CHAPTER II

Structure and Function of Prokaryotic and

Eukaryotic cells
Base competition:
analyze characteristics of microbe used to identify
microorganism
Indicators :
1. Compare and contrast of differentiation of
structure and function of prokaryotic and
eukaryotic cells
2. Explain of endospore formation
3. Demonstrate several different staining
procedures
 LEARNING OBJECTIVES
1st meeting
• Student can compare and contrast of differentiation of
structure and function of prokaryotic and eukaryotic
cells
• Student can differentiate among pili, fimbrie, flagella,
and glycocalyx
• Student can compare structure of gram-negative and
gram-positive bacteria
• Students are able to explain structure and function of
bacterial cell wall
• Students can explain the biosynthesis of bacterial cell
wall
EUKARYOTIC CELL STUCTURE
PROKARYOTIK CELL STUCTURE
Comparison of cells
Typical Bacterial Shapes
Typical Bacterial Arrangements
Typical Bacterial
Arrangements
 STRUCTURES OF BACTERIAL CELLS

STRUCTURES EXTERNAL
TO THE CELL WALL
a. Glycocalyx (capsule
or a slim layer)
b. flagella
c. axial filament
d. pili
glycocalyx
Capsules are more
regular and
gelatinous.

Slime Layers are

less regular and
more diffuse.
AXIAL FILAMENT
arise at the ends of the cell beneath the outer sheath and spiral around the
cell
Exmpl: Treponema pallidum (causative agent of syphilis)
Glycocalyx (capsule):
• a viscous (sticky) and gelatinous polymer that is
composed of polysaccharide and or polypeptide
• determined by using negative staining (Indian ink)
Function of capsule
• contributing to bacterial virulence (some spc); protect
pathogenic bacteria from phagocytosis
• to allow a bacterium to attach to various surfaces in
order to survive in natural environment.
• as a source of nutrition by breaking it down and utilizing
the sugar when energy stored are lower
FLAGELLA
Fimbriae and Pilli: shorter, straighter, and thinner
than flagella
Fimbriae
• allow a cell adhere to surfaces (Neisseria gonorrhea)
• They can number anywhere from a few to several hundred
per cell
Pilli
• usually longer, than fimbriae and number only one or two
per cell.
• function to join bacterial cells prior to the transfer of DNA
from one cell to another. For this reason, they sometimes
called sex pili.
STRUCTURE OF BACTERIAL CELL
Play Animation of bacterial envelope
Gram-Positive Cell Envelope
 The Prokaryotic Cell Wall

Determines cell In some cases

shape. recognized by host
immune system.

Prevents osmotic
Target for antibiotics.
lysis.

In Bacteria,
composed of Part of cell envelope.
Peptidoglycan.
 endotoxin
Gram-
Negative
Cell
Envelope

cell wall
External Membrane of Gram-Negative Bacteria
Outer membrane of Gram-
negative bacteria consists
of lipoproteins,
phospholipids, and
lipopolysaccharide (LPS).
This layer is lies out of
peptidoglycan

functions of the outer membrane:

a. because it has a large negative charge, outer membrane serves to
avoid phagocytosis and complement action (host immune
factors).
b. protects the cells of several antibiotics (penicillin), lysozyme, and
the cell wall breaking enzymes, heavy metals, bile salts, digestive
enzymes and some dyes.
lipopolysaccharide
LPS (Lipopolysaccharide) consists of:
lipid and polysaccharide

•two portions of polysaccharide are core

polysaccharide and O-polysaccharide

•Lipids on LPS, called lipid A, an endotoxin, which

is toxic when in blood vessels and digestive tract
that cause fever and shock
Porin (protein), in outer membrane of gram-
negative bacteria
•serves as a channel for out of the hydrophilic
substance with a small molecular weight
• Porins are nonspecific and permit the passage
of small molecules.
•Porin does not permeable to enzymes and
other large molecules.
Membrane permeability is also caused by the
presence of specific membrane proteins that can
only be traversed by a specific substance
(vitamin B12, iron, nucleotides, and maltose).
Periplasm:
• lies between the cytoplasmic membrane
and outer membrane
• contains a few proteins, such as hydrolytic
enzymes (degrading food), binding protein
(transport protein), and chemoreceptors
(chemotaxis response)
Cell wall Structures

