The classification of microorganism.

Morphology of Bacteria

• Bacteria are prokaryotic cells• The bacteria are single celled organism that

reproduce by simple division, i.e. binary fission.• Most are free living and contain the genetic

information , energy-producing and biosynthesis system necessary for the growth and reproduction

• Size of bacteria- very small in size, unit of measurement in bacteriology is the micron or micrometer (µm)

Shape of Bacteria• Cocci: cocci (from kokkos meaning berry) are spherical or

nearly spherical.• Bacilli: Bacilli (from baculus meaning rod) are relatively

straight, cylindrical cells. In some of the bacilli, the length of the cells may be equal to width, such bacillary forms are known as coccobacilli

• Vibrions : Vibrions are curved or comma shaped rods• Spirillas: spirilla are rigid or helical forms• Spirochetes : spirochetes (from speria meaning coil and

chaite meaning hair ) are flexuous spiral forms.• Mycoplasm: Mycoplasma are cell wall deficient bacteria and

hence do not possess a stable morphology.

Arrangement of bacterial cells

• Pathogenic bacterial species appear as sphere (cocci), rods (bacilli), spirals.

• Bacteria sometimes show characteristic cellular arrangement or grouping.

• The type of cellular arrangement is determined by the plane through which binary fission takes place by the tendency of the daughter cells to remain attached even after division.

• Cocci arrangment 1. Diplococci: cocci may be arranged in pairs when

cocci divide and remain together. Eg. Pnemococci, Gonococci

2. Long chains: long chains when cells adhere after repeatd divisions in one plane. Eg. Streptococcus, Enterococcus, Lactococcus

3. Grape like clusters: when cocci divide in random planes. Eg. Staphylococcus

4. Tetrads: square groups of four cells when cocci divide in two planes. Eg. Micrococcus

5. Cubical packets: cubical packets of eight cells when cocci divide in three planes. Eg. Sarcina

• Bacilli arrangementBacilli split only across their short axes, therefore

the patterns formed by them are limited.1. Chains : Eg. Streptobacilli2. Others are arranged at various angles to each

others, resembling the letter V presenting a cuneiform or Chinese letter. Eg. Cornybacterium diptheriae

Structure of Bacteria

Cell Wall

• The cell wall is the layer that lies just outside the plasma membrane.

• It is 10-25 nm thick, strong and relatively rigid, tough with some elasticity.

• It is openly porous, being freely permeable to solute molecules smaller than 10KDa in mass and 1 nm diameter.

• Marks the difference between gram +ve and gram –ve bacteria

Functions of Cell Wall

• To impart shape and rigidity to the cell.• It supports the weak cytoplasmic membrane against

the high internal osmotic pressure of the protoplasm.• Maintains the characteristics shape of the bacterium.• It takes part in cell division.• Provide specific protein and carbohydrates receptors

for the attachment of some bacterial viruses.• Also functions in interaction (e.g adhesion) with

other bacteria and with mammalian cells.

Chemical Structure of Bacterial Cell Wall• Chemically the cell wall is composed of mucopeptide

(peptidoglycan or murein) scaffolding formed by N-acetyl glucosamine and N-acetyl muramic acid molecules alternating in chains, which are crosslinked by peptide bonds.

• Peptidoglycan consists of three parts1. A backbone- composed of alternating N-acetyl glucosamine

and N- acetyl muramic acid.2. A set of identical tetrapeptide side chains attached to N-acetyl

muramic acid.3. A set of identical pentapeptide cross-bridges• In all bacterial species, the backbone is the same, however,

tetrapeptide side chains and pentapeptide cross-bridges vary from species to species.

Peptidoglycan in Gram positive bacteria

• Linked by polypeptides

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Difference Between Cell Wall of Gram-positive and Gram-negative Bcateria

Gram-positive Bacterial Cell Wall• It is about 80 nm thick and is composed mostly of several

layers of peptidoglycan.• Components of gram positve bacterial cell wall.1.Peptidoglycan – It constitutes 50-90 percent of the dry weight

of the wall and are thicker and stronger than those of gram negative bacteria.

2. Teichoic acid – the cell wall of gram positive bacteria contain teichoic acids, which consists primarily of an alcohol (such as glycerol or sorbitol) and phosphate. There are two types of techoic acid.

a. Cell wall techoic acid- covalently linked with peptidoglycan b. Membrane techoic acid (lipotechoic acid)- covalently linked

with membrane glycolipid and concentrated in mesosomes.

Gram-negative Cell Wall• It is different from that of gram- positive cell wall.• It consists of peptidoglycan, lipoprotein, outer membrane, and

lipopolisaccharide.1. Peptidoglycan layer • It is a single unit thick and constitutes 5-10 percent of the dry

weight of the wall of gram negative bacteria.• It is bonded to lipoproteins covalently in the outer membrane

and plasma membrane and is in the periplasmic, gel like fluid between the outer membrane and plasma membrane.

