Basics of Microbial existence:

Definition of microbiology:
 Study of living organisms of microscopic size,which includes bacteria, fungi, algae, protozoa
and viruses. It is concerned with their form, structure, reproduction, physiology, metabolism
and classification.
 It also includes study of their nature relationship between each and to other microorganism
their effect on human beings,animals,plants their abilities to make physical,chemical changes
in our environment and their reactions to physical and environmental changes.
 Some organisms are beneficial (eg. Penicillin,Lactic bacteria, Interferon, alcohol)and others
are detrimental(eg. Camphylobacter,Clostridium).
The word cell was coined by robert hook(1665) concept of cell theory by Matthias
Schledien,Theodor Schwann.
 Cell theory cell are the basic structural and functional units of all organism
 Unicellular organism:all life process are performed by a single cell.
 They can be grown in flasks and test tubes.form 100 generation within 24hrs
 Microorganism follow the same pattern like higher plant and animals do for metabolic
process
Eg., yeast utilizes glucose like other mammalian tissues,because they contain same enzymes.
Life process of organism:
1. Metabolizing
2. growing
3. reproducing
4. aging
5. dying
By modifying the environment we can alter the metabolic activities,regulate growth and change
some details of their genetic pattern without destroying the organism.Bacteriophages are the
viruses that infect bacteria.
STRUCTURE OF BACTERIAL CELLS:
Bacteria vary Significantly in their morphology that is their appearance.they range in size from
0.2um to 1um in diameter and 1um to 10um length.there ar 3 basic shapes of cells:
1. Spherical
2. Rod shaped
3. Spiral
A spherical or round bacterium is called as coccus
The are typically small cell they can occur single or in pairs(Diplococcus)in
clusters(Styphylococcus) or chains(Streptococcus). Frequently,the grouping of the cocci is
reflected in the naming of the organism.Hence Streptococcus pygenes is an organism with a
cellular arrangement consisting of chain of cocci.
A rod shaped or cylindrical bacterium is called a bacillus(Plural: Bacilli)
Most bacilli appear as a single cells,but a few are joined end to end to form diplobacilli or
streptobacilli some bacilli form endospores- a very resistant form of cell which is able to
withstand adverse environment conditions.
Spiral bacteria may have one of three shapes:
1. The spirochaetes have a crokscrew- like appearance and a strong axial filament propels the cell
along.
2. Spirilla are not as tightly coiled and move by means of a flagellum,propeller-like tail
3. Vibrios are slightly curved rod-shaped cells, resembling a comma or incomplete spiral.
Most species of bacteria always grow in their characteristic shape and this can be a useful means
of identification.however,the actual size of the cell can be influence by the availability of
nutirents in the medium.A few bacteria occur in more than one shape(eg. Some species of
Rhizobium and Corynebacterium), which makes identification more difficult.These bacteria are
termed pleomorphic.
Sometimes, group of cells remain together after cell division to form clutures or chains.Some
bacteria are motile;that is they are capable of movement, possessing one or more extracellular
appendages called flagella which enable them to swim.
STRUCTURE OF BACTERIA:
Cell wall:
The most characteristic structure of bacteria is their cell wall.It s a semi-rigid structure present in
almost all bacteria and is responsible for determining the shape of the cell as well as some of its
staining properties.It also provides mechanical support so that the cell does not burst when
exposed to conditions of osmotic pressure that allow movement of water into cell. Bacteria cell
walls contain varying amounts of different macromolecules. One of the major components is a
complex substance called peptidoglycan, which consists of repeating units of disaccharides
molecules.
The composition of the cell wall differs between bacteria and so is useful for classification.
Nearly all bacteria can be placed in one of two groups,gram-negative or gram positive,based on
the composition of the wall
Structure of gram positive cell walls
Cells classified as gram positive have a defined cell wall structure which determines their
reaction in the staining procedure developed by Danish microbiologist Hans Christians Gram.The
main component of gram positive cells walls is a thick layer of peptidoglycan. Attached to the
peptidoglycan are other complex polysaccharides called teichoic acids. These teichoic acids have
a strong negative charge which appears to influence the passage of materials in and out of the cell
and provides a large part of the antigenic specificity of the cells.
Structure of gram negative cell walls:
The group of gram negative have a complex outer structure than the gram positives. The cell wall
consists of a thin layer of peptidoglycan covered by an outer membrane containing
lipopolysaccharides,lipoproteins and phospholipids.the outer membrane has several functions.It
provides a barrier to the entry of various substance into the cell and also prevents the entry of
certain enzymes that break down the cell wall.The lipopolysaccharide component,called the o-
polysachharide or o-antigen, has a distinctive structure in each strain of bacteria.This is useful in
serological tests to distiguish between different strains of bacteria. The lipid portion known as