Play the animation

Composition and characteristic of cell wall
1. peptidoglycan (murein)
consist of N-acetylglucosamine
(NAG) and N-acetylmuramic
acid (NAM)
2. four or five amino acids
(tetrapeptide side chains):
comprises the amino acids l-
alanine, d-alanine, d-
glutamic acid and either l-
lysine or diaminopimelic
acid (DAP).
3. Techoic acid, an acidic polysaccharide:
connecting peptidoglycan layers
Lipoteichoic acid: connecting peptidoglycan to
plasma membrane
 Biosynthesis of Cell Wall
•Monomer of peptidoglycans are synthesized in
cytoplasm
•Two carrier molecules participate in peptidoglycans
synthesis:
a. uridine diphosphate
b. a lipid carrier (bactoprenol) : the lipid carrier is
C55 isoprenoid alcohol that is connected via
phosphodiester linkage to N-acetylmuramic acid
to which a pentapetide is athached. The
function of bactoprenol is to render sugar
intermediates sufficiently hydrophobic so that
they will pass through the hydrophobic
cytoplasmic membrane
Biosynthesis of Cell Wall

a. Synthesis of
monomer
peptidoglycans
(Madigan et al.,
2000)
b. Cross-linked peptidoglycans by
transpeptidase and inhibition of cell wall
synthesis (above)
biosynthesis stages of peptidoglycan!
1.Autolysis: cutting by autolysins of bounds connecting small areas of
preexisting peptidoglycans, at peptide bound and glycan
bounds
2. Synthesis and insertion of peptidoglycan monomer:
synthesis of monomere NAG and NAM in cytoplasm. Two carrier
molecules participate in peptidoglycan synthesis, uridine diphosphat
(UDP) and a lipid (bactoprenol), that is connected via phosphodiester
linkage to NAM to which a pentapeptide is attached. UDP carry the
peptidoglycan to bactoprenol, and then the bactoprenol render the
sugar intermediates sufficiently hydrophobic cytoplasm membrane.
The lipid carrier inserts the disaccharide peptide complex into the
glycan backbone and then move back inside the cell to pick up
another peptidoglycan precursor unit
3. Transglycosylasion: the assembly of
peptidoglycan monomer outside of cell
membrane
4. Transpeptidasion: The final step of biosynthesis
of peptidoglycan is formation the peptide
cross-link between adjacent glycan chain. This
step is involved an enzyme, transpeptidase,
Some Comparative Characteristics of Gram-Positive and Gram-Negative Bacteria

CHARACTERISTIC GRAM-POSITIVE GRAM NEGATIVE

Gram reaction Retain crystal violet dye and stain dark violet or purple Can be decolorized to accept counterstain (safranin)
and stain red

Peptidoglycan layer Thick (multilayered) Thin (single-layered)

Teichoic acid Present in many absent
Periplasmic gel Absent Present
Outer membrane Absent present
Lipopolysaccharide (LPS) content Absent present

Lipid and lipoprotein Low (acid-fast bacteria have lipids linked to High (due to presence of outer membrane)
peptidoglycan)

Toxins produced Primarily exotoxins Primarily endotoxin

Resistance to physical disruption High low
Cell wall disruption by lysozyme High Low (requires to pretreatment to destabilize the outer
membrane)

Susceptibility to penicillin and sulfonamide high low

Susceptibility to streptomycin, chloramphenicol, and Low high

tetracycline

Inhibition by basic dyes High low

Susceptibility anionic detergents High low
Resistance to sodium azide High low
Resistance to dyeing High low
Cellwall of Archaeobacteria
Cellwall of Archaeobacteria
REVIEW QUETIONS

1. Compare and contrast of differentiation of structure and

function of prokaryotic and eukaryotic cells
2. We have known that prokaryotic cells have no
mitochondria, how does they obtain the energy that
it needs?
3. In photosynthetic prokaryotes, chloroplasts are not
present. How do they carry out photosynthesis?
4. What is the plasmid?
5. differentiate among pili, fimbrie, flagella, and
glycocalyx
6. Describe the cell wall structures of prokaryotic
cells
7. compare and contrast structure of gram-negative
and gram-positive bacteria
8. Explain the biosynthesis stages of peptidoglycan!
Relationship of cell wall and Gram Stain
Gram stain, was developed by the Danish Hans Christian
Gram in 1884. This method differentiates bacteria into two
main groups, gram-positive and gram-negative.

