Download FMB - E

CLASS
SUBJECT
NEHRU ARTS AND SCIENCE COLLEGE
DEPARTMENT OF MICROBIOLOGY
E-LEARNING
: I M.Sc.
: FUNDAMENTALS OF MICROBIOLOGY & BIOINFORMATICS
SEMESTER I:
UNIT I
Basic concepts – Spontaneous generation- Germ theory of diseases – Cell theory – Contributions of
Antony van leuwenhoek – Joseph Lister – Robert Koch – Louis Pasteur
Edward Jenner – John Tyndall – Sergei N. Winogradsky – Salmon Awaksman – Alexander
Flemming- Paul Erlich – Fannie Hessie – Elie Metchnikoff - Kary Mullis. Development of pure
culture methods.
UNIT II
Sterilisation and disinfection – Definitions – Principles – Methods of sterilization -: Physical
methods – Heat –Filteration – Radiation and Chemical methods. Control of sterilization and Testing
of sterility. Microscopy – Principles, Light microscope, Phase Contrast, Dark field, Bright field,
Fluorescent – Interference microscope (Stereo microscope), Con focal scanning microscope –
Inverted microscope - Electron microscope – TEM, SEM. and Micrometry. Staining: Simple, Gram
staining, Negative staining, Capsule staining, Spore staining, Flagellar staining, Nuclear staining
and Acid fast staining.
UNIT III
Taxonomy – Principle and its types (Classical approach – Numerical, Chemical, Serological and
Genetic). Bacterial taxonomy – Bergey”s manual of Systematic Bacteriology (Eubacteria and
Archaebacterium)
UNIT IV
Fungal taxonomy – Alexopolus, Algal taxonomy – Classes, Ultra structure and general
characteristics. Outline classification of Protozoa – general characters and importance. Economic
importance of algae and fungi.
UNIT V
Introduction to bioinformatics – Classification of Biological data bases – Biological data formats –
Applications of Bioinformatics in various fields – Data retrieval – Entrez and SRS. Introduction to
sequence alignment. Data base search for similar sequences using FASTA and BLAST
programmes.
PART-A
What Is a Microorganism?
Microorganisms are the subject of microbiology, which is the branch of science that studies
microorganisms. A microorganism can be one cell or a cluster of cells that can be seen only by
using a microscope. Microorganisms are organized into six fields of study: bacteriology, virology,
mycology, phycology, protozoology, and parasitology.
How Small Is a Microorganism?
Microorganisms are measured using the metric system, which is shown below. In order to give you
some idea of the size of a microorganism, let’s compare a microorganism to things that are familiar
to you.
German shepherd
Human gamete (egg) from a female ovary
A human red blood cell
A typical bacterium cell
A virus
An atom
1 meter
1 millimeter
100 micrometers
10 micrometers
10 nanometers
0.1 nanometer
What is a microorganism?
(a) A microorganism is a small organism that takes in and breaks down food for energy and
nutrients, excretes unused food as waste, and is capable of reproduction.
(b) A microorganism is a small organism that causes diseases only in plants.
(c) A microorganism is a small organism that causes diseases only in animals.
(d) A microorganism is a term that refers to a cell.
What is a pathogenic microorganism?
(a) A microorganism that multiplies
(b) A microorganism that grows in a host
(c) A microorganism that is small
(d) A disease-causing microorganism
Why is a bacterium called a prokaryotic organism?
(a) A bacterium is a one-cell organism that does not have a distinct nucleus.
(b) A bacterium is a one-cell organism that has a distinct nucleus.
(c) A bacterium is a multicell organism that does not have a distinct nucleus.
(d) A bacterium is a multicell organism that has a distinct nucleus.
What is Germ Theory?
(a) Germ Theory states that a disease-causing microorganism should be present in animals infected
by the disease and not in healthy animals.
(b) Germ Theory states that a disease-causing microorganism should be present in healthy animals
and not in infected animals.
(c) Germ Theory states that a disease-causing microorganism should be destroyed.
(d) Germ Theory states that a disease-causing microorganism cannot be destroyed.
What is Edward Jenner’s contribution to microbiology?
(a) Edward Jenner discovered the Germ Theory.
(b) Edward Jenner discovered how to create vaccinations to trigger the body’s immune system to
develop antibodies that fight microorganisms.
(c) Edward Jenner discovered the compound microscope.
(d) Edward Jenner discovered the compound nomenclature system.
What magnification is used if you observe a microorganism with a microscope whose object is
100× and whose ocular lens is 10×?
(a) 1000× magnification
(b) 100× magnification
(c) 10× magnification
(d) 10,000× magnification
What is the function of an illuminator?
(a) To control the temperature of the specimen
(b) To keep the specimen moist
(c) An illuminator is the light source used to observe a specimen under a microscope
(d) To keep the specimen dry
What is the area seen through the ocular eyepiece called?
(a) The stage
(b) The objective
(c) The display
(d) The field of view
How do you maintain good resolution of a specimen at magnifications greater than 100?
(a) Display the specimen on a television monitor.
(b) Use a single ocular eyepiece.
(c) Immerse the specimen in oil.
(d) Avoid moving the specimen.
What is a micrograph?
(a) A microscopic photograph taken by an electron microscope
(b) A microscopic diagram of a specimen
(c) A microscopic photograph taken by a light microscope
(d) A growth diagram of a specimen
What is a smear?
(a) A smear is a preparation process in which a specimen is spread on a slide.
(b) A smear is a preparation process in which a specimen is dyed.
(c) A smear is a process in which a specimen is moved beneath a microscope.
(d) A smear is a process used to identify a specimen.
What process is used to cause a specimen to adhere to a glass slide?
(a) The heat fixation process
(b) Wet mount
(c) White glue
(d) Clear glue
Why is a specimen stained?
(a) A stain is used to label a specimen.
(b) A stain is used to determine the size of a specimen.
(c) A stain adheres to the specimen, causing more light to be reflected by the specimen into the
microscope.
(d) A stain is used to determine the density of a specimen.
When would you use a wet mount?
(a) A wet mount is used to observe a dead specimen under a microscope.
(b) A wet mount is used to observe a live specimen under a microscope.
(c) A wet mount is used to observe an inorganic specimen under a microscope.
(d) A wet mount is the first step in preparing a specimen.
What is a nanometer?
(a) 1/1,000,000,000 of a meter
(b) 1/100,000 of a meter
(c) 1/1,000,000 of a meter
(d) 1,000,000,000 meters
What is refractive index
Light waves that are reflected by the specimen are measured by the refractive index. The
refractive index specifies the amount of light waves that is reflected by an object. There is a low
contrast between a specimen and the field of view if they have nearly the same refractive index. The
further these refractive indexes are from each other, the greater the contrast between the specimen
and the field of view.
Sterilization
Sterilization is the destruction of all microorganisms and viruses, as well as endospores.
Sterilization is used in preparing cultured media and canned foods. It is usually performed by steam
under pressure, incineration, or a sterilizing gas such as ethylene oxide.
Antisepsis
Antisepsis is the reduction of pathogenic microorganisms and viruses on living tissue.
Treatment is by chemical antimicrobials, like iodine and alcohol. Antisepsis is used to disinfect
living tissues without harming them.
Commercial sterilization
Commercial sterilization is the treatment to kill endospores in commercially canned
products. An example is the bacteria Clostridium botulinum, which causes botulism.
Aseptic
Aseptic means to be free of pathogenic contaminants. Examples include proper hand
washing, flame sterilization of equipment, and preparing surgical environments and instruments.
Disinfection
Disinfection is the destruction or killing of microorganisms and viruses on nonliving tissue
by the use of chemical or physical agents. Examples of these chemical agents are phenols, alcohols,
aldehydes, and surfactants.
Degerming
Degerming is the removal of microorganisms by mechanical means, such as cleaning the
site of an injection. This area of the skin is degermed by using an alcohol wipe or a piece of cotton
swab soaked with alcohol. Hand washing also removes microorganisms by chemical means.
Pasteurization
Pasteurizationuses heat to kill pathogens and reducethe number of food spoilage
microorganisms in foods and beverages. Examples are pasteurized milk and juice.
Sanitation
Sanitation is the treatment to remove or lower microbial counts on objects such as eating
and drinking utensils to meet public health standards. This is usually accomplished by washing the
utensils in high temperatures or scalding water and disinfectant baths. acterostatic, fungistatic, and
virustastic agents— or any word with the suffix -static or -stasis—indicate the inhibition of a
particular type of microorganism. These are unlike bactericides or fungicides that kill or destroy the
organism. Germistatic agents include refrigeration, freezing, and some chemicals.
Microbial death
Microbial death is the term used to describe the permanent loss of a microorganism’s ability
to reproduce under normal environmental conditions.
What is a sterile culture medium?
(a) A hydroxyl radical medium
(b) A medium containing no living organisms
(c) A chemically defined medium
(d) A moisture-filled medium
What must chemically defined media contain?
(a) Growth factors
(b) Complex media
(c) Peptone complex
(d) Protein hydrolysis
What is agar?
(a) A nutrient
(b) Vitamins
(c) A solidifying agent
(d) Broth
What is an enrichment culture?
(a) Something that provides growth for all microorganisms
(b) Something that inhibits growth for all microorganisms
(c) An infectious culture
(d) Something that provides growth for a certain microorganism but not for others
What is an inoculating loop?
(a) A tool used to streak a microorganism in a pure culture
(b) A tool used to place agar in a pure medium
(c) A tool used to count colonies of microorganisms
(d) A tool used to view colonies of microorganisms
Sterilization
Sterilization [Latin sterilis, unable to produce offspring or barren] is the process by which
all living cells, viable spores, viruses, and viroids are either destroyed or removed from an object or
habitat. A sterile object is totally free of viable microorganisms, spores, and other infectious agents.
Classification
The arrangement of organisms into groups based on similar characteristics, evolutionary
similarity or common ancestry. These groups are also called taxa.
Nomenclature
The name given to each organism. Each name must be unique and should depict the
dominant characteristic of the organism.
Identification.
The process of observing and classifying organisms into a standard group that is recognized
throughout the biological community.
Taxonomy
Taxonomy is a subset of systemics. Systemics is the study of organisms in order to place
organisms having similar characteristics into the same group. Using techniques from other sciences
such as biochemistry, ecology, epidemiology, molecular biology, morphology, and physiology,
biologists are able to identify characteristics of a organism.
What is a natural classification?
The natural classification system may be a phenetic system, one that groups organisms
together based on the mutual similarity of their phenotypic characteristics. Although phenetic
studies can reveal possible evolutionary relationships, they are not dependent on phylogenetic
analysis.
What specific nutritional types exist among protozoa?
Most protozoa are chemoheterotrophic. Two types of heterotrophic nutrition are found in
the protozoa: holozoic and saprozoic. In holozoic nutrition, solid nutrients such as bacteria are
acquired by phagocytosis and the subsequent formation of phagocytic vacuoles. Some ciliates have
a specialized structure for phagocytosis called the cytostome (cell mouth). In saprozoic nutrition,
soluble nutrients such as amino acids and sugars cross the plasma membrane by pinocytosis,
diffusion, or carriermediated transport (facilitated diffusion or active transport).
What is a pseudopodium?
Pseudopodia [s., pseudopodium; false feet] are cytoplasmic extensions found in the
amoebae that are responsible for the movement and food capture.
PART-B
How Do Organisms Appear?
Italian physician Francesco Redi developed an experiment that demonstrated that an
organism did not spontaneously appear. He filled jars with rotting meat. Some jars he sealed and
others he left opened. Those that were open eventually contained maggots, which is the larval stage
of the fly. The other jars did not contain maggots because flies could not enter the jar to lay eggs on
the rotting meat. His critics stated that air was the ingredient required for spontaneous generation of
an organism. Air was absent from the sealed jar and therefore no spontaneous generation could
occur, they said (Fig). Redi repeated the experiment except this time he placed a screen over the
opened jars. This prevented flies from entering the jar. There weren’t any maggots on the rotting
meat. Until that time scientists did not have a clue about how to fight disease. However, Redi’s
discovery gave scientists an idea. They used Redi’s findings to conclude that killing the
microorganism that caused a disease could prevent the disease from occurring. A new
microorganism could only be generated by the reproduction of another microorganism. Kill the
microorganism and you won’t have new microorganisms, the theory went—you could stop the
spread of the disease. Scientists called this the Theory of Biogenesis. The Theory of Biogenesis
states that a living cell is generated from another living cell.
Describe the prefixes for the metric system and their equivalent in meters.
Prefix
Value in Meters
Kilo (km) (kilo = 1,000)
1,000 m
Deci (dm) (deci = 1/10)
0.10 m
Centi (cm) (centi = 1/100)
0.01 m
Milli (mm) (milli = 1/1000)
0.001 m
Nano (nm) (nano = 1/1,000,000,000)
0.000000001 m
Pico (pm) (pico = 1/1,000,000,000,000)
0.000000000001 m
Kilo (kg)
1,000 g
Hecto (hg)
100 g
Deka (dag)
10 g
Gram (g)
1g
Deci (dg)
0.1 g
Centi (cg)
0.01 g
Milli (mg)
0.001 g
Micro (μg)
0.000001 g
Nano (ng)
0.000000001 g
Pico (pg)
0.000000000001 g
Ameter is the standard for length in the metric system. Akilogram is the standard for mass in
the metric system. A gram uses the same prefixes as a meter to specify the number of grams that are
represented by a value. For example, a kilometer is 1,000 meters and a kilogram is 1,000 grams.
This makes it a lot easier to learn the metric system since the number of grams and meters are
indicated by the same set of prefixes.
Write about various parts of compound microscope
A microscope is a complex magnifying glass. In the 1600s, during the time of Antoni van
Leeuwenhoek, microscopes consisted of one lens that was shaped so that the refracted light
magnified a specimen 100 times its natural size. Other lenses were shaped to increase the
magnification to 300 times. However, van Leeuwenhoek realized that a single-lens microscope is
difficult to focus. Once Van Leeuwenhoek brought the specimen into focus, he kept his hands
behind his back to avoid touching the microscope for fear they would bring the microscope out of
focus. It was common in the 1600s for scientists to make a new microscope for each specimen that
wanted to study rather than try to focus the microscope. The single-lens magnifying lens or glass is
a thing of the past. Scientists today use a microscope that has two sets of lenses (objective and
ocular), which is called a compound light microscope. Fig. shows parts of a compound light
microscope. A compound light microscope consists of:
• Illuminator. This is the light source located below the specimen.
• Condenser. Focuses the light through the specimen.
• Stage. The platform that holds the specimen.
• Objective. The lens that is directly above the stage.
• Nosepiece. The portion of the body that holds the objectives over the stage.
• Field diaphragm. Controls the amount of light into the condenser.
• Base. Bottom of the microscope.
•Coarse focusing knob. Used to make relatively wide focusing adjustments to the microscope.
• Fine focusing knob. Used to make relatively small adjustments to the microscope.
• Body. The microscope body.
• Ocular eyepiece. Lens on the top of the body tube. It has a magnification of 10× normal vision.
Parts of
a
compou
nd light
microsc
ope.
How to
measur
e
magnifi
cation
in the
microsc
ope?
Explain
.
A
compou
nd
microscope has two sets of lenses and uses light as the source of illumination. The light source is
called an illuminator and passes light through a condenser and through the specimen. Reflected
light from the specimen is detected by the objective. The objective is designed to redirect the light
waves, resulting in the magnification of the specimen.
There are typically four objectives, each having a different magnification. These are 4×,
10×, 40×, and 100×. The number indicates by how many times the original size of a specimen is
magnified, so the 4× objective magnifies the specimen four times the specimen size. The eyepiece
of the microscope is called the ocular eyepiece and it, too, has a lens—called an ocular lens—that
has a magnification of 10×.
You determine the magnification used to observe a specimen under a microscope by
multiplying the magnification of the objective by the magnification of the ocular lens. Suppose you
use the 4× objective to view a specimen. The image you see through the ocular is 40× because the
magnification of the object is multiplied by the magnification of the ocular lens, which is 10×.
What is resolution? Explain about it.
The area that you see through the ocular eyepiece is called the field of view. Depending on
the total magnification and the size of the specimen, sometimes the entire field of view is filled with
the image of the specimen. Other times, only a portion of the field of view contains the image of the
specimen.
You probably noticed that the specimen becomes blurry as you increase magnification.
Here’s what happens. The size of the field of view decreases as magnification increases, resulting in
your seeing a smaller area of the specimen. However, the resolution of the image remains
unchanged, therefore you must adjust the fine focus knob to bring the image into focus again.
Resolution is the ability of the lens to distinguish fine detail of the specimen and is
determined by the wavelength of light from the illuminator. At the beginning of this chapter you
learned about the wave cycle, which is the process of the wave going up and then falling down time
and again. A wavelength is the distance between the peaks of two waves. As a general rule, shorter
wavelengths produce higher resolutions of the image seen through the microscope.
How to maintain good resolution at magnifications of 100× and greater? Explain
In order to maintain good resolution, the lens must be small and sufficient light must be
reflected from both the specimen and the stain used on the specimen. The problem is that too much light
is lost; air between the slide and the objective prevents some light waves from passing to the objective,
causing the fuzzy appearance of the specimen in the ocular eyepiece.
The solution is to immerse the specimen in oil. The oil takes the place of air and, since oil has
the same refractive index as glass, the oil becomes part of the optics of the microscope. Light that is
usually lost because of the air is no longer lost. The result is good resolution under high magnification.
Write about different types of specimen preparation
There are two ways to prepare a specimen to be observed under a light compound
microscope. These are a smear and a wet mount.
Smear
A smear is a preparation process where a specimen that is spread on a slide. Smear can prepare
using the heat fixation process:
1. Use a clean glass slide.
2. Take a loop of the culture.
3. Place the live microorganism on the glass slide.
4. The slice is air dried then passed over a Bunsen burner about three times.
5. The heat causes the microorganism to adhere to the glass slide. This is known as fixing
the microorganism to the glass slide.
6. Stain the microorganism with an appropriate stain.
Wet Mount
A wet mount is a preparation process where a live specimen in culture fluid is placed on a
concave glass side or a plain glass slide. The concave portion of the glass slide forms a cup-like
shape that is filled with a thick, syrupy substance, such as carboxymethyl cellulose. The
microorganism is free to move about within the fluid, although the viscosity of the substance slows
its movement. This makes it easier for you to observe the microorganism. The specimen and the
substance are protected from spillage and outside contaminates by a glass cover that is placed over
the concave portion of the slide.
What is stain? Explain about it
A stain is a chemical that adheres to structures of the microorganism and in effect dyes the
microorganism so the microorganism can be easily seen under a microscope. Stains used in
microbiology are either basic or acidic. Basic stains are cationic and have positive charge.
Common basic stains are methylene blue, crystal violet, safranin, and malachite green.
These are ideal for staining chromosomes and the cell membranes of many bacteria.
Acid stains are anionic and have a negative charge. Common acidic stains are eosin and
picric acid. Acidic stains are used to stain cytoplasmic material and organelles or inclusions.
Describe various chemical agents to control the growth of microorganisms
The growth of a microorganism can be controlled through the use of a chemical agent. A
chemical agent is a chemical that either inhibits or enhances the growth of a microorganism.
Commonly used chemical agents include phenols, phenolics, glutaraldehyde, and formaldehyde.
PHENOLS AND PHENOLICS
Phenols are compounds derived from pheno (carbolic acid) molecules. Phenolics disrupt the
plasma by denaturing proteins; they also disrupt the plasma membrane of the cell. As mentioned in
Chapter 1, Joseph Lister used phenol in the late 1800s to reduce infection during surgery. Alcohols
are effective against bacterial fungi and viruses. However, they, are not effective against fungal