lipid a is an endotoxin.It is responsible for some of the toxic effects.
Acid fast bacteria:
Not all bacteria can be grouped as either gram positive or gram negative some bacteria (eg.
Mycobacteria) have an outer layer composed of peptidoglycan similar to the gram positive
wall,but covered by a thick waxy layer that interferes with the gram staining procedure.the
presence bacteria (eg. Mycobacteria) the causative agent of tuberculosis, Mycobacterium
tuberculosis is usually detected in sputum or lung biopsises by using the ziehl-neelsen acid fast
stain. Mycobacteria are often referred to as acid fast bacteria.
Cell membrane:
All the bacteria have cell membrane or plasma membrane underneath the cell wall.The plasma
membrane, or the cell membrane, provides protection for a cell. It also provides a fixed
environment inside the cell, and that membrane has several different functions. One is to
transport nutrients into the cell and also to transport toxic substances out of the cell. bacterial
membranes have a structure that is essentially similar to the membrane of all living cells. plasma
membrane, will have proteins on it which interact with other cells.it consists of double layer of
phospholipd molecules. Prokaryotic cell membranes consists only protein and phospholipids but
eukaryotic cells in addition consists of sterols and carbohydrates.
The additional properties of eukaryotic membrane are
1. Polar water soluble hydrophilic head
2. Non polar hydrophobic tail
Arrangement:
1. The polarhydrophilic head are facing outside the water environment
2. Non polar hydrophobic tail to the interior of the membrane.
The widely accepted model of cell mebrane was proposed by singer and nicholson and usually
called as singer and nicholson model or fluid mosaic model.
Other functions of cell membrane:
Prokaryotic cell lack the functions of other cellular membrane
The plasma membrane in bacteria is the site of additional functions that are performed by the
organelles in eucaryotic cells.
These includes:
 Synthesis of cell wall components
 Cellular respiration and synthesis of ATP
 The secretion of proteins and enzymes,which are released from the cell as extracellular
enzymes or toxin.
The membrane is a vital part of the cell and as such is very susceptible to damage. Many
antimicrobial compounds exerrt their effect by disrupting the structure of the membrane. This
include disinfectents,such as alcohol and quaternary ammonium compounds and the polymyxin
group of antibiotics.
INTERNAL STRUCTURE OF BACTERIAL CELLS:
Cytoplasam:
The cytoplasam of bacterial cells is about 80% water and contains proteins,carbohydrates,lipids
and salts that are essential for activities of the cells. The nuclear area of the cell contains a single
ciecular chromosome,consisting of a double strand of DNA which carries the genetic information
necessary for the structure and function of the cell.bacteria often carry one or more extra pieces
of DNA,small circular structures called plasmids. Plasmids vary in size and replicate
independently of the main chromosomal DNA.They appear to carry information that is not
essential for the growth of the cell-such as genes for resistance to antibiotics or the production of
toxins.
Protoplast:
Cell membrane or plasma membrane with internal contents without cell wall is called
protoplast.Protoplast is metabolically active.protoplast remains intact till osmotic lyses does not
occur.The cell wall burst if protoplast is kept in an osmotic solution as plasma membrane is semi
permeable.Protoplast is sensitive to water,dilute salt and sugar concentration.
Ribosomes:
A small granular particles,consisting of 2 subunits, each containing proteins and ribonucleic
acid.Ribosomes of procaryotic cells are designated as 70S which refers to their rate of
sedimentation when subjected to ultracentifugation.They are slightly smaller and less dense than
the ribozomes of eucaryotic organisms which are designated as 80S.Ribosomes are the site of
protein synthesis in the cell.Messenger RNA(mRNA) attaches to the ribosomes to provide a site
for incorporation of amino acids into the growing peptide chain. Groups of ribosomes attached to
mRNA are called polyribosomes.
Granules or inclusions:
Can be identified by staining with simple dyes such as methylene blue or iodine.These inclusions
are usually polymers of cellular material and act as storage for the cell.polymers for glucose,such
as glycogen or starch, are also frequently present.occasionally,
Vesicles or Vacuoles:
cells contain vesicles or vacuoles.these are usually filled with gas to give the cell buoyancy.
Mitochondria (singular: mitochondria)
They are found in the cytoplasm of eucaryotic cells. They consist of a double membrane
structure, the inner one arranged in a series of folds called cristae. Mitochondria are often called
the -'powerhouses' of the cell as they are the main site of ATP production.
The Golgi complex is another membranous structure found in the cytoplasm. It consists of a
series of flattened sacs or vesicles and is responsible for the secretion of certain proteins, such as
enzymes and hormones.
Lysosomes are small organelles, bounded by a single membrane and found mainly in animal
cells. They consist of a sac containing strong digestive enzymes and appear to be important in
breaking down foreign material and microorganisms that have been ingested by the cell.
Mesosomes:
Extension of cell membrane present into cytoplasam as infloding and serves to increase the
