Microbio Lec 1 - Bacterial Morphology And Ultra Structure

MICROBIOLOGY LECTURE 1 – Bacterial Morphology and Ultrastructure – Dra. Garduque Notes from Lecture USTMED ’07 Sec C – AsM Only two types of cells are produced by all living organisms on earth. 1. Prokaryotes (pro. or primitive nucleus) do not have a membrane bound nucleus a. eubacteria (true bacteria) b. archaebacteria (ancient bacteria) 2. Eukaryotes (eu, or true nucleus) have a membrane bound nucleus a. Algae b. fungi c. protozoa d. plants e. animals PROKARYOTES

Size of cell

Nucleus

typically 10-100 um in diameter

usually < 5 um in diameter a single DNA molecule not bounded by a membrane

usually >10 um in diameter true nucleus within a membrane

DNA chains replicate and separate producing two nuclear bodies

divide by mitosis

single chromosome, circular, naked

always more than one chromosome

DNA not complexed with histones Cytoplasm no cytoskeleton or cytoplasmic streaming Cytoplasmic structures Endoplasmic Absent reticulum Golgi Absent apparatus Mitochondria Absent Lysosomes Absent Ribosomes 70 s free in the cytoplasm Plasma membrane Cell wall

Capsule Double membrane organelles Simple membrane organelles Locomotion Flagella

EUKARYOTES

typically 0.20 -2 um in diameter

sterols usually absent except in mycoplasma; no CHO chemically complex; mucopeptides containing muramic acid and DAP or lysine Frequently present Absent chlorobium; vesicles organelles chromatophores, thylakoids flagella that rotate; gliding motion submicroscopic; each equivalent to a single eukaryotic fibril consists of two protein building blocks

DNA complexed with histones cytoskeleton; cytoplasmic streaming present Present present present 80 s in the endoplasmic reticulum and free in the cytoplasm; 70 s in organelles contains sterols; CHO serve as receptors chemically simple; if present, consists of simple polysaccrides or inorganic substances(cellulose and chitin) Absent Mitochondria, chloroplasts Golgi membranes; lysosomes; endoplasmic reticulum; vacuoles flagella and cilia that undulate; ameboid motion microscopic, contain 20 fibrils (nine pairs surrounding two single fibrils) complex; consists of multiple microtubules

GENERAL PROPERTIES OF BACTERIA

1. 2.

Single celled organisms that reproduce by simple division, ie, binary fission Most are free living and contain the genetic information and energy producing and biosynthetic systems necessary for

growth and reproduction Obligate intracellular parasites- chlamydia and rickettsia Differs from eukaryotes a. do not contain 80 s ribosomes or membrane bound organelles (nucleus, mitochondria, lysosomes, endoplasmic reticulum or Golgi bodies) b. they lack the 9+2 fibril flagellum or cilia structures characteristic of eukaryotic cells 5. Have 70 s ribosomes and a naked single circular chromosomes (nucleiod) composed of DS DNA that replicates amitotically 6. Cytoplasmic membrane of eubacteria - contains esterlinked lipids and carries out transport, energy production specialized biosynthetic functions 7. Motility, if present, is conferred by single filament flagellar structures 8. Some produce external microfibrils(pili or fimbriae) that serve adhesive functions 9. Mycoplasmas do not possess cell walls, whereas other eubacteria produce envelope that contains a chemically similar cell wall peptidoglycan 10. Cell-wall producing eubacteria and archaebacteria may occur as spheres(cocci), rods(bacilli) or curved or spiralshaped cells 11. Chemical nature of envelope components and structuresimparts useful staining characteristics that classifies eubacteria into: gram positive, gram negative and acid fast organisms 12. Archaebacteria may differ from the eubacteria in their: a. chromosome components b. presence of ether linked membrane lipids c. absence of eubacterial peptidoglycan d. possession of different types of metabolism and cofactors 3. 4.

THE BACTERIAL CELL

1.