spores or bacterial endospores. Alcohols that are commonly used are isopropanol (rubbing alcohol)
and ethanol (the alcohol we drink).
Alcohols denature proteins and disrupt cytoplasmic membranes. Pure alcohol is not as
effective as 70 percent because the denaturing of proteins requires water. Alcohols are good to use
because they evaporate rapidly. A disadvantage is that they may not contact the microorganisms
long enough to be effective. Alcohol is commonly used in swabbing the skin prior to an injection.
Halogens are nonmetallic, highly resistive chemical elements. Halogens are effective against
vegetative bacterial cells, fungal cells, fungal spores, protozoan cysts, and many viruses. Halogencontaining antimicrobial agents include iodine, which inhibits protein function. Iodine is used in
surgery and by campers to disinfect water. An iodophur is an iodine-containing compound that is
longer-lasting than iodine and does not stain the skin. Other halogen agents include:
• Chlorine (Cl2). Used to treat drinking water, swimming pools, and in sewage plants to treat waste
water. Chlorine products such as sodium hypochlorite (household bleach) are effective
disinfectants.
• Chlorine dioxide (ClO2). A gas that can disinfect large areas.
• Chloroamines. Chemicals containing chlorine and ammonia. They are used as skin antiseptics and
in water supplies.
• Bromine. Used to disinfect hot tubes because it does not evaporate as quickly as chlorine in high
temperatures.
• Oxidizing agents. Fill microorganisms by oxidizing their enzymes, thus preventing metabolism.
Hydrogen peroxide, for example, disinfects and sterilizes inanimate objects, such as food
processing and medical equipment, and is also used in water purification.
Arsenic, zinc, mercury, silver, nickel, and copper are called heavy metals due to their high
molecular weights. They inhibit microbial growth because they denature enzymes and alter the
three-dimensional shapes of proteins that inhibit or eliminate the protein’s function. Heavy metals
are bacteriostatic and fungistatic agents.
An example is silver nitrate. At one time, hospitals required newborn babies to receive a one
percent cream of silver nitrate to their eyes to prevent blindness caused by Neisseria gonorhoeae,
which could enter the baby’s eyes while passing through the birth canal of a mother who was
infected. Today, antibiotic ointments that are less irritating are used. Another example is the use of
copper in swimming pools, fish tanks, and in reservoirs to control algae growth. Copper interferes
with chlorophyll, thus affecting metabolism and energy.
Aldehydes function in microbial growth by denaturing proteins and inactivating nucleic
acids. Two types are glutaraldehyde that is a liquid and formaldehyde that is a gas.
GLUTARALDEHYDE AND FORMALDEHYDE
Glutaraldehyde is used in a two percent solution to kill bacteria, fungi, and viruses on
medical and dental equipment. Healthcare workers and morticians dissolve gaseous formaldehyde
in water, making a 37 percent solution of formalin. Formalin is used in disinfecting dialysis
machines, surgical equipment, and embalming bodies after death.
Gaseous agents, such as ethylene oxide, propylene oxide, and beta-propiolactone, are used
on equipment that cannot be sterilized easily with heat, chemicals, or radiation. Certain items, like
pillows, mattresses, dried or powered food, plasticware, sutures, and heart-lung machines, are
placed in a closed chamber, then filled with these gases. Gaseous agents denature proteins.
SURFACTANTS
Surfactants are chemicals that act on surfaces by decreasing the tension of water and
disrupting cell membranes. Examples are household soaps and detergents.
Define thermal death point (TDP), thermal death time (TDT), decimal reduction time (D) or
D value, z value, and the F value.
Terms of thermal death point (TDP), the lowest temperature at which a microbial suspension is
killed in 10 minutes.
Thermal death time (TDT) is the shortest time needed to kill all organisms in a microbial
suspension at a specific temperature and under defined conditions.
The decimal reduction time is the time required to kill 90% of the microorganisms or spores in a
sample at a specified temperature.
The z value is the increase in temperature required to reduce D to 1/10 its value or to reduce it by
one log cycle when log D is plotted against temperature
The F value is the time in minutes at a specific temperature (usually 250°F or 121.1°C) needed to
kill a population of cells or spores.
Describe how an autoclave works. What conditions are required for sterilization by moist
heat, and what three things must one do when operating an autoclave to help ensure success?
Moist heat sterilization must be carried out at temperatures above 100°C in order to destroy
bacterial endospores, and this requires the use of saturated steam under pressure. Steam sterilization
is carried out with an autoclave (figure), a device somewhat like a fancy pressure cooker. The
development of the autoclave by Chamberland in 1884 tremendously stimulated the growth of
microbiology. Water is boiled to produce steam, which is released through the jacket and into the
autoclave’s chamber. The air initially present in the chamber is forced out until the chamber is filled
with saturated steam and the outlets are closed. Hot, saturated steam continues to enter until the
chamber reaches the desired temperature and pressure, usually 121°C and 15 pounds of pressure. At
this temperature saturated steam destroys all vegetative cells and endospores in a small volume of
liquid within 10 to 12 minutes. Treatment is continued for about 15 minutes to provide a margin of
safety. Of course, larger containers of liquid such as flasks and carboys will require much longer
treatment times.
M
oist heat
is
thought
to kill
so
effectiv
ely by
degradi
ng
nucleic
acids
and by
denaturi
ng
enzyme
s
and
other
essentia
l proteins. It also may disrupt cell membranes.
Autoclaving must be carried out properly or the processed materials will not be sterile. If all
air has not been flushed out of the chamber, it will not reach 121°C even though it may reach a
pressure of 15 pounds. The chamber should not be packed too tightly because the steam needs to
circulate freely and contact everything in the autoclave. Bacterial endospores will be killed only if
they are kept at 121°C for 10 to 12 minutes. When a large volume of liquid must be sterilized, an
extended sterilization time will be needed because it will take longer for the center of the liquid to
reach 121°C; 5 liters of liquid may require about 70 minutes. In view of these potential difficulties,
a biological indicator is often autoclaved along with other material. This indicator commonly
consists of a culture tube containing a sterile ampule of medium and a paper strip covered with
spores of Bacillus stearothermophilus or Clostridium PA3679. After autoclaving, the ampule is
aseptically broken and the culture incubated for several days. If the test bacterium does not grow in
the medium, the sterilization run has been successful. Sometimes either special tape that spells out
the word sterile or a paper indicator strip that changes color upon sufficient heating is autoclaved
with a load of material. If the word appears on the tape or if the color changes after autoclaving, the
material is supposed to be sterile. These approaches are convenient and save time but are not as
reliable as the use of bacterial endospores.
Give the advantages and disadvantages of ultraviolet light and ionizing radiation as sterilizing
agents. Provide a few examples of how each is used for this purpose.
Ultraviolet (UV) radiation around 260 nm is quite lethal but does not penetrate glass, dirt
films, water, and other substances very effectively. Because of this disadvantage,UV radiation is
used as a sterilizing agent only in a few specific situations. UV lamps are sometimes placed on the
ceilings of rooms or in biological safety cabinets to sterilize the air and any exposed surfaces.
Because UV radiation burns the skin and damages eyes, people working in such areas must be
certain the UV lamps are off when the areas are in use. Commercial UV units are available for
water treatment. Pathogens and other microorganisms are destroyed when a thin layer of water is
passed under the lamps.
Ionizing radiation is an excellent sterilizing agent and penetrates deep into objects. It will
destroy bacterial endospores and vegetative cells, both procaryotic and eucaryotic; however,
ionizing radiation is not always as effective against viruses. Gamma radiation from a cobalt 60
source is used in the cold sterilization of antibiotics, hormones, sutures, and plastic disposable
supplies such as syringes. Gamma radiation has also been used to sterilize and “pasteurize” meat
and other food. Irradiation can eliminate the threat of such pathogens as Escherichia coli O157:H7,
Staphylococcus aureus, and Campylobacter jejuni. Both the Food and Drug Administration and the
World Health Organization have approved food irradiation and declared it safe. A commercial
irradiation plant operates near Tampa, Florida. However, this process has not yet been widely
employed in the United States because of the cost and concerns about the effects of gamma
radiation on food. The U.S. government currently approves the use of radiation to treat poultry,
beef, pork, veal, lamb, fruits, vegetables, and spices. It will probably be more extensively employed
in the future.
What are the benefits of taxonomy?
Taxonomy organizes large amounts of information about organisms whose members of a
particular group share many characteristics. Taxonomy lets scientists make predictions and design a
hypothesis for future research on the knowledge of similar organisms. A hypothesis is a possible
explanation for an observation that needs experimentation and testing.
If a relative of an organism has the same properties, the organism may also have the same
characteristics. Taxonomy puts microorganisms into groups with precise names, enabling
microbiologists to communicate with each other in an efficient manner. Taxonomy is indispensable
for the accurate identification of microorganisms. For example, once a microbiologist or
epidemiologist identifies a pathogen that infects a patient, physicians know the proper treatment
that will cure the patient.
Write about taxonomic rank and file
A taxonomy has an overlapping hierarchy that forms levels of rank or category similar to an
organization chart. Each rank contains microorganisms that have similar characteristics. A rank can
also have other ranks that contain microorganisms.
Microorganisms that belong to a lower rank have characteristics that are associated with a
higher rank to which the lower rank belongs. However, characteristics of microorganisms of a
lower rank are not found in microorganisms that belong to the same higher rank as the lower-rank
microorganism.
Microbiologists use a microbial taxonomy, which is different from what biologists, who
work with larger organisms, use. Microbial taxonomy is commonly called prokaryotic taxonomy.
The widely accepted prokaryotic taxonomy is Bergey’s Manual of Systematic Bacteriology, first
published in 1923 by the American Society for Microbiology. David Bergey was chairperson of the
editorial board.
In the taxonomy of prokaryotes, the
most commonly used rank (in order from
most general to most specific) is:
The basic taxonomic group in microbial taxonomy is the species. Taxonomists working with
higher organisms define their species differently than microbiologists. Prokaryotic species are
characterized by differences in their phenotype and genotype. Phenotype is the collection of visible
characteristics and the behavior of a microorganism. Genotype is the genetic make up of a
microorganism
The prokaryotic species are collections of strains that share many properties and differ
dramatically from other groups or strains. A strain is a group of microorganisms that share
characteristics that are different from microorganisms in other strains. Each microorganism within a
strain is considered to have descended from the same microorganism.
For example, Biovars is a species that contains strains characterized by differences in its
biochemistry and physiology. Morphovars is also a species whose strains differ morphologically
and structurally. Serovars is another species that has strains that are characterized by distinct
antigenic properties (substances that stimulate the production of antibodies).
Microbiologists use the genus of the taxonomy to name microorganisms, which you learned
in Chapter 1. Microorganisms are given a two-part name. The first part is the Latin name for the
genus. The second part is the epithet. Together these parts uniquely identify the microorganism.
The first part of the name is always capitalized and the second part of the name is always lowercase.
Both parts are italicized.
For example, Escherichia coli is a bacterium that is a member of the Escherichia genus and
has the epithet coli. Sometimes the name is abbreviated such as E. coli. However, the abbreviation
maintains the same style as the full name (uppercase, lowercase, italic).
What is numerical taxonomy and why are computers so important to this approach?
The development of computers has made possible the quantitative approach known as
numerical taxonomy. Peter H. A. Sneath and Robert Sokal have defined numerical taxonomy as
“the grouping by numerical methods of taxonomic units into taxa on the basis of their character
states.” Information about the properties of organisms is converted into a form suitable for
numerical analysis and then compared by means of a computer. The resulting classification is based
on general similarity as judged by comparison of many characteristics, each given equal weight.
This approach was not feasible before the advent of computers because of the large number of
calculations involved.
The process begins with a determination of the presence or absence of selected characters in
the group of organisms under study. A character usually is defined as an attribute about which a
single statement can be made. Many characters, at least 50 and preferably several hundred, should
be compared for an accurate and reliable classification. It is best to include many different kinds of
data: morphological, biochemical, and physiological.
After character analysis, an association coefficient, a function that measures the agreement
between characters possessed by two organisms, is calculated for each pair of organisms in the
group. The simple matching coefficient (SSM), the most commonly used coefficient in
bacteriology, is the proportion of characters that match regardless of whether the attribute is present
or absent. Sometimes the Jaccard coefficient
(SJ) is calculated by ignoring any characters that both organisms lack (table 19.2). Both coefficients
increase linearly in value from 0.0 (no matches) to 1.0 (100% matches).
The simple matching coefficients, or other association coefficients, are then arranged to
form a similarity matrix. This is a matrix in which the rows and columns represent organisms, and
each value is an association coefficient measuring the similarity of two different organisms so that
each organism is compared to every other one in the table. Organisms with great similarity are
grouped together and separated from dissimilar organisms; such groups of organisms are called
phenons (sometimes called phenoms).
The results of numerical taxonomic analysis are often summarized with a treelike diagram
called a dendrogram. The diagram usually is placed on its side with the X-axis or abscissa
graduated in units of similarity. Each branch point is at the similarity value relating the two
branches. The organisms in the two branches share so many characteristics that the two groups are
seen to be separate only after examination of association coefficients greater than the magnitude of
the branch point value. Below the branch point value, the two groups appear to be one. The ordinate
in such a dendrogram has no special significance, and the clusters may be arranged in any
convenient order.
The significance of these clusters or phenons in traditional taxonomic terms is not always
evident, and the similarity levels at which clusters are labeled species, genera, and so on, are a
matter of judgment. Sometimes groups are simply called phenons and preceded by a number
showing the similarity level above which they appear (e.g., a 70-phenon is a phenon with 70% or
greater similarity among its constituents). Phenons formed at about 80% similarity often are
equivalent to species.
Numerical taxonomy has already proved to be a powerful tool in microbial taxonomy.
Although it often has simply reconfirmed already existing classification schemes, sometimes
accepted classifications are found wanting. Numerical taxonomic methods also can be used to
compare sequences of macromolecules such as RNA and proteins.
Clustering and Dendrograms in Numerical Taxonomy. (a) A small similarity matrix that
compares six strains of bacteria. The degree of similarity ranges from none (0.0) to complete
similarity (1.0). (b) The bacteria have been rearranged and joined to form clusters of similar strains.
For example, strains 1 and 2 are the most similar. The cluster of 1 plus 2 is fairly similar to strain 3,
but not at all to strain 4. (c) A dendrogram showing the results of the analysis in part b. Strains 1
and 2 are members of a 90-phenon, and strains 1–3 form an 80-phenon. While strains 1–3 may be
members of a single species, it is quite unlikely that strains 4–6 belong to the same species as 1–3.
Give the names and main distinguishing characteristics of the archea, bacteria and eucarya
What are the major ways in which gram-negative and gram-positive bacteria differ?
Distinguish mycoplasmas from other bacteria.
Write about economic importance of fungi
Useful aspects:
1. Directly used as a food in the form of mushroom
e.g., Morchella esculenta, Coprinus sp etc..
2. Saccharomyces cerevisiae is used in bread making industry and alcohol industry
3. Few fungi are used for processing of food
e.g., Penicillium camemberti is involved in the ripening of cheese.
4. Used for production of antibiotics.
e.g., Penicillin extracted from Penicillium notatum
5. Many important and useful enzymes have been synthesized from various fungi.
e.g., Amylase produced by Aspergillus niger
Harmful aspects:
1. Various parasitic fungi act as casual organisms and infect plants.
e.g., white rust of crucifer caused by Candida albugo
2. Decay of timber: many species of polyporus and Basidiomycetes cause the damage and
decay to the timber wood
3. Spoilage of food stuffs: Penicillium digitatum is responsible for the rotting of citrus fruits.
Mucor, Aspergillus and Fusarium causes damage to the milk and milk products
4. Several important human diseases caused by different species of fungi. For example,
Aspergillus fumigatus is responsible for causing Aspergillosis. The disease is mycoses.
Economic importance of Yeast
1. Yeast (Saccharomyces cereviseae) involved in sugar fermentation and produce alcohol. It
also used for beer and wine making industry.
2. Many types of yeast are responsible for the spoilage of cheese, tomato and other food
products.
3. A few species of yeasts are parasites for higher plants and cause diseases.
4. Several species of yeasts are pathogenic to man causing number of serious diseases. e.g.,
blastomycosis, torulopsis etc.. They attack central nervous system and skin of man.
5. S.cereviseae is used for bread making industry.
Write about economic importance of algae
Useful aspects
1. The algae are used as a direct source of food by several sea animals and fishes. e.g.,
Diatoms with dinoflagellates.
2. Sea weeds have been used as a food for human beings. Several fresh water algae have
also been utilized in the preparation of vitamin foods. The algae are the most important
part of the diet of Japan and China.
3. Agar is also obtained from several marine algae. Japan is the chief agar producing
country and it exports agar to most countries of the world. The agar is used for
preparation of jellies, ice cream etc.. The agar has constantly been used in laboratories
for media preparation.
4. Various countries prepared medicines from algal weeds. For example, antibiotic drug
Chlorellum obtained from algae.
5. The alginic acid is manufactured from the cell wall of Phaeophyceae. Sodium alginate
used in dyes, buttons, combs and many of such things.
6. Due to the presence of potassium chloride in sea weeds, they are used as fertilizers in
many countries.
7. Important part of the food chain in aquatic ecosystems because they fix carbon dioxide
into organic molecules that can be used by heterotrophs.
8. 80% of the earth’s oxygen is believed to be produced by planktonic algae.
9. Algal blooms are indicators of water pollution.
10. Petroleum and natural gas reserves were formed primarily from diatoms and plankton.
11. Many unicellular algae are symbionts in animals.
Harmful aspects:
1. Algae are found in water more abundantly that cause the whole water either green or
blue and cause death of fishes.
2. Some blue green algae have been reported poisonous and they directly cause the death
who drink this contaminated water.
3. The epiphytic algae which are found upon other plants and trees block photosynthesis
and indirectly harm the trees and plants.
4. Some algae form mucilaginous secretions which are the binding material for the harmful
bacteria causing human and animal diseases.
5. Some algae are attached to the ship (fouling of ships) which retards the speed of the
ship.
Economic importance of Diatoms:
1. Used as a food. It acts as a primary producer in food chain of aquatic animals.
2. Used as polishing material.
3. Used as filters in sugar refineries and brewing industries.
4. Used in the manufacture of dynamite.
5. Used in the manufacture of glass, porcelain paints and varnishes
6. Used in making of toothpaste and face powder.
7. Used for making light and heat resistant bricks.
What are the general characteristics of algae? Explain
1. Habitat
 Fresh, marine and brackish water
 Moist rocks, wood, trees, surface of moist soil
 Endosymbionts – protozoa, mollusks, worms and corals
2. Structure of the Plant Body
 Vegetative body – thallus
 Microscopic unicellular – Chlamydomonas
 Macroscopic multicellular - Macrocystis