surface area in photosynthetic prokaryotes.cyanobacteria is a photosynthetic one.
More common in gram positive bacteria and less common in gram negative bacteria. They are
thought to be involved in mechanism responsible for distribution of bacterial chromosome into
daughter cells during cell division. They also have the function analogous to the mitochondria of
the eukaryotic cell providing a membranous support for respiratory enzymes. Involved in the
ejection or excretion of materials from the cytoplasam to the exterior.
They also carry enzymes for aerobic respiration and increases surface area for aerobic respiration.
Do not contain enzymes for nitrogen fixation.
Nuclear material
The bacterial genome is composed of a single haploid circular chromosome containing double
stranded DNA.Nuclear membrane and the protein histone are, absent. The genes in the bacterial
chromosomes code for all the vital functions of the cell. Bacterial genomes vary in size
depending on the species. Because of its length, the bacterial chromosome is extensively folded
to form a dense body located at the centre of the cell as a nucleoid.The shape of nuclear area is
spherical dumb bell shaped or elongated.Chromosomes are attached with plasma membrane and
are responsible for the replication of DNA and segregation of new chromosomes to daughter
cells. During replication, the DNA helix unwinds and both daughter cells, produced by binary
fission,receive a copy of the original genome.
Plasmid
Bacteria also contain extrachromosomal DNA, small circular double stranded DNA called
plasmids. These are not connected with bacterial chromosomes and they are autonomously
replicating genetic elements. They contain 5-100 genes that are not crucial for survival of
bacteria. Under normal environmental conditions plasmids may be gained or lost without
harming the cell. Plasmids carry genes for lactose metabolism,citrate metabolism, antibiotic
resistance, tolerance to toxic metals. Production of toxins and enzyme synthesis. Plasmids can be
transferred from one bacteria to another. Plasmid DNA is used for gene manipulation in
biotechnology.Most of the plasmids in lactococci are cryptic that is no known functions is
associated with particular plasmid.
Difference between small and large plasmid
CHARACTERS SMALL PLASMID LARGE PLASMID
Size <20Mdal >20Mdal
Autonomous replication yes yes
Conjugative No No
Copy No/cell 10-40 1-5
Amplification sometimes yes No
Plasmid gives unique size and easier to use because of small size. Plasmid after
insertion can be judged by looking at its size.
EXTERNAL STRUCTURES OF BACTERIAL CELLS
Glycocalyx- Capsule
Some bacteria are surrounded by the glycocalyx, a sticky, polysaccharide-containing layer which
is secreted onto their cell surface. Depending on its structure and function its referred to as a
capsule or slime layer.
Barterial capsules are composed of polysaccharide and/or polypeptide molecules. They are
usually synthesized inside the cell and excreted to the cell surface where they form an organized
gelatinous layer firmly attached to the cell wall. An important function of the capsule is to help
the bacterium adhere to host cell surfaces. The capsule possesses specific molecules which bind
to complementary receptor molecules on the surface of the susceptible animal cell.The capsule
also has a protective function, allowing the bacterial cell to avoid phagocytosis (process by which
certain living cells called phagocytes ingest or engulf other cells or particles) apparently because
the phagocyte is unable to attach to the bacterial cell surface. The capsule is therefore considered
to contribute to the virulence (degree of pathogenicity) of the organism. For example,
encapsulated strains of Streptococcus pneumoniae are highly virulent whereas strains that lack a
capsule are unable to produce disease symptoms.
Slime layers are composed of glycoprotoins and polysaccharides and usually exist as loosely
attached, less defined structure that capsules.
Flagella
Many bacteria are motile, that is capable of movement in a water environment. The most
common way in which procaryotic cells move is by means of flagella (singular: flagellum) thin,
rigid filaments that are usually many times longer than the cell itself.Some bacteria have only one
flagellum located at one end of the cell; others have two,one at each end of the cell of the cell;
others have two, one at each end of the cell. A few bacteria have numerous flagella, located all
over the cell. Flagella consist of protein subunits arranged in a complex structure. They are
attached to the cell membrane and wall of the bacterium by a structure called a basal body, a
series of concentric protein rings which allow the flagellum to rotate.Movement of the bacterium
is achieved by rotation of the flagella with a propeller - like motion, a process requiring energy in
the form of ATP. Motile bacteria appear to respond to a stimulus in their environment and swim
towards it. This phenomenon is called chemotaxis.
The position at which flagella are inserted into a bacteria cell vary and may characteristic of a
genus or family which may be categorized as follows.
ATRICHOUS-bacteria with no flagella
MONORICHOUS-bacteria have a single flagellum on one end
LOPHOTRICHOUS-bacteria have a group of flagella at one pole
AMPHITRICHOUS-bacteria having flagella at both poles
PERITRICHOUS-bacteria are completely surrounded by flagella