Most bacteria produce a layered cell envelope that includes the plasma membrane, cell wall and associated proteins and polysaccharides. 2. Some bacteria produce capsules or slimes. 3. External filamentous Appendages(flagella and pili) may also occur. 4. The cell wall is a rigid structure that encloses and protects the protoplast from physical damage and conditions of low external osmotic pressure. 5. The cell wall also generally allows bacteria to tolerate a wide range of environmental conditions. 6. The protoplast comprises the naked cytoplasmic membrane and its contents. 7. Internally, bacteria are relatively simple cells. 8. Major cytoplasmic structures include a central fibrillar chromatin network surrounded by an amorphous cytoplasm that contains ribosomes. 9. Cytoplasmic inclusion bodies or energy storage granules vary in chemical nature according to species and in amounts depending on growth phase and environment. 10. Some cytoplasmic structures, such as endospores, are limited to only a few bacteria. 11. Typical gram positive and gram negative bacterial cells differ primarily in cell envelope organization. Size of bacteria Unit of microbial measurement

1. 2.

-

micrometers (um) o 1 um being 10-6 m or 0.000001 m(1/25,000 inch) nanometers o 1 nm being 10-9 or 0.000000001 m. Pathogenic bacterial species vary from approximately 0.4 to 2 um in size

1. 2. 3. 4. 5.

6. 7.

Plasma membrane or cytoplasmic membrane Cytoplasm Nuclear area Ribosomes Inclusions a. metachromatic granules or Babes Ernst granules b. polysaccharide granules c. lipid inclusions d. sulfur granules e. carboxysomes f. gas vacuoles Endospores mesosomes

Glycocalyx - a viscous(sticky), gelatinous polymer that is external to the cell wall composed of polysaccharide and polypeptide or both chemical composition varies widely with the specie made inside the cell and excreted to the cell surface 1. Slime layer - if the substance is unorganized and only loosely attached to the cell wall 2. Capsule o if the substance is organized and is firmly attached to the cell wall o found in both gram positive and Gram negative cells o presence denotes virulence o encapsulated cells form smooth or mucoid colonies o nonencapsulated cells form rough colonies o not seen in the usual stained smear because of their failure to retain the dye o capsular substance is antigenic functioning largely as a partial antigen or hapten A. Two simple methods to distinguish the capsule 1. India ink technique - most satisfactory method of demonstrating the capsule Bacteria is suspended in diluted India ink. Bacterial cells appear to lie in a lacunae. Chemical Composition of Bacteria 1. Water - 70% 2. Dry weight - 30% composed of: a. DNA - 5% MW 2,000,000,000 b. RNA - 12% c. protein- 70% found in: i. Ribosomes(10,000) – RNA ii. Protein particles - MW 3,000,000 iii. Enzymes iv. Surface structures d. polysaccharides - 5% e. lipids - 6% f. phospholipids - 4% Components of the Bacterial Cell A. Structures external to the cell wall 1. Glycocalyx a. Capsules b. slime layer 2. Flagella 3. Axial filaments 4. Pili or fimbriae B. C.

Cell Wall Structures internal to the cell wall

2.

Quellung reaction - Homologous antibody is added to a preparation of capsule.

•

microprecipitation at the periphery of the capsule altering its refractive index rendering the capsule to be visible

B. Functions of the Capsule 1. Protection 2. Identification 3. Vaccine preparation 4. Tissue attachment 5. Antibiotic barrier C. Medical Importance - rapid serological identification of:

1. 2. 3. 4. 5. 6.

Several groups of streptococci Meningococcus Hemophilus influenzae Klebsiella pneumoniae Some of the coliforms Yersinia and Bacillus specie Amphitrichous flagella

Flagella organ of locomotion - long filamentous appendage originating in a spherical body or basal granules - occur most commonly although not exclusively among the rod-shaped bacteria A.

Three morphological regions 1. Helical filament o long outermost region; composes up to 90% of its length o contains the globular (roughly spherical) protein flagellin arranged in several chains that intertwine and form a helix around a hollow core 2. Hooked or curved area o filament is attached; consists of a different protein 3. Basal body o terminal portion of the flagellum o anchors the flagellum to the cell wall and plasma membrane o composed of a central rod inserted into a series of rings a.

b. B.