There are four stages in this procedure.

1. The heat-fixed smear is covered with a basic purple dye,
usually crystal violet. Since the purple stain imparts its color
to all cell, it is referred to as primary stain.
2. After a short time, the purple dye is washed off, and the
smear is covered by iodine, a mordant. When the iodine is
washed off, both gram-positive and gram-negative appear a
dark violet or purple.
3. Next, the slide is washed with ethanol or an
ethanol-acetone solution. This solution is
decolorizing agent, which remove the purple from
the cell of some species but not from others.
4. The alcohol is rinsed off, and the slide is then
stained with safranin, a basic red dye. The slide is
washed again, blotted dry, and examined
microscopically.
If the cells are violet or purple, they are classified as
gram-positive. In contrast, if the cells are red, they
are classified as gram-negative.
 REVIEW QUETIONS

In the Gram stain, why do gram-positive

bacteria retain the purple stain after
decolorization step but gram-negative
bacteria do not and thus appear pink or red
from the counterstain
MEMBRANE SEL
DIFUSSION
OSMOSIS
MEMBRANE-TRANSPORTING SYSTEM
1. Simple Transport
 PHOSPHO
TRANSFERASE
(KIRI)

ABC
SYSTEM
(kanan)
Inclution Bodies of Bacteria
1. Carbon Strorage
Polymere: poly-b-
hydroxybutyrate
A bacterial (Bacillus
megaterium), showing the
nucleoid (blue), mesosome
(aqua), poly-b-hydroxybutyrate
inclusion body (pink), plasma
membrane (purple), and cell wall
(red). TEM X30,500
2. Sulfur Globules
A Sulfur bacterium, Thiomargarita,
was the small yellow spheres are
sulfur globules that are restricted to
the thin outer layer of the cell.
These bacteria oxidize sulfide using
nitrate, coupling the nitrogen and
sulfur cycles in the sediment.
(above)

CV, large central vacuole;

S, intracellular globules of
elemental sulfur (below)
3. Polyphosphate And
Lipid Inclusion
Bodies

Thin sections of strain K4.1T visualized by TEM showing

presumably polyphosphate and lipid inclusion bodies
(black and white arrowheads respectively).
 4.Acidocalcisomes
Acidocalcisomes in Agrobacterium
tumefaciens. (A) Arrow shows an
electron-dense material in the
periphery of an acidocalcisome.
White arrowhead shows an
electron-dense inclusion. (B)
Staining of acidocalcisomes with
DAPI (yellow). (C and D) Staining of
acidocalcisomes with antibodies
against a V-H+-PPase as detected
by immunofluorescence (C) or
immunoelectron microscopy

(D). Arrowheads in (C) show the acidocalcisomes in green. Inset is at higher

magnification. Arrowheads in (D) show the gold particles labeling the membrane of an
acidocalcisome (vg, volutin granule). Inset in (D) shows an immunoblot analysis of a
bacterial lysate. PPase, antibody against V-H+-PPase showing a band of 72 kDa. C,
control treated with preimmune serum. Bars, A = 0.1 μm; B, C = 0.5 μm; D = 40 nm.
5.Magnetosome
6. Gas vesicle of Cyanobacteri
STRUKTUR KROMOSOM BAKTERI
STAINING OF BACTEREIA
Overview
• Besides being very small, bacteria are also almost
completely transparent, colorless and featureless in
their natural states.
• Microscopy solve the size issue.
• Staining can make the structures of bacteria more
pronounced.
Tujuan pewarnaan
1. Mengamati morfologi kasar mikroorganisme
2. mengidentifikasi struktur sel mikroorganisme
3. Membedakan mikroorganism yg mirip/serupa
Langkah-langkah pewarnaan
1. Pembuatan smear/hapusan
2. Fikasai/pemubunuhan sel
3. Aplikasi pewarna
 Type of staining in Micro. lab
•1. Simple stain
•2. Differential Stain
Gram stain
Acid fast Stain
•3. Special stain
Capsular stain
Flagellar stain
Endospore stain
1. Simple stain
•In this exercise, we will use simple stains to show
the general structures of some bacteria. Usually, a
single basic stain is used in the procedure. Simple
stains do not usually provide any data for
identification of the bacterium; they simply make
the bacterium easier to see.

