practical notes by maruthappan sir

MICROBIOLOGICAL MEDIA PREPARATION
Bacteria and fungi are grown on or in microbiological media of various types. The
medium that is used to culture the microorganism depends on the microorganism that one is
trying to isolate or identify. Different nutrients may be added to the medium, making it higher
in protein or in sugar. Various pH indicators are often added for differentiation of microbes
based on their biochemical reactions: the indicators may turn one color when slightly acidic,
another color when slightly basic. Other added ingredients may be growth factors, NaCl, and
pH buffers which keep the medium from straying too far from neutral as the microbes
metabolize.
Materials Needed
 Sleeve of plastic petri plates
 Melted TSA (in 50○C water bath)
 Agar powder
 Erlenmeyer flask
 Graduated cylinder
 Glass pipette
Procedure
Pour your plates; using a liquified, sterile agar medium which has previously been
made (should be sitting in a water bath). The flasks of melted TSA are in the water bath
which is set to 45. This is to prevent them from solidifying, since agar solidifies at around 42○
C. Remove the flask from the water bath only when ready to pour your plates.
Set the petri dishes out, RIGHT SIDE UP (small side down), tops covering the dishes.
When ready to pour your plates, obtain your flask of agar and pour the plates. Depending on
whether you pour `heavy` or pour `light', you may use anywhere between 18-22 petri dishes.
Cover the bottom of each plate approximately 2/3 of the way across, put on the lid and gently
rotate the plate to distribute the agar across the entire dish bottom. Allow all plates to stand
until they are completely solidified. Once they have solidified, place them on the tray at the
instructor's table. Agar plates are always placed UPSIDE DOWN (bottom dish with agar on
top), definitely so when you are incubating your cultures.
(Why are agar plates incubated upside down? Two reasons:
• Although there may be air contaminants in the incubator, it will be more difficult for them
to get onto plates, and then move UP onto the agar to settle since gravity is working against
this movement.
• Often you will see a bit of water condensation on the petri dish cover. The water molecules
are cohesive and tend to run together. If the plate is sitting right side up, the water droplets
can fall onto the agar, creating a kind of little lake on the agar surface, messing up the plates.
Upside down plates prevent the condensation from dropping on the agar surface).
*****************
IDENTIFICATION OF BACTERIA USING GRAM’S STAINING
Aim: to identify the bacteria in the given bacterial samples using Gram’s staining.
Materials Required:
Clean glass slides, Inoculating loop, Bunsen burner, Bibulous paper, Microscope,
Lens paper and lens cleaner, Immersion oil, Distilled water and 18 to 24 hour cultures of
organisms
Reagents:
Primary Stain
- Crystal Violet
Mordant
- Grams Iodine
Decolourizer
- Ethyl Alcohol
Secondary Stain -
Safranin
Principle:
Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the
crystal violet-iodine complex that occurs during staining, while Gram-negative cells have
only a thin layer of peptidoglycan. Thus Gram-positive cells do not decolorize with ethanol,
and Gram-negative cells do decolorize.
counter stain safranin.
This allows the Gram-negative cells to accept the
Gram-positive cells will appear blue to purple, while Gram-negative
cells will appear pink to red.
Procedure:
Preparation of the glass microscopic slide
Grease or oil free slides are essential for the preparation of microbial smears. Grease
or oil from the fingers on the slides is removed by washing the slides with soap and water.
Wipe the slides with spirit or alcohol. After cleaning, dry the slides and place them on
laboratory towels until ready for use.
Labelling of the slides
Drawing a circle on the underside of the slide using a glassware-marking pen may be
helpful to clearly designate the area in which you will prepare the smear. You may also label
the slide with the initials of the name of the organism on the edge of the slide. Care should be
taken that the label should not be in contact with the staining reagents.
Preparation of the smear
Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth
culture on the slide. Spread by means of circular motion of the inoculating loop to about one
centimetre in diameter. Excessive spreading may result in disruption of cellular arrangement.
A satisfactory smear will allow examination of the typical cellular arrangement and isolated
cells.
Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or saline
solution on the slide. Sterilize and cool the loop again and pick up a very small sample of a
bacterial colony and gently stir into the drop of water/saline on the slide to create an
emulsion.
Swab Samples: Roll the swab over the cleaned surface of a glass slide.
Please note: It is very important to prevent preparing thick, dense smears which contain an
excess of the bacterial sample. A very thick smear diminishes the amount of light that can
pass through, thus making it difficult to visualize the morphology of single cells. Smears
typically require only a small amount of bacterial culture. An effective smear appears as a
thin whitish layer or film after heat-fixing.
Heat Fixing
Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and
allows the sample to more readily take up stains.
Allow the smear to air dry.
After the smear has air-dried, hold the slide at one end and pass the entire slide
through the flame of a Bunsen burner two to three times with the smear-side up.
Now the smear is ready to be stained.
