MICROBIOLOGY LECTURE 1 – Bacterial Morphology and Ultrastructure – Dra. GarduqueNotes 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. Algaeb. fungi c. protozoad. plants e. animals

PROKARYOTES EUKARYOTES

Size of cell typically 0.20 -2 um in diameter

usually < 5 um in diameter

typically 10-100 um

in diameter

usually >10 um in diameter

Nucleus a single DNA molecule not bounded by a membrane

DNA chains replicate and separate producing two nuclear bodies

single chromosome, circular, naked

DNA not complexed with histones

true nucleus within a membrane

divide by mitosis

always more than onechromosome

DNA complexed withhistones

Cytoplasm no cytoskeleton or cytoplasmic streaming

cytoskeleton; cytoplasmic streaming

Cytoplasmic structuresEndoplasmic reticulum

Absent present

Golgi apparatus

Absent Present

Mitochondria

Absent present

Lysosomes Absent presentRibosomes 70 s free in the

cytoplasm80 s in the endoplasmic reticulum and free in the cytoplasm; 70 s in organelles

Plasma membrane

sterols usually absent except in mycoplasma; no CHO

contains sterols;CHO serve as receptors

Cell wall chemically complex; mucopeptides containing muramic acid and DAP or lysine

chemically simple;if present, consistsof simple polysacc-rides or inorganicsubstances(cellulose and chitin)

Capsule Frequently present AbsentDouble membrane organelles

Absent Mitochondria, chloroplasts

Simple membrane organelles

chlorobium; vesicles organelles chromatophores, thylakoids

Golgi membranes;lysosomes; endoplasmic reticulum;vacuoles

Locomotion flagella that rotate; gliding motion

flagella and cilia that undulate; ameboid motion

Flagella submicroscopic; each equivalent to a single eukaryotic fibril

microscopic, contain 20 fibrils (nine pairs surrounding two single fibrils)

complex; consists of

consists of two protein building blocks

multiple microtubules

GENERAL PROPERTIES OF BACTERIA

1. Single celled organisms that reproduce by simple division, ie, binary fission

2. Most are free living and contain the genetic information and energy producing and biosynthetic systems necessary for growth and reproduction

3. Obligate intracellular parasites- chlamydia and rickettsia

4. Differs from eukaryotesa. 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 ester- linked 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 spiral- shaped cells

11. Chemical nature of envelope components and structures- imparts 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 componentsb. presence of ether linked membrane lipidsc. absence of eubacterial peptidoglycand. possession of different types of metabolism and

cofactors

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. micrometers (um)o 1 um being 10-6 m or 0.000001

m(1/25,000 inch)2. nanometers

o 1 nm being 10-9 or 0.000000001 m.

- Pathogenic bacterial species vary from approximately 0.4 to 2 um in size

Chemical Composition of Bacteria1. Water - 70%2. Dry weight - 30% composed of:

a. DNA - 5% MW 2,000,000,000b. RNA - 12%c. protein- 70% found in:

i. Ribosomes(10,000) – RNAii. Protein particles - MW 3,000,000iii. Enzymesiv. Surface structures

d. polysaccharides - 5%e. lipids - 6%f. phospholipids - 4%

Components of the Bacterial CellA. Structures external to the cell wall

1. Glycocalyxa. Capsulesb. slime layer

2. Flagella3. Axial filaments4. Pili or fimbriae

B. Cell WallC. Structures internal to the cell wall

1. Plasma membrane or cytoplasmic membrane2. Cytoplasm3. Nuclear area4. Ribosomes5. Inclusions

a. metachromatic granules or Babes Ernst granules

b. polysaccharide granulesc. lipid inclusionsd. sulfur granulese. carboxysomesf. gas vacuoles

6. Endospores7. 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

surface1. Slime layer - if the substance is unorganized and

only loosely attached to the cell wall2. 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 virulenceo encapsulated cells form smooth or

mucoid colonieso nonencapsulated cells form rough

colonieso not seen in the usual stained smear

because of their failure to retain the dyeo capsular substance is antigenic

functioning largely as a partial antigen or hapten

A. Two simple methods to distinguish the capsule1. 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.

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 Capsule1. Protection2. Identification3. Vaccine preparation4. Tissue attachment5. Antibiotic barrier