3. Reproduction
Vegetative, asexual and sexual
Vegetative – fragmentation / fission
Asexual – zoospores, aplanospores, akinetes, auxospores, endospores and cysts
Sexual – Isogamous, Anisogamous, Heterogenous, oogamous
4. Fertilization
 External – outside of gametangia
 Internal – inside of oogonium
5.
–
6.
Zygote
 Diploid
fusion
of two
gametes
Germination
 Direct –
Zygote
to new
plant
 Indirect – zygote to spore to new plant
How do protozoa reproduce asexually and sexually?
Most protozoa reproduce asexually, and some also carry out sexual reproduction. The most
common method of asexual reproduction is binary fission. During this process the nucleus first
undergoes mitosis, then the cytoplasm divides by cytokinesis to form two identical individuals
Protozoan Reproduction. Binary fission in Paramecium caudatum
The most common method of sexual reproduction is conjugation. In this process there is an
exchange of gametes between paired protozoa of complementary mating types (conjugants).
Conjugation is most prevalent among ciliate protozoa. A well-studied example is Paramecium
caudatum. At the beginning of conjugation, two ciliates unite, fusing their pellicles at the contact
point. The macronucleus in each is degraded. The individual micronuclei divide twice by meiosis to
form four haploid pronuclei, three of which disintegrate. The remaining pronucleus divides again
mitotically to form two gametic nuclei, a stationary one and a migratory one. The migratory nuclei
pass into the respective conjugates. Then the ciliates separate, the gametic nuclei fuse, and the
resulting diploid zygote nucleus undergoes three rounds of mitosis. The eight resulting nuclei have
different fates: one nucleus is retained as a micronucleus; three others are destroyed; and the four
remaining nuclei develop into macronuclei. Each separated conjugant now undergoes cell division.
Eventually progeny with one macronucleus and one micronucleus are formed.
Conjugation in Paramecium caudatum, Schematic Drawing. Follow the arrows. After the
conjugants separate, only one of the exconjugants is followed; however, a total of eight new
protozoa result from the conjugation.
PART-C
Give a detailed account on contribution in Microbiology by various scientists
1590 Zacharias Janssen Developed the first compound microscope.
1590 Robert Hooke Observed nonliving plant tissue of a thin slice of cork.
1668 Francesco Redi Discovered that microorganisms did not spontaneously appear. His
contribution led to the finding that killing the microorganism that caused the disease could prevent
the disease.
1673 Antoni van Invented the single-lens microscope, grinding the Leeuwenhoek microscope lens
to improve magnification. First person to view a living organism.
1798 Edward Jenner Developed vaccinations against disease-causing microorganisms.
1850s Mathias Schleiden, Theodore Schwann, Rudolf Virchow Developed cell theory.
1847 Ignaz Semmelweis Reported a dramatic decline in childbirth fever after physicians used
antiseptic techniques when delivering babies.
1864 Louis Pasteur Discovered that microorganisms were everywhere, living on organisms and in
nonliving things such as air. His work led to improved sterilization techniques called pasteurization.
One of the founders of bacteriology.
1867 Joseph Lister Reduced infections after surgery by spraying carbolic acid over the patient
before bandaging the
wound. This was the first surgical antiseptic.
1876 Robert Koch Discovered how microorganisms spread contagious diseases by studying
anthrax. Developed the Germ Theory. Developed techniques for cultivating microorganisms.
1870s John Tyndall, Discovered that some microorganisms are resistant to Ferdinand Cohn certain
sterilization techniques. One of the founders of bacteriology.
1884 Elie Metchnikoff Discovered that white blood cells (leukocytes) engulf and digest
microorganisms that invade the body. Coined the word phagocytes. Founded the branch of science
called immunology.
1887 Richard Petri Developed the technique of placing agar into a specially designed dish to grow
microorganisms, which was later called the Petri dish.
1890 Paul Ehrlich Developed the first drug to fight disease-causing microorganisms that had
already entered the body.
1928 Alexander Fleming Discovered Penicillium notatum, the fungus that kills staphylococcus
aureus, a microorganism that is a leading cause of infection.
Explain in detailed about the features of various types of microscopes
Bright-Field Microscope
The bright-field microscope is the most commonly used microscope and consists of two
lenses. These are the ocular eyepiece and the objective. Light coming from the illuminator passes
through the specimen. The specimen absorbs some light waves and passes along other light waves
into the lens of the microscope, causing a contrast between the specimen and other objects in the
field of view. Specimens that have pigments contrast with objects in the field of view and can be
seen by using the bright-field microscope. Specimens with few or no pigments have a low contrast
and cannot be seen with the bright-field microscope. Some bacteria have low contrast.
Dark Field Microscope
The dark-field microscope focuses the light from the illuminator onto the top of the
specimen rather than from behind the specimen. The specimen absorbs some light waves and
reflects other light waves into the lens of the microscope. The field of view remains dark while the
specimen is illuminated, providing a stark contrast between the field of view and the specimen.
Phase-Contrast Microscope
The phase-contrast microscope bends light that passes through the specimen so that it
contrasts with the surrounding medium. Bending the light is called moving the light out of phase.
Since the phase-contrast microscope compensates for the refractive properties of the specimen, you
don’t need to stain the specimen to enhance the contrast of the specimen with the field of view. This
microscope is ideal for observing living microorganisms that are prepared in wet mounted slides so
you can study a living microorganism.
Fluorescent Microscope
Fluorescent microscopy uses ultraviolet light to illuminate specimens. Some organism
fluoresce naturally, that is, give off light of a certain color when exposed to the light of different
color. Organisms that don’t fluoresce naturally can be stained with fluorochrome dyes. When these
organisms are placed under a fluorescent microscope with an ultraviolet light, they appear very
bright in front of a dark background.
Differential Interface Contrast Microscope (Nomanski)
The differential interface contrast microscope, commonly known as Nomanski, works in a
similar way to the phase-contrast microscope. However, unlike the phase-contrast microscope
(which produces a two-dimensional image of the specimen), the differential interface contrast
microscope shows the specimen in three dimensions.
THE ELECTRON MICROSCOPE
A light compound microscope is a good tool for observing many kinds of microorganisms.
However, it isn’t capable of seeing the internal structure of a microorganism nor can it be used to
observe a virus. These are too small to effectively reflect visible light sufficient to be seen under a
light compound microscope. In order to view internal structures of viruses and internal structures of
microorganisms, microbiologists use an electron microscope where specimens are viewed in a
vacuum. Developed in the 1930s, the electron microscope uses beams of electrons and magnetic
lenses rather than light waves and optical lenses to view a specimen. Very thin slices of the
specimen are cut so that the internal structures can be viewed. Microscopic photographs called
micrographs are taken of the specimen and viewed on a video screen. Specimens can be viewed up
to 200,000 times normal vision. However, living specimens cannot be viewed because the specimen
must be sliced.
Transmission Electron Microscope
The transmission electron microscope (TEM) has a total magnification of up to 200,000×
and a resolution as fine as seven nanometers. A nanometer is 1/1,000,000,000 of a meter. The
transmission electron microscope generates an image of the specimen two ways. First, the image is
displayed on a screen similar to that of a computer monitor. The image can also be displayed in the
form of an electron micrograph, which is similar to a photograph. Specimens viewed by the
transmission electron microscope must be cut into very thin slices, otherwise the microscope does
not adequately depict the image.
Scanning Electron Microscope
The scanning electron microscope (SEM) is less refined than the transmission electron
microscope. It can provide total magnification up to 10,000× and a resolution as close as 20
nanometers. However, a scanning electron microscope produces three-dimensional images of
specimen. The specimen must be freeze dried and coated with a thin layer of gold, palladium, or
other heavy metal.
What are the various types of stains used in microbiological laboratory? Add a note on it.
Types of Stains
There are two types of Stains: simple and differential. See Table 3-4 for a summary of
staining techniques.
Simple Stain
A simply stain has a single basic dye that is used to show shapes of cells and the structures
within a cell. Methylene blue, safranin, carbolfuchsin and crystal violet are common simple stains
that are found in most microbiology laboratories.
Differential Stain
A differential stain consists of two or more dyes and is used in the procedure to identify
bacteria. Two of the most commonly used differential stains are the Gram stain and the ZiehlNielsen acid-fast stain.
In 1884 Hans Christian Gram, a Danish physician, developed the Gram stain. Gram-stain is
a method for the differential staining of bacteria. Gram-positive microorganisms stain purple.
Gram-negative microorganisms stain pink. Staphylococcus aureus, a common bacterium that causes
food poisoning, is grampositive. Escherichia coli is gram-negative.
The Ziehl-Nielsen acid-fast stain, developed by Franz Ziehl and Friedrick Nielsen, is a red
dye that attaches to the waxy material in the cell walls of bacteria such as Mycobacterium
tuberculosis, which is the bacterium that causes tuberculosis, and Mycobacterium leprae, which is
the bacterium that causes leprosy. Microorganisms that retain the Ziehl-Nielsen acid-fast stain are
called acid-fast. Those that do not retain it turn blue because the microorganism doesn’t absorb the
Ziehl-Nielsen acid-fast stain.
Gramstain a
specime
n
1.
Prepare
the
specime
n using
the heat
fixation
process
2. Place a drop of crystal violet stain on the specimen.
3. Apply iodine on the specimen using an eyedropper. The iodine helps the crystal violet stain
adhere to the specimen. Iodine is a mordant, which is a chemical that fixes the stain to the
specimen.
4. Wash the specimen with ethanol or an alcohol-acetone solution, then wash with water.
5. Wash the specimen to remove excess iodine. The specimen appears purple in color.
6. Wash the specimen with an ethanol or alcohol-acetone decolorizing solution.
7. Wash the specimen with water to remove the dye.
8. Apply the safranin stain to the specimen using an eyedropper.
9. Wash the specimen.
10. Use a paper towel and blot the specimen until the specimen is dry.
11. The specimen is ready to be viewed under the microscope. Gram-positive bacteria appear purple
and gram-negative bacteria appear pink.
Ziehl-Nielsen acid-fast stain to a specimen.
1. Prepared the specimen (see “Smear” earlier in this chapter).
2. Apply the red dye carbol-fuchsin stain generously using an eyedropper.
3. Let the specimen sit for a few minutes.
4. Warm the specimen over steaming water. The heat will cause the stain to penetrate the cell wall.
5. Wash the specimen with an alcohol-acetone decolorizing solution consisting of 3 percent
hydrochloric acid and 95 percent ethanol. The hydrochloric acid will remove the color from non–
acid-fast cells and the background. Acid-fast cells will stay red because the acid cannot penetrate
the cell wall.
6. Apply methylene blue stain on the specimen using an eyedropper.
Special Stains
Special stains are paired to dye specific structures of microorganisms such as endospores,
flagella, and gelatinous capsules. One stain in the pair is used as a negative stain. A negative stain is
used to stain the background of the micro- organism, causing the microorganism to appear clear. A
second stain is used to colorize specific structures within the microorganism. For example, nigrosin
and India ink are used as a negative stain and methylene blue is used as a positive stain.
The Schaeffer-Fulton endospore stain is a special flagellar stain that is used to colorize the
endospore. The endospore is a dormant part of the bacteria cell that protects the acteria from the
environment outside the cell.
Schaeffer-Fulton endospore stain
1. Prepare the specimen (see “Smear” earlier in this chapter).
2. Heat the malachite green stain over a Bunsen burner until it becomes fluid.
3. Apply the malachite green to the specimen using an eyedropper.
4. Wash the specimen for 30 seconds.
5. Apply the safranin stain using an eyedropper to the specimen to stain parts of the cell other than
the endospore.
6. Observe the specimen under the microscope.
Briefly explain how the effectiveness of antimicrobial agents varies with population size,
population composition, concentration or intensity of the agent, treatment duration,
temperature, and local environmental conditions.
Destruction of microorganisms and inhibition of microbial growth are not simple matters
because the efficiency of an antimicrobial agent (an agent that kills microorganisms or inhibits
their growth) is affected by at least six factors.
1. Population size. Because an equal fraction of a microbial population is killed during each
interval, a larger population requires a longer time to die than a smaller one. The same principle
applies to chemical antimicrobial agents.
2. Population composition. The effectiveness of an agent varies greatly with the nature of the
organisms being treated because microorganisms differ markedly in susceptibility. Bacterial
endospores are much more resistant to most antimicrobial agents than are vegetative forms, and
younger cells are usually more readily destroyed than mature organisms. Some species are able to
withstand adverse conditions better than others. Mycobacterium tuberculosis, which causes
tuberculosis, is much more resistant to antimicrobial agents than most other bacteria.
3. Concentration or intensity of an antimicrobial agent. Often, but not always, the more
concentrated a chemical agent or intense a physical agent, the more rapidly microorganisms are
destroyed. However, agent effectiveness usually is not directly related to concentration or intensity.
Over a short range a small increase in concentration leads to an exponential rise in effectiveness;
beyond a certain point, increases may not raise the killing rate much at all. Sometimes an agent is
more effective at lower concentrations. For example, 70% ethanol is more effective than 95%
ethanol because its
activity is enhanced by the presence of water.
4. Duration of exposure. The longer a population is exposed to a microbicidal agent, the more
organisms are killed. To achieve sterilization, an exposure duration sufficient to reduce the
probability of survival to 10–6 or less should be used.
5. Temperature. An increase in the temperature at which a chemical acts often enhances its activity.
Frequently a lower concentration of disinfectant or sterilizing agent can be used at a higher
temperature.
6. Local environment. The population to be controlled is not isolated but surrounded by
environmental factors that may either offer protection or aid in its destruction. For example,
because heat kills more readily at an acid pH, acid foods and beverages such as fruits and tomatoes
are easier to pasteurize than foods with higher pHs like milk. A second important environmental
factor is organic matter that can protect microorganisms against heating and chemical disinfectants.
Biofilms are a good example. The organic matter in a surface biofilm will protect the biofilm’s
microorganisms; furthermore, the biofilm and its microbes often will be hard to remove. It may be
necessary to clean an object before it is disinfected or sterilized. Syringes and medical or dental
equipment should be cleaned before sterilization because the presence of too much organic matter
could protect pathogens and increase the risk of infection. The same care must be taken when
pathogens are destroyed during the preparation of drinking water. When a city’s water supply has a
high content of organic material, more chlorine must be added to disinfect it.
How are pasteurization, flash pasteurization, ultrahigh temperature sterilization, and dry
heat sterilization carried out? Give some practical applications for each of these procedures.
Many substances, such as milk, are treated with controlled heating at temperatures well
below boiling, a process known as pasteurization in honor of its developer Louis Pasteur. In the
1860s the French wine industry was plagued by the problem of wine spoilage, which made wine
storage and shipping difficult. Pasteur examined spoiled wine under the microscope and detected
microorganisms that looked like the bacteria responsible for lactic acid and acetic acid
fermentations. He then discovered that a brief heating at 55 to 60°C would destroy these
microorganisms and preserve wine for long periods. In 1886 the German chemists V. H. and F.
Soxhlet adapted the technique for preserving milk and reducing milktransmissible diseases. Milk
pasteurization was introduced into the United States in 1889. Milk, beer, and many other beverages
are now pasteurized. Pasteurization does not sterilize a beverage, but it does kill any pathogens
present and drastically slows spoilage by reducing the level of nonpathogenic spoilage
microorganisms.
Milk can be pasteurized in two ways. In the older method the milk is held at 63°C for 30