Please Note: Take care to prevent overheating the slide because proteins in the specimen can
coagulate causing cellular morphology to appear distorted.
Gram Stain Procedure
 Place slide with heat fixed smear on staining tray.
 Gently flood smear with crystal violet and let stand for 1 minute.
 Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.
 Gently flood the smear with Gram’s iodine and let stand for 1 minute.
 Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle. The smear will appear as a purple circle on the slide.
 Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the
alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be
careful not to over-decolorize.
 Immediately rinse with water.
 Gently flood with safranin to counter-stain and let stand for 45 seconds.
 Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.
 Blot dries the slide with bibulous paper.
 View the smear using a light-microscope under oil-immersion.
Observation:
Observe the following gram type bacteria: 1. Gram – negative cocci.
2. Gram – negative rods.
3. Gram – positive cocci.
4. Gram – positive rods.
Inference:
Typical Gram-negative bacteria:
Bordetella pertusis, the causative agent of whooping cough. Salmonella typhi, the
causative agent of typhoid. Vibrio cholera, the causative agent of cholera. Escherichia coli,
the normally benign, ubiquitous, gut-dwelling bacteria
Typical Gram-positive bacteria:
Staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which
is a common cause of boils. Streptococci such as the many species of oral streptococci,
Streptococcus pyogenes which causes many a sore throat and scarlet fever and Streptococcus
pneumoniae which causes lobar pneumonia. Clostridia such as Clostridium tetani, the
causative agent of tetanus (lockjaw). Actinomyces such as Actinomyces odontolyticus which
is found in mouth. Species of the genus Bacillus such as Bacillus subtilis which are common
microbes living in soil. Generally cocci are Gram-positive but there are exceptions. The most
significant from a clinical point of view is the gonococcus, Neisseria gonorrhoea which
typically appears as a Gram-negative diplococcus looking very much like a pair of kidney
bean.
**************************
IDENTIFICATION OF BACTERIA USING SIMPLE STAINING
Aim: to identify the bacteria in the given bacterial samples using simple staining.
Materials Required:
Clean glass slides, Inoculating loop, Bunsen burner, Bibulous paper, Microscope,
Lens paper and lens cleaner, Immersion oil, Distilled water and 18 to 24 hour cultures of
organisms
Reagents:
Methylene blue, Crystal violet and Carbol fuchsin.
Principle :
In order to observe most bacterial cells using bright field microscopy the cells must be
dark enough to see, that is they must have contrast to the light. To create contrast a simple
stain can be used. Simple stains use basic dyes which are positively charged. These positive
dyes interact with the slightly negatively charged bacterial cell wall thus lending the color of
the dye to the cell wall.
Procedure:
 Clean and dry microscope slides thoroughly.
 Flame the inoculating loop.
 Touch the inoculating loop to the inside of the tube to make sure it is not so hot that it will
distort the bacterial cells; then pick up a pinhead size sample of the bacterial growth
without digging into the agar.
 Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over
an area the size of a dime. It should be a thin, even smear.
 Allow the smear to air dry thoroughly.
 Stain the smear by flooding it with one of the staining solutions and allowing it to remain
covered with the stain for the time designated below.
o Methylene blue - 1 minute
o Crystal violet - 30 seconds
o Carbol fuchsin - 20 seconds
 During the staining the slide may be placed on the rack or held in the fingers.
 At the end of the designated time rinse off the excess stain with gently running tap water.
Rinse thoroughly.
 Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper
towel.
 Place the stained smear on the microscope stage smear side up and focus the smear using
the 10X objective.
 Choose an area of the smear in which the cells are well spread in a monolayer. Centre the
area to be studied, apply oil directly to the smear, and focus the smear under oil with the
100X objective.
 Draw the cells observed.
Observation:
Cocci were observed and have been stained with crystal violet.
****************************
IDENTIFICATION OF BACTERIA USING NEGATIVE STAINING
Aim: to identify the bacteria in the given bacterial samples using negative staining.
Materials Required:
Clean glass slides, Inoculating loop, Bunsen burner, Bibulous paper, Microscope,
Lens paper and lens cleaner, Immersion oil, Distilled water and 18 to 24 hour cultures of
organisms
Reagents:
Nigrosin
Principle:
The negative stain uses the dye nigrosin, which is an acidic dye.
By giving up a
proton (as an acid) the chromophore of the dye becomes negatively charged. Because the cell
wall is also negatively charged only the background around the cells will become stained,
leaving the cells unstained
Procedure:
 Place a very small drop (more than a loop full--less than a free falling drop
from the dropper) of nigrosin near one end of a well-cleaned and flamed
slide.
 Remove a small amount of the culture from the slant with an inoculating
loop and disperse it in the drop of stain without spreading the drop.
 Use another clean slide to spread the drop of stain containing the organism
using the following technique.