Peritrichous flagella

Gram negative - 2 pairs of rings i. Outer pair - anchored to the outer membrane (Lring) and peptidoglycan layer (P ring) ii. Inner pair - anchored to the plasma membrane (SM ring) Gram positive - only inner pair is present

Types of arrangement of flagella 1. Atrichous - no flagella 2. Monotrichous - one polar flagellum 3. Lophotrichous - tuft of several polar flagella 4. Amphitrichous - flagella at both poles 5. Peritrichous - flagella over the entire cell surface

C.

Motility - presumptive evidence that bacteria possesses flagella although it gives no indication of the number or rearrangement of the flagella 1.

2.

Types of bacterial motility a. run or swim - when a bacterium moves in one direction for a length of time b. tumbles - periodic, abrupt random changes in direction c. swarming - rapid wavelike growth across a solid culture medium Two ways by which motility can be demonstrated a. direct or microscopic o Hanging drop preparation or wet mount preparation o Distinguishes:

i.

Brownian movement - when the bacteria show molecular movement with no apparent effort to change their position and direction to the field ii. true motility - if a bacterium describes a rotatory, undulatory or sinuous movement b. indirect or macroscopic o Stab inoculation of the semisolid media i. nonmotile - growth is limited at the point of inoculation ii. motile - growth is diffuse or moves away from the line of inoculation; turbidity of the medium c.

D.

Monotrichous flagella

Lophotrichus flagella

Mechanism of flagellar movement - relative rotation of the rings in the basal body of the flagellum

Antigenicity - flagellar or H antigen - useful in the serological identification of serotypes of Salmonella organisms

Axial filaments - bundles of fibrils that arise at the ends of the cell beneath the outer sheath and spiral around the cell

-

a.

rotation moves an opposing movement of the outer sheath that propels the spirochetes by causing them to move like corkscrews

teichoic acid - polymeric complexes of ribitol phosphate or glycerol phosphate o Functions:

i. ii. iii.

iv. Found in Spirochetes and are similar to flagella, but are located between the cell wall and an outer sheath, and are attached to one end of the organism.

v.

b.

o

Fimbriae and pili - hairlike appendages that are shorter, straighter and thinner than flagella used for attachment rather than for motility - consist of a protein called pilin arranged helically around a central core A.

Fimbriae - can occur at the poles of the bacterial cell or they can be evenly distributed over the entire surface of the cell allow a cell to adhere to surfaces

B.

Pili usually longer than fimbriae and number only one or two per cell - genetically determined by a fertility factor called F factor which is carried on an episome (sex or F pilus) -

Two Types:

1.

Sex or conjugation pili for the transfer of extrachromosomal DNA between donor and recipient

2.

Attachment pili or fimbriae. There are many and are used for attachment to surfaces.

-

Functions of Pili: Sites of adsorption for RNA and DNA viruses Act as a means of genetic transfer between similar or related Gram negative enteric species Provide the channel through which DNA from the donor (male) cell is transferred to the recipient (female cell)

1. 2. 3.

Cell wall major structural component of all microorganisms responsible for the shape of the microorganism - a strong and rigid structure that protects and supports the weaker and biochemically more active parts of the cell - high tensile strength is attributed to a layer composed of a substance variously referred to as murein, mucopeptide or peptidoglycan layer A.

Composition 1. Peptidoglycan layer - component in both gram positive and gram negative cells that provides the structural support for the cell wall Components of the peptidoglycan layer: a. backbone of polysaccharide - consists of alternating residues of amino sugars, Nacetylglucosamine and N-acetylmuramic acid held by beta 1,4 linkages b. a set of identical tetrapeptide side chains attached to N- acetylmuramic acid c. a set of identical peptide cross bridges 2. -

-

The gram positive cell wall thickness of the cell wall varies from 150 to 500 A many layers of peptidoglycan forming a thick rigid structure peptidoglycan layer - 50 to 90% of the wall rest of the wall is made up of:

c.

o o

3.

