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Chapter 3
Observing Microorganisms
Through a Microscope
- continued -
Differential Stains: Acid-Fast Stain
• Primary stain (carbolfuchsin) binds strongly
only to bacteria that have a waxy material in
their cell wall.
• Used to identify genus Mycobacteria
– M. tuberculosis causes TB
– M. leprae causes leprosy or Hansen’s disease
Differential Stains: Acid-Fast Stain
Primary stain = Carbolfuchsin (stains cells red)

Slide is heated gently to drive stain into cell wall

Decolorizer = Acid alcohol

Counterstain = Methylene blue (stains cells blue)
Differential Stains: Acid-Fast Stain
• Cells that retain a basic stain in the presence of acidalcohol are called acid-fast. = red cells
• Non–acid-fast cells lose the basic stain when rinsed with
acid-alcohol, and are usually counterstained (with a
different color basic stain) to see them. = blue cells
Figure 3.11
Special Stains
• Used to color and isolate specific parts of
microorganisms
– e.g. capsules, endospores, and flagella
• Flagella are used for movement.
• Capsules contribute to the microorganism’s
virulence (the degree to which a pathogen
can cause disease).
• Capsules are hard to stain because it is nonionic and soluble in water.
negative stain
Special Stains:
• Endospores: a special resistant, dormant
structure formed within a cell that protects a
bacteria from adverse environmental
conditions. (survival mechanism)
– genera Bacillus and Clostridium
– Cannot be stained by ordinary staining method
• Use Schaeffer-fulton endospore stain
– Malachite green (heat), water wash, & safranin
– Endospores stain green (malachite green)
Special Stains
• Negative staining is
useful for capsules.
– Nigrosin + basic dye
• Heat is required to
drive a stain into
endospores.
• Flagella staining
requires a mordant
to make the flagella
wide enough to see.
Figure 3.12a-c
Chapter 4
Functional Anatomy of
Prokaryotic and Eukaryotic Cells
Part 1
Prokaryotes and Eukaryotes
• All living cells can be classified into two
groups based on their ultrastructure.
– Prokaryotes means prenucleus in Greek
(bacteria, and archaea)
– Eukaryotes means true nucleus in Greek (fungi,
protozoa, algae, plants, and animals)
• Distinguished by the structure of cell walls
and membranes, and the lack of organelles
The Prokaryotic Cell
• Most prokaryotes are unicellular organisms
• Majority of prokaryotes are included in the
bacteria.
• Bacteria are differentiated by morphology
(shape), chemical composition (often
detected by staining reactions), nutritional
requirements, biochemical activities, and
source of energy (sunlight or chemicals).
Bacterial Cell
Fig. 4.6
The Size, Shape, and Arrangement
of Bacterial Cells
• Most bacteria range from 0.2 - 2.0 m in
diameter and from 2 - 8 m in length
• Three basic shapes of bacterial cells
– Coccus (spherical), pl. cocci
– Bacillus (rod-shaped), pl. bacilli
– Spiral
Cocci
Bacilli
Spiral
Spirillum
Fig. 4.1 a
Coccobacilli
Fig. 4.2 a & d
Spirochete
Fig. 4.4 b & c
• Unusual shapes
– Star-shaped Stella
– Square Haloarcula (halophilic archaea)
• Most bacteria are monomorphic (maintain a
single shape)
• A few are pleomorphic (can have many shapes)
Figure 4.5
Arrangements
• Pairs: diplococci,
diplobacilli
• Clusters:
staphylococci
• Chains:
streptococci,
streptobacilli
Fig. 4.1 a & d
Fig. 4.2 b
Structures External to the Cell Wall
Capsule
Cell wall
Plasma membrane
Capsule
Cell Wall
Plasma
membrane
Flagella
Fimbriae
Fig. 4.6
Glycocalyx
• Glycocalyx: a gelatinous polymer
surrounding a cell (outside cell wall)
– Secreted by many prokaryotes
– Viscous (sticky), composed of polysaccharide,
polypeptide, or both
– Can protect a cell against dehydration and its
viscosity may inhibit the movement of nutrients
out of the cell
Glycocalyx
• Capsule: organized and firmly attached to
the cell wall
– Contribute to bacterial virulence
– Protect pathogen from phagocytosis
– Allows bacteria to adhere to and colonize
• Slime layer: unorganized and only loosely
attached to the cell wall
• Extracellular polysaccharide (EPS) allows
bacteria to attach to various surfaces.
Flagella
• Long filamentous appendages that propel
bacteria
– Bacteria with flagella are motile (have the ability
to move on their own)
• Three basic parts of a flagellum
– Filament (made of chains of flagellin)
– Hook (made of a protein), where filament is
attached
– Basal body (made of pair of rings), anchors the
flagellum to the cell wall and plasma membrane
Flagella
• Rotation of a
flagellum is either
clockwise or
counterclockwise.
– As the flagella
rotate, form a
bundle that
pushes against
the surrounding
liquid and propels
the bacterium.
Figure 4.8
Flagella Arrangement
Figure 4.7
Flagella
• Motility allows a bacterium to move toward
a favorable environment or away from a
adverse one.
– move toward or away from stimuli (taxis)
– chemotaxis: movement in response to the
presence of a chemical
– phototaxis: movement in response to the
presence of light
Flagella
• A bacterium exhibits “run/swim” or
“tumble” movement
– run/swim: move in one direction for a length of
time
– tumble: periodic, abrupt, random changes in
direction (caused by a reversal of flagella
rotation)
– bacteria move toward attractant with many runs
and few tumbles
– bacteria move away from repellent with
frequent tumbles
Flagella
Figure 4.9
Flagella
• Flagella proteins are H antigens.
– Useful for distinguishing among serovars
