highly stringent conditions with adapter specific
primers that have at their 3' ends an extension of one to three nucleotides running into the
unknown chromosomal restriction fragment. Alternative AFLP typing procedures are
based on one enzyme with a single adapter and analysis by agarose gel electrophoresis. A
major improvement has been obtained by switching from radioactive to fluorescently
labeled primers for detection of fragments in an automatic sequence. In addition, it has
been shown that for small bacterial and fungal genomes a single PCR amplification with
one and two selective nucleotides, respectively, on both primers are sufficient.
7. Random Amplified Polymorphic DNA (RAPD)
The technically demanding method of RAPD has been applied to the study of crayfish
plague fungus, Astacus astaci . RAPD uses a single primer in low-stringency polymerase
chain reactions. Random binding of primers results in different sizes of fragments from
samples with nonidentical DNA. Application of the RAPD technique grouped different
isolates of the fungus and provides the means to carry out epidemiological investigations
(Lilley et al., 1997; Oidtmann et al., 1999). The method has also been used to examine
another Aphanomyces species that has resulted in serious losses in both farmed and wild
fish in Asia (Lilley et al., 1997). Other fish pathogens have been studied using RAPD, but
problems with reproducibility and risks of contamination render the method unsuitable as
a stand-alone method of diagnosis. However, RAPD can be a useful technique as a first
step in the development of specific primers or probes and has been used in such a way in
the study of bacteria.
8. In Situ Hybridization

In situ PCR has become a powerful molecular tool in research as well as clinical practice.
This technique has resulted in an increased understanding of infectious and neoplastic
diseases and improvements in diagnosis of disease. In situ RT-PCR gives more detailed
information by allowing for highly sensitive detection of low abundance gene expression
in a given cell while providing anatomical information. The usefulness of these
techniques has been hampered by low detection sensitivity, poor reproducibility, and high
backgrounds. Moreover, many of the methods used to visualize the results of PCR
amplification within cells and tissues employ radioactive tracers, making performance of
the techniques cumbersome and costly. Researchers have developed a method for specific
fluorescent detection of gene expression using in situ RT-PCR. This method enables the
researcher or clinician to detect low levels of gene expression within tissues with very
low background interference while addressing many of the other existing drawbacks to
using in situ RT-PCR. Potential Areas of Application are detection and diagnosis of
viruses and other infectious agents in specific cell types within tissues, detection, and
characterization of tumor cells within a tissue, detection, and diagnosis of genetic
mutations in inherited diseases, and detection of genes and gene expression in tissue.
Fluorescence in situ hybridization, or FISH, is a method used to label cells or
chromosomes according to the sequences of nucleic acids contained within them. In
microbiology, the nucleic acid that is labelled as RNA or DNA of the ribosomes and the
target is usually whole cells. The process works by taking fluorescently labelled pieces of
DNA or RNA called probes that are around 20 nucleotides in length. The probes are
incubated in the presence of cells munder appropriate conditions to permit specific
hybridization of probe to target nucleic acid. Cells types that contain ribosomes with
complementary RNA sequences become labeled by the binding of the fluorescent probe
in situ. These labelled cells can then be visualized by flow cytometric or fluorescence
microscopy.
9. DNA microarrays
There are a number of ways of using DNA microarrays for the detection of unique DNA
(or RNA) sequences. One method is to fluorescently label all the DNA sequences in the
test sample. The sample DNA that hybridizes to a specific location on the microarray can
be detected by fluorescent array detection and the data analyzed by computer programs.
Often more practical is to use competitive hybridization in which the test sample
competes for hybridization to the tethered oligonucleotide, on the chip, with a fluorescent
labeled competitor oligonucleotide. When the test DNA is perfectly complimentary to the
tethered oligonucleotide, it will hybridize to the chip. When the test DNA is not perfectly
complementary to the tethered oligonucleotide, the fluorescent labeled competitor
oligonucleotide will bind to the tethered oligonucleotide on the chip and displace the test
DNA. A fluorescent microarray detector and computer program can then analyze the
fluorescent array for the presence or absence of the species/strain specific DNA sequence.