• To observe basic external structures of cell with

brightfield scope (cellular morphology)
Method of simple stain
1. Obtain broth cultures of the bacteria.
2. Using an inoculating loop, remove a loopful of
suspension from one of the tubes. Remember to use
sterile technique.

3. Smear the bacteria across the center of the slide with

the loop. If the bacterial suspension is very thick, add a
drop of water and mix the bacteria and the water on
the slide.
Method continued
4. Allow the smear to completely air dry.
• Air dry first to prevent lysis (boiling)

5. Heat-fix the smear by quickly passing the slide

through a Bunsen burner flame three times. This
causes partial melting of the cell walls and
membranes of the bacteria, and makes them stick to
the slide. Do not overheat the slide as this will
destroy the bacteria.
• Heat Fixing
• Kill
• Stops autolysis
• Adherence to slide
Method continued

6. Cover the smear with a few drops of one of the

stains. Allow the stain to remain for the
following periods of time:
Carbolfuchsin- 15-30 seconds.
Methylene blue- 1-2 minutes.
Nigrosin- 20-60 seconds.
Method continued
7. Gently rinse the slide by holding its surface
parallel to a gently flowing stream of water.
8. Gently blot the excess water from the slide
with bibulous paper. Do not wipe the
slide. Allow the slide to air dry.
• Observe the slide under the microscope
with air and oil lenses. A coverslip is not
required. Repeat this process with the
other bacteria and stains. Note the
differences between the various types of
stains and their appearances
Summary of simple stain
Gram stain
• Differential stain (Hans Christian Gram, a Danish
doctor )
• The most important stain
• Differentiate bacteria into two large groups (the
Gram Positive and the Gram negative)

• Almost all bacteria are described by their Gram

stain characteristics.
• Based on differences of Cell wall structures
Theory behind Gram stain
Reagents for Gram Stain 1. Crystal Violet (purple)
Primary stain; positive
stain. Stains cell wall
purple
2. Iodine. As A Mordant
Combines with CV to form an insoluble complex that gets
trapped in thicker peptidoglycan layers
What happens if you skip this step?
3. Ethanol. As Decolorizer
CV-I complex washed out of Gram negative organisms
because it cannot be trapped by peptidoglycan layer;
flows right through outer membrane
4. Safranin (pink). As Counterstain
Simple positive stain that provides contrasting dye for
decolorized cells (Gram negative)
Stains all cells, but only the negative ones actually appear
pink.
 III

I II IV
Procedures and events
Gram positive bacilli
Gram Positive cocci
Gram negative Cocci

Yeast
 Capsule
Important
•Older cultures are more likely to exhibit capsule
production. When performing a capsule stain on
your unknown, be sure the culture you take your
sample from is at least five days old
 Procedures: of capsule stain
1. Use an inoculating needle to suspend the organism in a
drop of India Ink at one end of the slide.
2. Place the short end of a clean microscope slide into the
suspension and spread the mixture across the slide to
form a thin layer.
3. Allow to air dry. Do not heat fix.
4. Cover the smear with methylene blue for 2-3 minutes.
Rinse gently with water and allow to air dry.
5. Examine with oil immersion.
6. Diagram the appearance of the organism
capsul cell
Endospores staining
Importen
Resting structures formed by some bacteria for survival
during adverse environmental conditions
•The endospore is a highly resistant differentiated
bacterial cell that are highly resistant to heat, and
drying out and are difficult to destroy
•Endospores can remain dormant indefinitely but germinate
quickly when the appropriate trigger is applied
Endospores differ significantly from the vegetative, or
normally functioning cells
•Formed by Gram-positive bacteria (e.g. Bacillus,
Clostridium
BACTERIAL ENDOSPORE
When essential nutrients are depleted, or when water is not available, some gram-
positive bacteria, such as Corynebacterium and Bacillus, form specialized ‘resting’
cell called endospore,

