SBS2013 : LECTURE 2

MICROSCOPY AND STAINING;

CHARACTERISTICS OF PROKARYOTIC
AND EUKARYOTIC CELLS
CHAPTER 3 & 4
DR. HANINA BINTI MOHD NOOR
1
 Topics
•Size of Microbes & Microscopy
•Technique of Light Microscopy:
Preparation of Specimen & Staining
Methods
•Basic Cell Types: Prokaryote VS
Eukaryote
•Characteristics of Prokaryotic Cells
(Bacteria cells)
•Characteristics of Eukaryotic Cell
•Evolution by Endosymbiosis
•Endocytosis & Exocytosis
 Size of Microbes
• Microbes are generally described as being microscopic in size.
• They are smaller than a human eye can see.
• The size of microbes are so small.
• Microbe sizes – μm to nm
• Microbes tend to be smaller than animal cells.
• They are about 1/10th the size of a typical human cell.
• Microbes are generally measured in the scale of one millionth of a meter - a micrometer.
Types of Microscopy
• Light Microscopy / Bright-Field Microscopy
• Dark-Field Microscopy
• Phase-Contrast Microscopy
• Nomarski Microscopy
• Fluorescence Microscopy
• Confocal Microscopy
• Digital Microscopy
• Transmission Electron Microscopy
• Scanning Electron Microscopy
• Scanning Tunneling Microscopy
 Compound Light Microscopy
• Base

• Condenser

• Iris diaphragm

• Objective lenses

• Ocular lens(es)

• Mechanical Stage

• Coarse adjustment

• Fine adjustment
 Technique of Light Microscope: Specimen
Preparation

• Wet Mounts – to view living microorganisms

 A drop of medium containing the microorganisms is
placed on a microscope slide
 Hanging drop technique – to determine the microbial
motility

• Smears – to view killed microrganisms

 Placement of cells – microrganisms from aloopful
of medium are spread onto the surface of glass
slide Hanging drop technique:
to observe motility
 Allowed to air dry completely
 Passed through an open flame - Heat fixation 
kills the microorganisms, causing them to adhere to
the slide and ready to accept stains
 Technique of Light Microscope: Staining
Principles & Methods
 Stain / dye – is a molecule that can bind to a structure and give it colour
 Two commonly used dye:
• Anionic (-ve) / Acidic – e.g eosin & picric acid
Attracted to any positively charged cell materials
• Cationic (+ve) / Basic - e.g methylene blue, crystal violet, safranin & malachite green
Attracted to any negatively charged cell components

 Types of Staining:
• Simple Staining – e.g methylene blue, safranin, carbolfuchsin & crystal violet
 single dye; reveals basic cell shapes & arrangements

• Differential Staining – Gram stain, Ziehl-Neelsen asid fast stain & Schaefer-Fulton spore
stain
 Two or more dyes & distinguish between two kinds or microorganisms or between two
different parts of an organism.

• Special Staining – negative staining; flagellar staining; Endospore staining

 Identify various specialized structure
Gram Stain
Types of Staining
Ziehl-Neelsen Acid Fast Stain

Produces red colour in

acid-fast organisms, e.g
Mycobacterium leprae

Endospore staining

Negative staining

Endospores of Bacillus megatarium

The capsule reveals a clear
area (does not accept stain)
in a dark background of
india ink & crystal violet
counterstain
are visible as green, oval structures
inside & outside the rod shape
cells; vegetative cells which
represent a non-spore-forming
stage and cellular regions without
spores stain red
 Basic Cell Types
• Prokaryote: single-celled
organisms, and all are
bacteria.
• Eukaryote: single-celled or
multi-cellular organisms
• Pro = before
• Eu = true
• Karyon = nucleus
• Similarities: Plasma
membrane, DNA and cell
wall (plant cells)
• Differences:
 Eukaryotic DNA is in a
nucleus surrounded by a
nuclear membrane
 Prokaryotic DNA is in a
nuclear region not
surrounded by a membrane
 Prokaryote & Eukaryote
Eukaryote
Prokaryote
Cellular Characteristics
 Prokaryotic Cells: Size