Motility in bacteria
Many bacteria move by means of hair like structure called flagella. As a result, they can move
towards favorable environments (nutrients, light for photpsynthetic bacteria and so on) and away
from less favorable ones such as toxic substances :
Chemotaxis is the word used to describe this behavioral response.Bacterial flagella are thin
(20nm diameter) and rigid, coiled in a spiral fashion and rotate very much like a ship's propella.
They cannot be seen under the light microscope unless special stains are used to increase their
diameter.Bacterial flagella are made up of a protein called flagellia, which is organized into
flagella subunits. It has three main components structure :filament, hook and basal body.
Rotation of the hook and filament causes the cell to move. The based body acts as motor to bring
about the rotation.The basal anchors the flagellum to the cytoplasmic membranes and cell wall.
The energy required for rotation of the flagellum comes from the proton motive force generated
during either respiratory or photosynthetic electron transport through the action of the basal body,
each flagellum is caused to rotate in an anticlockwise direction .Motile bacteria can move into
suitable microenvironment in response to physical or chemical stimuli. Flagella can be
demonstrated by electron microscopy, by light microscopy using special methods or by serology
using antibodies specific for flagellar antigens (H antigen).
PILI AND FIMBRIAE
Pili (singular: pilus) and fimbriae (singular: fimbria) appear as hair-like appendages on the
outside of gram-negative bacterial cells. Each has a distinct function. The thicker sex pili (1 to 10
per cell) are located on the surface of the cell; they are involved in the joining together of
bacterial cells to allow transfer of the genetic material, DNA, from one cell to another during
conjugation. There are several thousand fimbriae per cell (sometimes called attachment pili).
They are thinner than pili and concerned mainly with the attachment of the bacteria to the surface
of mucosal cells, so that the organism can adhere to the host cell and colonise it. Thus the
presence of fimbriae contributes to the pathogenicity of the organism.
ENDOSPORES
When environmental conditions are unfavourable (Decreased alanin content), certain
Vegetative cells are converted to endospores. It takes 7hrs for a bacteria to form spore.Spores
have some essential protein, Ca=Dipiclonic acid.Outer Protein Coat Layer,Peptidoglycan
Layer,Inner layer.Bacterial sporulation does not occur when cells are growing rapidly, but only
when growth ceases due to the exhaustion of essential nutrients. An endospore is a specialised
type of resting cell which is formed inside the bacterial cell membrane. It is surrounded by a
spore coat consisting of thick layers of peptidoglycan and protein.The endospore develops inside
the bacterial cell membrane and can be located by special staining procedures. The spore has a
lower water content than vegetative cells and does not carry out metabolic reactions. It does,
however, contain the cellular nucleic acid and various other enzymes and substances that are
essential if spore germination is to occur when conditions become favourable. When the spore is
placed in a favourable medium, water enters, germination occurs in the vegetative cell that is
produced resumes normal growth and metabolism. Endospore formation is not a method of
reproduction. One bacterial cell gives rise to only one endospore, which then germinates into a
single cell again. Bacterial endospores have been shown to survive in the soil for many years.
Many of the Gram-positive bacteria that form endospores are important pathogens. These
include members of the genus Clostridium. Endospores survive conditions that kill most bacteria,
such as boiling for up to five hours, freezing, desiccation (drying out) and exposure to chemicals
and radiation. In the food industry, spores may survive the normal cooking process and cause
food Poisoning
SPORE GERMINATION
Spore turns to vegetative state by a process called germination., when an endospore is
reactivated, the Outer Coat of the endospore breaks, the water surrounding endospore enters and
metabolism resumes germination. It occurs in three stages namely activation, initiation and
outgrowth.
Activation may occur in response to factors such as brief exposure to heat, abrasion of the spore
coat or environmental acidity.
If other environmental conditions including the presence of adequate nutrients are favourble,
initiation of germination will occur. The spore cortex and coat are degraded, water is absorbed,
calcium dipicolinate is released and outgrowth develops. One vegetative cell gives rise single
spores which after germination remains as one. Hence, it is not a method of Reproduction
Bacterial endospores are highly resistant to heat, partly because of their comparatively low water
content. A temperature of 121℃ for 15 minutes in an autoclave is required to kill endospores.
The great environmental stability of the bacterial endospore appear to be related to three major
factors 1. The dehydrated state of the spore 2. High calcium content 3. Presence of a unique
chemical known as dipicolinic acid.
Dipicolinic acid is not found in any other living organisms.
In addition to that endospore contains small acid soluble DNA binding proteins which saturate
spore DNA and protect it from beat radiation, desiccation and chemicals