minutes. Large quantities of milk are now usually subjected to flash pasteurization or hightemperature short-term (HTST) pasteurization, which consists of quick heating to about 72°C for 15
seconds, then rapid cooling. The dairy industry also sometimes uses ultrahigh-temperature
(UHT) sterilization. Milk and milk products are heated at 140 to 150°C for 1 to 3 seconds. UHTprocessed milk does not require refrigeration and can be stored at room temperature for about 2
months without flavor changes. The small coffee creamer portions provided by restaurants often are
prepared using UHT sterilization.
What are depth filters and membrane filters, and how are they used to sterilize liquids?
Describe the operation of a biological safety cabinet.
Filtration is an excellent way to reduce the microbial population in solutions of heatsensitive material, and sometimes it can be used to sterilize solutions. Rather than directly
destroying contaminating microorganisms, the filter simply removes them. There are two types of
filters. Depth filters consist of fibrous or granular materials that have been bonded into a thick
layer filled with twisting channels of small diameter. The solution containing microorganisms is
sucked through this layer under vacuum, and microbial cells are removed by physical screening or
entrapment and also by adsorption to the surface of the filter material. Depth filters are made of
diatomaceous earth (Berkefield filters), unglazed porcelain (Chamberlain filters), asbestos, or other
similar materials.
Membrane filters have replaced depth filters for many purposes. These circular filters are
porous membranes, a little over 0.1 mm thick, made of cellulose acetate, cellulose nitrate,
polycarbonate, polyvinylidene fluoride, or other synthetic materials. Although a wide variety of
pore sizes are available, membranes with pores about 0.2 _m in diameter are used to remove most
vegetative cells, but not viruses, from solutions ranging in volume from 1 ml to many liters. The
membranes are held in special holders (figure) and often preceded by depth filters made of glass
fibers to remove larger particles that might clog the membrane filter. The solution is pulled or
forced through the filter with a vacuum or with pressure from a syringe, peristaltic pump, or
nitrogen gas bottle, and collected in previously sterilized containers. Membrane filters remove
microorganisms by screening them out much as a sieve separates large sand particles from small
ones. These filters are used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils,
antibiotics, and other heat-sensitive solutions.
Membr
ane
Filter
Steriliz
ation. A
membra
ne filter
outfit
for
sterilizi
ng
medium
volumes
of
solution. (a) Cross section of the membrane filtering unit. Several membranes are used to increase
capacity. (b) A complete filtering setup. The solution to be sterilized is kept in the Erlenmeyer
flask, 1, and forced through the filter by a peristaltic pump, 2. The solution is sterilized by flowing
through a membrane filter unit, 3, and into a sterile container. A wide variety of other kinds of
filtering outfits are also available.
Air also can be sterilized by filtration. Two common examples are surgical masks and
cotton plugs on culture vessels that let air in but keep microorganisms out. Laminar flow
biological safety cabinets employing high-efficiency particulate air (HEPA) filters, which
remove 99.97% of 0.3 _m particles, are one of the most important air filtration systems. Laminar
flow biological safety cabinets force air through HEPA filters, then project a vertical curtain of
sterile air across the cabinet opening. This protects a worker from microorganisms being handled
within the cabinet and prevents contamination of the room (figure). A person uses these cabinets
when working with dangerous agents such as Mycobacterium tuberculosis, tumor viruses, and
recombinant DNA. They are also employed in research labs and industries, such as the
pharmaceutical industry, when a sterile working surface is needed for conducting assays, preparing
media, examining tissue cultures, and the like.
A Laminar Flow Biological Safety Cabinet. A schematic diagram showing the airflow pattern.
Describe each of the following agents in terms of its chemical nature, mechanism of action,
mode of application, common uses and effectiveness, and advantages and disadvantages:
phenolics, alcohols, halogens (iodine and chlorine), heavy metals, quaternary ammonium
compounds, aldehydes, and ethylene oxide.
Phenolics
Phenol was the first widely used antiseptic and disinfectant. In 1867 Joseph Lister employed
it to reduce the risk of infection during operations. Today phenol and phenolics (phenol derivatives)
such as cresols, xylenols, and orthophenylphenol are used as disinfectants in laboratories and
hospitals. The commercial disinfectant Lysol is made of a mixture of phenolics. Phenolics act by
denaturing proteins and disrupting cell membranes. They have some real advantages as
disinfectants: phenolics are tuberculocidal, effective in the presence of organic material, and remain
active on surfaces long after application. However, they do have a disagreeable odor and can cause
skin irritation.
Hexachlorophene has been one of the most popular antiseptics because it persists on the skin
once applied and reduces skin bacteria for long periods. However, it can cause brain damage and is
now used in hospital nurseries only in response to a staphylococcal outbreak.
Alcohols
Alcohols are among the most widely used disinfectants and antiseptics. They are
bactericidal and fungicidal but not sporicidal; some lipid-containing viruses are also destroyed. The
two most popular alcohol germicides are ethanol and isopropanol, usually used in about 70 to 80%
concentration. They act by denaturing proteins and possibly by dissolving membrane lipids. A 10 to
15 minute soaking is sufficient to disinfect thermometers and small instruments.
Halogens
A halogen is any of the five elements (fluorine, chlorine, bromine, iodine, and astatine) in
group VIIA of the periodic table. They exist as diatomic molecules in the free state and form
saltlike compounds with sodium and most other metals. The halogens iodine and chlorine are
important antimicrobial agents. Iodine is used as a skin antiseptic and kills by oxidizing cell
constituents and iodinating cell proteins. At higher concentrations, it may even kill some spores.
Iodine often has been applied as tincture of iodine, 2% or more iodine in a water-ethanol solution of
potassium iodide. Although it is an effective antiseptic, the skin may be damaged, a stain is left, and
iodine allergies can result. More recently iodine has been complexed with an organic carrier to form
an iodophor. Iodophors are water soluble, stable, and nonstaining, and release iodine slowly to
minimize skin burns and irritation. They are used in hospitals for preoperative skin degerming and
in hospitals and laboratories for disinfecting. Some popular brands are Wescodyne for skin and
laboratory disinfection and Betadine for wounds.
Chlorine is the usual disinfectant for municipal water supplies and swimming pools and is
also employed in the dairy and food industries. It may be applied as chlorine gas, sodium
hypochlorite, or calcium hypochlorite, all of which yield hypochlorous acid (HClO) and then
atomic oxygen. The result is oxidation of cellular materials and destruction of vegetative bacteria
and fungi, although not spores. Death of almost all microorganisms usually occurs within 30
minutes. Since organic material interferes with chlorine action by reacting with chlorine and its
products, an excess of chlorine is added to ensure microbial destruction. One potential problem is
that chlorine reacts with organic compounds to form carcinogenic trihalomethanes, which must be
monitored in drinking water. Ozone sometimes has been used successfully as an alternative to
chlorination
in Europe and Canada.
Chlorine is also an excellent disinfectant for individual use because it is effective,
inexpensive, and easy to employ. Small quantities of drinking water can be disinfected with
halazone tablets. Halazone (parasulfone dichloramidobenzoic acid) slowly releases chloride when
added to water and disinfects it in about a half hour. It is frequently used by campers lacking access
to uncontaminated drinking water.
Chlorine solutions make very effective laboratory and household disinfectants. An excellent
disinfectant-detergent combination can be prepared if a 1/100 dilution of household bleach (e.g., 1.3
fl oz of Clorox or Purex bleach in 1 gal or 10 ml/liter) is combined with sufficient nonionic
detergent (about 1 oz/gal or 7.8 ml/liter) to give a 0.8% detergent concentration. This mixture will
remove both dirt and bacteria.
Heavy Metals
For many years the ions of heavy metals such as mercury, silver, arsenic, zinc, and copper
were used as germicides. More recently these have been superseded by other less toxic and more
effective germicides (many heavy metals are more bacteriostatic than bactericidal). There are a few
exceptions. A 1% solution of silver nitrate is often added to the eyes of infants to prevent
ophthalmic gonorrhea (in many hospitals, erythromycin is used instead of silver nitrate because it is
effective against Chlamydia as well as Neisseria). Silver sulfadiazine is used on burns. Copper
sulfate is an effective algicide in lakes and swimming pools. Heavy metals combine with proteins,
often with their sulfhydryl groups, and inactivate them. They may also precipitate cell proteins.
Quaternary Ammonium Compounds
Detergents [Latin detergere, to wipe off or away] are organic molecules that serve as
wetting agents and emulsifiers because they have both polar hydrophilic and nonpolar hydrophobic
ends. Due to their amphipathic nature, detergents solubilize otherwise insoluble residues and are
very effective cleansing agents. They are different than soaps, which are derived from fats.
Although anionic detergents have some antimicrobial properties, only cationic detergents
are effective disinfectants. The most popular of these disinfectants are quaternary ammonium
compounds characterized by a positively charged quaternary nitrogen and a long hydrophobic
aliphatic chain. They disrupt microbial membranes and may also denature proteins.
Cationic detergents like benzalkonium chloride and cetylpyridinium chloride kill most
bacteria but not M. tuberculosis or endospores. They do have the advantages of being stable,
nontoxic, and bland but they are inactivated by hard water and soap. Cationic detergents are often
used as disinfectants for food utensils and small instruments and as skin antiseptics. Several brands
are on the market. Zephiran contains benzalkonium chloride and Ceepryn, cetylpyridinium chloride.
Aldehydes
Both of the commonly used aldehydes, formaldehyde and glutaraldehyde, are highly
reactive molecules that combine with nucleic acids and proteins and inactivate them, probably by
crosslinking and alkylating molecules. They are sporicidal and can be used as chemical sterilants.
Formaldehyde is usually dissolved in water or alcohol before use. A 2% buffered solution of
glutaraldehyde is an effective disinfectant. It is less irritating than formaldehyde and is used to
disinfect hospital and laboratory equipment. Glutaraldehyde usually disinfects objects within about
10 minutes but may require as long as 12 hours to destroy all spores.
Sterilizing Gases
Many heat-sensitive items such as disposable plastic petri dishes and syringes, heart-lung
machine components, sutures, and catheters are now sterilized with ethylene oxide gas. Ethylene
oxide (EtO) is both microbicidal and sporicidal and kills by combining with cell proteins. It is a
particularly effective sterilizing agent because it rapidly penetrates packing materials, even plastic
wraps. Sterilization is carried out in a special ethylene oxide sterilizer, very much resembling an
autoclave in appearance, that controls the EtO concentration, temperature, and humidity. Because
pure EtO is explosive, it is usually supplied in a 10 to 20% concentration mixed with either CO2 or
dichlorodifluoromethane. The ethylene oxide concentration, humidity, and temperature influence
the rate of sterilization. A clean object can be sterilized if treated for 5 to 8 hours at 38°C or 3 to 4
hours at 54°C when the relative humidity is maintained at 40 to 50% and the EtO concentration at
700 mg/liter. Extensive aeration of the sterilized materials is necessary to remove residual EtO
because it is so toxic.
Betapropiolactone (BPL) is occasionally employed as a sterilizing gas. In the liquid form it
has been used to sterilize vaccines and sera. BPL decomposes to an inactive form after several
hours and is therefore not as difficult to eliminate as EtO. It also destroys microorganisms more
readily than ethylene oxide but does not penetrate materials well and may be carcinogenic. For
these reasons, BPL has not been used as extensively as EtO.
Recently vapor-phase hydrogen peroxide has been used to decontaminate biological safety
cabinets.
Describe about the history of taxonomy
In the mid-1700s, Swedish botanist Carl Linnaeus was one of the first scientists to develop a
taxonomy for living organisms. It is for this reason that he is known as the father of taxonomy.
Linnaeus’ taxonomy grouped living things into two kingdoms: plants and animals.
By the 1900s, scientists had discovered microorganisms that had characteristics that were
dramatically different than those of plants and animals. Therefore, Linnaeus’ taxonomy needed to
be enhanced to encompass microorganisms. In 1969 Robert H. Whitteker, working at Cornell
University, proposed a new taxonomy that consisted of five kingdoms. These were monera,
protista, plantae (plants), fungi, and animalia (animals). Monera are organisms that lack a nucleus
and membrane-bounded organelles, such as bacteria. Protista are organisms that have either a
single cell or no distinct tissues and organs, such as protozoa. This group includes unicellular
eukaryotes and algae. Fungi are organisms that use absorption to acquire food. These include
multicellular fungi and single-cell yeast. Animalia and plantae include only multicellular
organisms. Scientists widely accepted Whitteker’s taxonomy until 1977 when Carl Woese, in
collaboration with Ralph S. Wolfe at the University of Illinois, proposed a new six-kingdom
taxonomy. This came about with the discovery of archaea, which are prokaryotes that lives in
oxygen-deprived environments.
Whitte
ker’s
fivekingdo
m
taxono
my.
B
efore
Woese’
s sixkingdo
m
taxono
my,
scientist
s
grouped
organisms into eukaryotes animals, plants, fungi, and one-cell microorganisms (paramecia)— and
prokaryotes (microscopic organisms that are not eukaryotes). Woese’s six-kingdom taxonomy
consists of:
• Eubacteria (has rigid cell wall)
• Archaebacteria (anaerobes that live in swamps, marshes, and in the intestines of mammals)
• Protista (unicellular eukaryotes and algae)
• Fungi (multicellular forms and single-cell yeasts)
• Plantae
• Animalia
Woese determined that archaebacteria and eubacteria are two groups by studying the rRNA
sequences in prokaryotic cells.
Woese used three major criteria to define his six kingdoms. These are:
• Cell type. Eukaryotic cells (cells having a distinct nucleus) and prokaryotic cell (cells not having a
distinct nucleus)
• Level of organization. Organisms that live in a colony or alone and one-cell organisms and
multicell organisms.
• Nutrition. Ingestion (animal), absorption (fungi), or photosynthesis (plants).
In the 1990s Woese studied rRNA sequences in prokaryotic cells (archaebacteria
and eubacteria) proving that these organisms should be divided into two distinct groups. Today
organisms are grouped into three categories called domains that are represented as bacteria, archaea,
and eukaryotes.
The domains are placed above the phylum and kingdom levels. The term archaebacteria
(meaning from the Greek word archaio “ancient”) refers to the ancient origin of this group of
bacteria that appears to have diverged from eubacteria. The archaea and bacteria came from the
development of eukaryotic organisms.
The evolutionary relationship among the three domains is:
• Domain Bacteria (eubacteria)
• Domain Archaea (archaebacteria)
• Domain Eulcarya (eukaryotes)
Different classifications of organisms are:
• Bacteria
• Eubacteria
• Archaea
• Archaebacteria
• Eukarya
• Protista
• Fungi
• Plantae
• Animalia
The three domains are archaea, bacteria, and eukarya .
• Archaea lack muramic acid in the cell walls.
• Bacteria have a cell wall composed of peptidoglycan and muramic acid. Bacteria also have
membrane lipids with ester-linked, straight-chained fatty acids that resemble eukaryotic membrane
lipids. Most prokaryotes are bacteria. Bacteria also have plasmids, which are small, double-stranded
DNA molecules that are extrachromosomal.
• Eukarya are of the domain eukarya and have a defined nucleus and membrane bound organelles.
Threedomain
taxono
my
Summa
rize the
advant
ages of
using
each
major
group
of
charact
eristics
(morph
ological, physiological/metabolic, ecological, genetic, and molecular) in classification and
identification. How is each group related to the nature and expression of the genome? Give
examples of each type of characteristic.
Morphological Characteristics
Morphological features are important in microbial taxonomy for many reasons. Morphology
is easy to study and analyze, particularly in eucaryotic microorganisms and the more complex
procaryotes. In addition, morphological comparisons are valuable because structural features
depend on the expression of many genes, are usually genetically stable, and normally (at least in
eucaryotes) these do not vary greatly with environmental changes. Thus morphological similarity
often is a good indication of phylogenetic relatedness.
Many different morphological features are employed in the classification and identification