• The periplasmic contains a high concentration of degradive enzymes and transport proteins.

• The periplasmic space is approximately 20 to 40 percent of cell volume.

2. Lipoprotein• Lipoprotein or murein lipoprotein seemingly attach to the

peptidoglycan by their protein portion and to the outer membrane by their lipid component.

• It helps to stabilize the outer membrane and anchor it to the peptidoglycan layer.

3. Outer membrane• External to the peptidoglycan, and attached to it by lipoproteins is the

outer membrane.• It is bilayered structure.• Its inner leaflets is composed of phospholipid while phospholipids of

outer leaflet are replaced by lipopolysaccharides molecules.• It helps as protective barrier which prevent the entry of salts,

antibiotics and other toxic substances.• Outer membrane also consists of porins or transmembrane proteins

which helps in transportation of nutrition's to the cell.

4. Lipopolysaccharides (LPS)• A structural component that is unique to the gram negative

outer membrane is lipopolysaccharide (LPS).• It is a large complex molecule that contains lipids and

carbohydrates and consists of three components.a. Lipid A- is the lipid portion of LPS and is embedded in the top

layer of the outer membrane. When gram negative bacteria die they releases Lipid A which functions as an endotoxin.

b. The core polysaccharide- is attached to lipid A and terminal series of repeat unit contains unusual sugars. It role is to provide stability.

c. O polysaccharides –extend outward from the core polysaccharides and is composed of sugar molecules. O polysaccharides function as antigens and are useful for distinguishing species of gram negative bacteria.

Demonstration of Cell Wall

• Plamolysis- when placed in a hypertonic solution the cytoplasm loses water by osmosis and shrinks while cell wall retains its original shape and size.

• Microdissection• Exposure to specific antibody• Mechanical rupture of the cell• Differential staining procedures• Electron Microscopy

Damage to the cell wall• (Gram –ve): Lysozyme digests disaccharide in

peptidoglycan• (Gram +ve): Penicillin inhibits peptide bridges in

peptidoglycan• Autolysin : bacteria posses autolysin enzyme, able to

hydrolyse their own cell• Protoplast is a wall-less gram positive cell • Spheroplast is a wall-less gram negative cell

- Protoplasts and spheroplasts are susceptible to osmotic lysis

• L forms are wall-less cells that swell into irregular shapes

Cytoplasmic Membrane• Cytoplasmic membrane is a thin, elastic

semipermeable layer.• It lies beneath cell wall and separating it from the cell

wall.• It is 5-10 nm is thick.• It has lipid bilayer with their hydrophilic polar region

externally aligned and inner layer hydrophobic.• Presence of two proteins externally i.e external

peripheral protein and inner integral protein.• It is in fluid mosaic in structure.

Function of Cytoplasmic Membrane• Protective outer covering for the cell. • Cell membrane anchors the cytoskeleton (a cellular 'skeleton' made of protein

and contained in the cytoplasm) and gives shape to the cell.• Responsible for attaching the cell to the extracellular matrix (non living

material that is found outside the cells), so that the cells group together to form tissues.

• Transportation of materials needed for the functioning of the cell organelles without using cellular energy.

• The protein molecules in the cell membrane receive signals from other cells or the outside environment and convert the signals to messages, that are passed to the organelles inside the cell.

• In some cells, the protein molecules in the cell membrane group together to form enzymes, which carry out metabolic reactions near the inner surface of the cell membrane.

• The proteins help very small molecules to get transported through cell membrane, provided, the molecules are traveling from a region with lots of molecules to a region with less number of molecules.

Capsule • Many bacteria synthesize large amount of extracellular

polymer in their natural environments.• When the polymer forms a condensed, well defined layer

closely surrounding the cell, it is called the capsule.• It is thin in nature and can be seen only by electron

microscope.• If the polymer is easily washed off and does not appear to

be associated with the cell in definite fashion, it is referred as a slim layer.

• Capsule are composed of polysaccharide or of polypeptide.• Organism which produce capsule they are mucoid growth

on solid agar.

Capsulated Bacteria• Streptococcus pneumoniae, Nesseria meningitidis,

Klebsiella spps, Hemophillus influenza, Yersinia, and Bacillus

• Demonstration of Capsule1. Gram stain2. Special capsule staining technique3. India ink staining4. Electron microscope5. Serological method

Functions of Capsule

• Virulence factor- capsule often act as a virulence factor by protecting the bacterium from ingestion by phagocytosis, and nocapsulated mutant of these bacteria are non virulent.