CLASSFICATION OF MICROORGANISMS
Improvements in microscopes enabled scientists to study the 'invisible' organisms - that is, those
too small to be seen with the naked eye. They were able to show that certain of these microscopic
creatures are responsible for the infectious diseases of higher organisms. Most of them are
harmless, some are essential for life and some are used in the manufacture of products that are
of benefit to humans.
Swedish botanist Carl Linnaeus, who divided all living organisms into two kingdoms: plants and
animals. He then devised a careful naming system for each species within the kingdoms, a system
still widely used today.As knowledge increased, it became apparent that the Linnaeus system did
not allow for the strange organisms that were visible only with a microscope and which did not
really fit into the description of either plant or animal. Nor did it allow for 'plants' such as fungi
which could not convert the energy from photosynthesis into food.
In 1866, the German scientist Ernst Haeckel divided all living organisms into three groups - the
Animalia and Plantae of Linnaeus,and a third group, the Protista. Protista consisted of single-
celled organisms such as bacteria and protozoa, together with some fungi and algae.
As more microorganisms were discovered, and more was learnt about their structure, habitats and
nutritional requirements, scientists felt the need to make further refinements to this classification;
so, in 1969, R.H.WHITTAKER proposed a five-kingdom classification. This system places
organisms in the following groups:
1.Monera, or Procaryota - single-celled organisms comprising the eubacteria, or true bacteria, and
the archaebacteria, or ancient bacteria.
2.Protista - including slime moulds, protozoa and some algae.
3.Fungi - single-celled yeasts, multicellular moulds and mushrooms.
4.Plantae - some algae together with mosses, ferns, gymnosperms (conifers) and angiosperms
(flowering plants).
5.Animalia - invertebrates (worms), arthropods (insects) and vertebrates.
This five-kingdom classification is used today as a convenient way of grouping living organisms.
The five-kingdom classification is based on microscopic observations of morphology and on the
method of reproduction.
Further in 1990 the five kingdom classification is converted to 3 domine
Bacteria, Archaea and Eukarya.