 Prokaryotes are among the smallest of all organisms

 Prokaryotes range from 0.5 – 2.0 µm in diameter and from
1.0 – 60 µm in length
 Prokaryotic Cells: Shape
• Three basic shapes: spherical,
rodlike & spiral
• Basic Cell Types:
• Spherical bacterium – coccus
(plural: cocci) common bacterial shapes
• Rodlike bacterium – bacillus
(plural: bacilli)
• Short rods intermediate –
coccobacilli
• Spiral bacterium: curve shape
 comma-shaped bacterium –
vibrio
 Rigid,wavy-shaped – spirillum
(plural:spirilla)
 Corkscrew-shaped -
spirochete
 Prokaryotic Cells: Arrangement
• Cocci in pairs (diplococci):
Neisseria sp.
• Cocci in chains
(streptococci): Streptococcus
sp.
• Cocci in four (tetrad)
• Cocci in eight (sarcinae)
• Cocci in cluster (staphylo):
Staphylococcus sp.
• Bacilli / rods in chains
(streptobacilli): Lactobacillus
sp.
• Bacilli in rossete, attach by
stalk to a substrate
• Star-shaped bacteria
• Square-shaped bacteria
 Prokaryote (Bacteria): An Overview of
Structure
• Structurally, bacterial cells
consist of the following:
 Cell Wall
 Cell membrane - usually
surrounded by a cell wall
 Internal structure:
cytoplasm with ribosomes,
nuclear region, and in some
cases granules and/or
vesicles
 External Structure -
Capsules, flagella, and pili
 Prokaryote: The Cell Wall
• Lies outside the cell • Cell wall components:
membrane in nearly all
bacteria  Peptidoglycan

• Two important  Outer Membrane

functions:
 Maintains the
characteristic shape  Lipolysaccharide A (LPS)

 Prevents the cell from  Periplasmic space

bursting when fluids flow
into the cell by osmosis
 Components of Bacterial Cell Walls
• Peptidoglycan (murein):
The single most
important component

• This polymer is made up

of two alternating sugar
units:
 N-acetylglucosamine
(gluNAc)
 N-acetylmuramic acid
(murNAc)

• The sugars are joined

(cross-linked) by short
peptide chains that
consist of four amino
acids (tetrapeptides)
 Gram Positive Cell Walls
• Peptidoglycan polymer - gluNAc alternate with MurNac - 40 layers; 3rd amino acid – lysine

• Teichoic Acids
 An additional component found in cell walls of gram-positive bacteria
 Consists of glycerol, phosphates, and ribitol (sugar alcohol)
 This polymer extends beyond the rest of the cell wall
 Two functions:
1. Attachment site for bacteriophages
2. Passageway for movement of ions in/out of cell
 Gram Negative Cell Walls
• Peptidoglycan polymer - gluNAc alternate with MurNac; 3rd amino acid –
diaminopimelic acid acid

• Outer Membrane
 A bilayer membrane found in gram-negative bacteria
 Forms the outermost layer of the cell wall; is attached to the peptidoglycan by a
continuous layer of lipoprotein molecules
 Proteins called porins form channels through the OM
 OM has surface antigens and receptors
 Gram Negative Cell Walls
• Lipopolysaccharide (LPS) • Periplasmic Space

 An important component of the OM  The area between the

cytoplasmic membrane and
 Also called endotoxin; used to the plasma membrane in
identify Gram-negative bacteria gram-negative bacteria
 Released when the cell walls of  Active area of cell metabolism
bacteria are broken down
 Contains the cell wall,
 Consists of polysaccharides and Lipid digestive enzymes and
A transport proteins
 Lipopolysaccharide layer (LPS) also
called as endotoxin - bacteria Gram-  Gram-positive bacteria lack
both an OM and a periplasmic
negative space
 Other Cell Walls
Acid Fast Cell Wall
 Found in bacteria that belong to the genus, Mycobacterium sp.

