Chapter 2. Bacterial Morphology and Structure. Methods to study bacterial morphology and structure...

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Chapter 2. Bacterial Morphology and Structure
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Transcript of Chapter 2. Bacterial Morphology and Structure. Methods to study bacterial morphology and structure...

  • Chapter 2. Bacterial Morphology and Structure

  • Methods to study bacterial morphology and structure The light microscope100-power objective lens with a 10-power ocular lens magnifying the specimen 1000 timesThe electron microscopesTransmission electron microscope (TEM) can resolve particles with 0.001 m in sizeScanning electron microscope (SEM) is particularly providing three-dimensional images of the surface structure of microscopic objectsPhase microscopeobserve living cellsOptical 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 methodsDifferential staining methods (Gram stain, Acid-fast stain) Special staining methods The spore staining method The flagella staining method The capsule staining Negative stainingMethods to study bacterial morphology and structureStaining methods

  • Unit for measurement: Micron or micrometer, m: 1m=10-3 mm Size: Varies with kinds of bacteria, and also related to their age and external environment.Size of bacteria

  • Shape of bacteriaSpiral (Spiral bacteria)

  • Spherical bacteria 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 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 shapedSpirillum: helical rigid (spirillum) or flexible (spirochete)Helicobacterium: curved rod-shapedSpiral-shaped bacteria

  • Structure of Bacteria

  • Bacteria are prokaryotesHave a nucleoid (nuclear body) rather than an enveloped nucleus Except ribosomes, lack membrane-bound cytoplasmic organellesThe 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.

  • Important bacterial structures

  • Cell envelopeThe 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.

  • Simplified diagram and electronic microscopy pictures of the cell envelope of G+ and G- bacteria

  • Cell wallThe cell wall consists of the peptidoglycan (murein) layer and attached structures. Situation: outmost portion. 15-30 nm in thickness10%-25% of dry weight.

  • peptidoglycanIt 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) .

  • peptidoglycanPeptidoglycan 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: characteristics 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: characteristics 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 membrane of a G- bacterium contains lipopolysaccharide (LPS), Phospholipids and lipoproteins.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) The O antigen (somatic antigen) is a specific polysaccharide region composed of repeating units by some specific monosaccharides. Highly variableThe core polysaccharide is similar within a single genus.lipid A contains 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?

  • Maintaining a bacterial characteristic shapePossessing the resistance to osmotic pressureProviding a platform for surface appendages such as flagella and pili.Providing a pathogenic function to adhere host cellsCell wall: functionFor 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 bacteriophagesPlay an essential role in cell divisionParticipating the bacterial material exchangeBe the sites of major antigenic determinants of the cell surface. Cell wall: functionFor 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 formL-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 StaphylococcusBecause 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 structures

  • Cell membraneSite of biosynthesis of DNA, cell wall polymers and membrane lipids.Selective permeability and transport of solutes into cellsElectron transport and oxidative phosphorylationExcretion of hydrolytic exoenzymes

  • MesosomesMesosomes are specialized structures folded invaginations in the plasma membrane of bacteriadivided into septal and lateral mesosome.

  • Functions 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 structures

  • Cytoplasm Composed largely of water, together with proteins, nucleic acid, lipids and small amount of sugars and saltsRibosomes: Plasmids: extrachromosomal genetic elements capable of autonomous replicationInclusions: sources of stored energy, e,g volutin

  • Ribosomes15-20 nm in diameter with 70S, distributed throughout the cytoplasmsensitive to streptomycin and erythromycin a workbench for protein synthesis, numerous the target of antimicrobial agents: Streptomycin Tetracyline

  • PlasmidsPlasmids 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

  • 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.Inclusions

  • Important bacterial structures

  • caryoplasmLacking nuclear membrane, absence of nucleoli, hence known as nucleic material or nucleoid, one to several per bacterium.

  • Special 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 structures

  • Flagellum: 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: types Three types of arrangement are known:monotrichous single polar flagellumlophotrichousin one plane with cellperitrichousflagella distributed over the entire cellmonotrichouslophotrichousperitrichousElectron graphFlagella stainLight microscopy graph

  • 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: functionMotility 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 structures

  • PilusPili 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 piliShorter, thinner, numerous for one bacteriumRelative to bacterial adhesion (adhering to host cells)Contribute to virulence of some pathogenic bacteria Sex piliLonger, coarser, only 1-4 for one bacteriumRelative 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. Pilus

  • Electron graph of pili

  • Endospores (spores) Special bacterial structures

  • 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) 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 endosporeCorespore wall /coreCortexCoatexosporium

  • Process of endospore formation: Sporogenesis/sporulation

  • Classification of bacteriaSince 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 bacteriaFamily (familia) EnterobacteriaceaeGenus EscherichiaSpecies E. coliVariety or type Serovar O157:H7Strain xyzIn formal terms, the prokaryotes are classified in phyla, classes, orders, families, genera, and species, plus subtaxa if any:

  • nomenclature of bacteriaScientifically, 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.

  • OxygenObligate aerobe an organism that grows only in the presence of oxygenMicroaerophile an organism that requires a low concentration of oxygen for growthFacultative anaerobe an organism that grows with or without oxygenOblige anaerobe an organism that grows only in the absence of oxygen

  • TemperaturePsychrophile (15-20 ) an organism that grows best at cold temperaturesMesophile an organism that grows best at moderate temperaturesThermophile (50-60 ) an organism that grows best at high temperatures

  • pHAcidophile an organism that grows best at a pH below 6Neutrophile an organism that grows best at a pH between 6 and 8Alkalophile an organism that grows best at a pH above 8

  • Some other features that have been used to classify bacteriaGram stain (G+/G-) Cell shape (coccus/ bacillus/ spiral bacterium)Ability to form spores (spore-forming/non-spore-forming clostridia)

  • 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

    nanometerThey were first observed in the samples prepared for electron microscopy in 1953, but in 1980s, it is proved that mesosomes are artifacts formed trhough damage to the membrane during the process of chemical fixation and do not occur in living cells.

    that function in the biosynthesis of DNA, cell wall polymers, and membrane lipids;

    reverseMole percent G+C in the genome Electron acceptors for respiration Ability to use various carbon and nitrogen sources Special nutritional requirements (e.g., vitamins)Motility (Flagellum)