CHARACTERISTICS OF PROKARYOTIC AND EUKARYOTIC CELLS.

CHARACTERISTIC PROCARYOTIC EUCARYOTIC
Size 0.4 to 2.0 pm diameter 5 to 100 pmdiameter.
Cell wall Always present Bounded by wall or
 membrane
Nucleus No defined region' Contains DNA,bounded by double
membrane.
Genetic material One circular chromosome of Double strand of DNA associated with
double stranded DNA; proteins (histones) to form pairs; DNA
plasmids in some cells of also present in mitochondria and
chromosomes chloroplasts.
Sub-cellular Absent Present - include
organelles mitochondria,
chloroplasts,
Golgiapparatus,
lysosomesand
endoplasmic reticulum.
Motilty Some are motile, use flagella Some are motile, e.g.
Protozoa; most are non-motile.
Plasma membrane Present - selectively permeable Present - contains sterols; selectively
permeable.
Cytoplasm Contains all enzymes and Has cytoskeleton, exhibit chemicals; no
defined structures streaming moving of
cytoplasmic fluid.
Endoplasmic Absent Present.
reticulum
Ribosomes Present, smaller 70s Present, larger 80s.
Reproduction Mainly asexual binary fission Sexual or asexual.

FUNGI:
 They exixt either in yeast or mold forms
 The smallest of yeast are similar to the size of bacteria,but most are larger (2 to 12um) and
multiply by budding
 Molds form tubular extensions called hyphae,which when linked together in a branched
network,form the fuzzy structure seen on neglected bread.
 Fungi are eukaryotic,and both yeast and molds have a rigid external cell wall composed of
their own unique polymers called glucan,mannan and chitin
 There genome may exist in a diploid or haploid state and replicate by meiosis or simple
mitosis.
 Most fungi are free living and widely distributed in nature.
 Generally,fungi grow more slowly than bacteria,although their growth rates sometimes
overlap
 The fungi probably represent an evolutionary off shoot of the protozoa they are unreleated to
actinomycetes,mycelial bacteria that they superficially resemble
 The major subdivisions (phyla) of fungi are: Chytridiomycota, Zygomycota (zygomycetes),
Ascomycota(the ascomycetes), Basidiomycota(Basidiomycetes) and the Deuteromycetes (or
imperfect fungi)
ALGAE:
1. The term algae has long been used to denote all organisms that produce O 2 as aproduct of
photosynthesis.
2. One major subgroup of these organisms-the blue-green bacteria or cyanobacteria-are
prokaryotic and no longer are termed algae.
3. This classification is reserved exclusively for photosynthetic membrane of their subcellular
chloroplast
4. Many algal species are unicellular microorganisms.other algae may form extremely large
multicellular structures.
5. Brown algae sometimes are several hundred meters in length
6. A number of algae produces toxins that are poisonous to human and other animals.
7. Dinoflagellates, a unicellular algae,causes algal blooms,or red tides,in the ocean.red tide
caused by dinoflagellate Gonyaulax species are serious as this organism produces
neurotoxins such as saxitoxin and gouyautoxins, which accumulate in shellfish(eg, clams,
mussels,scallops and oysters) that feed on this organism.
8. Ingestion of this shellfish by human results in symptoms of paralytic shellfish poisioning and
lead to death.
PROTOZOA:
1. Protozoa are unicellular non photosynthetic protists.
2. The most primitive protozoa appear to be flagellated forms that in many respects resemble
representatives of algae.
3. It seems likely that the ancestors of these protozoa were algae that became hetrotrophs-the
nutritionak requirements of such organisms are met by organic compound.adaptation to a
 heterotrophic mode of life was sometimes accomponied by loss of chloroplast,and algae thus
gives rise to closely related protozoa.
4. From flagellated protozoa appear to have evolved the ameboid and the ciliated
types;intermediate forms are known that have flagella at one stage in the life cycle and
pseudopodia(Characteristics of the ameba) at another stage
5. A fourth major group of protozoa the sporozoa are strict parasities that are usually immobile
most of which reproduce sexually and asexually in alternate generations by means of spores.\
VIRUSES:
1. Viruses are strict intercellular parasities of the other living cells,not only of mammalian and
plant cells , but also of simple unicellular organisms,including bacteria(the Bacteriophages).
2. Viruses are simple forms of replicating, bioloogically active particles that carry genetic
information in either DNA or RNA molecules enclosed in a protein coat or capsid.
3. Proteins-frequently glycoproteins-in the capsid determine the specificity of interaction of a
virus with its host cell.
4. The capsid protects the nucleic acid and facilitates attachment and penetration of the host cell
by virus.
5. Inside the cell,viral nucleic acid redirects the hosts enzymatic machinery to functions
associated with replication of the virus.in some cases,genetic information from the virus ca
be incorporated as DNA into a host chromosome.
6. In other instances,viral genetic information can serve as a basis for cellular manufacture and
release of copies of the virus.this process calls for replication of the viral nucleic acid and
production of specific viral proteins.
BACTERIA:
1. Bacteria are the smallest (0.1 to 10um) living cells.they have cytoplasmic membrane
surrounded by a cell wall; a unique interlinking polymer called peptidoglycan makes the wall
rigid.
2. The simple prokaryotic plan includes no mytochondria,lysosomes,endoplasmic recticulam,
or other organelles.most bacteria are about the size of mitochondria
3. Their cytoplasm contains only ribosomes and a single,double standered DNS Chromosomes.
4. Bacteria have no nuclues but all the chemical element s of nucleic acid and protein synthesis
are present.
5. Although their nutritional requirement vary greatly,most bacteria are free-living if given an
appropriate energy source
6. They divide by binary fission and can be grown in artificial culture,often in less than 1 day.
Archaebateria differ radically from other bacteria in structure and metabolic processes they
live in environments human consider hostile(eg., hot springs,high salt areas) but are not
associated with disease.
NAMING OR TAXONOMY OF MICROORGANISMS
Linnaeus proposed that each group of organisms should be placed into a genus (plural: genera)
the name of which is always spelt with a capital letter. Within the genera there are different
species which have discrete but related characteristics. A species can be further divided into
different strains that exhibit specific properties.
Genera Named after individuals and species named after organism in which it was found:
For example, under the Linnaean system a bacterium would be named Escherichia (genus) coli
(species), K12 (strain). The names assigned to organisms may reflect the place where the
organism is found (e.g. coli = of the colon), the name of the researcher who first described the
organism (e.g. Escherich).
Genera Named after Microbe Shape:
Vibrio Cholera:
Bacteria is comma Shaped which causes cholera
Staphylococcus epidermidis:
Staphylo means clusters; coccus means spheres.
Genera Named after Attribute of the microbie:
Saccharomyces cerevisiae:
Saccharo - sugar,Myces-fungus,cerevisiae-beer.the bacteria which converts sugar in the
sample to alcohol
Serotypes - Development of serological techniques that differentiate between strains of
organisms on the basis of their antigenic properties.,In the higher kingdoms of plants and
animals, the genera are grouped into families, orders, classes and phyla.