 Cell wall is mainly composed of lipid

 Lipid component is mycolic acid

 Acid-fast bacteria stain Gram-positive

 Distinguishing Bacteria by Cell Walls
• Certain properties of cell walls – produce different
staining reactions: Gram +ve; Gram –ve & acid fast
bacteria
 Gram-positive Bacteria - have a relatively thick layer of
peptidoglycan (60-90%)

 Gram-negative Bacteria - have a more complex cell wall

with a thin layer of peptidoglycan (10-20%)

 Acid-fast Bacteria - is thick, like that of gram-positive

bacteria, but has much less peptidoglycan and about 60%
lipid
 Controlling Bacteria by
Damaging cell walls
• The antibiotic penicillin -
blocks the final stages of
peptidoglycan synthesis
• The enzyme lysozyme, found
in tears and other human
body secretions - digests
peptidoglycan
Wall-Deficient Organisms
• Bacteria that belong to the
genus Mycoplasma have no
cell walls
• They are protected from
osmotic swelling and bursting
by a strengthened cell
membrane that contains
sterols
• Wall deficient strains are
called L-forms
 Prokaryote: Plasma Membrane
• Cell membrane/plasma membrane/cytoplasmic membrane
– boundary between cells & its environment
• Unit membranes – consist phospholipids & proteins
• Main function – regulate the movement of materials into &
out of cells
• Other functions – synthesizes cell wall components; assists
with DNA replication; secretes proteins; carries on
respiration; captures energy as ATP
• Contains appendages – flagella
Phospholipids – 2 long
• Proteins in cell membrane – respond to chemical fatty acid tails of
substances in the environment hydrocarbon: tails –
hydrophorbic (water-
Fluid Mosaic model – phospholipids forming a bilayer & proteins interspersed
fearing) do not interact
in a mosaic pattern
with water & form
barrier to water-
soluble substances;
head consists of a
charge phosphate
group & hydrophilic
(water-loving) can
interact with watery
environment .
 Prokaryote: Internal structures
• Cytoplasm
 Semifluid substance inside the cell
membrane (four-fifths water & one-fifth
substances dissolved in water)
 Substances – enzymes, other proteins,
carbohydrates, lipids & inorganics ion
 Anbolic and catabolic
• Nuclear region / nucleoid
 Prokaryote – absence of nucleus
bounded by nuclear membrane –
nuclear region / nucleoid
 This centrally located nuclear region
consists mainly of DNA, but also
contains RNA and protein
 DNA: Usually one large, circular
chromosome
 Vibrio cholerae: Two chromosomes,
one large and one small
 Plasmids: Extrachromosomal pieces of
smaller, circular DNA
• Ribosome
 Consist of ribonucleic acid and
protein
 Abundant in the cytoplasm of
bacteria - often grouped in long
chains called polyribosomes
 Serve as sites for protein synthesis
 Spherical, stain densely, contain large
& small subunits
 Size - 70S (small subunit – 30S & large
subunit – 50S)
 Streptomycin & Erythromycin bind
specifically to 70S ribosomes and
disrupt bacterial protein synthesis
BACTERIAL NUCLEAR REGION
Nuclear area not
surrounded by a
membrane
(Murray 1968)
 Prokaryote: Internal structures
• Internal Membrane Systems
 Photosynthetic bacteria and
cyanobacteria; nitrifying bacteria
contain internal membrane systems –
chromatophores
 Derived from the cell membrane and
contain the photosynthetic pigments
• Inclusions
 Within the bacterial cytoplasm are a
variety of small bodies - inclusions:
 Granules - Not membrane bound and
contain densely compacted substances
(glycogen or polyphosphate)

• Endospores  Vesicles - Specialized membrane-

 A specialized resting structure found in enclosed structures that contain gas or
bacteria such as Bacillus sp. and Clostridium poly-B-hydroxybutyrate (lipid)
sp.
 Helps the bacterial cell survive when
conditions become unfavorable
 Highly resistant to heat, drying, acids, bases,
certain disinfectants and radiation
 External Prokaryotic Structures
• Many bacteria have structures that extend Pili
beyond or surround the cell wall