-

-

-

Binds and regulate the movement of cations (positive ions) into and out of the cell Assumes a role in cell growth Storage of phosphorus Regulates the activity of autolysins thereby preventing extensive wall breakdown and possible cell lysis (rupturing) Provides much of the wall’s antigenic specificity making it possible to identify bacteria by serological means Polysaccharides Streptococci - C- CHO - immunologically active substance present in the cell wall; responsible for their serological differences and the basis of Lancefield Classification (A to U) proteins not an internal component of the cell wall but maybe in a microcapsular or capsular layer Ex. M proteins of streptococci glutamyl polypeptides of Bacillus specie

The Gram negative cell wall more complex than that of Gram positive cell wall peptidoglycan layer makes up only a portion of the wall - 5 to 10% one or very few layers of peptidoglycan layer and an outer membrane peptidoglycan layer is bounded to the lipoproteins in the outer membrane peptidoglycan is embedded in a soft material, the periplasmic gel, which contains a high concentration of degradative enzymes and transport proteins does not contain teichoic acid more susceptible to mechanical breakage due to a small amount of peptidoglycan layer outer membrane consists of: a.

lipoproteins o Composed of protein covalently linked to lipid o Firmly bind the outer membrane to the underlying peptidoglycan layer via covalently bonds

b.

Lipopolysaccharides i. Polysaccharide portion of LPS - composed of sugars called O polysaccharides- functions as antigens and are useful for determining species of gram negative bacteria ii. Lipid portion of LPS - Lipid A - referred to as endotoxin and is toxic when in the host’s bloodstream or gastrointestinal tract

c.

phospholipids

 

difficult to cultivate



capable of reverting to the normal bacillary form upon the removal of the inducing stimulus

produced more readily by penicillin than with lysozyme



2.

-

1. 2. 3.

4. 5. 6.

B.

Functions of the outer membrane Effective hydrophobic barrier to the passage of a large number of molecules including fatty acids, lysozymes, detergents and in particular antibiotics Blocks the escape of hydrolytic enzymes which accumulate between the peptidoglycan layer and the outer membrane An intact outer membrane carrying the O antigens helps prevent the interaction of complement and antibiotics on its surface Makes bacteria vulnerable to attack by providing attachment sites for viruses and bacteriocins Permeability due to porins - form channels; permit the passage of molecules such as nucleotides, peptides, amino acids, vitamin B12 and iron Its strong negative charge is an important factor in evading phagocytosis and the action of complement, two components of the defenses of the host.

Functions of the cell wall 1. Maintains the cell’s characteristic shape - the rigid wall compensates for the flexibility of the phospholipids membrane and keeps the cell from assuming a spherical shape 2. Countering the effects of osmotic pressure – the strength of the wall is responsible for keeping the cell from bursting when the intracellular osmolarity is much greater than the extracellular teichoic osmolarity 3. Provides attachment sites for bacteriophages – teichoic acids attached to the outer surface of the wall are like landing pads for viruses that infect bacteria 4. Provides a rigid platform for surface appendages -flagella, fimbriae, and pili all emanate from the wall and extend beyond it

C.

Atypical cell walls naturally occurring wall less microorganisms 1. Genus Mycoplasma and related organism o smallest known bacteria that can grow and reproduce outside living host cells o pass through most bacterial filters and were first mistaken for viruses o require cholesterol or sterols for growth o cultivated in the laboratory only in hypertonic media 2. Archaebacteria o lack walls or may have unusual walls composed of polysaccharides and proteins but not peptidoglycan o walls contain N-acetylalosaminuronic acid but lacks the D amino acids found in the bacterial cell walls

D.

Damage to the cell wall 1. Hydrolysis with lysozyme a. Protoplast

b.

c.



