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INTRODUCTION the kingdom protista has been classified into prokaryotes and eukaryotes. Bacteria are prokaryotes, white fungi, algae, protozoa are eukaryotes. MACROSCOPIC MORPHOLOGY Size of bacteria Prokaryotic cells are the smallest of the unicellular organisms. Usually it varies from 1-15 µm wide and 2 – 6 µm long.

• SHAPE OF THE BACTERIA • When viewed under the microscope, most clinically significant bacteria appear in three basic shapes, they are ; • Cocci (from Kokkes meaning berry) are spherical or oral. • Bacilli (from bacillus meaning rod) are rod shaped. • Spiral • Other then three basic bacterial shapes some bacterial are square, filaments and large number produces buds, lancet (spneumoid bean – shaped (Neisseria gonnorhea).

• Cocci • These spherical bacteria vary in diameter between 0.5µm - 2µm. • They occur as single cells (micro cocci) • Pairs of cocci (Diplococci) • Chains (Streptococci) • Packets (Staphylococci) • Cube – shaped cellular packets (Sarcinae) • Bacilli • This morphotype consists of cylindrical cells, which are either large or small rods. • The bacilli are usually 0.2 – 1 µm in diameter, with lengths of approximately 10µm being common. • Most rods occur single, but some form long chains called as stretptobaccilli.

• Spiral • In reality, the spirilla are bacilli twisted into helix. • the rigid spirilla are surrounded by a cell wall and are represented by genera spirillum and vibrio. • Flexible spirilla with more than one way are typical of the spirochaetes, represented by the genera treponema, spirocheta, leptespira, borrelia and cristispira. • Actinomycetes are branching filamentous bacteria, so called because of fancied resembled to the radiating rays of sun when seen in tissue lesion (from actis meaning ray and mykes meaning fungus). •

BACTERIAL ANATOMY • The outer layer or cell envelope consists of two components – A rigid cell wall and beneath is a cytoplasmic or plasma membrane.

THE CELL WALL • • • • • •

It may be demonstrated by plasmolysis. Cell wall may be demonstrated by micro dissection. Functions Cell wall gives the cell its shape and protects cytoplasm from osmotic pressure differences between the intracellular and extra cellular environment. Cell wall also serves as primary sieve in the transport molecules into the cell. It also functions as an anchor for the capsule, as well as pilli, fimbriae, flagella. It is primary receptor for the absorption of specific viruses and bacteriophages

• • • •

Cell walls of many pathogens have components that contribute to their pathogenecity. Protects a cell from toxic substances & it is the site of several antibiotic. Frequently a space in seen between electron micrographs between the plasma membrane and outer membrane this space is the periplasmic space.

• peptidoglycan structure • The polymer contains two sugar derivatives , N – acetyl glucosamine N – acetyl muramic acid alternating in chains which are crosslinked by peptide chains. • Gram positive cell wall • Normally thick, homogeneous call wall of gram positive bacteria composed primarily of peptidoglycan. • However gram positive cell walls, usually contain large amounts of teichoic acids, polymers of glycerol joined by phosphate group. • Lipids and proteins are usually absent, •

• Functions of teichoic acids • They may contribute to the overall negative change density of cell surface. • This negative surface charge and lipoteichoic acids are strong adhesions, especially in the attachment to mammalian tissues, and adherence to red blood cells, tooth surfaces and other positively charged surfaces. • Gram negative cell walls • This membrane consists of two layers, they are • An outer layer (containing lipopolysaccharides) • A thin peptidoglycan layer. • A small protein associated lipid or lipoproteins called the murein lipoprotein, forms link with outer membrane.