Compared with traditional nucleic acid hybridization with membranes, microarrays offer
the additional advantages of high density, high sensitivity, rapid detection, lower cost,
automation, and low background levels. Microarrays may provide a better option for
largescale diagnostic testing and can survey a sample for a multitude of sequences
simultaneously. Since most of the pathogens genetic sequences are available in the
GenBank, oligonucleotide probes complementary to all pathogens can be made and
inserted into microarray so that variety of microbes could be detected by a single
microarray chip. As a result, microarray-based technology is potentially well suited for
identifying fish pathogens in fish populations. The microarray techniques does not
require such sequence conservation, however, because all of the diverse gene sequences
from different populations of the same functional group can be fabricated on arrays and
used as probes to monitor contagious fish disease especially during the asymptomatic
period of the diseases. Microarrays are already proving valuable for assaying gene
expression. A large number of probes on an array can reveal which genes are expressed or
are present in the sample. This type of array would be particularly useful in studies of
pathogens, where the presence of certain genes or gene products indicate whether the
organism is pathogenic or not. Set up cost for the use of DNA microarrays is high.
However, once the equipment is available and microarrays have been prepared, cost per
unit of sample analyzed will be low. Furthermore, analysis time is extremely short. DNA
microarray technology will be used in the future for fish diseases diagnosis especially
during the asymptomatic period of diseases.
10. Loop Mediated Isothermal Amplification (LAMP)
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification
method that amplifies DNA with high specificity, efficiency and rapidity under
isothermal conditions (Natomi et al, 2000). When combined with reverse transcription,
this method can also amplify RNA sequences with high efficiency. The method relies on
auto-cycling strand displacement DNA synthesis using a DNA polymerase with a high
strand displacement activity and a set of four specially designed primers. These four
primers, termed as inner and outer primers, recognize six distinct sequences of the target
DNA, which improves the specificity of the reaction. The reaction is carried out at
isothermal condition, as the denaturation of strands takes place by strand displacement.
I.

LAMP Reaction: In the initial stages of LAMP reaction all four primers are involved,
however, in the later cycling reaction only the inner primers are used for strand
displacement DNA synthesis. The LAMP reaction is initiated by an inner primer
containing sequences of sense and anti-sense strands of the target DNA. This is followed
by the release of a single-stranded DNA through the priming by an outer primer. This
single-stranded DNA will serve as a template for DNA synthesis primed by the second
inner and outer primers that can hybridize at the other end of the target. This process will

result in the formation of a stem-loop DNA structure. In the subsequent step of LAMP
cycling, one inner primer will hybridize to the loop on the product and initiate strand
displacement DNA synthesis which will result in the original stem-loop DNA and a new
stem-loop. Cycling continues for a period of approximately 1 h and results in the
accumulation of 109 copies the target. The final products of the reaction are stem-loop
DNA with several inverted repeats of the target and cauliflower-like structures with
multiple loops.
II.

Visualization of Amplified Products: Several methods can be employed to visualize the

end products of LAMP reaction. The most common method of visualization is by agarose
gel electrophoresis. The agarose gel is stained with intercalating dyes such as ethidium
bromide or SYBR Green I. Since the end products of LAMP consist of stem-loop DNA
and cauliflower-like structures with multiple loops of various lengths, the agarose gel
electrophoresis will reveal the products from the minimum length of the target DNA to
the loading well, which appears as smear at the top and bands at the base of the gel.
Since, one of the characteristics of the LAMP reaction is its ability to synthesize
extremely large amount of DNA, addition of intercalating dye, SYBR Green I, into the
reaction tube itself would help in visualizing the product under a UV- transilluminator.
This method is useful in the field-level application, where gel electrohoresis will be a
limiting factor. Another method is also based on the accumulation large amount of
byproduct of the reaction. In the LAMP reaction large amount of byproduct,
pyrophosphate ion is produced, which will yield white precipitate of magnesium
pyrophosphate in the reaction mixture. Hence, detection of presence or absence of white
precipitate will provide an easy distinction of whether the target DNA is amplified during
the reaction. Further, since increase in the turbidity of the reaction mixture according to
the production of the precipitate correlates to the amount of target DNA synthesized, a
colorimetric estimation of the turbidity in real-time is also being used as an efficient
method of visualizing the amplified product.

III.

Advantages of LAMP: LAMP amplifies the target DNA under isothermal amplification
with high efficiency; The detection limit of LAMP is a few copies and comparable to
PCR; No significant influence of the co-presence of non-target DNA; LAMP allows
simple, easy and selective detection; Lamp is highly specific for the target sequence, as it
employs four primers targeting multiple sequences; LAMP is simple and easy to perform,
as it requires (after appropriate primers are prepared) only a regular laboratory water bath
or heat block for the reaction; BY incorporating reverse transcription, LAMP can be used
for amplifying RNA as well.

