Chapter 2.

Bacterial Morphology and Structure

Methods to study bacterial morphology and Methods to study bacterial morphology and structurestructure

• The light microscope 100-power objective lens with a 10-power

ocular lens magnifying the specimen 1000 times

• The electron microscopes

Transmission electron microscope (TEM)

can resolve particles with 0.001 μm in size

Scanning electron microscope (SEM)

is particularly providing three-dimensional images of

the surface structure of microscopic objects

• Phase microscope observe living cells

Optical methodsOptical methods

Treponemes are recognized by their characteristic

corkscrew shape and deliberate forward and

backward movement with rotation about the

longitudinal axis.

Electron micrograph of Spirillum serpens, showing

lophotrichous flagellation (9000 x ).

•Simple staining methods

•Differential staining methods (Gram stain, Acid-fast stain)

•Special staining methods

The spore staining method

The flagella staining method

The capsule staining

Negative staining

Methods to study bacterial morphology and Methods to study bacterial morphology and structurestructure

Staining methodsStaining methods

• Unit for measurement: Micron or micrometer, μm: 1μm=10-3 mm

• Size: Varies with kinds of bacteria, and

also related to their age and external environment.

Size of bacteriaSize of bacteria

Shape of bacteriaShape of bacteria

• Spherical (Cocci, sing. Coccus )

• Rods (Bacilli, sing. Bacillus)

• Spiral (Spiral bacteria)

vibrio helicobacteriumspirillum

Spherical bacteriaSpherical bacteria

DiplococciDiplococci: Pair of cells divide in one plane

StreptococciStreptococci: Chain of cells formed by dividing in one plane several times

TetradTetrad: Divide in two planes

SarcinaeSarcinae: Divide in three planes

StaphylococciStaphylococci: Divide in many planes and remain together as a cluster

Different arrangements depending on the plane of division

Considerable variation in length and diameter: 0.5-

1 um in width and 2-5 um in length.

Most of rod-shaped bacteria are single

arrangement.

Rod-shaped bacteria Rod-shaped bacteria

Diplobacilli: Bacilli that remain in pairs after they

divide.

Streptobacilli: Bacilli that remain in chains after they

divide.

Coccobacilli: A short Bacilli that nearly looks like a

cocci.

Divided into:

Vibrio: comma shaped

Spirillum: helical rigid (spirillum) or

flexible (spirochete)

Helicobacterium: curved rod-shaped

Spiral-shaped bacteriaSpiral-shaped bacteria

Structure of BacteriaStructure of Bacteria

Bacteria are prokaryotesBacteria are prokaryotes

•Have a nucleoid (nuclear body) rather than an enveloped nucleus

•Except ribosomes, lack membrane-bound cytoplasmic organelles

•The plasma membrane performs many of the functions carried out by membranous organelles in eukaryotes

•Despite their lack of complexity compared to eukaryotes, a number of bacterial structures may be defined:

•Only plasma membrane, which belonging to cell envelope, is the essential component for any bacteria.

Basic structure: Cell wall Cell membrane Cytoplasm Nucleoid

Specific structure: Flagellum Pilus Capsules and slime layers Endospores (spores)

Important bacterial structuresImportant bacterial structures

Cell envelopeCell envelope

•The cell envelope may be defined as the cell membrane and

cell wall.

•Usually, bacterial cell envelopes fall into two major categories:

Gram positive and Gram negative.

•This is based on Gram staining characteristics that reflect major structural

differences between the two bacterial groups.

1884: Christian Gram: First publication for the Gram stain method

Editor's note: I would like to testify that I have found the Gram method to be one of the best and for many cases the best method which I have ever used for staining Schizomycetes.

Gram-positive cocciGram-positive cocci Gram-negative bacilliGram-negative bacilli

Simplified diagram and electronic microscopy

pictures of the cell envelope of G+ and G- bacteria

periplasmic space

Cell wall

The cell wall consists of the peptidoglycan (murein) layer and attached structures.

Situation: outmost portion. 15-30 nm in thickness10%-25% of dry weight.

peptidoglycanpeptidoglycan

•It is huge (billions in molecular weight).

•Peptidoglycan is found in all eubacteria except Chlamydia and

Mycoplasma.

•The peptidoglycan is a single bag-shaped, highly cross-linked macromolecule that surrounds the bacterial cytoplasmic membrane and provides rigidity (which decides the shape of a bacterium) .

peptidoglycan

•Peptidoglycan consists of a glycan (polysaccharide) backbone consisting of alternatively residues of N-acetyl muramic acid and N-acetyl glucosamine connected by -1,4 linkage.