• Protection of the cell wall- in protecting the cell wall attack by various kinds of antibacterial agents. i.e bacteriophage, lysozyme, lytic enzymes etc.

• Identification and typing of bacteria- capsular antigens is specific for bacteria and can be used for identification and typing of bacteria.

Flagella• Motile bacteria, except spirochets posses one or more unbranched,

long, sinuous filaments called flagella.• They are the organ of locomotion.• They are long, hollow, helical filaments, usually several times the

length of the cell..• They are 3-20 µm long and are of uniform diameter (0.01-0.013 µm).• It is originates in the bacterial protoplasm and extruded through the

cell wall.• Flagella consists of largely or entirely of protein, flagellin.• Flagella are highly antigen and induce specific antibodies in high

titer.• Flagellar antibodies are not protective but are useful in

serodiagnosis.

Parts and Composition• Each flagellum consists of three parts;a. Filamentb. Hookc. Basal Body

a. FilamentThe filament is the longest and most obvious portion which

extends from the cell surface to the tip.b. HookThe hook is a short, curved segment which links the filament to

its basal body and functions as universal joint between the basal body and the filament

C. Basal body• The basal body is embedded in the cell (cytoplasmic

membrane)• In the gram negative bacteria the basal body has four

rings connected to a central rod ( L, P, S and M).• The outer L and P rings associated with the

lipopolysaccharides and peptidoglycan layers respectively.

• S ring is located just above the cytoplasmic membrane and inner M ring contacts the cytoplasmic membrane.

• Gram positive bacteria have only two basal body rings, inner ring connected to the cytoplasmic mambrane and an outer one probably attached to peptidoglycan.

P ring

L ring

S ringM ring

Arrangements/Types

• Atrichous- without flagella.e.g Staphylococcus spps.• Monotrichous- single polar flagellum. E.g Vibrio

cholera• Amphitrichous- single flagellum at both ends.e.g

Alcaligens faecalis• Lophotrichous- tufts of flagella at one or both ends.

E.g spirilla• Peritrichous- flagella surrounding the cell. E.g

salmonella thyphoid

Demonstration of flagella

• By using Dark field microscope• Flagellar staining method• Electron microscopy• By using hanging drop method for detection of

motility of microorganism• By spread of bacterial growth as a film over

agar• Turbidity spreading through semisolid agar

Fimbria or pilli

• Many gram negative bacteria have short, fine, hair like appendages called fimbria or pilli.

• They are shorter and thinner than flagella.• They originate from cell membrane but emerge from

the cell wall.• Single cells have been seen to be covered with as few

as 10 fimbriae to as many as 100.• It is composed of structural protein called pillin.• They act as virulence factor by attaching to host cell.• Antigenicity of pilli is useful for the serodiagnosis,

preparation of antisera.

Function of fimbriae

• A. ordinary (common) pilli- it functions as organ of adhesion that allow attachment of bacterial cell to other cell surface.

• B. sex pilli- sex pilli are similar to fimbriae but they are functionally different. These are longer and fewer in numbers. They are genetically determined by sex factors or conjugation plasmids and are appeared to be involved in the transfer of DNA during congugation.

• It posses antigen hence can be used for serodiagnosis.

Demonstration of fimbriae

• Electron microscopy• Haemagglutination- attachment to red blood

cells and forms agglutination

Cytoplasm

• The cytoplasm of bacterial cell is viscous watery solution or gel.

• It contains a variety of organic and inorganic solutes, and numerous ribosomes and polysomes.

• The cytoplasm may contain granules or inclusion such as starch, glycogen, poy-β- hydroxy/alkanoates, sulphur globules, vesicles and endospores.

Ribosomes

• It is small, electron-dense particles situated in cytoplasm of bacterial cell.

• The ribosomes are the location for all bacterial protein synthesis.

• In bacterial cell there are presence of 70S ribosomes being composed of 30S and 50S subunit

• Ribosomes are associated with different ribonucleic acid (mRNA, rRNA, tRNA) for the synthesis of protein.

• Some drugs like streptomycin, tetracyclin acts on ribosome and inhibit the synthesis of protein.

Mesosomes • These are convoluted or multilaminated membranous

bodies formed as invaginations of the plasma membrane.• Mesosomes develop sometimes in relation to the nuclear

body and often from the sites of cross wall formation in gram positive bacteria.

• Types of mesosomeA. septal mesosome-they function in the formation of cross-

wall during cell divisionB. lateral mesosomes- they are present in a more random

fashion

Function of Mesosomes

• Compartmenting of DNA- involved in mechanism responsible for the compartmenting of DNA at cell division and sporulation.

• Sites of the respiratory enzymes- they provide increased membrane surface and are the principle sites of the respiration enzyme in bacteria

Intracytoplasmic inclusion bodies

• Depend upon the nutritional availability and environmental condition cell form inclusion bodies.