Endospores structure
exosporium • exposporium, outerrmost layer, a
thin layer, made of a protein.
spore coat • spore coats,:composed of layers of
DNA
spore-specific proteins.
• cortex, which consist of loosely
ribosome
cross-linked peptidoglycan,
cortex • core or protoplast, which contain
core wall
usual cell wall (core wall),
cytoplasm, nucleoid, and so on.
Thus the structure of the spores
differs from vegetative cells,
especially in the outer core wall.
Medically significant spore formers

Bacteria disease

Bacillus anthracis anthrax

Clostridium botulinum botulism

Clostridium perfringens gas gangrene

Clostridium tetani tetanus

Spores are resistant to harsh environments
• Heat (owing to high concentration of calcium dipicolinic
acid)
• UV radiation (increased cysteine amino acids)
• Dessication
• Disinfectants (impermeable cell coat)
• Mechanical stress
Sporulation and Germination
Sporulation Animation
Staining procedures of endospore
1. Malachite green is the primary stain .which is placed
on blotting paper over the smear gently heating
over a warm water bath to penetrate the spore
coat.
2. The bacteria are decolorized with water. leaves the
endospores green as the stain is driven into the
endospore . The malachite green is washed out of
the vegetative cells with the water.
3. It is then counterstained with safranin.
• Do not allow the stain evaporate. to prevent
formation of metallic sheet
Illustration
Under the microscope
Free spores and Endospre
Core consist of:
• a high calcium ion (10% of the dry weight), most of
which combine with dipiolinic.
• dipicolinic acid, characteristic in spores but not present
in vegetative cells
• contains only 10-30% of the water content of the vegetative
cells, consequently and make pH of the core cytoplasm is
about one unit lower then that the vegetative cell
• small acid-soluble spore proteins (SASPs): made during the
sporulation process,
SASPs bind tightly to DNA in the core and protect
it from potential damage from ultraviolet
radiation, dessication, and dry heat. However,
ASPs function as a carbon and energy sources for
outgrowth of a new vegetative cells from
endospore (germination). During spore
formation, vegetative cells do not grow and
become more resistant to heat.
Sporogenesis
Sporulation (spore formation) occurs when the major nutrients, such as
sources of carbon, nitrogen, and water undergoes limit. There will be
many changes in the genetic command to the cell from the vegetative to
the sporulation. Sporulation process called sporogenesis.
sporogenesis in Bacillus substilis
a. bacterial chromosome replication and unequal division of
cytoplasm separated by spore septum formation.

b. the septum will surround the core or protoplast (as if swallowing /

engulfment), so the cores inside the cells 'parent', producing a
forespore that has a two-layered membrane,

c. cortex (composed of peptidoglycan) is formed between the two

membranes, and exsosporium began to appear. Process drought
continues, the accumulation of Ca+ ions, production SASPs, and
dipicolinict acid, thus forming a protective layer (coat).

d. After the spores mature (coat becomes thick and formed

exosporium outside it), the cells will lyse and the spores will be
released into the environment.
SPOROGENESIS
 Form inside of
vegetative cells
(hence “endo”).
Endospores

Characteristic of
many soil bacteria,
e.g., Bacillus spp. &
Clostridium spp.

Highly resistant to
heat, U.V.,
desiccation, etc.
 REVIEW QUETIONS
•Why are the discovery of bacterial endospores
very important in microbiology (for the
development of methods of sterilization, not
only to the culture medium but also in health
and food and perishable products)?
•What are the differences between endospore
in prokaryotic cells and eukaryotic cells?
The acid-fast stain (modified Ziel-Neelsen method).

• The acid-fast stain is another differential staining method.