Gram positive cell to a wall less form

 

no cell wall material is retained

spherical and is capable of carrying on metabolism spheroplast  Gram negative cell to a wall less form  some cell wall materials are retained L forms  tiny mutant bacteria with defective cell walls

infections are resistant to antibiotic treatment since they become sequestered in the protective regions interference in the formation of peptide cross bridges of peptidoglycan thus preventing the formation of a functional cell wall Ex. Penicillin

Plasma (Cytoplasmic membrane or inner membrane) - a thin structure lying inside the cell wall and enclosing the cytoplasm of the cell - consists primarily of phospholipids which are the most abundant in the membrane and proteins A. Structure - Electron microscopy - two layered structure; phospholipid bilayer; phospholipid molecules arranged in two parallel rows 1. Phospholipid molecule a. polar head - composed of a phosphate group and glycerol that is hydrophilic(water loving) and soluble in water b. nonpolar tail - composed of fatty acids that are hydrophobic (water fearing) and are insoluble in water; lies in the interior of the bilayer 2. Protein molecule a. peripheral proteins o easily removed from the membrane by mild treatment o lie at the inner or outer surface of the membrane o Functions: i. Act as enzymes that catalyze chemical reactions ii. Act as a scaffold for support iii. Act as mediators of changes in membrane shape during movement b. integral proteins



can be removed from the membrane only after disrupting the bilayer

 

penetrate the membrane completely contain channels through which substances enter and exit the cell

2. 3.

Ribosomes Inclusions

Nuclear area or nucleoid A. Bacterial chromosomes - single long, circular molecules of double stranded DNA 1. Carries all the information required for the cell structures and functions 2. Bacterial chromosomes do not include histones and are not surrounded by a nuclear envelope (membrane) 3. Attached to plasma membrane, the proteins of which are responsible for the replication of DNA and segregation of the new chromosomes to daughter cells in cell division B. Plasmids - small, circular, double stranded DNA molecules 1. Extrachromosomal genetic elements 2. Replicate independently of chromosomal DNA 3. Associated with plasma membranes 4. Usually contain from five to 100 genes 5. Maybe gained or lost without harming the cell 6. May carry genes for such activities as: a. antibiotic resistance b. tolerance to toxic metals c. production of toxins d. synthesis of enzymes 7. Can be transferred from one bacterium to another Ribosomes sites for protein synthesis - composed of two subunits, each subunit being composed of protein and a type of RNA called ribosomal RNA (rRNA) prokaryotic ribosomes are called 70s ribosomes MA: inhibition of protein synthesis on the ribosomes Ex. Streptomycin, tetracyclines Inclusions reserve deposits A. B. Functions of the plasma membrane 1. Holds the intracellular contents within the cytoplasm and prevents their leakage 2. Concentrates nutrients by effecting their transport from the external environment of the cell to the cytoplasm of the cell 3. Provides the enzymes necessary for capsules, cell wall and cell membrane synthesis 4. Serves as the site for enzymes involved in electron transport and energy metabolism C. Associated structures 1. Chromatophores or thylakoids o infoldings of the plasma membrane that extend into the cytoplasm o contains the pigments and enzymes involved in photosynthesis 2. Mesosomes - one or more large, irregular folds in the plasma membrane; believed to be artifacts - Functions of Mesosomes: a. site for energy metabolism and respiration b. site for attachment of DNA to membrane and are the site of growing septum formation c. contain enzymes instrumental in the synthesis of the cell wall, cell membrane and other components external to the cytoplasmic membrane d. site of exoenyzme synthesis and secretion for penicillinase Cytoplasm - the internal matrix of the cell contained inside the plasma membrane thick, aqueous, semitransparent and elastic A. Composition 1. Water - 80% 2. Proteins (enzymes) 3. Carbohydrates 4. Lipids 5. Inorganic ions 6. Low molecular weight compounds B. Major Structures 1. DNA

B.

C.

D.

E.