• The bodies are usually for storage and reduce osmotic pressure by trying up molecules in particulate form.

• They consists of volutine (polyphosphate), lipid, glycogen, starch or sulfur

• They are mostly found in diptheria bacillus, plague bacillus, and mycobacterium tuberculosis.

Demonstration of inclusion bodies

• Methylene blue staining- they stain a red violet color for inclusion bodies and blue staining for protoplasm

• Albert staining- it stains inclusion bodies with dark blue stain

• Acid-fast staining- inclusion bodies resist decoloration by 1% sulfuric acid

• Wet flim- they are more refractile than the protoplasm • Electron microscopy- they appear as very opaque,

clear demarcated bodies

Bacterial Nucleus

• The genetic material of a bacterial cell is contained in single, long molecule of double stranded deoxyribonucleic acid (DNA).

• It appears as closed circular thread about 1 mm long.• It occurs tightly coiled like as skein of woollen thread.• It consists of haploid chromosomes and replicates by

simple fission (binary fission).• It does not posses nuclear membrane, nucleolus, and

deoxyribonucleoprotein.

Demonstration of Nucleus

• Electron microscopy• Light microscopy

Plasmid • It is circular, extrachromosomal genetic in addition to

chromosomal DNA elements.• It consists of a circular piece of double-stranded DNA.• It replicates autonomously (independent replicons).• They are found mostly in bacteria but also in some

eukaryotic microorganism.• Types of plasmid• A. conjugative plasmid- such plasmid that contains the

information for self transfer to another cell by conjugation• B. non conjugative plasmid- those plasmid which do not

posses information for self transfer to another cell .

Uses of Plasmid

• As vectors in genetic engineering• To transfer drug resistant gene• To produce bacteriocin• To enhance pathogenicity• To produce enterotoxin• To clean up of environmental waste

Bacterial Spore

• Dormant cell.• Produced when starved.• Resistant to adverse conditions, ultraviolet

radiation, high temperatures, extreme freezing and chemical disinfectants.

• Contain calcium dipicolinate, dipicolinic acid (DPA).

• Mostly gram positive bacteria (Bacillus and clostridium).

Structure of Bacterial Spore

• Core- fully developed spore has core which is the spore protoplast containing the normal cell structures but is metabolically inactive.

• Core wall- the innermost layer surrounding the inner spore membrane. It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell.

• The cortex -is the thickest layer of the spore envelope. It contains an unusual type of peptidoglycan. Cortex peptidoglycan is extremely sensitive to lysozyme, and its autolysis plays a role in spore germination.

• The coat- is composed of a keratin-like protein containing many intramolecular disulfide bonds. The impermeability of this layer confers on spores their relative resistance to antibacterial chemical agents.

• The exosporium- is a lipoprotein membrane containing some carbohydrate.

Sporulation (Spore formation/ Sporogenesis)1. Spore septum • In the first observable stage of sporulation, a newly replicated bacterial

chromosome and a small portion of cytoplasm are isolated by an ingrowth of plasma membrane called spore septum.

2. Forespore• The spore septum becomes a double layered membrane that

surrounds the chromosome and cytoplasm.• Structure entirely enclosed within the orginal cell, is called forespore.

3.Spore coat• The forespore is subsequently completely encircled by dividing septum

as a double layered membrane. • The two spore membranes now engage in active synthesis of various

layers of the spore.

• The inner layers becomes the inner membrane. Between two layers is laid spore cortex and outer layer is transformed into spore coat which consists of several layers.

• In some species from outer layer also develops exosporium which bears ridges and folds.

4. Free endospores• finally exosporium disintegrates and endospore is freed.

Shape and position of spores

• The shape and position of the spore and its size is relative to the parent cell are species characteristics.

• Spores may be central (equatorial), sub-terminal(close to one end), or terminal.

• The appearance may be spherical, ovoid, or elongated and being narrower than the cell or border and bulging it.

• The diameter of spore may be same or less than the width of bacteria (Bacillus) or may be wider than the bacillary body producing a distension or bulge in the cell (clostridium).

Resistance

• Due to presence of calcium dipicolinate and acid soluble protein stabilization of DNA, protoplast dehydration, the spore coat, the greater stability of the cell protein, hence it is resistance to heat.

• It can resist boiling point and can survive for decades of times.

• Spores are resistant to Disinfectants used for disinfection

DemonstrationUnstained. Being recognized by its greater refractivity.

Gram’s stain. Appearing as a clear space within the stained cell protoplasm.

The spore stain

Uses of Spores

• Importance in food, industrial and medical microbiology.

• Sterilization control- Bacillus stearothermophilus for autoclaveBacillus subtilis susp.niger for hotair oven