•with tetrapeptide side chains usually containing D- and L- amino acid residuals, and in some instances containing diaminopimelic acid (DAP) residual.

•The side chains are cross-linked by peptide bridges. These peptide bridges vary in structure among bacterial species (gram-negative bacteria have no peptide bridges)

•Lysozyme can block the -1,4 linkage. •So lysozyme can kill G+ and G- bacteria by destroying their glycan backbone .

•Penicillin can block the linkage of tetrapeptide side chains and peptide bridges.• So penicillin can kill G+ bacteria by inhibiting their peptidoglycan synthesis.

Cell wall: Cell wall: characteristics of gram-positive bacteriacharacteristics of gram-positive bacteria

•A gram-positive bacterium has a thick cell wall (20-80 nm).

•It may also include special components such as teichoic acids and proteins.

•Membrane teichoic acids are anchored in the cytoplasmic membrane. Lipoteichoic acids

•Teichoic acids are water-soluble, a polymer of polyphosphates.

•Wall teichoic acids are linked to the peptidoglyacan.

•The peptidoglycan layer of it is thick (15-50 layers).

Cell wall: Cell wall: characteristics of gram-negative bacteriacharacteristics of gram-negative bacteria

•A gram-negative cell has a thin (10-15 nm) cell wall but it is more complex than gram-positive cell walls, both structurally and chemically.

•The peptidoglycan layer of it is thin (1-2 layers).

•It has no any teichoic acids but has an unique outer membrane which is external to the peptidoglycan layer.

•The space between cytoplasm and outer membrane is periplasmic space. It contains transport, degradative, and cell wall synthesis proteins.

Outer membraneOuter membraneOuter membrane of a G- bacterium contains lipopolysaccharide (LPS), Phospholipids and lipoproteins.

phospholipids

A group of transmembrane proteins is known as porins which form channels to allow passage of small hydrophilic nutrients (such as sugars, amino acids and certain ions) through the outer membrane.

Lipopolysaccharide (LPS)Lipopolysaccharide (LPS)

• The O antigen (somatic antigen) is a specific

polysaccharide region composed of repeating

units by some specific monosaccharides.

Highly variable

•The core polysaccharide is similar within a

single genus.

•lipid A contains β hydroxy fatty acids

(uncommon in nature). This molecule

displays endotoxin activity.

LPS is also called endotoxin because it is poisonous to mammal cells.

It consists of 3 regions: an external O antigen, a middle core, and an inner lipid A.

What is the difference between

G+ cell wall and G- cell wall?

Property Gram positive Gram negative

Number of layers in wall 1 2

Peptidoglycan content >50% 10-20%

Teichoic acid + -

Outer membrane - +

lipopolysaccharide - +

Sensitive to penicillin yes Less sensitive

Digested by lysozyme yes weakly

• Maintaining a bacterial characteristic shape

• Possessing the resistance to osmotic pressure

• Providing a platform for surface appendages such as flagella and pili.

• Providing a pathogenic function to adhere host cells

Cell wall: Cell wall: function

For G+ bacteria, the major adhering molecular is teichoic acids.

For G- bacteria, the major adhering molecular is the pili as well as

some of the outer membrane proteins).

• Providing attachment sites for bacteriophages

• Play an essential role in cell division

• Participating the bacterial material exchange

• Be the sites of major antigenic determinants of the cell surface.

Cell wall: Cell wall: function

For G+ bacteria, the major surface antigen is the teichoic acids and then

the polysaccharides and the peptidoglycan.

For G- bacteria, the major surface antigen is the LPS and then the outer

membrane proteins.

• When bacteria are treated with 1) enzymes that are lytic for the cell wall

e.g. lysozyme or 2) antibiotics that interfere with biosynthesis of

peptidoglycan, wall-less bacteria are often produced.

• Usually these treatments generate non-viable organisms. Wall-less

bacteria that can not propagate are referred to as spheroplast (G-) or

protoplast ( G+).

• Occasionally, some wall-less bacteria can propagate (so called as L forms

of bacteria).

Wall-less forms of bacteria

Bacterial L form•L-forms of bacteria are artificial cell wall-less organisms (means they are

different from Mycoplasma) first found by the Lister institute in 1935.•Can be produced from normal bacteria by damaging the cell wall (by

Penicillin, salt solutions, antisera etc.).•L-form cells are difficult to cultivate and usually require a medium with a

right osmotic strength. •They can reconvert to wild type. Rarely they can develop to stable L-

forms.•May cause chronic infections which are relatively resistant to antibiotic

treatment and difficulty to detectable using routing serological diagnosis

methods, because the target sites of some antibiotics (penicillin) and somatic

antigens (O antigen) are absent..