 Rest one end of the clean slide on the center of the slide with the stain. Tilt
the clean slide toward the drop forming an acute angle and draw that slide
toward the drop until it touches the drop and causes it to spread along the
edge of the spreader slide. Maintaining a small acute angle between the
slides, push the spreader slide toward the clean end of the slide being stained
dragging the drop behind the spreader slide and producing a broad, even,
thin smear.
5. Allow the smear to dry without heating.
 Focus a thin area under oil immersion and observe the unstained cells
surrounded by the gray stain.
Observation:
Negatively Stained Bacillus and Coccus: (A) Vegetative Cell (B) Endospore was
observed.
******************
ISOLATION OF BACTERIA
Aim:
To explain the steps involved in the isolation of bacteria. Isolation of bacteria forms a
very significant step in the diagnosis and management of the illness. Isolation of bacteria
involves various steps: Specimen collection and preservation and transportation of specimen
Specimen collection
Many different specimens are sent for microbiological examination from patients with
suspected bacterial infection. Common specimens include urine, faeces, wound swabs, throat
swabs, vaginal swabs, sputum, and blood. Less common, but important specimens include
cerebrospinal fluid, pleural fluid, joint aspirates, tissue, bone and prosthetic material (e.g. line
tips). Some types of specimen are normally sterile e.g. blood, CSF. These samples are usually
obtained via a percutaneous route with needle and syringe, using appropriate skin disinfection
and an aseptic technique. The culture of bacteria from such specimens is usually indicative of
definite infection except if they are skin contaminants (bacteria inhabitants of normal skin).
It is preferred to obtain the samples for bacteriological culture before antibiotic
therapy is started. This maximizes the sensitivity of the investigations and reduces false-
negative results. Similarly, samples of tissue or pus are preferred over swabs, to maximize the
recovery of bacteria in the laboratory. Specimens must be accurately labelled and
accompanied by a properly completed requisition form, indicating the nature of the specimen,
the date of sample collection, relevant clinical information, the investigations required, and
details of antibiotic therapy, if any.
This allows the laboratory to perform the correct range of tests, and helps in the
interpretation of results and reporting. Along with clinical specimens, medical microbiology
laboratories also process samples of food, water and other environmental samples (e.g. air
sampling from operating theatres) as part of infection control procedures.
High-risk samples
Certain bacterial infections are a particular hazard to laboratory staff and specimens
that might contain these pathogens should be labelled as ‘high risk’ to allow for additional
safety measures if necessary. For example - blood cultures from suspected typhoid
(Salmonella
typhi)
or
brucellosis
Mycobacterium tuberculosis.
(Brucella
species) and
samples from suspected
Preservation and Transport of specimen
Most specimens are sent to the laboratory in sterile universal containers. Swabs are
placed in a suitable transport medium (eg. charcoal medium) otherwise it leads to false
negative reporting.
CULTURE METHOD OF ISOLATION OF BACTERIA
The specimens received in the laboratory are plated on the culture media. The
appropriate culture media is selected depending upon the bacteria suspected. The following
precautions need to be taken into consideration when the culture methods are processed.
Optimal atmospheric conditions
Optimal temperature
Growth requirement of the bacteria
Atmospheric conditions:
Colonies of bacteria are usually large enough to identify after 18–24 hours of
incubation (usually at 37°C), but for some bacteria longer incubation times are required (from
2 days to several weeks). Culture plates are incubated (1) in air, (2) in air with added carbon
dioxide (5%), (3) anaerobically (without oxygen) or (4) micro-aerophilically (a trace of
oxygen) according to the requirements of the different types of bacteria that may be present
in specimens. In case of Mycobacteria especially the scotochromogen the culture bottles are
placed in dark or the bottles are covered with black paper and kept for incubation at 37°C.
Temperature:
Most of the bacteria require a temperature of 37°C for optimal growth. This
temperature is provided placing the inoculated culture plates in the incubator set at 37°C
temperature.
Growth requirement of the bacteria
Different
bacteria
have
different growth requirements.
For
eg Streptococcus
pneumoniae requires factor V and factor X for its growth, which are found in chocolate agar.
Thus for sample suspected of S. pneumoniae the samples are plated on chocolate agar.
Similarly depending upon the growth requirements the appropriate culture media are used.
CULTURE ON SOLID MEDIA
The principal method for the detection of bacteria from clinical specimens is by
culture on solid culture media. Bacteria grow on the surface of culture media to produce
distinct colonies. Different bacteria produce different but characteristic colonies, allowing for
early presumptive identification and easy identification of mixed cultures. There are many
different types of culture media. Agar is used as the gelling agent to which is added a variety
of nutrients (e.g. blood, peptone and sugars) and other factors (e.g. buffers, salts and
indicators).
Some culture media are nonselective (e.g. blood agar, nutrient agar) and these will
grow a wide variety of bacteria. While some e.g. MacConkey agar are more selective (in this
case through the addition of bile salts selecting for the ‘bile-tolerant’ bacteria found in the
large intestine such as Escherichia coli and Enterococcus faecalis). MacConkey agar also
contains lactose and an indicator system that identifies lactose-fermenting coliforms (e.g.