 Flagella and pili extend from the cell membrane

through the cell wall and beyond

 Capsules and slime layers surround the cell wall

 Spirochetes – have axial filaments / endoflagella

Flagellae Slime layer

Axial filaments
 External Prokaryotic Structures: Flagella
• Arrangements of Bacterial Flagella:
 Monotrichous: Bacteria with a single polar flagellum
located at one end (pole)
 Peritrichous: Bacteria with flagella all over the surface
 Atrichous: Bacteria without flagella
 Cocci shaped bacteria rarely have flagella Polar, amphitrichous (Spirullum)

Polar, Monotrichous (Pseudomonas) Lophootrichous (Spirullum)

Peritrichous (Salmonella)

Peritrichous (Proteus)
 Bacterium & Flagella: Chemotaxis & Motility
Sometimes bacteria move toward or away
from substances in their
environment by this nonrandom
process
• Positive chemotaxis: net result is
movement towards the attractant
(nutrients)
• Negative chemotaxis: net result is
movement away from the repellent

• Flagella rotate in a counterclockwise

direction – flagella bundle together &
push the bacterium in a fairly
straight, forward movement called
run

• Flagella reverse & rotate in a
clockwise direction – the bundle
comes apart, each flagellum acts
independently & cells tumble about
in random directions, a movement
called tumble
The cell swims forward (run) only when flagella
bundled & the bacterium changes direction
following a tumble; when nothing attracts or
repels a bacterium – it has frequent tumbles &
short runs, resulting in random movement
 External Prokaryotic Structures: Pili
• Pilus (singular)

• Tiny, hollow projections

• Used to attach bacteria to

surfaces

• Not involved in movement

 Long conjugation pili (F-pili)

 Short attachment pili (fimbriae)

• Conjugation pili – Conjugation  transfer of genetic material

• Attachment pili - Adhesion; helps pathogenicity, forms pellicle

 External Prokaryotic Structures: Glycocalyx
Glycocalyx - refer to all polysaccharide/polypeptide-containing substances found external to
the cell wall:
• Capsule

 Protective structure outside the cell wall of the

organism that secretes it
 Only certain bacteria are capable of forming
capsules
 Chemical composition of each capsule is unique
to the strain of bacteria that secreted it
Capsule / glycocalyx (negative staining)
 Encapsulated bacteria are able to evade host
defense mechanisms (phagocytosis)

• Slime Layer
 Less tightly bound to the cell wall and is usually
thinner than a capsule

 Protects the cell against drying, traps nutrients

and binds cells together (biofilm) Slime Layer: Bacteria growing on tooth
enamel
 Eukaryotic Cells Structure
• Structurally, eukaryotic
cells consist of the
following:
 Plasma membrane

 Internal structure - cell

nucleus, mitochondria,
chloroplasts, endoplasmic
reticulum, golgi apparatus,
lysosomes, peroxisomes,
vacuoles, cytoskeleton
(microfilaments &
microtubules)

 External Structure – cilia,

pseudopodia
 Eukaryotic Cells: Core Cellular Components
• Plasma membrane – almost identical to prokaryotic
cells, except its contain sterols Pores through the
cell nucleus
• Cytoplasm – smaller portion of eukaryotic cells than
prokaryotic cells  nucleus & organelles; contain
water, dissolve substances & cytoskeleton (give
larger cells shape & support)

• Nucleus – distinct organelle enclosed with nuclear

envelope; contain nucleoplasm, nucleoli &
chromosomes
 nuclear pores in the envelope allow RNA to leave
the nucleoplasm & participate in protein synthesis The nucleus
 nucleoli (singular: nucleolus) – contain RNA & serve stores the
hereditary
as site for ribosomes assembly
material of the
 Chromosomes – contain DNA & protein (histone) cell
 Divide by mitosis

• Ribosomes
 Larger than prokaryotic cells; 60% RNA & 40%
protein Ribosomes are
 Size – 80S (large subunit 60S & small subunit 40S) responsible for protein
 Provide sites for protein synthesis synthesis
 Eukaryotic Cells: Mitochondria and Chloroplasts
• Mitochondria (singular: mitocondrion)