Electron micrograph of StaphylococcusElectron micrograph of Staphylococcus

A: L-formA: L-form B: Reconvert to wild typeB: Reconvert to wild type

•Because of the lack of a rigid cell wall, L-form cells generally

show various shapes (such as filar) compared to the original

shapes and gram-negative staining.

Important bacterial structuresImportant bacterial structures

Cell membrane

Site of biosynthesis of DNA, cell wall polymers and membrane lipids.Selective permeability and transport of solutes into cells

Electron transport and oxidative phosphorylation

Excretion of hydrolytic exoenzymes

Mesosomes

Mesosomes are specialized structures folded invaginations in

the plasma membrane of bacteria

divided into septal and lateral mesosome.

Functions of the cytoplasmic membraneFunctions of the cytoplasmic membrane

(1) selective permeability and transport of solutes;

(2) electron transport and oxidative phosphorylation, in aerobic

species;

(3) excretion of hydrolytic exoenzymes;

(4) bearing the enzymes and carrier molecules that function in the

biosynthesis of DNA, cell wall polymers, and membrane lipids;

(5) bearing the receptors and other proteins of the chemotactic and

other sensory transduction systems.

Important bacterial structuresImportant bacterial structures

Cytoplasm

• Composed largely of water, together with proteins, nucleic acid, lipids and small amount of sugars and salts

• Ribosomes:

• Plasmids: extrachromosomal genetic elements capable of autonomous replication

• Inclusions: sources of stored energy, e,g volutin

RibosomesRibosomes

•15-20 nm in diameter with 70S, distributed throughout the cytoplasm

•sensitive to streptomycin and erythromycin

• a workbench for protein synthesis, numerous

• the target of antimicrobial agents: Streptomycin Tetracyline

PlasmidsPlasmids

•Plasmids are small, circular / line, extra-

chromosomal double-stranded DNA.

•Usually present in multiple copies and

are capable of self-replication.

•Often code for pathogenic factors and

antibiotic resistant factors.

Are not essential for bacterial survival

Inclusions of Bacteriagranulose

are aggregates of various compounds that are normally involved in

storing energy reserves or building blocks for the cell. Inclusions

accumulate when a cell is grown in the presence of excess nutrients and

they are often observed under laboratory conditions.

InclusionsInclusions

Important bacterial structuresImportant bacterial structures

caryoplasm

• Lacking nuclear membrane,

absence of nucleoli, hence

known as nucleic material or

nucleoid, one to several per

bacterium.

Special bacterial structuresSpecial bacterial structures

•Are structures surrounding the outside of the cell envelope.

•Usually, slime layer is thinner than capsule.

•They are usually demonstrated by the negative staining or “capsule stain” which gives color to the background.

Capsules and slime layers

•They are usually composed of polysaccharide, however, in certain bacilli they are composed of a polypeptide.

• Some strains within a species can produce a capsule, whereas the others can not.

•They are not essential to bacterial viability.

•Capsules are often lost during in vitro culture.

•The capsules contribute to the invasiveness (virulence) of pathogenic bacteria by protecting them from phagocytosis by phagocytes.

Capsules and slime layers

Special bacterial structuresSpecial bacterial structures

Flagellum: general descriptionFlagellum: general description

• a thin, threadlike appendage on many bacterial cells that is responsible for

their motility• Flagellum consist of a number of proteins including flagellin• Are embedded in the cell membrane, extend through the cell envelope and project as a long strand.

• The diameter of a flagellum is thin (20 nm) and long with a length 10

times of the bacterial cell diameter. Due to their small diameter,

flagellum cannot be seen under light microscope unless a special stain is

applied.

Flagellum: typesFlagellum: types• Three types of arrangement are known:

monotrichous single polar flagellumlophotrichous in one plane with cellperitrichous flagella distributed over the entire cell

monotrichous lophotrichous peritrichous

Electron graph

Flagella stainLight microscopy graph

Flagellum: composition Flagellum: composition • Composition: protein

A bacterial flagellum is made up of several thousand molecules of a protein

subunit called as flagellin. They move the bacterial cell by rotating with a

propeller like action.

Flagellins are highly

antigenic (H antigens).

Flagella: function: function

•Motility of bacteria: Flagella enable bacteria to taste their environment and respond to

specific chemical foodstuffs or toxic materials and move towards or away from

them (chemotaxis).

•Identification of bacteria: According to the mobility and antigenicity of flagellins (H antigen).