• Outer membrane • The outer membrane functions as a molecular sieve, regulating the transfer of molecules with 700 Daltons into the cells. • This membrane also contains receptor molecules place a role in adherence to the other bacteria. • The outer membrane has both important physiological and mechanical functions, it contains a variety of proteins that either traverse the membrane or embedded in it. • Protein that traverse the outer membrane are referred to us transmembrane protein or porins and function in the transport of molecules. •

• • • • • • • •

The most notable chemical component of outer membrane is the lipo polysaccharide (LPS). LPS is an amphipathic molecule, i.e one end is hydrophilic and other end is hydrophobic. Chemically, LPS has three main components O – specific polysaccharide The core polysaccharide Lipid A The core polysaccharide joins to lipid A to the O – specific polysaccharide and consists of unique molecule, 2 keto – 3 – deoxy octanolate (KDO). Lipid A is highly hydrophobic and embedded in the outer leaflet of the outer membrane.

• It contains hydroxy fatty acids thought to function as the endotoxin. • Functionally, the LPS protects the cell from the action of antibody and complement, is a bacteriophage receptor, and one of major endotoxic molecules of gram negative bacteria. • It also functions as activators of variety of inflammatory mediators including interleukin – 1, prostaglandin, even tumour research factor. • Hence, it is considered an important virulence factors of gram negative bacteria.

• Cytoplasmic membrane • The cytoplasmic membrane is a layer lining the inner surface of the cell wall separating it from the cytoplasm. • It consists of three layers – outer, middle and inner layer, chemically, both inner and outer layers are composed of 70%, protein, the middle (hydrophobic) layer contains 30% lipid in addition to fatty acids, phospholipids, transmembrane proteins (permeases). • • In summary, functionally, the cytoplasmic membrane is one of most important components of the bacterial cell, which is site of anabolic and catabolic metabolism • and regulates the transport of molecules into without of the cell.

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The cytoplasm and its inclusions The bacterial cytoplasm consists of central nuclear region surrounded by ribosome’s mixed in a sea of enzymes, cofactors, amino acids, and vitamin, as well as multitude of other bone and molecules. It differs from eukaryotic cytoplasm in not exhibiting internal mobility and the absence of endoplasmic reticulum and mitochondria. These serve as building blocks for cellular biosynthesis and energy production. The cytoplasm contains ribosome’s, mesosomes, inclusions and vacuoles. Ribosome’s The ribosome’s are the most abundant inclusion in bacterial cytoplasm. They are called 70S ribosomes. It consists of 40% protein and 60% RNA. Eukaryotic ribosome’s are larger than bacterial ribosome’s. Three types of RNA exist ribosomal RNA (rRNA), Transfer RNA (tRNA), messenger RNA (mRNA)).

Mesosomes (Chondroids) • These are vesicular, convoluted or multilaminated structures formed as invaginations of the plasma membrane into cytoplasm’s. • They are more prominent in gram positive bacteria. • They are principal sites of respiratory enzymes in bacteria and are analogous to the mitochondria of eukaryotes. • Mesosomes are often seen in relation to the nuclear body and site of synthesis of cross wall septa, suggesting that they coordinate nuclear and cytoplasmic division during binary fission.

Nucleus •

Bacterial nuclei can be demonstrated by acid or ribo nuclease hydrolysis and subsequent staining for nuclear material

Bacterial nuclei have no nuclear membrane or nucleolus. The nuclear DNA is not associated with basic protein. The genome consists of single molecule of DNA arranged in the form of circle.

The bacterial chromosome is haploid and replicates by simple fission instead of by mitosis as in higher cells.

Bacteria may posses extranuclear genetic elements consisting of DNA. These cytoplasmic carries of genetic information ore termed as plasmid or episomes.

These may account for drug resistance which may constitute a survival advantage.

Bacterial storage granules and inclusion •

• • • •

During their growth and metabolism, bacteria accumulate variety of cytoplasmic storage products, as a result of the environment and metabolism by the cell of large number of carbohydrates, lipids, proteins. These inclusions, includes both membrane limited and Non membrane limited. Two major types of inorganic inclusion bodies are seen. Polyphosphate granules or volutin granules Magnetosome.