 Respiratory enzymes & cytochromes

 Examples: NADH2 dehydrogenase; cytochrome b, c,

a3
 Carry out the oxidative reactions that capture
energy in ATP for cell activities

• Chloroplasts

 Carry out photosynthesis

 Internal membranes called thylakoids –

contain the pigment chlorophyll  captures
energy from light

Mitochondria & chloroplasts – contain

DNA & can replicate independently
 Eukaryotic Cells: Membranous Networks &
Organelles / Inclusions

If the endoplasmic reticulum (ER) has ribosomes

The Golgi apparatus sorts and packages cellular products
attached to it, it is called rough ER; if it does not,
then it is called smooth ER.

Peroxisomes are Cytoskeleton – a network of

membrane-bound protein fibers made of
organelles that contain microtubules (hollow tubes)
an abundance of & microfilaments (fliemntous
enzymes for detoxifying Vacuole – membrane-
Lysosomes digest enclosed structures that fibers); Supports & gives
harmful substances and rigidity & shape to a cell.
foreign substances that store materials e.g starch,
lipid metabolism.
might harm the cell. glycogen, or fat for energy
 Eukaryotic Cells: External Structure & Motility
• Flagella – larger & more complex than
those in prokaryotes, consists of 2
central microtubules & 9 pairs of
peripheral microtubules (9+2
arrangement) surrounded by a
membrane.

• Cilia – shorter & more numerous than

flagella, but have same chemical
composition & basic arrangement of
The Ciliated
microtubules; mainly found among
Protozoan
ciliated protozoa.

• Pseudopods (singular: pseudopodium)

– temporary projections of cytoplasm
associated with amoeboid movement
Amoeba
(occurs only cells without walls, e.g engulfing
amoebas & some white blood cells) food
particles
 Eukaryotic Cells: Cell Wall components
 Unicellular eukaryotic organisms

• Algae
 mainly consists cellulose; some consist other
polysaccharides

• Fungi
 consist cellulose or chitin, or both

• Protozoans
 have flexible external coverings called pellicles:
cell walls give cells rigidity & protect from bursting
due to water movement from the environment
 Evolution of Endosymbiosis
• Endosymbiotic theory – the organelles of eukaryotic cells
arose from prokaryotic cells that had developed a symbiotic • Mitochondria
relationship with eukaryote-to-be
• Symbiosis – is a relationship between two different kinds of
organisms that live in close contact
• Endosymbiosis – if one lives inside the other
Size of mitochondria & chloroplast similar to bacteria; 1
Mitochondria & chloroplast have their own DNA
DNA filaments inside the
Ribosome of 70s in cells similar to bacteria in contrast with mitochondrion of a frog cell
eukaryote with 80s
• Chloroplasts
DNA of the organelles synthesize protein similar to bacteria
Antibiotic effecting bacteria (70s) also acts on
mitochondria/chloroplast
divide independently of the eukaryotic cell cycle by means
of binary fission
Double membrane of chloroplast & mitochondria resembles
to G –ve bacteria
DNA filaments (5-6 m in length)
Chloroplast resembles to a prokaryotic cyanobacteria isolated from the mitochondrion
 Endocytosis & Exocytosis
• Occur only in eukaryotic cells
• Involve formation of vesicles from fragments of plasma membrane & fusion of vesicles
with the plasma membrane
• Allow the movement of relatively large quantities of materials across plasma membranes

• Engulfment (Endocytosis)
 The process of taking materials into the cell
 The vesicle enters the cell, as a phagocytosis

• Phagocytosis - endocytosis
 Large vacuoles called phagosomes form arround
microorganisms & debris from tissue injury
 The phagosomes fuse with lysosomes, which
release enzyme that degrade the vacuolar contents
 Reusable components are absorbed into the cell, &
debris is released by exocytosis

• Extrusion (Exocytosis)
 The process of releasing materials from cell
 The vesicle leaves the cell, as in secretion