C. Medical Importance - rapid serological identification of:

1. Several groups of streptococci2. Meningococcus3. Hemophilus influenzae4. Klebsiella pneumoniae5. Some of the coliforms6. Yersinia and Bacillus specie

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 regions1. 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 protein3. Basal body

o terminal portion of the flagellum o anchors the flagellum to the cell wall and

plasma membraneo composed of a central rod inserted into a

series of rings

a. Gram negative - 2 pairs of ringsi. Outer pair - anchored to the

outer membrane (Lring) and peptidoglycan layer (P ring)

ii. Inner pair - anchored to the plasma membrane (SM ring)

b. Gram positive - only inner pair is present

B. Types of arrangement of flagella1. Atrichous - no flagella2. Monotrichous - one polar flagellum3. Lophotrichous - tuft of several polar flagella4. Amphitrichous - flagella at both poles5. Peritrichous - flagella over the entire cell surface

Monotrichous flagella Lophotrichus flagella

Amphitrichous flagella Peritrichous flagella

C. Motility - presumptive evidence that bacteria possesses

flagella although it gives no indication of the number or rearrangement of the flagella

1. Types of bacterial motilitya. run or swim - when a bacterium moves in

one direction for a length of timeb. tumbles - periodic, abrupt random changes

in directionc. swarming - rapid wavelike growth across a

solid culture medium2. Two ways by which motility can be demonstrated

a. direct or microscopico 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 movementb. indirect or macroscopico 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. Mechanism of flagellar movement - relative rotation of the rings in the basal body of the flagellum

D. 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

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

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.

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:1. Sites of adsorption for RNA and DNA viruses 2. Act as a means of genetic transfer between similar

or related Gram negative enteric species3. Provide the channel through which DNA from the

donor (male) cell is transferred to the recipient (female cell)

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. Composition1. 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, N-acetylglucosamine 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:

a. teichoic acid - polymeric complexes of ribitol phosphate or glycerol phosphateo Functions:i. Binds and regulate the movement of

cations (positive ions) into and out of the cell

ii. Assumes a role in cell growthiii. Storage of phosphorus iv. Regulates the activity of autolysins

thereby preventing extensive wall breakdown and possible cell lysis (rupturing)

v. Provides much of the wall’s antigenic specificity making it possible to identify bacteria by serological means

b. Polysaccharideso 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)

c. proteins o not an internal component of the cell wall

but maybe in a microcapsular or capsular layer

o Ex. M proteins of streptococci glutamyl polypeptides of Bacillus specie

3. 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

lipido Firmly bind the outer membrane to the

underlying peptidoglycan layer via covalently bonds

b. Lipopolysaccharidesi. 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

- Functions of the outer membrane1. Effective hydrophobic barrier to the passage of a

large number of molecules including fatty acids, lysozymes, detergents and in particular antibiotics

2. Blocks the escape of hydrolytic enzymes which accumulate between the peptidoglycan layer and the outer membrane

3. An intact outer membrane carrying the O antigens helps prevent the interaction of complement and antibiotics on its surface

4. Makes bacteria vulnerable to attack by providing attachment sites for viruses and bacteriocins

5. Permeability due to porins - form channels; permit the passage of molecules such as nucleotides, peptides, amino acids, vitamin B12 and iron

6. Its strong negative charge is an important factor in evading phagocytosis and the action of complement, two components of the defenses of the host.

B. Functions of the cell wall1. 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 organismo smallest known bacteria that can grow

and reproduce outside living

host cellso pass through most bacterial filters and

were first mistaken for viruseso require cholesterol or sterols for growtho cultivated in the laboratory only in

hypertonic media2. 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 wall1. Hydrolysis with lysozyme

a. Protoplast Gram positive cell to a wall less

form no cell wall material is retained spherical and is capable of

carrying on metabolismb. spheroplast

Gram negative cell to a wall less form

some cell wall materials are retained

c. L forms tiny mutant bacteria with

defective cell walls difficult to cultivate produced more readily by

penicillin than with lysozyme capable of reverting to the

normal bacillary form upon the removal of the inducing stimulus

infections are resistant to antibiotic treatment since they become sequestered in the protective regions