• • • •

Capsules & Slimes

Depending upon the overall consistency of this material, this layer referred as either a capsule or slime layer. Capsule – usually of uniform consistency and integrity Slime layer – are of ill defined and loosely formed. Streptococcus salivarius and streptococcus mutans form large capsules consisting of glucan (dextran) or Fructan (Levan) depending on the source on which or in which they are grown.

Chemical composition • • •

Capsules are compared of either carbohydrate or protein depending on the species. Capsules have been identified that contain pectin, cellulose, and mixed carbohydrate polymers. Several of the bacterial capsules have been formulated into effective vaccines. A potential capsular vaccine has been produced streptococcus mutans that may be effective against dental caries.

Functions of Capsules

• Capsules are important in permitting bacteria to express their virulence capabilities, as well as providing them with ability to survive the numerous host defense mechanisms. • Capsules also produces or provide an immunologic specifying encapsulated bacteria produce smooth (S) colonies and are immunologically characterized by as “S-type” colonies, white bacteria that have had their capsules removed produce rough (R) colonies and are termed “R – type” colonies. • In addition to capsules providing immunologic specificity, and functioning as virulence factors, capsules also serve as osmotic barrier, which prevents the too – rapid influx of water into or out of the cell.

Flagella and Motility • • • • • • • • •

Flagella are organelles of motility / organs of locomotion. Each flagellum consists of three distinct parts, The filament – external to the cell and connected to the hook at the cell surface. Hook – connects the basal body to the distal portion of filament. The basal body – which is embedded in the cytoplasmic membrane and portions of cell wall. Gram positive - 4 rings. Gram negative – 2 rings. Chemically, flagella are made up of protein flagellin similar to keratin or myosin. Flagellin structure of each bacterial species is sufficiently unique to confer immunologic specificity and useful in microbiological identification of specific microorganisms.

• • • • • • • • • • •

Types of flagellar arrangement 1. Flagella arranged all round the cell (peritrichous) eg. Typhoid 2. Monotrichous – single flagella situated at one end eg. Vibrio cholera 3. Lopotrichous – Tufts of flagella at one end eg. Spirilla 4. Amphitrichous – Flagella at the both poles Motility Motility can be observed by noting the spreading type of growth on a semisolid agar medium.

Pilli or Fimbriae • Both Pilli and Fimbriae are attached to the outer most surface of cell. These hair like appendages. • They are confined to gram negative bacteria and to selected gram positive bacteria (i.e, streptococcus, actinomyces). • The pilli are distinguished from flagella in being approximately one half their diameter as well as being distinctively straight or stiff in appearance.

Functions of Pilli or Fimbriae

These structures function primarily in the transfer of genetic material between the bacteria, as well as adherence.

Pilli that help in transfer of genetic material are referred as F or sex Pilli.

F Pilli form physical bridges between the donor and recipient cells known as conjugation bridges, which permit the transfer of DNA between them.

The Pilli are also bacterophage receptors, specific bacteriophage will absorb to the type of the Pilli, which are then retracted into the cell.

• This reaction brings the virus into close contact with bacterial cell surface, where a second receptor in the outer membrane transfers the virus into the cytoplasm. • Pilli are also adherence structures, their adherence specificity for certain bacterial cells gives them their type specific character. • The type specific fimbraie are also involved in cellcell aglutina, an antigens, as well as in the agglutinaous of erythrocytes. • Importantly, the ability of several of the gramnegative pathogens to express their virulence in a susceptible host is regulated by the presence or absence of fimbriae.


Some bacteria have the ability to form highly resistant resting stages called spores.

Each bacterium forms one spore, which on germination forms a single vegetative cell.

An bacterial spores are formed inside the parent cell, they are called endospores.

Sporulation occurs after a period of vegetative growth and is presumed to be related to the depletion of exogenous nutrient. Sporulation is initiated by the appearance of clear area, usually near one end of the cell, which gradually becomes more opaque to form forespore.

The fully developed spore has at its core the nuclear body, surrounded by the spore wall, a delicate membrane from which the cell wall of the future vegetative bacterium will develop.