Escherichia coli, Klebsiella) from lactose-non fermenting coliforms (e.g. Morganella,
Salmonella). Media can be made even more selective by the addition of antibiotics or other
inhibitory substances, and sophisticated indicator systems can allow for the easy detection of
defined bacteria from mixed populations.
Method of inoculating the solid culture media
Method used for inoculating the solid media depends upon the purpose of inoculation-
whether to have isolated colonies or to know the bacterial load of the sample (quantitative
analysis). For obtaining the isolated colonies streaking method is used, the most common
method of inoculating an agar plate is streaking.
Streak plates
1. A small amount of sample is placed on the side of the agar plate (either with a swab, or as
a drop from an inoculating loop).
2. A sterile loop is then used to spread the bacteria out in one direction from the initial site of
inoculation. This is done by moving the loop from side to side, passing through the initial
site.
3. The loop is then sterilised (by flaming) again and the first streaks are then spread out
themselves.
4. This is repeated 2-3 times, moving around the agar plate as shown in the figure.
In this method single bacterial cells get isolated by the streaking, and when the plate is
incubated, forming discrete colonies that will have started from just one bacterium each. For
quantitative analysis or semi quantitative analysis of the sample for example in case of
urinary tract infection. In fact E .coli is implicated as the causative organism in urinary tract
infection only if there are >105Colony forming units per milliliter of urine. The method of
inoculating the solid culture media is as shown in the figure.
Inoculation methods
Inference:
The identification is required so as to cure the illness or the infection caused due to
these bacteria,
using appropriate antibiotics. Identification also holds significance for
epidemiological purposes. We would learn about identification of bacteria and the ways to
contain the infections caused by them.
*************************
ESTIMATION OF BACTERIA IN THE GIVEN SUSPENSION USING
HEAMOCYTOMETER
Aim: To estimate the number of bacteria in the given suspension using haemocytometer.
Materials:
The necessary elements to perform a cell count with Neubauer chamber are as
follows:
a. cellular dilution to measure
b. hemocytometer or Neubabuer chamber
c. optical microscope
d. cover glass
e. pipette / micropipette with disposable tips.
f.
Dilution buffer / PBS (if needed)
Procedure:
Sample preparation
Depending on the type of sample, a preparation of a dilution with a suitable
concentration should be prepared for cell counting. Typically, the concentration range for a
cell count with Neubauer chamber is between 250.000 cells / ml and 2.5 million cells / ml.
It is recommended for the dilution concentration to be around 106 cells / ml (1 million cells /
ml) applying the required dilutions. With concentrations below 250.000 cells per ml,
(2,5 * 105 cells / ml) the amount of cells counted will not be enough to obtain a fair
estimation of the original concentration.
Introducing the sample into the Neubauer chamber
Take 10 μl of dilution prepare in sample preparation with the micropipette.
1) Put the glass cover on the Neubauer chamber central area. Use a flat surface to place the
chamber, like a table or a workbench.
2) Put a disposable tip at the end of the micropipette.
3) Adjust the micropipette to suck 10 μl. You can adjust it by turning the upper plunger
roulette to select the required pipetting volume.
4) Introduce the micropipette tip on the dilution previously prepared (sample preparartion)
5) Push the pipette plunger slowly until you feel it has arrived to the end of its travel.
6) Remove the pipette tip from the dilution, and bring it to the Neubauer chamber. When the
pipette is loaded, it must always be held in vertical position.
7) Place pipette tip close to the glass cover edge, right at the centre of the Neubauer chamber.
8) Release the plunger slowly watching how the liquid enters the chamber uniformly, being
absorbed by capillarity. See Fig.
9) In case of the appearance of bubbles, or that the glass cover has moved, repeat the
operation.
Microscope set up and focus
1. Place the Neubauer chamber on the microscope stage. If the microscope has a fixing
clamp, fix the Neubauer chamber.
2. Turn on the microscope light.
3. Focus the microscope until you can see a sharp image of the cells looking through the
eyepiece and adjusting the stage.
4. Look for the first counting grid square where the cell count will start. In this example, 5
big squares from a Neubauer-Improved chamber will be counted. See Fig.
5. Start counting the cells in the first square.
6. Write down the amount of cells counted in the first square.
7. Repeat the process for the remaining squares, writing down the counting results from all of
them. The higher the number of cells counted, the higher the accuracy of the measurement.
Concentration calculation
We apply the formula for the calculation of the concentration
Number of cells
Concentration (cel / ml) = -------------------
Volume (in ml)
The number of cells will be the sum of all the counted cells in all squares counted.
The volume will be the total volume of all the squares counted.