•Possible Pathogenesis: In the past, flagella were considered not to be relative to bacterial

pathogenicity. In the recent data, the flagella of some bacterial species have

adhering ability to host cells.

Special bacterial structuresSpecial bacterial structures

PilusPilus•Pili are hair-like appendages of many bacterial cells.•shorter and thinner than flagella, only visible under electron microscope. •receptors for certain bacterial viruses. Chemical nature is pilin

•Pili are composed of structural protein subunits termed pilins.•Two types can be distinguished: Common pili

•Shorter, thinner, numerous for one bacterium•Relative to bacterial adhesion (adhering to host cells)•Contribute to virulence of some pathogenic bacteria

Sex pili•Longer, coarser, only 1-4 for one bacterium•Relative to bacterial conjugation (a pattern of bacterial genetic material exchanges)•The recent data revealed that sex pili of some bacteria has the ability to adhere host cells.

PilusPilus

Common pili

Sex pili

Donor bacterium

Recipient

Electron graph of piliElectron graph of pili

Endospores (spores)

Special bacterial structuresSpecial bacterial structures

Endospores (Spores)Endospores (Spores)

• Under adverse conditions, such as nutrient/water depletion, some bacteria form a thick wall inside the cytoplasmic membrane leading to a resting stage known as spores.

Endospores (spores)Endospores (spores)• Spore is an intracellular body which found in some species of bacteria. One spore-forming bacterium can only produce one spore. Spore can be seen after staining with dyes by heating the preparation. Sometimes, it can also be seen as a colorless area by using conventional bacterial staining methods.

Endospores (Spores)•A highly resistant resting structure produced within a bacterium. It enables the bacterium in soil to survive many years. It can withstand heating, freezing, chemicals and radiation.

•Spores are commonly found gram-positive bacilli in the genera Bacillus and Clostridium.

•The different sizes, shapes and positions of spores will help us to identify spore-forming bacteria.

•Under favorable conditions, one spore germinates into one vegetative bacterial cell (propagation / multiplication). But spore has no ability for proliferation.

•Spore has no ability for propagation (multiplication).

Contents of endospore

Corespore wall /coreCortexCoatexosporium

Process of endospore formation: Sporogenesis/sporulation

Classification of bacteria

•Since there is the diversity of organisms including bacteria,

it is necessary to group similar organisms together and

organize these groups in a non-overlapping hierarchical

arrangement.

•Taxonomy is the science of biological classification, which

includes the two disciplines of classification and

nomenclature.

The bacteria are classified in a hierarchic system

based on phenotypic characteristics (morphological,

physiological, and chemical characteristics).

The basic unit is the species. Similar and related

species are classified in a single genus and related

genera are placed in a single family.

Classification of bacteria

Classification of bacteria

Family (familia) Enterobacteriaceae

Genus Escherichia

Species E. coli

Variety or type Serovar O157:H7

Strain xyz

•In formal terms, the prokaryotes are classified in phyla,

classes, orders, families, genera, and species, plus subtaxa

if any:

nomenclature of bacteria

StaphylococcusStaphylococcus aureusaureus

S. aureusS. aureus

Genus Genus species species

•Scientifically, species is named by a Latin binominal. Each species receives a name of two parts, and the first is the genus name and the second is the species name.

•The genus name is always capitalized but the species name is not. Both species and genus are usually in italics.

•The genus name may be abbreviated by just using its initial letter.

•While described in Chinese, the genus is followed by species name.

Oxygen• Obligate aerobe

an organism that grows only in the presence of oxygen

• Microaerophile an organism that requires a low concentration of oxygen for

growth

• Facultative anaerobe an organism that grows with or without oxygen

• Oblige anaerobe an organism that grows only in the absence of oxygen

Temperature• Psychrophile (15-20 ℃ )

an organism that grows best at cold temperatures

• Mesophile

an organism that grows best at moderate temperatures

• Thermophile (50-60 ℃ )

an organism that grows best at high temperatures

pH

• Acidophile

an organism that grows best at a pH below 6

• Neutrophile

an organism that grows best at a pH between 6 and 8

• Alkalophile

an organism that grows best at a pH above 8

Some other features that have been used to classify bacteria

• Gram stain (G+/G-)

• Cell shape

(coccus/ bacillus/ spiral bacterium)

• Ability to form spores

(spore-forming/non-spore-forming clostridia)

Summary Summary

Structure of bacteria include essential structures of cell wall, cell membrane, cytoplasm, and nuclear material (nucleoid).

Some bacteria also have one or more of the particular structures of capsule, flagella, pili, endospores.

The difference between G+ cell wall and G- cell wall

Medical importance of four special structures