PRINCIPLES OF DIFFERENT STAINING:

In their natural state, most of the cells and microorganisms that we observe under the
microscope lack color and contrast. This makes it difficult, if not impossible, to detect important
cellular structures and their distinguishing characteristics without artificially treating specimens.
Certain techniques involving stains and fluorescent dyes, were suggested and in this section we
will discuss specific techniques for sample preparation in greater detail. Indeed, numerous
methods have been developed to identify specific microbes, cellular structures, DNA sequences,
or indicators of infection in tissue samples, under the microscope. Here, we will focus on the
most clinically relevant techniques.

Preparing Specimens for Light Microscopy

In Microbial laboratory, light microscopes are the most commonly used microscopes. There are
two basic types of preparation used to view specimens with a light microscope: wet mounts and
fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed on the slide
in a drop of liquid. Some specimens, such as a drop of Milk, are already in a liquid form and can
be deposited on the slide using a dropper. Solid specimens, such as a Meat scraping, can be
placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid
used is simply water, but often stains are added to enhance contrast. Once the liquid has been
added to the slide, a coverslip is placed on top and the specimen is ready for examination under
the microscope.
The second method of preparing specimens for light microscopy is fixation. The “fixing”
of a sample refers to the process of attaching cells to a slide. Fixation is often achieved either by
heating (heat fixing) or chemically treating the specimen. In addition to attaching the specimen to
the slide, fixation also kills microorganisms in the specimen, stopping their movement and
metabolism while preserving the integrity of their cellular components for observation.
To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear),
and the slide is then briefly heated over a heat source. Chemical fixatives are often preferable to
heat for tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde
(formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize
cell structures in tissue samples.
In addition to fixation, staining is almost always applied to color certain features of a
specimen before examining it under a light microscope. Stains, or dyes, contain salts made up of
a positive ion and a negative ion. Depending on the type of dye, the positive or the negative ion
may be the chromophore (the colored ion); the other, uncolored ion is called the counterion. If the
chromophore is the positively charged ion, the stain is classified as a basic dye; if the negative
ion is the chromophore, the stain is considered an acidic dye.
Dyes are selected for staining based on the chemical properties of the dye and the
specimen being observed, which determine how the dye will interact with the specimen. In most
cases, it is preferable to use a positive stain, a dye that will be absorbed by the cells or organisms
being observed, adding color to objects of interest to make them stand out against the
background. However, there are scenarios in which it is advantageous to use a negative stain,
which is absorbed by the background but not by the cells or organisms in the specimen. Negative
staining produces an outline or silhouette of the organisms against a colorful background.

Figure 2.32 (a) These Bacillus anthracis cells have absorbed crystal violet, a basic positive stain.
(b) This specimen of Spinoloricus, a microscopic marine organism, has been stained with rose
bengal, a positive acidic stain. (c) These B. megaterium appear to be white because they have not
absorbed the negative red stain applied to the slide.
Because cells typically have negatively charged cell walls, the positive chromophores in
basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly used
basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue,
and safranin typically serve as positive stains. On the other hand, the negatively charged
chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative
stains. Commonly used acidic dyes include acid fuchsin, eosin, and rose bengal.
Some staining techniques involve the application of only one dye to the sample; others
require more than one dye. In simple staining, a single dye is used to emphasize particular
structures in the specimen. A simple stain will generally make all of the organisms in a sample
appear to be the same color, even if the sample contains more than one type of organism. In
contrast, differential staining distinguishes organisms based on their interactions with multiple
stains. In other words, two organisms in a differentially stained sample may appear to be different
colors. Differential staining techniques commonly used in clinical settings include Gram staining,
acid-fast staining, endospore staining, flagella staining, and capsule staining.

Gram Staining
The Gram stain procedure is a differential staining procedure that involves multiple
steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective
method to distinguish between bacteria with different types of cell walls, and even today it
remains one of the most frequently used staining techniques. The steps of the Gram stain
procedure are listed below.