Since the volume of 1 big square is:
0.1 cm x 0.1 cm = 0.01 cm2 of area counted. Since the depth of the chamber is 0.1mm
x 0.1 mm = 0.01 cm
0.01 cm2 *0.01 cm = 0.0001 cm2 = 0.0001ml = 0.1 μl
So, for the Neubauer chamber, the formula used when counting in the big squares.
Number of cells x 10.000
Concentration = --------------------------------
Number of squares
In case a dilution was applied, the concentration obtained should be converted to the
original concentration before the dilution. In this case, the concentration should be divided by
the dilution applied.
The
7.3 ANTIGEN ANTIBODY REACTIONS
formula will be:
Number of Cells x 10.000
Concentration = ----------------------------------
Number of square x dilution
Example:
For a 1 : 10 dilution.
Dilution = 0.1
For a 1 : 100. Dilution = 0.01
Result:
The number of bacterial cells in the given sample =
-------------------------cells/ml.
******************
ANTIGEN-ANTIBODY REACTIONS USING PRECIPITATION METHOD
Aim:
To perform the antigen-antibody reaction in the human blood using precipitation
method.
Material required
1. Glass slides/white tile
2. Monoclonal Antisera A and Antisera B
3. Glass rod for mixing
4. Lancet
Principle
This can be performed in emergency or outdoor camps but must not be performed as a
routine test. Agglutination is the clumping of red cells. It occurs when sensitized cells come
into contact with each other resulting in formation of bridges between them and formation of
aggregates. It is the most common procedure in blood banking. The ABO grouping system is
subdivided into 4 types based on the presence or absence of antigens A and B on the red cell
surface as shown below. Red cells that only have antigen A are called group A. Those that
only have B antigen are called group B. Cells that have both A and B antigens are group AB.
Cells that lack both antigens are O. The ABO antibodies; anti-A and anti-B are naturally
occurring antibodies and are present in the sera of individuals who lack the corresponding
antigen. Cells with A antigen will have anti-B in the serum. Cells with B antigen will have
anti-A in the serum and cells with AB antigens will not have any antibody. Group O
individuals will have both anti-A and anti-B antibodies. These antibodies are IgM in nature.
Procedure
1. Mark one side of the glass slide as A and the other side as B.
2. Put one drop of antisera A on the side marked as A and one drop of antisera B on the side
marked as B.
3. Add one drop of test blood sample to each antisera.
4. Mix the blood with the reagent using a clean stick. Spread the mixture over
15mm diameter.
5. Gently rock the slide to and fro and look for agglutination.
6. Record the result.
Observation
Agglutination if present indicates a positive result.
an area of
Inference
Antigen is a substance which when introduced into an individual leads to the
production of antibody. On the red cell surface there is presence of glycoproteins and
glycolipids
which
act
as
antigen,
they
are
called
group
antigen.
Antibodies are
Immunoglobulins present in the serum and are IgG, IgM, IgD, IgA and IgE. Certain
antibodies occur without antigenic stimulus and are called naturally occurring antibodies like
ABO antibodies. ABO grouping system is subdivided into 4 types based on the presence or
absence of Antigen A and B on the red cell surface.
*********************
ONLINE BIOINFORMATICS TOOLS AND THEIR APPLICATION
Bioinformatics tools are software programs that are designed for extracting the
meaningful information from the mass of molecular biology/biological databases and to carry
out sequence or structural analysis. Factors that must be taken into consideration when
designing bioinformatics tools, software and programmes are: The end user (the biologist)
may not be a frequent user of computer technology and these software tools must be made
available over the internet given the global distribution of the scientific research community.
Major categories of Bioinformatics Tools:
There are both standard and customized products to meet the requirements of
particular projects. There are data-mining software that retrieves data from genomic sequence
databases and also visualization tools to analyze and retrieve information from proteomic
databases. These can be classified as homology and similarity tools, protein functional
analysis tools, sequence analysis tools and miscellaneous tools. Here is a brief description of
a few of these, everyday bioinformatics is done with sequence search programs like BLAST,
sequence analysis programs, like the EMBOSS and Staden packages, structure prediction
programs like THREADER or PHD or molecular imaging/modelling programs like RasMol
and WHATIF.
BLAST:
BLAST (Basic Local Alignment Search Tool) comes under the category of homology
and similarity tools. It is a set of search programs designed for the Windows platform and is
used to perform fast similarity searches regardless of whether the query is for protein or
DNA. Comparison of nucleotide sequences in a database can be performed. Also a protein
database can be searched to find a match against the queried protein sequence. NCBI has also
introduced the new queuing system to BLAST (Q BLAST) that allows users to retrieve
results at their convenience and format their results multiple times with different formatting
options. Depending on the type of sequences to compare, there are different programs:
 blastp compares an amino acid query sequence against a protein sequence database.
 blastn compares a nucleotide query sequence against a nucleotide sequence database.
 blastx compares a nucleotide query sequence translated in all reading frames against a
protein sequence database.
 tblastn compares a protein query sequence against a nucleotide sequence database
dynamically translated in all reading frames.
 tblastx compares the six-frame translations of a nucleotide query sequence against the
six-frame translations of a nucleotide sequence database.