(variations within a species) of gram-negative
bacteria
e.g., E. coli O157:H7 (causes bloody diarrhea)
Axial Filaments (Endoflagella)
• Bundles of fibrils that arise at the ends of
the cell beneath an outer sheath and spiral
around the cell
• Used by spirochetes for motility
– e.g. Treponema pallidum (cause syphilis)
Borrelia burgdorferi (cause Lyme disease)
• Anchored at one end of the spirochete
– have a structure similar to that of flagella
Axial Filaments (Endoflagella)
• Rotation of the
filaments produces
a movement of the
outer sheath that
propels the
spirochetes in a
spiral motion.
Figure 4.10a
Fimbriae and Pili
• Found in many gram-negative bacteria
– hairlike appendages that are shorter, straighter,
and thinner than flagella
– consist of a protein called pilin arranged
helically around a central core
• Finbriae are used to attach and colonize
e.g. Neisserai gonorrhoeae (causes gonorrhea)
– can be polar or evenly distributed over the
entire surface of the cell
– number from a few to several hundred per cell
Fimbriae and Pili
• Pili (sex pili) are
used to transfer
DNA from one
cell to another
– usually longer
than fimbriae
– number only one
or two per cell
Figure 4.11
The Cell Wall
• Almost all prokaryotes have cell walls
– A complex, semirigid structure
• Protect bacterial cells from osmotic lysis
– Lysis: destruction caused by rupture of the
plasma membrane and the loss of cytoplasm
• Maintains bacterial cell shape and serves as
a point of anchorage for flagella
• Enable some species of bacteria to cause
disease and is the site of action of some
antibiotics (e.g. Penicillin)
The Cell Wall:
Composition and Characteristics
• Bacterial cell wall is composed of
peptidoglycan (murein)
– a macromolecular network consisting of a
repeating disaccharide attached by polypeptides
to form a lattice that surrounds and protects the
entire cell
• Penicillin interferes with the cell wall
(peptidoglycan) synthesis
– Cell lysis due to weakened cell wall
Peptidoglycan
– Polymer of disaccharide N-acetylglucosamine (NAG)
& N-acetylmuramic acid (NAM)
– Linked by polypeptides
Figure 4.13a
Gram-positive
cell walls
• Thick
peptidoglycan
– Provides rigidity
• Teichoic acids
• In acid-fast cells,
contains mycolic
acid (waxy lipid)
Gram-negative
cell walls
• Thin
peptidoglycan
– Susceptible to
mechanical
breakage
• No teichoic acids
• Outer membrane
Figure 4.13b, c
Gram-positive cell walls
• Teichoic acids:
– Consists primarily of an alcohol and phosphate
– Two classes of Teichoic acids:
• Lipoteichoic acid spans the peptidoglycan layer and is
links to plasma membrane
• Wall teichoic acid links to peptidoglycan layer
Figure 4.13b
Gram-positive cell walls
– Teichoic acid (negatively charged) may
regulate movement of cations in and out of the
cell
– May assume a role in cell growth, preventing
extensive wall breakdown and possible cell
lysis
– provide antigenic specificity
• Polysaccharides on cell wall of Grampositive streptococci provide antigenic
variation
Gram-negative cell walls
• Peptidoglycan is bonded to lipoproteins in
the outer membrane and is in the periplasm.
• Periplasm is a fluid-filled space between the
outer membrane and the plasma membrane.
– high concentration of degradative enzymes and
transport proteins in periplasm
Gram-Negative Outer Membrane
• Consists of Lipopolysaccharides (LPS),
lipoproteins, & phospholipids.
Figure 4.13c
Gram-Negative Outer Membrane
• Has strong negative charge
• Protection from phagocytes, complement,
antibiotics, lysozyme, detergents, heavy
metals, bile salts, and certain dyes
• Porins (proteins) form channels through
membrane for small molecules to pass through
• LPS component
– O polysaccharide = antigen, e.g., E. coli O157:H7.
– Lipid A = endotoxin
Gram Stain Mechanism
• Based on differences in cell wall structures
• Crystal violet-iodine crystals form in cell
• Gram-positive
– Alcohol dehydrates peptidoglycan
– CV-I crystals do not leave
• Gram-negative
– Alcohol dissolves outer membrane and leaves small
holes in peptidoglycan
– CV-I washes out
Gram Stain Mechanism
• Reasons for Gram-positive bacteria to give
gram-negative response
– Cells are dead
– When bacteria culture is older than 24 hours
• Bacillus, Clostridium, and Mycobacterium becomes
gram variable
Atypical Cell Walls
• Mycoplasmas
– smallest known free-living bacteria; pass through
most bacterial filters
– Lack cell walls
– Sterols in plasma membrane (unique among bacteria)
• Archaea
– Lack cell walls, or
– Walls composed of polysaccharides & proteins (no
peptidoglycan), containing peptidoglycan-like
pseudomurein (lack NAM and D amino acids)
Damage to Cell Walls
• Cell wall synthesis is the target for some
antimicrobial drugs because bacterial cell
wall is made of chemicals unlike those in
eukaryotic cells.
• Lysozyme digests disaccharide in
peptidoglycan, making gram-positive
bacteria susceptible to lysis.
• Penicillin inhibits peptide bridges in
peptidoglycan (effective for gram-positive)
Damage to Cell Walls
• Protoplast: a gram-positive or plant cell treated
(e.g. lysozyme) to remove the cell wall.
• Spheroplast: a gram-negative bacterium treated
(e.g. lysozyme) to damage the cell wall.
• L forms are wall-less cells that swell into
irregular shapes. e.g. some members of Proteus
• Protoplasts and spheroplasts are susceptible to
osmotic lysis.
– Osmotic lysis: rupture of the plasma membrane
resulting from movement of water into the cell