• Young spores are seen attached to the parent cell. The shape and position of the spore and its size relative to the parent cell are species characteristics. • Spores may be central (equatorial), terminal or subterminal. They may or may not distend the bacillary body. • Bacterial spores constitute some of most resistant forms of life, they are extremely resistant to desiccation and relatively to chemicals and heat. • They can be destroyed by autoclaving at 120°C for 15 minutes

Bacterial Growth and Multiplication •

By Definition Growth is the orderly increase or synthesis of al cellular constituents from extra cellular nutrients the ultimate result of this increase being division or replication

Transverse or Binary cell division •

Growth of bacteria can be separated into two distinct stages

An increase in cell mass such as elongation of a bacillus, or an increase in size or volume such as in occurs

The division of these cells into two daughter cells

The most common mechanism of bacterial cell division which can be described as Binary or transverse division.

Ideally, binary fission consists of at least three steps •

During, the initial growth or division period, bacilli increase in both length and volume, while cocci increase in volume. The nuclear material separates into two approximately equal halves, with one of the nuclear segments being attached to the cytoplasmic membrane.

Late in the initial division period, division septa or cross walls develop.

The septum routinely forms at the approximately center of the dividing cell or a result of the in growth of the cell wall. Septum formation at the approximate center of the cell is common among rods and bacilli.

While in gram –ve bacteria were originally thought to have a “constrictive division” (a pinching of the central region of the cell to form two identical daughter cells), the outer membrane actually is maintained as a rigid layer, with septum formation occurring as an in growth of the cytoplasmic membrane and

peptidoglycan. Therefore, the completed septum consists of two “lamellae” of peptidoglycan that are separated by a gap.

In gram positive bacteria, cell separation or binary. Fission occurs as the result of hydrolytic cleavage of the in growing peptido glycan by specific hydrolytic enzymes, or amidases.

While in gram –ve bacteria cell separation occurs by the ingrowth of the outer membrane after septum formation has been completed. This ingrowth of the outer membrane results in physical separation of cells into two identical daughter cells.

Other mechanism of bacteria cell division • Bacteria such as hyphomicrobium, corynebacterium and mycobacteria divide by budding. • The budding cell originates as an outgrowth of the original, or mother cell, which eventually reach a size equal to that of the mother cell. the bud then separates to undertake an independent existence several of the actinomycetes form filamentous cells that reproduce by the formation of spores from a chain of cells or by a simple fragmentation of the original filament into new cells.

• The bacterial growth curve • When bacterium is seded into a suitable liquid medium and incubated its growth shows a definite course. • If bacterial consists are made at intervals after inoculation and plated in relation to time, a growth curve is obtained. The curve shows the following phases. • Lag phase • This phase of growth occurs when cells are transferred from one medium to another or one environment to another • It is a phase of adjustment and represents the period required for the adaptation of the cells to new environment. • The cells in this phase often increase in total volume almost two or threefold, but they do not divide. • These cells are rapidly synthesizing DNA, new protein and new enzymes as a prerequisite to division.

• Exponential phase – or log phase • In this phase, cells are dividing at both a constant geometric rate as well as maximum rate. • Cellular component such as RNA, protein, dryweight, and cell wall polymers are also increasing at a constant rate, they are much smaller in diameter than cells in the lag phase. • The exponential growth phase usually comes to an end because of depletion of essential nutrients, depletion of oxygen in an acrostic culture or the accumulation of toxic products.

• Stationary phase • During this phase, there is a rapid decrease in the rate of cell division. Eventually, the total number of dividing cells will equal the number of dying cells, a true stationery cell population occurs. • The cells then direct their resources towards survival energy generation is harnessed to maintain osmotic barriers, mobility, and the repair and synthesis of essential macromolecules. • The energy required to maintain cells in stationery phase is called. Maintenance energy and is obtained from the degradation of cellular storage products. • After all of the storage products are exhausted, the cells then degrade their cellular components, which leads to death phase.