FASTA:
The program is one of the many heuristic algorithms proposed to speed up sequence
comparison. The basic idea is to add a fast pre-screen step to locate the highly matching
segments between two sequences, and then extend these matching segments to local
alignments using more rigorous algorithms such as Smith-Waterman.
EMBOSS:
EMBOSS (European Molecular Biology Open Software Suite) is a software-analysis
package. It can work with data in a range of formats and also retrieve sequence data
transparently from the Web. Extensive libraries are also provided with this package, allowing
other scientists to release their software as open source. It provides a set of sequence-analysis
programs, and also supports all UNIX platforms.
Clustalw:
It is a fully automated sequence alignment tool for DNA and protein sequences. It
returns the best match over a total length of input sequences, be it a protein or a nucleic acid.
RasMol:
It is a powerful research tool to display the structure of DNA, proteins, and smaller
molecules. Protein Explorer, a derivative of RasMol, is an easier to use program.
PROSPECT:
PROSPECT (PROtein Structure Prediction and Evaluation Computer ToolKit) is a
protein-structure prediction system that employs a computational technique called protein
threading to construct a protein's 3-D model.
PatternHunter:
PatternHunter, based on Java, can identify all approximate repeats in a complete
genome in a short time using little memory on a desktop computer. Its features are its
advanced patented algorithm and data structures, and the java language used to create it. The
Java language version of PatternHunter is just 40 KB, only 1% the size of Blast, while
offering a large portion of its functionality.
COPIA:
COPIA (COnsensus Pattern Identification and Analysis) is a protein structure analysis
tool for discovering motifs (conserved regions) in a family of protein sequences. Such motifs
can be then used to determine membership to the family for new protein sequences, predict
secondary and tertiary structure and function of proteins and study evolution history of the
sequences.
Application of Programmes in Bioinformatics:
JAVA in Bioinformatics :
Since research centers are scattered all around the globe ranging from private to
academic settings, and a range of hardware and OSs are being used, Java is emerging as a key
player
in
bioinformatics.
Physiome
Sciences'
computer-based
biological
simulation
technologies and Bioinformatics Solutions' PatternHunter are two examples of the growing
adoption of Java in bioinformatics.
Perl in Bioinformatics :
String
manipulation,
regular
expression
matching,
file
parsing,
data
format
interconversion etc are the common text-processing tasks performed in bioinformatics. Perl
excels in such tasks and is being used by many developers. Yet, there are no standard
modules designed in Perl specifically for the field of bioinformatics. However, developers
have designed several of their own individual modules for the purpose, which have become
quite popular and are coordinated by the BioPerl project.
Bioinformatics Projects:
BioJava:
The BioJava Project is dedicated to providing Java tools for processing biological data
which includes objects for manipulating sequences, dynamic programming, file parsers,
simple statistical routines, etc.
BioPerl:
The BioPerl project is an international association of developers of Perl tools for
bioinformatics and provides an online resource for modules, scripts and web links for
developers of Perl-based software.
BioXML:
A part of the BioPerl project, this is a resource to gather XML documentation, DTDs
and XML aware tools for biology in one location.
Biocorba:
Interface objects have facilitated interoperability between bioperl and other perl
packages such as Ensembl and the Annotation Workbench. However, interoperability
between bioperl and packages written in other languages requires additional support software.
CORBA is one such framework for interlanguage support, and the biocorba project is
currently implementing a CORBA interface for bioperl. With biocorba, objects written within
bioperl will be able to communicate with objects written in biopython and biojava (see the
next
subsection).
For
more
information,
see
the
biocorba
project
website
at
http://biocorba.org/ . The Bioperl BioCORBA server and client bindings are available in the
bioperl-corba-server and bioperl-corba-client bioperl CVS repositories respecitively. (see
http://cvs.bioperl.org/ for more information).
Ensembl:
Ensembl is an ambitious automated-genome-annotation project at EBI. Much of
Ensembl\'s code is based on bioperl, and Ensembl developers, in turn, have contributed
significant pieces of code to bioperl. In particular, the bioperl code for automated sequence
annotation has been largely contributed by Ensembl developers. Describing Ensembl and its
capabilities is far beyond the scope of this tutorial The interested reader is referred to the
Ensembl website at http://www.ensembl.org/.
bioperl-db:
Bioperl-db is a relatively new project intended to transfer some of Ensembl's
capability
of
integrating
bioperl
syntax
with
a
standalone
Mysql
database
(http://www.mysql.com) to the bioperl code-base. More details on bioperl-db can be found in
the bioperl-db
CVS
directory at http://cvs.bioperl.org/cgi-bin/viewcvs/viewcvs.cgi/bioperl-
db/?cvsroot=bioperl. It is worth mentioning that most of the bioperl objects mentioned above
map directly to tables in the bioperl-db schema. Therefore object data such as sequences,
their features, and annotations can be easily loaded into the databases, as in $loader->store
($newid,$seqobj) Similarly one can query the database in a variety of ways and retrieve
arrays
of
Seq
objects.