1. First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the
cells a purple color.
2. Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or
stabilize stains or dyes; in this case, Gram’s iodine acts like a trapping agent that
complexes with the crystal violet, making the crystal violet–iodine complex clump and
stay contained in thick layers of peptidoglycan in the cell walls.
3. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells
that have thick peptidoglycan layers in their cell walls are much less affected by the
decolorizing agent; they generally retain the crystal violet dye and remain purple.
However, the decolorizing agent more easily washes the dye out of cells with thinner
peptidoglycan layers, making them again colorless.
4. Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized
cells pink and is less noticeable in the cells that still contain the crystal violet dye.
The purple, crystal-violet stained cells are referred to as gram-positive cells, while the
red, safranin-dyed cells are gram-negative . However, there are several important considerations
in interpreting the results of a Gram stain. First, older bacterial cells may have damage to their
cell walls that causes them to appear gram-negative even if the species is gram-positive. Thus, it
is best to use fresh bacterial cultures for Gram staining. Second, errors such as leaving on
decolorizer too long can affect the results. In some cases, most cells will appear gram-positive
while a few appear gram-negative.This suggests damage to the individual cells or that decolorizer
was left on for too long; the cells should still be classified as gram-positive if they are all the
same species rather than a mixed culture.
Besides their differing interactions with dyes and decolorizing agents, the chemical
differences between gram-positive and gram-negative cells have other implications with clinical
relevance. For example, Gram staining can help microbiologist classify bacterial pathogens in a
sample into categories associated with specific properties. Gram-negative bacteria tend to be
more resistant to certain antibiotics than gram-positive bacteria.
Acid-Fast Stains
Acid-fast staining is another commonly used, differential staining technique that can be
an important diagnostic tool. An acid-fast stain is able to differentiate two types of gram-
positive cells: those that have waxy mycolic acids in their cell walls, and those that do not. Two
different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun
technique. Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the
carbolfuchsin even after a decolorizing agent (an acid-alcohol solution) is applied. A secondary
counterstain, methylene blue, is then applied, which renders non–acid-fast cells blue.
The fundamental difference between the two carbolfuchsin-based methods is whether heat is used
during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the
carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both
techniques are important diagnostic tools because a number of specific diseases are caused
by acid-fast bacteria (AFB). If AFB are present in a tissue sample, their red or pink color can be
seen clearly against the blue background of the surrounding tissue cells
Capsule Staining
Certain bacteria and yeasts have a protective outer structure called a capsule. Since the presence
of a capsule is directly related to a microbe’s virulence (its ability to cause disease), the ability to
determine whether cells in a sample have capsules is an important diagnostic tool. Capsules do
not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is
typically used for capsule staining. The dye stains the background but does not penetrate the
capsules, which appear like halos around the borders of the cell. The specimen does not need to
be heat-fixed prior to negative staining.
One common negative staining technique for identifying encapsulated yeast and bacteria is to add
a few drops of India ink or nigrosin to a specimen. Other capsular stains can also be used to
negatively stain encapsulated cells. Alternatively, positive and negative staining techniques can
be combined to visualize capsules: The positive stain colors the body of the cell, and the negative
stain colors the background but not the capsule, leaving halo around each cell.

Endospore Staining
Endospores are structures produced within certain bacterial cells that allow them to
survive harsh conditions. Gram staining alone cannot be used to visualize endospores, which
appear clear when Gram-stained cells are viewed. Endospore staining uses two stains to
differentiate endospores from the rest of the cell. The Schaeffer-Fulton method (the most
commonly used endospore-staining technique) uses heat to push the primary stain (malachite
green) into the endospore. Washing with water decolorizes the cell, but the endospore retains the
green stain. The cell is then counterstained pink with safranin. The resulting image reveals the
shape and location of endospores, if they are present. The green endospores will appear either
within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are
present, then only the pink vegetative cells will be visible
Endospore-staining techniques are important for identifying Bacillus and Clostridium, two genera
of endospore-producing bacteria that contain clinically significant species. Among others, B.
anthracis (which causes anthrax) has been of particular interest because of concern that its spores
could be used as a bioterrorism agent. C. difficile is a particularly important species responsible
for the typically hospital-acquired infection known as “C. diff.”

Flagella Staining
Flagella (singular: flagellum) are tail-like cellular structures used for locomotion by some
bacteria, archaea, and eukaryotes. Because they are so thin, flagella typically cannot be seen
under a light microscope without a specialized flagella staining technique. Flagella staining
thickens the flagella by first applying mordant (generally tannic acid, but sometimes potassium
alum), which coats the flagella; then the specimen is stained with pararosaniline (most
commonly) or basic fuchsin.
Though flagella staining is uncommon in clinical settings, the technique is commonly
used by microbiologists, since the location and number of flagella can be useful in classifying
and identifying bacteria in a sample. When using this technique, it is important to handle the
specimen with great care; flagella are delicate structures that can easily be damaged or pulled off,
compromising attempts to accurately locate and count the number of flagella.
Preparing Specimens for Electron Microscopy
Samples to be analyzed using a TEM must have very thin sections. But cells are too soft to cut
thinly, even with diamond knives. To cut cells without damage, the cells must be embedded in
plastic resin and then dehydrated through a series of soaks in ethanol solutions (50%, 60%, 70%,
and so on). The ethanol replaces the water in the cells, and the resin dissolves in ethanol and
enters the cell, where it solidifies. Next, thin sections are cut using a specialized device called
an ultramicrotome. Finally, samples are fixed to fine copper wire or carbon-fiber grids and
stained—not with colored dyes, but with substances like uranyl acetate or osmium tetroxide,
which contain electron-dense heavy metal atoms.

Factors affecting spoilage of foods