• Death Phase • In this stage, the cells reproduce more slowly, and death overtakes them in increasing numbers. • The cells eventually enter the logarithmic death phase, during which the decrease in the number of viatie cells occurs at a regular, unchanging rate, a rate that approximates that of the exponential growth phase but is of negative slope.

Basic Nutritional Requirements of Bacteria •

For growth and multiplication of bacteria, the minimum nutritional requirements are water, a source of carbon, nitrogen and some in organic rates.

At least ten elements are considered essential for growth which are usually supplied from the environment (Tube from sots).

Amino acids, pur, pyramidines and vitamin are also required for cell growth.

AA are required for the synthesis of cellular protein purinices and pyramides are needed for synthesis of nucleic acid biosynthesis as well as participating as activating cervical molecule in a biosynthesis reaction (e.g. peptidoglycan, LPS, polysaccharide synthesis). The vitamins function primarily as components of co enzymes in the numerous enzyme driven chemical reactions that occur in cells.

Oxygen requirement • Depending on the influence of oxygen on growth and viability, bacteria are divided into aerobes and anaerobes. • Obligate aerobes-Which will grow only in the presence of oxygen. • Facilitative anaerobes – which are ordinarily aerobic but can also grow in the absence of oxygen. • Obligate anaerobes- which will grow in absence of oxygen. • Microaerophilic – are those that grow best in the presence of low oxygen tension.

• Aerobic bacteria obtain their energy and intermediates only through oxidation involving oxygen as the ultimate hydrogen receptor. • while anaerobes use hydrogen acceptors other than oxygen. • Facilitative anaerobes may act in both ways. • In case anaerobes electron acceptor is atmospheric oxygen the carbon and energy source may be completely oxidized to carbon dioxide and water. This process is called oxidative phophorylation. • Anaerobic bacteria use as electron acceptor compounds as nitrates or sulphates instead of oxygen. A more common process in anaerobic bacteria is series of oxidoreduction

• Carbon Dioxide • All bacteria require small amounts of carbon dioxide for growth • This requirement is met by carbondioxide present in the atmosphere, or produced endogenously by metabolism. • Temperature • Bacteria vary in their requirements of temperature for growth • For each species, there is a temperature range and growth does not occur above maximum or below the minimum of the range

• Temperature at which growth occurs best is known as optimum temperature, which is in case of most pathogenic bacteria is 37°C. • Bacteria which grow at temperatures 25-40°C are called mesophilic bacteria there that grow at temperature below 20°C – psychrophilic. Bacteria those grow at higher temperature between 55 – 80°C thermophilts. (Nonperotogenic). • Heat is an important method for the destruction of microorganism moist heat causing coagulation is denaturation of proteins and dry heat causing oxidation and charming. • Moist heat is more lethal than dry heat. The lowest temperature that kills bacterium under standard conditions in a given time is known as the thermal death point.

• • • • • • • • • •

H-ion concentration Bacteria are sensitive to variations in pH. Each species has a pH range, above or below which it does not survive, and an optimum pH at which it grows best. The majority of pathogenic bacteria grow best at neutral or alkaline reaction (pH – 7.2 – 7.6). Light Bacteria grow well in the dark. They are sensitive to ultraviolet light and other radiations. Bacterial Virulence Factors A pathogenic microorganism is capable of producing disease in a susceptible host. Bacteria that cause decrease are virulent microorganisms. A virulence factor is a properly or characteristic of a pathogenic that allows it to cause disease. The sum total of virulence factors of a pathogenic bacterium includes the mechanisms by which the bacterium evades the hosts defensive mechanism, establishes an infection and damages host cells.

• Infection, defined here is an invasion or colonization of the host by pathogenic microorganism or its products, resulting in disease in the host. • Bacteria may establish an infection by two mechanisms – Colorization and invasion of tissues of the host – The production of toxins that may dissemitate and produce cytotoxic effects on tissues distant from the initial site of infection. Therefore for an infection to occur, the infecting organism must be able to • • • •

Gain access to host tissues Multiply on or in host tissue Resist host defense mechanism Damage host tissues

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