See
biodatabases.pod,
Bio::DB::SQL::SeqAdaptor,
Bio::DB::SQL::QueryConstraint, and Bio::DB::SQL::BioQuery for examples.
Biopython and biojava:
Biopython and biojava are open source projects with very similar goals to bioperl.
However their code is implemented in python and java, respectively. With the development
of interface objects and biocorba, it is possible to write java or python objects which can be
accessed by a bioperl script, or to call bioperl objects from java or python code. Since
biopython and biojava are more recent projects than bioperl, most effort to date has been to
port bioperl functionality to biopython and biojava rather than the other way around.
However, in the future, some bioinformatics tasks may prove to be more effectively
implemented in java or python in which case being able to call them from within bioperl will
become more important. For more information, go to the biojava http://biojava.org/ and
biopython http://biopython.org/ websites.
*********************
FROG EARLY CLEAVAGE
The frog egg contains a moderate amount of yolk, which affects the pattern of
cleavage segmentation. The first cleavage furrow occurs about 3 hours after fertilization and
passes from the animal pole to the vegetal pole through an area of the egg called the gray
crescent. The second furrow occurs at right angles to the first. Due to the higher
concentration of yolk, the third pair of cleavages occurs slightly toward the animal pole. The
regularity of the cleavage pattern is lost after about the fifth division. Notice the uneven size
of the blastomeres. Although the entire egg cleaves, the blastomeres of the yolk laden vegetal
pole will divide more slowly than those of the animal pole. Thus, early in cleavage the
amphibian embryo can be divided into smaller blastomeres (micromeres) at the animal pole
and larger blastomeres (macromeres) in the vegetal pole. This pattern of cleavage is termed
unequal holoblastic.
FROG LATE CLEAVAGE
The rate of division also varies between the micromeres and megameres. It has been
seen that the micromeres divide at a faster rate than the megameres. Initially the continued
division of blastomeres forms a ball like structure which is solid. It is called the morula stage,
as this has superficial resem-blance to a mulberry fruit. Very soon however the morula stage
gives rise to a stage called the blastula which is a hollow ball like structure. In the thirty two
cell stage, the blastula consists of a single layer of cells and is called the early blastula. The
pigmented cells (micromeres) are found in the anterior half while the yolky megameres are
present in the posterior half. As has been already pointed out, the blastocoel lies entirely in
the anterior half. The blastula of frog is hollow and has a very well developed blastocoel. It is
said to be a coeloblastula.
As segmentation proceeds, the number of cells in the blastula increase; so also the
blastocoel. The floor of the blastocoel is flat while its top portion is arched. The roof (top) is
made up of three to four layers of pigmented micromeres while the floor is formed by yolky
megameres. Between the micromeres and the megameres and along the equator is found a
group of cells which are intermediate in size (between megameres and micromeres). These
cells constitute the germ ring. The germ ring is formed in the region of the grey crescent.
FROG - BLASTULA
As cleavage continues, the blastomeres become arranged around the outside, with a
central fluid filled cavity, the blastocoel. Be sure you can identify the blastocoel. Note the
different sizes of cells at the animal and vegetal poles. Remember that this size difference
results from the uneven division of the blastomeres at the animal and vegetal poles. The high
magnification image is labeled.
FROG GASTRULA
The first indication of gastrulation is the appearance of a small groove on the surface
of the blastula just ventral to the grey crescent. This slit is the beginning of the blastopore and
represents the area where the cells forming the surface of the blastula move toward the inside
to form the primary germ layers. This slide is a section through an embryo which is just
beginning gastrulation. The section passes through the blastopore, which is visible as an
indentation near the vegetal pole. The dorsal lip of the blastopore can be seen as a finger like
projection pointing toward the vegetal pole. Initial cell migration occurs at this site. Slightly
later in gastrulation, cells will begin to move inward over the ventral lip, and the blastopore
will become a round opening. Movement of cells inward will result in the formation of the
gastrocoele (or archenteron). The archenteron will expand dorsally toward the animal pole
obliterating blastocoel in the process.
FROG YOLK PLUG
This embryo is near the end of gastrulation. Most cells have finished their movement
to the inside, and the last yolk cells are moving through the now circular blastopore, forming
the yolk plug. You should be able to identify the newly formedgastrocoele and the remnants
of the blastocoel. Although they will not look different, the ectodermal cells directly
overlying the archenteron roof at the yolk plug gastrula stage constitute theneural ectoderm
(visible in low-magnification view only), which will have an important role in the next stage
of development.