of microorganisms. Although the light microscope has always been a very important tool, its
resolution limit of about 0.2 _m reduces its usefulness in viewing smaller microorganisms and
structures. The transmission and scanning electron microscopes, with their greater resolution, have
immensely aided the study of all microbial groups.
Some Morphological Features Used in Classification and Identification
Physiological and Metabolic Characteristics
Physiological and metabolic characteristics are very useful because they are directly related
to the nature and activity of microbial enzymes and transport proteins. Since proteins are gene
products, analysis of these characteristics provides an indirect comparison of microbial genomes.
Some Physiological and Metabolic Characteristics Used in Classification and Identification
Molecular Characteristics
Some of the most powerful approaches to taxonomy are through the study of proteins and
nucleic acids. Because these are either direct gene products or the genes themselves, comparisons of
proteins and nucleic acids yield considerable information about true relatedness. These more recent
molecular approaches have become increasingly important in procaryotic taxonomy.
Comparison of Proteins
The amino acid sequences of proteins are direct reflections of mRNA sequences and
therefore closely related to the structures of the genes coding for their synthesis. For this reason,
comparisons of proteins from different microorganisms are very useful taxonomically. There are
several ways to compare proteins. The most direct approach is to determine the amino acid
sequence of proteins with the same function. The sequences of proteins with dissimilar functions
often change at different rates; some sequences change quite rapidly, whereas others are very stable.
Nevertheless, if the sequences of proteins with the same function are similar, the organisms
possessing them are probably closely related. The sequences of cytochromes and other electron
transport proteins, histones, heat-shock proteins, transcription and translation proteins, and a variety
of metabolic enzymes have been used in taxonomic studies. Because protein sequencing is slow and
expensive, more indirect methods of comparing proteins frequently have been employed. The
electrophoretic mobility of proteins is useful in studying relationships at the species and subspecies
levels. Antibodies can discriminate between very similar proteins, and immunologic techniques are
used to compare proteins from different microorganisms.
The physical, kinetic, and regulatory properties of enzymes have been employed in
taxonomic studies. Because enzyme behavior reflects amino acid sequence, this approach is useful
in studying some microbial groups, and group-specific patterns of regulation have been found.
Nucleic Acid Base Composition
Microbial genomes can be directly compared, and taxonomic similarity can be estimated in
many ways. The first, and possibly the simplest, technique to be employed is the determination of
DNA base composition. DNA contains four purine and pyrimidine bases: adenine (A), guanine (G),
cytosine (C), and thymine (T). In double-stranded DNA, A pairs with T, and G pairs with C. Thus
the (G +C )/(A_T) ratio or G +C content, the percent of G +C in DNA, reflects the base sequence
and varies with sequence changes as follows:
The base composition of DNA can be determined in several ways. Although the G +C
content can be ascertained after hydrolysis of DNA and analysis of its bases with high-performance
liquid chromatography (HPLC), physical methods are easier and more often used. The G +C
content often is determined from the melting temperature (Tm) of DNA. In double-stranded DNA
three hydrogen bonds join GC base pairs, and two bonds connect AT base pairs (see section 11.2).
As a result DNA with a greater G +C content will have more hydrogen bonds, and its strands will
separate only at higher temperatures—that is, it will have a higher melting point. DNA melting can
be easily followed spectrophotometrically because the absorbance of 260 nm UV light by DNA
increases during strand separation. When a DNA sample is slowly heated, the absorbance increases
as hydrogen bonds are broken and reaches a plateau when all the DNA has become single stranded.
The midpoint of the rising curve gives the melting temperature, a direct measure of the G +C
content. Since the density of DNA also increases linearly with G +C content, the percent G +C can
be obtained by centrifuging DNA in a CsCl density gradient.
The G +C content of many microorganisms has been determined (table 19.5). The G +C
content of DNA from animals and higher plants averages around 40% and ranges between 30 and
50%. In contrast, the DNA of both eucaryotic and procaryotic microorganisms varies greatly in G
+C content; procaryotic G _C content is the most variable, ranging from around 25 to almost 80%.
Despite such a wide range of variation, the G +C content of strains within a particular species is
constant. If two organisms differ in their G +C content by more than about 10%, their genomes
have quite different base sequences. On the other hand, it is not safe to assume that organisms with
very similar G +C contents also have similar DNA base sequences because two very different base
sequences can be constructed from the same proportions of AT and GC base pairs. Only if two
microorganisms also are alike phenotypically does their similar G +C content suggest close
relatedness.
A DNA Melting Curve. The Tm is indicated.
G_C content data are taxonomically valuable in at least two ways. First, they can confirm a
taxonomic scheme developed using other data. If organisms in the same taxon are too dissimilar in
G +C content, the taxon probably should be divided. Second, G +C content appears to be useful in
characterizing prokaryotic genera since the variation within a genus is usually less than 10% even
though the content may vary greatly between genera. For example, Staphylococcus has a G _C
content of 30 to 38%, whereas Micrococcus DNA has 64 to 75% G +C ; yet these two genera of
gram-positive cocci have many other features in common.
Nucleic Acid Hybridization
The similarity between genomes can be compared more directly by use of nucleic acid
hybridization studies. If a mixture of singlestranded DNA formed by heating dsDNA is cooled and
held at a temperature about 25°C below the Tm, strands with complementary base sequences will
reassociate to form stable dsDNA, whereas noncomplementary strands will remain single. Because
strands with similar, but not identical, sequences associate to form less temperature stable dsDNA
hybrids, incubation of the mixture at 30 to 50°C below the Tm will allow hybrids of more diverse
ssDNAs to form. Incubation at 10 to 15°C below the Tm permits hybrid formation only with almost
identical strands.
Nucleic Acid Melting and Hybridization.
.
In one of the more widely used hybridization techniques, nitrocellulose filters with bound
nonradioactive DNA strands are incubated at the appropriate temperature with single-stranded DNA
fragments made radioactive with 32P, 3H, or 14C. After radioactive fragments are allowed to
hybridize with the membrane-bound ss- DNA, the membrane is washed to remove any
nonhybridized ssDNA and its radioactivity is measured. The quantity of radioactivity bound to the
filter reflects the amount of hybridization and thus the similarity of the DNA sequences. The degree
of similarity or homology is expressed as the percent of experimental DNA radioactivity retained
on the filter compared with the percent of homologous DNA radioactivity bound under the same
conditions. Two strains whose DNAs show at least 70% relatedness under optimal hybridization
conditions and less than a 5% difference in Tm often are considered members of the same species.
Representative G + C Contents of Microorganisms
If DNA molecules are very different in sequence, they will not form a stable, detectable
hybrid. Therefore DNA-DNA hybridization is used to study only closely related microorganisms.
More distantly related organisms are compared by carrying out DNA-RNA hybridization
experiments using radioactive ribosomal or transfer RNA. Distant relationships can be detected
because rRNA and tRNA genes represent only a small portion of the total DNA genome and have
not evolved as rapidly as most other microbial genes. The technique is similar to that employed for
DNA-DNA hybridization: membrane-bound DNA is incubated with radioactive rRNA, washed,
and counted. An even more accurate measurement of homology is obtained by finding the
temperature required to dissociate and remove half the radioactive rRNA from the membrane; the
higher this temperature, the stronger the rRNA-DNA complex and the more similar the sequences.
Nucleic Acid Sequencing
Despite the usefulness of G _ C content determination and nucleic acid hybridization
studies, genome structures can be directly compared only by sequencing DNA and RNA.
Techniques for rapidly sequencing both DNA and RNA are now available; thus far RNA
sequencing has been used more extensively in microbial taxonomy.
Most attention has been given to sequences of the 5S and 16S rRNAs isolated from the 50S
and 30S subunits, respectively, of procaryotic ribosomes (see sections 3.3 and 12.2). The rRNAs
are almost ideal for studies of microbial evolution and relatedness since they are essential to a
critical organelle found in all microorganisms. Their functional role is the same in all ribosomes.
Furthermore, their structure changes very slowly with time, presumably because of their constant
and critical role. Because rRNA contains variable and stable sequences, both closely related and
very distantly related microorganisms can be compared. This is an important advantage as distantly
related organisms can be studied only using sequences that change little with time.
There are several ways to sequence rRNA. Ribosomal RNAs can be characterized in terms
of partial sequences by the oligonucleotide cataloging method as follows. Purified, radioactive 16S
rRNA is treated with the enzyme T1 ribonuclease, which cleaves it into fragments. The fragments
are separated, and all fragments composed of at least six nucleotides are sequenced. The sequences
of corresponding 16S rRNA fragments from different procaryotes are then aligned and compared
using a computer, and association coefficients (Sab values) are calculated. Complete rRNAs now
are sequenced using procedures like the following. First, RNA is isolated and purified. Then,
reverse transcriptase is used to make complementary DNA (cDNA) using primers that are
complementary to conserved rRNA sequences. Next, the polymerase chain reaction amplifies the
cDNA. Finally, the cDNA is sequenced and the rRNA sequence deduced from the results.
Write about Bergey’s manual of systametic bacteriology
In 1923, David Bergey, professor of bacteriology at the University of Pennsylvania, and
four colleagues published a classification of bacteria that could be used for identification of
bacterial species, the Bergey’s Manual of Determinative Bacteriology. This manual is now in its
ninth edition. The first edition of Bergey’s Manual of Systematic Bacteriology, a more detailed
work that contains descriptions of all procaryotic species currently identified, also is available. The
first volume of the second edition has been published recently. This section briefly describes the
current edition of Bergey’s Manual of Systematic Bacteriology (or Bergey’s Manual) and then
discusses at more length the new second edition.
The First Edition of Bergey’s Manual of Systematic Bacteriology
Because it has not been possible in the past to classify prokaryotes satisfactorily based on
phylogenetic relationships, the system given in the first edition of Bergey’s Manual of Systematic
Bacteriology is primarily phenetic. Each of the 33 sections in the four volumes contains procaryotes
that share a few easily determined characteristics and bears a title that either describes these
properties or provides the vernacular names of the procaryotes included. The characteristics used to
define sections are normally features such as general shape and morphology, Gram-staining
properties, oxygen relationship, motility, the presence of endospores, the mode of energy
production, and so forth. Procaryotic groups are divided among the four volumes in the following
manner: (1) gramnegative bacteria of general, medical, or industrial importance; (2) gram-positive
bacteria other than actinomycetes; (3) gramnegative bacteria with distinctive properties,
cyanobacteria, and archaea; and (4) actinomycetes (gram-positive filamentous bacteria).
Gram-staining properties play a singularly important role in this phenetic classification; they
even determine the volume into which a species is placed. There are good reasons for this
significance. As noted in chapter 3, Gram staining usually reflects fundamental differences in
bacterial wall structure. Gram-staining properties also are correlated with many other properties of
bacteria. Typical gram-negative bacteria, gram-positive bacteria, and mycoplasmas (bacteria
lacking walls) differ in many characteristics, as can be seen in. For these and other reasons, bacteria
traditionally have been classified as gram positive or gram negative. This approach is retained to
some extent in more phylogenetic classifications and is a useful way to think about bacterial
diversity.
The Second Edition of Bergey’s Manual of Systematic Bacteriology
There has been enormous progress in procaryotic taxonomy since 1984, the year the first
volume of Bergey’s Manual of Systematic Bacteriology was published. In particular, the sequencing
of rRNA, DNA, and proteins has made phylogenetic analysis of prokaryotes feasible. As a
consequence, the second edition of Bergey’s Manual will be largely phylogenetic rather than
phonetic and thus quite different from the first edition. Although the new edition will not be
completed for some time, it is so important that its general features will be described here.
Undoubtedly the details will change as work progresses, but the general organization of the new
Bergey’s Manual can be summarized.
The second edition will be published in five volumes. It will have more ecological
information about individual taxa. The second edition will not group all the clinically important
prokaryotes together as the first edition did. Instead, pathogenic species will
be placed phylogenetically and thus scattered throughout the following five volumes.
Volume 1—The Archaea, and the Deeply Branching and
Phototrophic Bacteria
Volume 2—The Proteobacteria
Volume 3—The Low G _ C Gram-Positive Bacteria
Volume 4—The High G _ C Gram-Positive Bacteria
Volume 5—The Planctomycetes, Spirochaetes, Fibrobacteres, Bacteroidetes, and Fusobacteria
(Volume 5 also will contain a section that updates descriptions and phylogenetic arrangements that
have been revised since publication of volume 1.)
The second edition’s five volumes will have a different organization than the first edition.
The greatest change in organization of the volumes will be with respect to the gram-negative
bacteria. The first edition describes all gram-negative bacteria in two volumes. Volume 1 contains
the gram-negative bacteria of general, medical or industrial importance; volume 3 describes the
archaea, cyanobacteria, and remaining gram-negative groups. The second edition describes the
gram-negative bacteria in three volumes, with volume 2 reserved for the proteobacteria. The two
editions treat the gram-positive bacteria more similarly. Although volume 2 of the first edition does
have some high G _ C bacteria, much of its coverage is equivalent to the new volume 3. Volume 4
of the first edition describes the actinomycetes and is similar to volume 4 of the second edition
(high G _ C gram-positive bacteria), although the new volume 4 will have broader coverage. For
example, Micrococcus and Corynebacterium are in volume 2 of the first edition and will be in
volume 4 of the second edition. summarizes the planned organization of the second edition and
indicates where the discussion of a particular group may be found in this textbook depicts the major
groups and their relatedness to each other
Discuss in detail about the classification of fungi
Non-vascular, achlorophyllous plants
Multicellular eukaryotic
Reproduce by spores
Lack-root, stem and leaves
Little tissue differentiation
Heterotrophic
Saprophytes or parasites
THE BODY PLAN OF FUNGI
Vegetative body consists of mycelia made up of networks of hyphae
Hyphae - Long treads of cells designed to maximize surface area and also transport
nutrients
Fungus-like protists:
 Lack this body structure
 Lack cell walls of chitin
HYPHAE
Hyphae are designed to increase the surface area of fungi and thus facilitate absorption
May grow fast, up to 1 km per day, as they spread throughout a food source
Haustoria - Specialized structures budding off hyphae of parasitic fungi which penetrate
host cells to absorb nutrients
May be coenocytic, having no septa between cells, or septa may be present with pores
through which cytoplasm can flow moving nutrients through out the fungus
Hyphae
Pores
Septa
Coenocytic
MYCETAE
DIVISION
II. MASTIGOMYCOTA
I. GYMNOMYCOTA
III. AMASTOGOMYCOTA
SUBDIVISION
1. Acarisio - ina
CLASS
a) Acarisiomycetes
EXAMPLE
-- Dictyostelium
2. Plasmodio - ina
a) Protosteliomycetes
-- Nematostelium
b) Myxomycetes
-- Ceratomyxa
1. Haplo - ina
2. Diplo - ina
a) Chytridiomycetes
a) Oomycetes
-- Synchitrydium
-- Saproleginia
b) Hypochytridium
-- rhizidiomycetes
c) Plasmodiophoromycetes
-- Plasmodiophora
1. Zygo --ina
2. Asco - ina
a) Zygomycetes
Deutromycetes
a) Ascomycetes
-- Rhizopus
Fusarium
-- Penicillium
b) Trichomycetes
3. Basidio -ina
a) Basidiomycetes
-- Agaricus
4. Deutro - ina
a)
--
-- Harpella
Division I: GYMNOMYCOTA
Phogotrophic mode of nutrition
cell wall absent – somatic structure
Two subdivisions
Subdivision 1: Acarisiogymnomycotina (Cellular slime moulds)