F.

metachromatic granules or volutin or Babes Ernst granules spherical granules that are not membrane bound - contains relatively large amounts of trichloroacetic acidinsoluble polymetaphosphate or polymerized phosphoric acid or polymerized polymetaphosphate or volutin generally formed by cells that grow in phosphate rich environments found in algae, fungi and protozoans as well as bacteria stain red with certain blue dyes such as methylene blue characteristic of Corynebacterium diptheriae Polysaccharide granules- not usually enclosed by a membrane consist of glycogen and starch glycogen granules - reddish brown with iodine starch granules - blue with iodine Lipid inclusions - appear in various species of Mycobacterium, Bacillus, Azobacter, Spirillum polymer polybetahydroxybutyric acid - storage material sulfur granules - sulfur bacteria – (Genus Thiobacillus) deposit sulfur granules in the cell, where they serve as an energy reserve Carboxysomes polyhedral and hexagonal inclusions - contain the enzyme ribulose 1,5 diphosphate carboxylase - required by bacteria for carbon dioxide fixation during photosynthesis - Ex. Nitrifying bacteria, cyanobacteria, thiobacilli Gas vacuoles - hollow cavities found in many aquatic prokaryotes - consist of rows of several individual gas vesicles which are hollow cylinders covered by protein - Function: maintain buoyancy so that the cells can remain at the depth in the water appropriate for them to receive sufficient amounts of oxygen, light and nutrients

Endospores a refractile oval body formed within the bacterial cell found intracellularly and extracellularly in the usual stained smear found in all species of Family Bacillaceae o Genus Bacillus - aerobic sporeforming rods o Genus Clostridium - anaerobic sporeforming

rods found also in Sporosarcinae- Gram positive coccus Coxiella Burneti

A.

2.

3.

4.

5.

Core or spore protoplast – contains: a. complete nucleus b. all of the components of the protein synthesizing apparatus c. energy generating systen on glycolysis- energy for germination- stored as 3 phosphoglycerate Spore wall a. innermost layer surrounding the inner spore membrane b. contains the normal peptidoglycan c. Becomes the cell wall of the germinating vegetative cell Cortex a. thickest layer of the spore envelope with many fewer cross links than are found in cell wall peptidoglycan b. Cortex peptidoglycan is extremely sensitive to lysozyme c. Contains dipicolinic acid, mucopeptide and calcium, all of which are significant in spore resistance Coat - composed of keratin like portion containing many intramolecular disulfide bonds Exosporium - lipoprotein membrane containing some carbohydrate

2.

Zoonoitic organisms Enteric & related organisms

Curved Aerobic Anaerobic 3. Acid fast

Haemophilus Bordetella Leigionella Brucella Francisella Pasteurella Escherichia Serratia Klebsiella Salmonella Shigella Proteus Camphylobacter Vibrio Pseudomonas Bacteroides -Mycobacteria

B. Non-free living (Obligate intracellular parasites) -Rickettsia -Chlamydia II. Flexible, thin walled cells (Spirochetes) -Treponema -Borellia -Leptospira III. Wall-less cells

Medical Importance of Spores 1.

Neisseria Facultative Straight Respiratory organisms

Structure of the endospores

1.

B.

Cocci Rods

-Mycoplasma

Sporeforming pathogens may cause human illness. a. Clostridium botulinum – botulism b. Clostridium tetani – tetanus c. Clostridium perfringens- gas gangrene d. Bacillus anthracis – anthrax e. Coxiella burneti - Q fever Sporulation process provides medicine a number of antibiotics a. Bacitracins b. Gramicidins c. Tyrocidins

Classification of Medically Important bacteria

1.

I. Rigid, thick walled cells

2. 3. 4. 5.

A. Free living(extracellular) 1. Gram positive Cocci Sporeforming rods Aerobic Anaerobic Nonsporeforming rods Nonfilamentous Filamentous 2. Gram negative

Identification of Bacteria

6.

Isolation of organisms in pure (axenic) Culture Bacterial Colony Morphology Microscopic Morphology and Staining Reactions Biochemical Characteristics Antibiotic susceptibility Epidemiologic Phage and Serotyping

Streptococcus Staphylococcus

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Bacillus Clostridium

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Corynebacterium Listeria Actinomyces Nocardia