FROG NEURAL PLATE
At the beginning of neurulation, the cells of the neural ectoderm thicken to form the
neural plate. This thickening is induced by mesodermal cells in the roof of the archenteron;
this process is called induction. With formation of the neural plate, the gastrula is transformed
into a neurula. This slide is a cross section through an embryo just beginning neurulation. The
thickened region at the dorsal surface is the neural plate. Identify the neural plate and
archenteron. In the high magnification view, can you find the notochord, which appears as a
midline rod of mesodermal cells ventral to the neural plate or neural tube? It is labeled in one
image.
FROG NEURAL TUBE
This slide contains four cross sections, taken from various places along the embryo.
At this stage the neural folds have fused to form the neural tube. In the high magnification
images you should be able to clearly see the ectoderm, neural tube, notochord, mesoderm and
endoderm.
INSECT EGG
In most insects, life begins as an independent egg. Each insect species produces eggs that are
genetically unique and often physically distinctive as well -- spherical, ovate, conical,
sausage-shaped, barrel-shaped, or torpedo-shaped. An egg's cell membrane is known as the
vitelline membrane.
It is a phospholipid bilayer similar in structure to most other animal
membranes. It surrounds the entire contents of the egg cell, most of which consists of yolk
(food for the soon-to-develop embryo). The cell's cytoplasm is usually distributed in a thin
band just inside the vitelline membrane (where it is commonly called periplasm) and in
diffuse strands that run throughout the yolk (cytoplasmic reticulum).
The egg cell's nucleus
(haploid) lies within the yolk, usually close to one end of the egg. Near the opposite end, the
oosome (a region of higher optical density) may be visible as a dark region in the more
translucent yolk.
The egg's anterior/posterior polarity is determined by the relative positions
of the nucleus and the oosome. In most insects the egg is covered by a protective "shell" of
protein secreted before oviposition by accessory glands in the female's reproductive system.
This egg shell, called the chorion, is often sculptured with microscopic grooves or ridges that
may be visible only under the high magnification of an electron microscope. The chorion is
perforated by microscopic pores (called aeropyles) that allow respiratory exchange of oxygen
and carbon dioxide with relatively little loss of water. The micropyle, a special opening near
the anterior end of the chorion, serves as a gateway for entry of sperm during fertilization.
******************
HEN EGG
Shell - The outer eggshell is made almost entirely of calcium and has as many as 8,000 tiny
pores. It is semi-permeable, so it lets gas exchange occur, but keeps other substances from
entering the egg. The thickness of an eggshell is determined by the amount of time it spends
in the shell gland (uterus) and the rate of calcium deposition during egg shell formation.
Inner and outer shell membrane - These two membranes, the inner and outer, are just
inside the shell surrounding the albumen (white). The two membranes provide an efficient
defense against bacterial invasion and are made partly of keratin. The outer membrane sticks
to the egg shell while the inner membrane sticks to the albumen. When an egg is first laid, it
is warm. As it cools, the contents contract and the inner shell membrane separate from the
outer shell membrane to form the air cell.
Chalazae - Are twisted in opposite directions and serve to keep the yolk centered.
Outer thin albumen - The outer thin albumen is a narrow fluid layer next to the shell
membrane. It is the watery part of the egg white which is located farthest from the yolk
Inner thick albumen - The inner thick white (chalaziferous layer) is a dense, matted, fibrous
capsule of albumen around the membrane located nearest the yolk. In high-quality eggs, the
inner thick albumen stands higher and spreads less than thin white. In low-quality eggs, it
appears thin white.
Yolk membrane - The clear casing that encloses the egg yolk.
Germinal disk - A small, circular, white spot on the surface of the yolk is where the sperm
enters the egg. The embryo develops from this disk, and gradually sends blood vessels into
the yolk to use it for nutrition as the embryo develops.
Yolk - The yellow yolk is a major source of vitamins, minerals, almost half of the protein,
and all of the fat and cholesterol. The yolk contains less water and more protein than the
white, some fat, and most of the vitamins and minerals of the egg. These include iron,
vitamin A, vitamin D, phosphorus, calcium, thiamine, and riboflavin. The yolk is also a
source of lecithin, an effective emulsifier. Yolk color ranges from just a hint of yellow to a
magnificent deep orange, according to the feed and breed of the hen. The yolk contains the
food that will nourish the embryo as it grows.
Air cell - An air space forms when the contents of the egg cool and contract after the egg is
laid. The air cell usually rests between the outer and inner membranes at the egg’s larger end.
As the egg ages, moisture and carbon dioxide leave through the pores of the shell, air enters
to replace them and the air cell becomes larger.
Cuticle (also called bloom) - The shell is produced by the shell gland (uterus) of the oviduct,
and has an outer coating called the bloom or cuticle. When the cuticle or bloom is deposited
by the hen on the shell this acts as a barrier to keep bacteria from entering the egg. These
barriers provide a good line of defense against invading bacteria. Most of the protective
covering is removed from eggs when they are mechanically washed.

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