Somatic structure are seen as myxamoeba

They fuse together – pseudopodium

Sporocarp develops from pseudopodium

Single class
Class a) Acarasiomycetes

flagellated cells are not seen except one species

Sporocarp usually stalked

Sexual reproduction through macrocysts
 e.g., Dictyostelium, Polysphondylium
Subdivision 2: Plasmodiogymnomycotina (True slimemoulds)
 Somatic structure – simple myxamoeba
 Fuse to form true pseudopodium
 Two classes
Class a: Protostelomycetes
 Sporocarp develops from myxamoeba /
plasmodia
 No sexual reproduction
 e.g., Nematostelium
Class b: Myxomycetes
 Myxamoeba / flagellated cells fuse – Zygotes
 Mytotic division – true plasmodium
 Sexual reproduction seen
 e.g., Ceratomyxa, Stemonitis
Division II: MASTOGOMYCOTA
Have centriole
Flagellated cells are produced during life cycle
Nutrition – absorptive type
Asexual – Zoospore formation
Two subdivisions
Subdivision 1: Haplomastigomycotina

Uni / biflagellate

Life cycle – haplobiontic haploid / diplobiontic diploid

Three classes
Class a: Chitridiomycetes
 Single posterior flagella – whiplash type
 e.g., Synchytridium
Class b: Hypochridiomycetes
 Aquatic habitats
 Single anterior flagella – tinsel type

e.g., Rhizidiomycetes
Class c: Plasmodiophoromycetes
 Parasitic
 Two anterior flagella – whiplash type
 e.g., Plasmodiophora
Subdivision 2: Diplomastigomycotina

Biflagellated zoosporres

Life cycle – haplobiontic diploid

Singleclass
Class a: Oomycetes (water moulds)
 Cell wall – glucans and cellulose
 Twoflagella (anterior) – one whiplash and one tinsel
 e.g., Sporolegnia
Division III: AMASTOGOMYCOTA
Lack centriole
No motile cells
Mycelium – septate / aseptate
Asexual – budding, fragmentation, conidia formation
Four subdivisions
Subdivision 1: Zygomycotina

Saprobic, parasitic or predatory fungi

Asexual - sporangiospores

Sexual – unequal gametangia fuse - Zygospore

Two classes
Class a: Zygomycetes
 Terriatrial
 Saprophytes / parasites
 Asexual - aplanospores
 e.g., Rhizopus, Mucor
Class b: Trichomycetes
 Symbionts  No sexual reproduction

e.g., Harpella
Subdivision 2: Ascomycotina

Parasitic or Saprophyticy fungi

Uni / Multi cellular, Multi - septate

Meospores (Ascospores) – sac like cells

Single class
Class a: Ascomycetes
 Sexual reproduction - conidia
 e.g., Saccharomyces, Penicillium
Subdivision 3: Basidiomycotina

Meiospores (Basidiospores) - basidiocarp

Asexual - sporangiospores

Single class
Class a: Basidiomycetes
 Long diacaryotic somatic phase
 e.g., Agaricus, Pleurotus
Subdivision 4: Deutromycotina