• In this case, the target cells are usually members of the
genus Mycobacterium.
• The cell walls of these bacteria contain an unusually high
concentration of waxy lipids, thus making conventional
simple stains and Gram stains useless.
• The genus Mycobacterium contains two important human
pathogens, M. tuberculosis and M. leprae, which cause
tuberculosis and leprosy, respectively.
• Carbolfuchsin, a phenolic stain, is the primary stain in the
acid-fast test. It is soluble in the lipids of the mycobacterial
cell wall.
• Heating the specimen, or adding a wetting agent such as
Tergitol, increases the penetration of the carbolfuchsin.
• Following application of the carbolfuchsin, the specimen is
cooled and decolorized with a solution of 3% hydrochloric
acid and 95% ethanol (acid-alcohol).
• Since carbolfuchsin is more soluble in waxy cell lipids than
in acid-alcohol, the acid-alcohol removes the carbolfuchsin
from non-acid-fast organisms, but not from acid-fast
organisms. Following decolorization, the sample is
counterstained with methylene blue.
 Acid Fast Reagents
• Carbolfuchsin (red)
• Primary stain
• Lipid soluble; phenol
• Acid alcohol
• Decolorizer
• Removes carbol fuchsin that has not bound to a
mycolic acid.
• Methylene blue
• Counterstain
• Cannot penetrate mycolic acid; provides contrast to
non acid fast cells.
Procedures
•1. Prepare a smear of each organism and a
combined smear of both organisms on separate
glass slides. When making the combined smear,
be careful not to cross-contaminate the stock
cultures.
•2. Allow the slides to air dry, and then heat fix
the organisms.
•3. Apply enough of carbolfuchsin with Tergitol to
cover the bacteria. Allow it to set for five
minutes.
Procedures
•4. (Alternate) If Tergitol is not available, apply
enough carbolfuchsin to cover the bacteria. Place
the slide on a pre-warmed hot plate set on low for
five minutes. Do not allow the stain to evaporate
or Boil. Add additional stain, if necessary.
Remove the slide and allow it to cool.
•5. Rinse the slide with acid-alcohol, drop by drop,
just until the alcohol runs clear.
•6. Gently rinse the slide with water
Procedures

• 7. Apply enough methylene blue to cover the bacteria. Allow it to set

for two minutes.
• 8. Gently rinse the slide with water.
• 9. Blot (don't wipe) the slide dry with bibulous paper. Allow the
slide to air dry.
• 10. Examine the slide under oil immersion. Positive organisms will
appear pink or red; negative organisms will appear blue
Summary of Acid fast stain
 Under the microscope

Acid Fast bacilli (red)

Non Acid Fast bacilli (blue)

Acid Fast bacilli (red) mixed with non acid fast (blue cocci
Archaeobacteria vs eubacteria
Size and shape
Both archae and eubacteria are similar in shape and size.
They are both found occurring as rods, cocci, spirals, plates,
coiled etc.
Difference in Cell structure
The general cell structure of archae and bacteria are the same
but composition and organization of some structures differ in
archae. Similar to bacteria archae do not have
interior membranes but both have cell wall and use flagella to
swim. Archae differ in the fact that their cell wall does not
contain peptidoglycan and cell membrane uses ether linked
lipids as opposed to ester linked lipids in bacteria.
Flagella
Archae flagella evolved from bacterial type IV pili while bacterial
flagella evolved from type III secretion system. Bacterial flagellum is
like a stalk which is hollow and is assembled by subunits that are free
to move up the central pore adding on to tip of flagella while in
archael flagella subunits are added on to the base.
Reproduction and growth
Archae reproduce asexually by the process of binary fission,
budding and fragmentation. Eubacteria reproduceasexually
through binary fission, budding, fragmentation, but eubacteria
have the unique ability to form spores to remain dormant over
years, a trait that is not exhibited by Archae. Bacteria growth
follows in three phases, the lag phase when cells adapt to new
environment, log phase marking exponential growth and
stationary phase when nutrients get depleted.
Habitat
Archae can survive in extreme and harsh environments like
hot springs, salt lakes, marshlands, oceans, gut of
ruminants and humans. Eubacteria are ubiquitous and are
found in soil, hot springs, radioactive waste water, Earth's
crust, organic matter, bodies of plants and animals etc.

pseudomurein

N-acetyltalosaminuronic acid
 Like bacteria, archaea cell membranes are usually
bounded by a cell wall and they swim using one or
more flagella. Structurally, archaea are most similar
to Gram-positive bacteria. Most have a single plasma
membrane and cell wall, and lack a periplasmic space

Membrane structures. Top, an archaeal phospholipid: 1,

isoprene chains; 2, ether linkages; 3, L-glycerol moiety; 4,
phosphate group. Middle, a bacterial or eukaryotic
phospholipid: 5, fatty acid chains; 6, ester linkages; 7, D-
glycerol moiety; 8, phosphate group. Bottom: 9, lipid
bilayer of bacteria and eukaryotes; 10, lipid monolayer of
some archaea.