2. 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 proteinsA. 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 moleculea. peripheral proteinso easily removed from the membrane by

mild treatmento lie at the inner or outer surface of the

membraneo Functions:

i. Act as enzymes that catalyze chemical reactions

ii. Act as a scaffold for supportiii. 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

B. Functions of the plasma membrane1. Holds the intracellular contents within the

cytoplasm and prevents their leakage2. 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 structures1. 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. Composition1. Water - 80%2. Proteins (enzymes)3. Carbohydrates4. Lipids5. Inorganic ions6. Low molecular weight compounds

B. Major Structures1. DNA

2. Ribosomes3. Inclusions

Nuclear area or nucleoidA. Bacterial chromosomes - single long, circular

molecules of double stranded DNA1. Carries all the information required for the cell

structures and functions2. 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 molecules1. Extrachromosomal genetic elements2. Replicate independently of chromosomal DNA3. Associated with plasma membranes4. Usually contain from five to 100 genes5. Maybe gained or lost without harming the cell6. May carry genes for such activities as:

a. antibiotic resistanceb. tolerance to toxic metalsc. production of toxinsd. 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. metachromatic granules or volutin or Babes Ernst granules- spherical granules that are not membrane bound- contains relatively large amounts of trichloroacetic

acid- insoluble 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 diptheriaeB. Polysaccharide granules- not usually enclosed by a

membrane- consist of glycogen and starch- glycogen granules - reddish brown with iodine- starch granules - blue with iodine

C. Lipid inclusions- appear in various species of Mycobacterium,

Bacillus, Azobacter, Spirillum- polymer polybetahydroxybutyric acid - storage

materialD. sulfur granules

- sulfur bacteria – (Genus Thiobacillus) deposit sulfur granules in the cell, where they serve as an energy reserve

E. 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

F. 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. Structure of the endospores

1. Core or spore protoplast – contains:a. complete nucleusb. all of the components of the protein

synthesizing apparatusc. energy generating systen on glycolysis-

energy for germination- stored as 3 phosphoglycerate

2. Spore wall a. innermost layer surrounding the inner

spore membraneb. contains the normal peptidoglycanc. Becomes the cell wall of the germinating

vegetative cell3. 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

4. Coat - composed of keratin like portion containing many intramolecular disulfide bonds

5. Exosporium - lipoprotein membrane containing some carbohydrate

B. Medical Importance of Spores

1. Sporeforming pathogens may cause human illness.

a. Clostridium botulinum – botulismb. Clostridium tetani – tetanusc. Clostridium perfringens- gas gangrened. Bacillus anthracis – anthraxe. Coxiella burneti - Q fever

2. Sporulation process provides medicine a number of antibiotics

a. Bacitracinsb. Gramicidinsc. Tyrocidins

Classification of Medically Important bacteria

I. Rigid, thick walled cells

A. Free living(extracellular)

1. Gram positive

Cocci

StreptococcusStaphylococcus

Sporeforming rodsAerobic BacillusAnaerobic Clostridium

Nonsporeforming rods

Nonfilamentous

CorynebacteriumListeria

Filamentous ActinomycesNocardia

2. Gram negative

Cocci

Neisseria

RodsFacultative

StraightRespiratoryorganisms

HaemophilusBordetellaLeigionella

Zoonoitic organisms

BrucellaFrancisellaPasteurella

Enteric &relatedorganisms

EscherichiaSerratiaKlebsiellaSalmonellaShigellaProteus

Curved CamphylobacterVibrio

Aerobic PseudomonasAnaerobic Bacteroides

3. Acid fast -Mycobacteria

B. Non-free living (Obligate intracellular parasites)-Rickettsia-Chlamydia

II. Flexible, thin walled cells (Spirochetes)-Treponema-Borellia-Leptospira

III. Wall-less cells -Mycoplasma

Identification of Bacteria

1. Isolation of organisms in pure (axenic) Culture2. Bacterial Colony Morphology3. Microscopic Morphology and Staining Reactions4. Biochemical Characteristics5. Antibiotic susceptibility6. Epidemiologic Phage and Serotyping

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