Parasitic, saprophytic / symbionts some times

Mycelium - septate

Asexual – conidia

Sexual – unknown

Single class
Class a: Deutromycetes
 e.g., Fusarium
Discuss in detail about the classification of algae
Fritsh – 1935
Criteria
1. Organization of membrane bound cell organelles
2. Composition of cell wall
3. Types of photosynthetic pigments
4. Types, number, arrangement and orientation of flagella
5. types of reserve food material
6. types of sexual reproduction
Classes
1. Chlorophyceae (grass-green)
2. Xanthophyceae (yellow-green)
3. Chrysophysea (brown / orange)
4. Bacillarophyceae (yellow / golden brown)
5. Cryptophyceae (nearly brown)
6. Dinophyceae (dark yellow / brown)
7. Chloromonadiae (bright green)
8. Euglininae (pure green)
9. Phaeophyceae (brown)
10. Rhodophyceae (red)
11. Myxophyceae / Cyanophyceae (blue green)
1. Chlorophyseae
Pigments: Chlorophyll a & b + two yellow pigments (carotenoids)
Res food: starch
Structure: motile - 2 to 4 flagella (isokontae), heterotrichous filaments, cell wall –
cellulose, Pyrenoids – surrounded by starch sheath
Reproduction: isogamous to oogamous type (Sexual)
Habitats: mostly fresh water, few marine
e.g., Chlamydomonas, Stigeoclonium
2. Xanthophyseae
Pigments: Yellow xanthophyll found abundantly
Res food: oil
Structure: unicellular motile to simple filamentous, cell wall – petin rich (two equal /
unequal pieces with overlapping edges), Flagella – Heterokontae, pyrenoid absent
Reproduction: isogamous (Sexual-rare)
Habitats: mostly fresh water, few marine
e.g., Botrydium
3. Chrysophyceae
Pigments: brow / orange color chromatophores, accessory pigments – phycochrysin
Res food: Fat, Lucosin
Structure: unicellular motile to branched filamentous, flagella – unequal, anterior
Habitats: colder fresh water, few marine
e.g., Phaeothmnion
4. Bacillarophyceae
Pigments: yellow / golden yellow chromatophores
Res food: fat & volutin
Structure: Unicellular / colonial, cell wall – silica & pectin, two halves, ornamented
Reproduction: Diploid, Special type – fusion of two protoplast
Habitats: Fresh water, sea, soil and terrestrial
e.g., Pinnularia
5. Cryptophyceae
Pigments: diverse with brown shade
Res food: Solid carbohydrates, starch
Structure: motile – slightly unequal flagella, coccoid mostly
Reproduction: Isogamous type
Habitats: Both marine and fresh water
E.g., Cryptomonas
6. Dinophyceae
Pigments: dark yellow, discoid
Res food: starch / oil
Structure: unicellular, motile – flagella two furrows
Reproduction: Isogamous type, rare
Habitats: Sea water plantons, few fresh water forms
e.g., Gyrodinium
7. Chloromonadinea
Pigments: bright green, excess xanthophylls, numerous, discoid
Res food: oil
Structure: motile, flagella – almost equal
Habitats: fresh water forms only
8. Eugleninae
Pigments: Pure green, several in each cell
Res food: Polysaccharide, paramylon
Structure: anterior flagella – arise from base of canal, complex vascular system,
predominant nucleus
Habitats: Only fresh water forms are known
9. Phaeophyceae
Pigments: Brow with yellow – fucoxanthin
Res food: Alcohols (mannitol), polysaccharide (laminarin), fat
Structure: simple filamentous to bulky paranchymatous form, several attain giant sized
with ext and int differentiation
Reproduction: Isogamous to oogamous type, male gamete- two laterally attached flagella
e.g., Sargassum
10. Rhodophyceae
Pigments; red (phycoerythrin), blue (phycocyanin)
Res food: Floridean starch, a polysaccharide similar to starch
Structure: Simple filamentous to complex, motile not known
Reproduction: Advanced oogamous type, male – non motile gametes, female – long
receptive neck, carpospores produced
Habitats: mostly marine, few freshwater
e.g., Gracillaria
11. Myxophyceae
Pigments: Chlorophyll, carotin, phycocyanin and phycoerythrin
Res food: sugar and glycogen
Structure: Simple to filamentous, filaments shows false branching, no motile stages
known, chromatophores diffused all over cytoplasm
Reproduction: no sexual reproduction
Habitats: mostly fresh water
e.g., Nostoc, Anabena
Diagrammatic Algal Bodies: (a) unicellular, motile, Cryptomonas; (b) unicellular,
nonmotile, Palmellopsis; (c) colonial, Gonium; (d) filamentous, Spirotaenia; (e)
bladelike kelp, Monostroma; (f) leafy tubular axis, branched tufts or plumes,
Stigeoclonium; (g) unicellular, nonmotile, Chrysocapsa.
Chlorophyta (Green Algae); Light Micrographs. (a) Chlorella, a unicellular nonmotile
green alga. (b) Volvox, a typical green algal colony. (c) Spirogyra, a filamentous green
alga. Four filaments are shown. Note the ribbonlike, spiral chloroplasts within each
filament. (d) Ulva, commonly called sea lettuce, has a leafy appearance. (e) Acetabularia,
the mermaid’s wine goblet. (f ) Micrasterias, a large desmid.
Chlamydomonas: The Structure and Life Cycle of This Motile Green Alga. During
asexual reproduction, all structures are haploid; during sexual reproduction, only the
zygote is diploid.
Euglena. A Diagram Illustrating the Principal Structures Found in This Euglenoid.
Notice that a short second flagellum does not emerge from the anterior invagination. In
some euglenoids both flagella are emergent.
Chrysophyta (Yellow-Green and Golden-Brown Algae; Diatoms). (a) Scanning
electron micrograph of Mallomonas, a chrysophyte, showing its silica scales. The scales
are embedded in the pectin wall but synthesized within the Golgi apparatus and
transported to the cell surface in vesicles. (b) Ochromonas, a unicellular chrysophyte.
Diagram showing typical cell structure. (c) Scanning electron micrograph of a diatom,
Cyclotella meneghiniana. (d) Assorted diatoms as arranged by a light microscopist.
Phaeophyta (Brown Algae). Diagram of the parts of the brown alga, Nereocystis.
Due to the holdfast organ, the heaviest tidal action and surf seldom dislodge brown algae
from their substratum. The stipe is a stalk that varies in length; the bladder is a gas-filled
float.
Rhodophyta (Red Algae). These algae (e.g., Corallina gracilis) are much smaller
and more delicate than the brown algae. Most red algae have a filamentous, branched
morphology as seen here.
Dinoflagellates. (a) Ceratium. (b) Scanning electron micrograph of Gymnodinium.
Notice the plates of cellulose and the two flagella: one in the transverse groove and the
other projecting outward.
Write about the outline classification of protozoa
Drawings of Some Representative Protozoa. (a) Structure of the flagellate,
Trypanosoma brucei rhodesiense. (b) The structure of the amoeboid protist, Amoeba
proteus. (c) Structure of an apicomplexan sporozoite. (d) Structure of the ciliate
Paramecium caudatum.
Explain in detail about the classification of biological data
• Collection of both experimental & theoretical data in an organized manner
so that its contents can be easily
– accessed
– managed
– updated
– Retrieved
Broad classification of Biological Databases
Sequence databases
• Common data bases
• Familiar with molecular biologist
Sequence databases
Annotate
d
E.g. Genebank,
Low-Annotated
E.g. EST
Specialize
E.g.dRDP, rRNA
EMBL, TrEMBL
and SWISS-PROT
Gene Bank
• created in 1988 as part of the National Library of Medicine at NIH,
Bethesda, Maryland
• collection of nucleotide and protein sequences publicly available
• maintained by NCBI (www.ncbi.nlm.nih.gov)
• Release 127.0 on December 15, 2001- approx 15, 850, 000, 000 bases in 105,
000 different organisms
• Release every 2 months
EMBL ,TrEMBL & DDBJ
• EMBL – div EBI, Hinxton Hall, U.K.
 Release 69 on 1st December 2001 – 14, 366, 182 sequences
• DDBJ, Mishma, Japan
 Release 48 in January 2002 – 15, 016, 100 sequences
SWISS-PROT (1986)
• maintained by Amos Bairoch’s group, Geneva, Switzerland along with EBI
• Manually created data base
• Simple keyword searching
• Release 40.9 on 31st January 2002 – 104,948 sequences.
PIR
• National Biotechnology Research Foundation, America
• Independent protein data base
• differ from Swiss-Prot
• Less convenient for text searching
EST (Expressed Sequence Tags)
– obtained by single pass sequencing of 3’ to 5’ end of cDNAs
– Incomplete, poor quality with frequent sequencing errors
– Useful to identify new genes and new functions
Ribosomal RNA databases
• rRNA very useful molecule in evolution study
• This sequence can be aligned on basis of its secondary structure to produce
multiple sequence alignments – phylogenetic tree can be built
• Useful for characterizing and classifying a new bacteria
• RDP (Michigan State University, USA)
• rRNA database (University of Antwerp, Belgium)
Structural databases
•
PDP – major repository of protein structures & some extend of nucleic acid
structures
• This database stored 3 dimensional atomic coordinators of protein and
nucleic acids
• Data obtained by experimental mehtods like X-ray crystallography, NMR or
computer modeling
• records similar to Genebank entries
• Seq similarity search such as BLAST also be used
• Entry also contains secondary structure information like location of helices
and strands and disulfide bonds
• Position of the atoms in various axes (i.e., X, Y and Z) also given in three
dimensional structure
• RasMol (by Roger Sayle) programs – allows drawing includes spacefill, ball
and stick, wireframe views, ribbon diagrams and cartoon diagrams which
emphasize secondary structure
• Others – SCOP (Structural Classification of Proteins) – classify the protein
according to the structural similarity and evolutionary relationship.
Motif databases
• Pattern of conservation in a group of sequences – reflects function
• Consensus sequences – allowed / not
• Hidden Markov models (HMMs) – statistical analysis
• TRANSFAC
• PROSITE
Genome databases
• E.g., GDB (Human), Flybase (Drosophila), AceDB (C.elegans), SGD
(S.cerevisiae)
Proteome databases
• E.g., SWISS 2D PAGE
RNA expression databases
Change in specific mRNA content under certain condition
Others
• There are many databases that cannot be classified in the categories listed
previously;
• Examples: ReBase (restriction enzymes), TRANSFAC (transcription factors), OGLYCBASE (O-linked sugars), Protein-protein interactions db (DIR),
biotechnology patents db, etc.;
• As well as many other resources concerning any aspects of macromolecules and
molecular biology.
• GenBank database (http://www.ncbi.nih.gov/Genbank/)
• EMBL nucleotide sequence database (http://www.ebi.ac.uk/embl/)
• SWISS-PROT (http://us.expasy.org/sprot/sprot-top.html)
• PDB (http://www.pdb.org)
What is blast? Explain in detail about it
In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an
algorithm for comparing primary biological sequence information, such as the aminoacid sequences of different proteins or the nucleotides of DNA sequences.
A BLAST search enables a researcher to compare a query sequence with a library
or database of sequences, and identify library sequences that resemble the query sequence
above a certain threshold.
For example, following the discovery of a previously unknown gene in the mouse,
a scientist will typically perform a BLAST search of the human genome to see if humans
carry a similar gene; BLAST will identify sequences in the human genome that resemble
the mouse gene based on similarity of sequence.
Algorithms and software

BLAST is about 50 times faster than the dynamic programming; however, it
cannot guarantee to find the best alignments of the query and database sequences
as in the dynamic programming. BLAST is more time efficient than FASTA by
searching only for the more significant patterns in the sequences.

The main idea of BLAST is that there are often high-scoring segment pairs (HSP)
contained in a statistically significant alignment.
BLAST searches for high scoring sequence alignments between the query
sequence and sequences in the database using a heuristic approach that
approximates the Smith-Waterman algorithm.
The exhaustive Smith-Waterman approach is too slow for searching large
genomic databases such as GenBank. Therefore, the BLAST algorithm uses a
heuristic approach that is less accurate than the Smith-Waterman but over 50
times faster.
The algorithm of BLASTP (a protein to protein search) is introduced to present
the concept of BLAST.



1. Remove low-complexity region or sequence repeats in the query sequence.
Low-complexity region means a region of a sequence is composed of few kinds of
elements. These regions might give high scores that confuse the program to find the
actual significant sequences in the database, so they should be filtered out. The
regions will be marked with an X (protein sequences) or N (nucleic acid sequences)
and then be ignored by the BLAST program.
To filter out the low-complexity regions, the SEG program is used for protein
sequences and the program DUST is used for DNA sequences. On the other hand, the
program XNU is used to mask off the tandem repeats in protein sequences.
2. Make a k-letter word list of the query sequence.
Take k=3 for example, we list the words of length 3 in the query protein sequence (k
is usually 11 for a DNA sequence) “sequentially”, until the last letter of the query
sequence is included. The method can be illustrated in figure as given below.
The method to establish the k-letter query word list.
3. List the possible matching words.



This step is one of the main differences between BLAST and FASTA.
FASTA cares about all of the common words in the database and query
sequences; however, BLAST cares about only the high-scoring words.
The scores are created by comparing the word in the list in step 2 with all the
3-letter words. By using the scoring matrix to score the comparison of each
residue pair, there are 20^3 possible match scores for a 3-letter word. For
example, the score obtained by comparing PQG with PEG and PQA is 15 and
12, respectively. For DNA words, a match is scored as +5 and a mismatch as 4.
After that, a neighborhood word score threshold T is used to reduce the
number of possible matching words. The words whose scores are greater than
the threshold T will remain in the possible matching words list, while those
with lower scores will be discarded. For example, PEG is kept, but PQA is
abandoned when T is 13.
4. Scan the database sequences for exact match with the remaining high-scoring
words.

The BLAST program scans the database sequences for the remaining highscoring word, such as PEG, of each position. If an exact match is found, this
match is used to seed a possible ungapped alignment between the query and
database sequences.
5.Stastical scores

Stastical scores is useful to evaluate identified HSP sequence is more significant
or not bacause it represents a real biological or evolutionary relationship rather
than a chance of similarity between unrelated sequences.
p value is a value between unmatched sequences of identified similarity word.
The p value is less than the word score (S) indicates more significant.
A related quantity that is reported my most search software is the E (Expect)
value. E value is a value between matched sequences of identified similarity
word. The E value is more than word score indicates more significant.