IV.

Disadvantages: Because amplification of the target DNA is so high at the final stage it is
vulnerable to contamination in subsequent amplifications; Multiplexing is not possible
with LAMP.

Advantages of Molecular Methods

Apart from the sensitivity and rapidity of diagnosis, principal advantage of molecular diagnostic
methods is in the detection of non-culturable agents; DNA amplification can assist in detecting
the pathogens that are present in low numbers and also in handling a tiny volume of specimen;
Can be sued to detect latent infection and thereby identify the reservoir hosts of infection that is
significant in epizootiology; Can be used to differentiate antigenically similar pathogens.

Disadvantages of Molecular methods

These methods are cost-intensive procedures; These tests cannot detect unsuspected samples;
Molecular methods will have difficulty in detecting new pathogens as the exclusive use of these
would overlook such infections.

Conclusion
Diagnostic methods for aquatic animal pathogens have advanced from microscopic
characterization and morphological descriptions to molecular characterization and probe-based
diagnosis. Molecular tools are increasingly relevant to fish diseases. The sequencing of the
complete genomes of pathogens is allowing great advances in studying the biology, and
improving diagnosis and control of pathogens. Using nucleic acid as targets, and new methods of
analyzing polymorphism in this nucleic acid, can improve specificity, sensitivity, and speed of
diagnosis and offer means of examining the relationships between genotype and phenotype of
various pathogens. Progress in techniques aids epidemiological studies as well as identifying
causes of disease outbreaks or the presence of pathogens. Therefore, molecular biology can be a
routine tool in the search for improved methods of diagnosis and control of fish pathogens and
the epidemiology of infectious fish diseases.
References

K Riji John & Rosalind George, 2004, Fin Fish & Shell Fish Diseases (Practical
Manual). Fisheries College & Research Institute, Thoothukudi.

S Felix, Riji John, Prince Jeyaseelan & V Sundararaj, 2001, National Traning Course
On Fish Disease Diagnosis & Health Management, Fisheries College & Research
Institute, Thoothukudi.

K M Shankar & C V Mohan, 2002, Fish And Shellfish Health Management, College Of
Fisheries, Mangalore.

Ilhan Altinok & Ilknur Kurt, 2004, Molecular Diagnosis Of Fish Diseases: A Review,
Turkish Journal Of Fisheries And Aquatic Sciences 3: 131-138

National Standard Methods, Center For Infection, Wales.

Erlich, H.A. PCR Technology Principles And Applications For DNA Amplification.
Stockton Press, New York, 1989.

PCR Technology: Current Innovations, 2nd Edition. Edited By Thomas Weissensteiner Et

Al.

Edawards K., Logan J., Saunders N. (Eds.). 2004. Real-Time PCR: An Essential Guide.
Horizon Bioscience, Norfolk, UK, P. 346.

Durand S.V., Lightner D.V. 2002. Quantitative Real Time PCR For The Measurement Of
White Spot Syndrome Virus In Shrimp. J. Fish Dis. 25, 381-389.

La Fauce K.A., Layton R., Owens L. 2007. Taqman Real-Time PCR For Detection Of
Hepatopancreatic Parvovirus From Australia. J. Virol. Methods, 140, (1-2), 10-16.

Mouillesseaux K.P., Klimpel K.R., Dhar A.K. 2003. Improvement In The Specificity And
Sensitivity Of Detection For The Taura Syndrome Virus And Yellow Head Virus Of
Penaeid Shrimp By Increasing The Amplicon Size In SYBR Green Real-Time RT-PCR. J.
Virol. Methods, 111 (2), 121-127.

Rajendran K.V., Jeff A. Cowley, Russell J. Mcculloch, Peter J. Walker. 2006. A Taqman
Real-Time RT-PCR For Quantifying Mourilyan Virus Infection Levels In Penaeid Shrimp
Tissues. J. Virol. Methods 137, 265-271.

Tang K.F., Wang J., Lightner D.V. 2004. Quantification Of Taura Syndrome Virus By
Real-Time RT-PCR With A Taqman Assay. J. Viro. Methods 11, 109-114.