6.Sencitivity and Specificity
Sensitivity and specificity is uaed to evaluate the results of data bases.
Relationship of sensitivity can be calcualted by
Sn = ntp / (ntp + nfn)
Where,
tp
is the true positive hits, fn is the false negative hits
Relationship of specificity can be calcualted by
Sp = ntp / (ntp + nfp)
Where,
tp
is the true positive hits, fn is the false positive hits
7.Database types
Programme
Query sequence
Database type
name
1
Blastp
Protein
Protein
2
Blastn
Nucleic acid
Nucleic acid
3
Blastx
Nucleic acid (translated)
Protein
4
Tblastn
Protein
Nucleic acid (translated)
5
Tblastx
Nucleic acid (translated)
Nucleic acid (translated)
6
Fasta
Protein or Nucleic acid
Protein or Nucleic acid
7
Tfastax
Protein
Nucleic acid (translated)
8
Fastx
Nucleic acid (translated)
Protein
8.BLAST alignment corresponding to high scoring similarity
S.NO.
GENE ID: 2855831 bla | protein coding
[Bacillus thuringiensis serovar konkukian str. 97-27] (10 or fewer
PubMed links)
Score = 469 bits (1208),
matrix adjust.
Expect = 5e-131, Method: Compositional
Identities = 226/246 (91%), Positives = 236/246 (95%), Gaps = 0/246
(0%)
Query 1
MKNTLLKLGVCVSLLGITPFVSTISSVQAERTVEHKVIKNETGTISISQLNKNVWVHTEL
60
MKNTLLKLGVCVSLLGITPFVSTISSVQAERTVEHKVIKNETGTISISQLNKNVWVHTEL
Sbjct 1
MKNTLLKLGVCVSLLGITPFVSTISSVQAERTVEHKVIKNETGTISISQLNKNVWVHTEL
60
Query 61
GYFSGEAVPSNGLVLNTSKGLVLVDSSWDDKLTKELIEMVEKKFKKRVTDVIITHAHADR
GYF+GEAVPSNGL+LNTSKGLVLVDSSWDDKLTKELI+M EKKFK
RVTDVIITHAHADR
Sbjct 61
GYFNGEAVPSNGLILNTSKGLVLVDSSWDDKLTKELIDMAEKKFKNRVTDVIITHAHADR
Query 121
IGGMKTLKERGIKAHSTALTAELAKKNGYEEPLGDLQSVTNLKFGNMKVETFYPGKGHTE
IGG+KTLKERGIK HST LTAELAKKNGYEEPLGDLQ++T
LKFGNMKVETFYPGKGHTE
Sbjct 121
IGGIKTLKERGIKTHSTTLTAELAKKNGYEEPLGDLQAITKLKFGNMKVETFYPGKGHTE
120
120
180
180
Query 181
DNIVVWLPQYQILAGGCLVKSASSKDLGNVADAYVNEWSTSIENVLKRYGNINLVVPGHG 240
DNIVVWLPQY +L GGCLVKSAS+KDLGN+ DAYVNEWSTSIENVLKRY NIN
VVPGHG
Sbjct 181
DNIVVWLPQYNMLVGGCLVKSASAKDLGNITDAYVNEWSTSIENVLKRYENINFVVPGHG 240
Query
241
Sbjct
241
EVGDRG
EVGD+G
EVGDKG
246
246
Write about the data retrival system
A large number of data available in www but its widely distributed. Therefore
certain efficient mechanism is needed to retrieve data. There are a number of data
retrieval tools can be used to access information. The most widely used systems are
Entrez and SRS.
ENTREZ
It is a WWW-based data retrieval system developed by NCBI. This system
integrates information held in all NCBI databases. These data bases include nucleotide
sequence data bases, protein sequences, macromolecular structures and whole genomes.
The resources linked in the NCBI can also searched using Entrez. These include Online
Mendelian Inheritance in Man (OMIM) and the literature database MEDLINE, through
PubMed. Entrez can be accessed via NCBI web site using following URL.
http:/ / www.ncbi.nlm.nih.gov/Entrez/.
The databases covered by Entrez
SNO
1.
Category
Nucleic acid sequences
2.
Protein sequences
3.
4.
5.
3D structures
Genomes
PopSet
6.
7.
8.
9.
10.
OMIM
Taxonomy
Books
Probe set
3D domains
11.
Literature
Databases
Entrez nucleotides: sequences obtained from gene
bank, Ref sequence and PDB
Entrez protein: sequences obtained from SWISSPROT, PIR, PRF, PDB and translation from
annotated coding region in GeneBank and Refseq
Entrez molecular modeling data base (MMDB)
Complete genome assembly from many sources
From GeneBank, set of DNA sequences that have
been to analyze the evolutionary relatedness of a
population
Online Mendilian Inheritance in Man
NCBI Taxonomy Data Base
Bookshelf
Gene Expression Omnibus (GEO)
Domain from the Entrez Molecular Modeling
Database
PubMED
Getting started with NCBI and Entrez
Entrez is a common front-end to all the data bases maintained by the NCBI. It is
extremely easy system to use. The main page of Entrez (similar in NCBI pages) is
undemanding in its browser requirements and downloads quickly. Part of the front page
illustrated in figure given below
The data bases available for the searching can be accessed by hyperlinks at the top
of the page or by using the drop-down menu. Once data bases are selected, a search term
then entered in the space provided. Clicking on ‘Go’ initiates the search. Hits the selected
data base are displayed (these are known as neighbors) and the matching records in other
Entrez databases are also shown (these are known as links). Further more, the following
URL provides an overview of Entrez and useful tutorial: http:/ /
www.ncbi.nlm.gov/Database/index.html.
SRS (Sequence Retrival System)
This retrieval tool developed by European Bioinformatics Institute (EBI). It
integrates over 80 molecular biology data bases. SRS can be accessed using following
URL.
http:/ / srs.ebi.ac.uk/
The difference between SRS and Entrez, SRS is that SRS is open source
software that can be downloaded and installed locally.
The important databases covered by SRS
SRS description
Literature
Sequence
InterPro & Related
SeqRelated
TransFac
User Owned Databanks
Application Results
Protein 3D struct
Genome
Mapping
Mutations
SNP
Examples
MEDLINE, GO, GOA
EMBL, EMBLNEW, SWISSPROT, SPTREMBL,
REMTREMBL, TREMBLNEW, ENSEMBL, PATENT,
PRT, USPO_PRT, IMGTLIGM, IMGTHLA
INTERPRO, IPRMATCHES, PROSITE,
PROSITEDOC.BLOCKS, PRINTS, PFAMA, PFEMA,
PFAMSEED, PRODOM
UTR, UTRSITE, TAXONOMY, GENETICCODE, EPD,
CPGISLAND, EMBLALIGN, EMESTLIB
TFSITE, TFFACTOR, TECELL, TFCLASS, TFMATRIX,
TFGENE
USERDNA, USERPROTEIN
FASTA, FASTX, FASTY, NFASTA, BLASTP, BLASTN,
CLUSTALW, NCLUSTALW, PPSEARCH,
RESTRICTION MAP
PDB, DSSP, HSSP, FSSP, PDBFINDER, RESID
MOUSE2HUMAN, LOCUSLINK, HGNC, HSAGENES
RHDB, RHDBNEW, RHEXP, RHMAP, RHPANEL,
OMIMMAP
OMIMALLELLE, MUTRES, SWISSCHANGE,
EMBLCHANGE, MUTRESSTATUS, OMIM,
OMIMOFFSET, HUMUT, P53LINK
MITSNP, dbSNPSubmitter, dbSNPAssay, dbSNPSNP,
HGBASE
Metabolic Pathways
Others
System
LENZYME, LCOMPOUND, PATHWAY, ENZYME,
EMP, MPW, UPATHWAY, UREACTION, UENZYME,
UCOMPOUND, UIMAGEMAP
REBASE, SRSFAQ, BIOCATAL
PRISMASTATUS
Using SRS
To start SRS, open SRS home page using URL http://srs6.ebi.ac.uk. On the www
version, the top page lists 17 classifications. By clicking adjacent ‘+’ symbol, each
classification can be expanded, reveal the associated data bases, and by clicking on the ‘‘symbol, the classification can be collapsed.
Note that the SRS classifications are grouped by the type of data but entrez
classification are grouped by the type of molecule. Therefore, SRS sequence libraries
covers all sequences (nucleotide and protein).
What is fasta format? Explain
In bioinformatics, FASTA format is a text-based format for representing either
nucleic acid sequences or peptide sequences, in which base pairs or amino acids are
represented using single-letter codes. The format also allows for sequence names and
comments to precede the sequences.
The simplicity of FASTA format makes it easy to manipulate and parse sequences
using text-processing tools and scripting languages like Python and Perl.
Format
A sequence in FASTA format begins with a single-line description, followed by
lines of sequence data. The description line is distinguished from the sequence data by a
greater-than (">") symbol in the first column. The word following the ">" symbol is the
identifier of the sequence, and the rest of the line is the description (both are optional).
There should be no space between the ">" and the first letter of the identifier. It is
recommended that all lines of text be shorter than 80 characters. The sequence ends if
another line starting with a ">" appears; this indicates the start of another sequence. A
simple example of one sequence in FASTA format:
>gi|5524211|gb|AAD44166.1| cytochrome b [Elephas maximus maximus]
LCLYTHIGRNIYYGSYLYSETWNTGIMLLLITMATAFMGYVLPWGQMSFWGATVITNLFSAIPYIGTNLV
EWIWGGFSVDKATLNRFFAFHFILPFTMVALAGVHLTFLHETGSNNPLGLTSDSDKIPFHPYYTIKDFLG
LLILILLLLLLALLSPDMLGDPDNHMPADPLNTPLHIKPEWYFLFAYAILRSVPNKLGGVLALFLSIVIL
GLMPFLHTSKHRSMMLRPLSQALFWTLTMDLLTLTWIGSQPVEYPYTIIGQMASILYFSIILAFLPIAGX
IENY
Header line
The header line, which begins with '>', gives a name and/or a unique identifier for
the sequence, and often lots of other information too. Many different sequence databases
use standardized headers, which helps when automatically extracting information from
the header.
In the original Pearson FASTA format, one or more comments, distinguished by a
semi-colon at the beginning of the line, may occur after the header. Most databases and
bioinformatics applications do not recognize these comments and follow the NCBI
FASTA specification. An example of a multiple sequence FASTA file follows:
>SEQUENCE_1
MTEITAAMVKELRESTGAGMMDCKNALSETNGDFDKAVQLLREKGLGKAAKKADRLAAEG
LVSVKVSDDFTIAAMRPSYLSYEDLDMTFVENEYKALVAELEKENEERRRLKDPNKPEHK
IPQFASRKQLSDAILKEAEEKIKEELKAQGKPEKIWDNIIPGKMNSFIADNSQLDSKLTL
MGQFYVMDDKKTVEQVIAEKEKEFGGKIKIVEFICFEVGEGLEKKTEDFAAEVAAQL
>SEQUENCE_2
SATVSEINSETDFVAKNDQFIALTKDTTAHIQSNSLQSVEELHSSTINGVKFEEYLKSQI
ATIGENLVVRRFATLKAGANGVVNGYIHTNGRVGVVIAAACDSAEVASKSRDLLRQICMH
Sequence representation
After the header line and comments, one or more lines may follow describing the
sequence: each line of a sequence should have fewer than 80 characters. Sequences may
be protein sequences or nucleic acid sequences, and they can contain gaps or alignment
characters (see sequence alignment). Sequences are expected to be represented in the
standard IUB/IUPAC amino acid and nucleic acid codes, with these exceptions: lowercase letters are accepted and are mapped into upper-case; a single hyphen or dash can be
used to represent a gap character; and in amino acid sequences, U and * are acceptable
letters (see below). Numerical digits are not allowed but are used in some databases to
indicate the position in the sequence.
The nucleic acid codes supported are:
Nucleic Acid Code
Meaning
A
Adenosine
C
Cytosine
G
Guanine
T
Thymidine
U
Uracil
R
G A (puRine)
Y
T C (pYrimidine)
K
G T (Ketone)
M
A C (amino group)
S
G C (Strong interaction)
W
A T (Weak interaction)
The amino acid codes supported are:
Amino Acid Code
Meaning
A
Alanine
B
Aspartic acid or Asparagine
C
Cysteine
D
Aspartic acid
E
Glutamic acid
F
Phenylalanine
G
Glycine
H
Histidine
I
Isoleucine
K
L
M
N
O
P
Q
R
S
T
U
V
W
Y
Z
X
*
-
Lysine
Leucine
Methionine
Asparagine
Pyrrolysine
Proline
Glutamine
Arginine
Serine
Threonine
Selenocysteine
Valine
Tryptophan
Tyrosine
Glutamic acid or Glutamine
any
translation stop
gap of indeterminate length
Sequence identifiers
The NCBI defined a standard for the unique identifier used for the sequence
(SeqID) in the header line. The formatdb man page has this to say on the subject:
"formatdb will automatically parse the SeqID and create indexes, but the database
identifiers in the FASTA definition line must follow the conventions of the FASTA
Defline Format."
However they do not give a definitive description of the FASTA defline format. An
attempt to create such a format is given below.
GenBank
EMBL Data Library
DDBJ, DNA Database of Japan
NBRF PIR
Protein Research Foundation
SWISS-PROT
Brookhaven Protein Data Bank (1)
Brookhaven Protein Data Bank (2)
Patents
GenInfo Backbone Id
General database identifier
NCBI Reference Sequence
Local Sequence identifier
gi|gi-number|gb|accession|locus
gi|gi-number|emb|accession|locus
gi|gi-number|dbj|accession|locus
pir||entry
prf||name
sp|accession|name
pdb|entry|chain
entry:chain|PDBID|CHAIN|SEQUENCE
pat|country|number
bbs|number
gnl|database|identifier
ref|accession|locus
lcl|identifier
The vertical bars in the above list are not separators in the sense of the BackusNaur form, but are part of the format.
File extension
There is no standard file extension for a text file containing FASTA formatted
sequences. FASTA format files often have file extensions like .fa, .mpfa, .fna, .fsa, .fas or
.fasta
There are several pitfalls to the traditional FASTA format that this format is meant to
solve:





Definition lines vary widely for no good reason. This causes problems for end
users who want to use these files with protein identification tools. The creators of
these tools are faced with a significant challenge of either supporting all of these
variations or enabling a user to cope with them.
Same database processed in different search engines -> different identifiers ->
difficult to map (P00761 vs. ALBU_HUMAN)
Same protein in different databases can have very different identifiers (P00761 vs
gi|3446572|sp|p00761 vs IPI:12345678)
The information extracted from the fasta formats is heterogeneous: parsability
issues. Should come from the DB
Description and availability of taxonomy (Latin names, common names, NCBI
TaxID)
Give a detailed sccount on sequence allignment
Definition:
A sequence alignment is a way of arranging the primary sequences of DNA,
RNA, or protein to identify regions of similarity that may be a consequence of functional,
structural, or evolutionary relationships between the sequences.
Aligned sequences of nucleotide or amino acid residues are typically represented
as rows within a matrix. Gaps are inserted between the residues so that residues with
identical or similar characters are aligned in successive columns.
A sequence alignment, produced by ClustalW between two human zinc finger
proteins identified by GenBank accession number.
If two sequences in an alignment share a common ancestor, mismatches can be
interpreted as point mutations and gaps as indels (that is, insertion or deletion mutations)
introduced in one or both lineages in the time since they diverged from one another. In
protein sequence alignment, the degree of similarity between amino acids occupying a
particular position in the sequence can be interpreted as a rough measure of how
conserved a particular region or sequence motif is among lineages. The absence of
substitutions, or the presence of only very conservative substitutions (that is, the
substitution of amino acids whose side chains have similar biochemical properties) in a
particular region of the sequence, suggest that this region has structural or functional
importance. Although DNA and RNA nucleotide bases are more similar to each other
than to amino acids, the conservation of base pairing can indicate a similar functional or
structural role.
I.Global and local alignments
Global alignments, which attempt to align every residue in every sequence, are
most useful when the sequences in the query set are similar and of roughly equal size.
(This does not mean global alignments cannot end in gaps.) A general global alignment
technique is called the Needleman-Wunsch algorithm and is based on dynamic
programming. Local alignments are more useful for dissimilar sequences that are
suspected to contain regions of similarity or similar sequence motifs within their larger
sequence context. The Smith-Waterman algorithm is a general local alignment method
also based on dynamic programming. With sufficiently similar sequences, there is no
difference between local and global alignments.
II. Pairwise alignment
Pairwise sequence alignment methods are used to find the best-matching
piecewise (local) or global alignments of two query sequences. Pairwise alignments can
only be used between two sequences at a time, but they are efficient to calculate and are
often used for methods that do not require extreme precision (such as searching a
database for sequences with high homology to a query).
1.Dot-matrix methods
2.Dynamic programming
3.Word methods
1.Dot-matrix methods
A DNA dot plot of a human zinc finger transcription factor (GenBank ID NM_002383)
The dot-matrix approach, which implicitly produces a family of alignments for
individual sequence regions, is qualitative and simple, though time-consuming to analyze
on a large scale. It is very easy to visually identify certain sequence features—such as
insertions, deletions, repeats, or inverted repeats—from a dot-matrix plot. To construct a
dot-matrix plot, the two sequences are written along the top row and leftmost column of a
two-dimensional matrix and a dot is placed at any point where the characters in the
appropriate columns match—this is a typical recurrence plot. Some implementations vary
the size or intensity of the dot depending on the degree of similarity of the two characters,
to accommodate conservative substitutions. The dot plots of very closely related
sequences will appear as a single line along the matrix's main diagonal.
Dot plots can also be used to assess repetitiveness in a single sequence. A
sequence can be plotted against itself and regions that share significant similarities will
appear as lines off the main diagonal. This effect can occur when a protein consists of
multiple similar structural domains.
2.Dynamic programming
The technique of dynamic programming can be applied to produce global
alignments via the Needleman-Wunsch algorithm, and local alignments via the SmithWaterman algorithm. In typical usage, protein alignments use a substitution matrix to
assign scores to amino-acid matches or mismatches, and a gap penalty for matching an
amino acid in one sequence to a gap in the other. DNA and RNA alignments may use a
scoring matrix, but in practice often simply assign a positive match score, a negative
mismatch score, and a negative gap penalty. (In standard dynamic programming, the
score of each amino acid position is independent of the identity of its neighbors, and
therefore base stacking effects are not taken into account. However, it is possible to
account for such effects by modifying the algorithm.). The BLAST and EMBOSS suites
provide basic tools for creating translated alignments.
3.Word methods
Word methods, also known as k-tuple methods, are heuristic methods that are not
guaranteed to find an optimal alignment solution, but are significantly more efficient than
dynamic programming. These methods are especially useful in large-scale database
searches where it is understood that a large proportion of the candidate sequences will
have essentially no significant match with the query sequence. Word methods are best
known for their implementation in the database search tools FASTA and the BLAST
family. Word methods identify a series of short, nonoverlapping subsequences ("words")
in the query sequence that are then matched to candidate database sequences. The relative
positions of the word in the two sequences being compared are subtracted to obtain an
offset; this will indicate a region of alignment if multiple distinct words produce the same
offset. Only if this region is detected do these methods apply more sensitive alignment
criteria; thus, many unnecessary comparisons with sequences of no appreciable similarity
are eliminated.
III. Multiple sequence alignment
Multiple sequence alignment is an extension of pairwise alignment to incorporate
more than two sequences at a time. Multiple alignment methods try to align all of the
sequences in a given query set. Multiple alignments are often used in identifying
conserved sequence regions across a group of sequences hypothesized to be
evolutionarily related. Such conserved sequence motifs can be used in conjunction with
structural and mechanistic information to locate the catalytic active sites of enzymes.
Alignments are also used to aid in establishing evolutionary relationships by constructing
phylogenetic trees. Multiple sequence alignments are computationally difficult to produce
and most formulations of the problem lead to NP-complete combinatorial optimization
problems. Nevertheless, the utility of these alignments in bioinformatics has led to the
development of a variety of methods suitable for aligning three or more sequences.
IV. Structural alignment
Structural alignments, which are usually specific to protein and sometimes RNA
sequences, use information about the secondary and tertiary structure of the protein or
RNA molecule to aid in aligning the sequences. These methods can be used for two or
more sequences and typically produce local alignments; however, because they depend
on the availability of structural information, they can only be used for sequences whose
corresponding structures are known (usually through X-ray crystallography or NMR
spectroscopy). Because both protein and RNA structure is more evolutionarily conserved
than sequence, structural alignments can be more reliable between sequences that are
very distantly related and that have diverged so extensively that sequence comparison
cannot reliably detect their similarity.
Structural alignments are used as the "gold standard" in evaluating alignments for
homology-based protein structure prediction because they explicitly align regions of the
protein sequence that are structurally similar rather than relying exclusively on sequence
information. However, clearly structural alignments cannot be used in structure
prediction because at least one sequence in the query set is the target to be modeled, for
which the structure is not known. It has been shown that, given the structural alignment
between a target and a template sequence, highly accurate models of the target protein
sequence can be produced; a major stumbling block in homology-based structure
prediction is the production of structurally accurate alignments given only sequence
information.