Microbiology

Microbiology/Definition
 Science that study microorganisms
 Microorganisms – single cell particle that need
a light or electronic microcope to be seen
 Type of cells
 Eukariotic cells: fungi and protozoa
 Prokariotic cells: bacteria and blue-green algae

 Viruses: infective particle that need a viable

cell for their replication and can be seen using
EM
 Microbiology
Bacteria structures and functions
 Definition
 Bacteria are living forms that are
microscopical in size (1-10 µ m) and
relatively simple, unicellular, in structure
 a microscope is therefore necessary for their
observation.
 Structure
 Each cell consists of a body of protoplasm, the
protoplast, enclosed by a thin, semipermeable
membrane, the cytoplasmic membrane and
also, in most cases, by an outer, relatively rigid
cell wall.
The bacterial structures may be divided into
three categories:

 1. Essential structures, present in all bacteria

 2. Structures present in some species
(primary taxonomic characters)
 3. Structures present in some strains of some
species
 Essential structures, present in all
bacteria

 Protoplast (cytoplasm and nuclear body)

 Cytoplasmic membrane
 Cell wall
Bacteria’structure
2. Structures present in some species
(primary taxonomic characters):

 flagella;
 spores;
 inclusion granules.
Flagellum
 3. Structures present in some strains of
some species:
 fimbriae;
 sex pili;
 glicocalix (capsule, microcapsule, loose
slime).
Fimbriae (pili), sex pili, capsule
 Structure
 The protoplast is differentiated into a major
part,
 the cytoplasm, and an inner body,
 the nuclear body, which contains the
hereditary determinants of character, the
genes, borne on chromosome.
Schematic presentation
 Bacteria = prokaryotic cells
 The bacteria are prokaryotic cells.
 It is useful to draw a clear distinction between
relatively primitive (prokaryotic) and more
advanced (eukaryotic) cells.
 The main distinguishing features of the
prokaryotic cell are:

 Its nucleus appears as a simple, homogeneous

body, containing a single chromosome, not
possessing a nuclear membrane separating it
from the cytoplasm, nor a nucleolus, nor a
spindle, nor a number of separate non-identical
chromosomes.
 It reproduces by binary fission, not by mitotic
division.
Nucleus - DNA
 The main distinguishing features of the
prokaryotic cell
 It lacks the internal membranes isolating the respiratory
and photosynthetic enzyme systems in specific organelles.
Thus, the respiratory enzymes in bacteria are located mainly
in the peripheral cytoplasmic membrane.
 The cytoplasm is a soft gel and has no internal mobility.
Prokaryotes have 70 Svedberg unit ribosomes.
 The main distinguishing features of the
prokaryotic cell
 Its rigid cell wall contains as its main
strengthening element a specific
peptidoglycan (substance not found in
eukaryotic organisms).
 Steroids are absent in the procaryotic cell wall
(exception, mycoplasmas).
PROKARYOTIC CELL STRUCTURE

 The protoplast is bounded peripherally by a

very thin, elastic and semipermeable
cytoplasmic membrane.
 Outside, and closely covering this, lies the
rigid, supporting cell wall, which is porous and
relatively permeable.
Cell membrane and cell wall
1. Essential structures, present in all
bacteria
 the cytoplasm containing ribosomes;
 the cytoplasmic membrane (or plasma
membrane);
 the rigid cell wall (exception, mycoplasmas).
2. Structures present in some species
(primary taxonomic characters):

 flagella;
 spores;
 inclusion granules.
Flagella (electron microscopy)
Spores (Clostridium tetani)
Granules (inslusion bodies)
 3. Structures present in some strains of
some species:
 fimbriae;
 sex pili;
 glicocalix (capsule, microcapsule, loose
slime).
Fimbriae, sex pili, capsule
Bacterial nuclear body (DNA)
 BACTERIAL DNA

 The bacterial cell lacks a nuclear membrane;

instead, the DNA is concentrated in the
cytoplasm as a nuclear body.
 The nuclear body consists of a single
chromosome (1 mm length) of double-
stranded, circular, covalently closed, super-
coiled DNA.
 Size
 The cell solves the problem of packaging this
enormous DNA molecule (2-3 x 109 kDa) by
condensing and looping it into the supercoiled
state.
DNA molecule and plasmid
 Plasmids
 In many bacteria, a small portion of DNA
persists as extrachromosomal elements
referred to as plasmids, which are also
circular, but are much smaller than bacterial
chromosomes.
Plasmids - extrachromosomal
elements
 Plasmids’ functions
 Plasmids encode variable numbers of genes
(mainly responsible for the bacterial survival
in the environment) and often determine
virulent behavior or antibiotic resistance.
 Nuclear body
 The nuclear body is constantly present in all
cells and under all conditions of culture.
 It replicates by growth and simple fission,
and not by mitosis. Electron micrographs
reveal the absence of the outer nuclear
membrane separating it from the cytoplasm,
and of the nucleolus.
 Bacterial DNA has a similar structure to that
of eukaryotic chromatin (see genetic).
 Nuclear body’s observation
 The nuclear body can’t be seen with the light
microscope in bacteria stained by usual
methods because it is covered by the multitude
of RNA molecules from the bacterial
cytoplasm;
 it is observed on stained films after RNA
hydrolysis.
 Functions
 The bacterial DNA stores the genetic
information.
 It is involved in autoreplication (by which copies
of genetic information are transmitted to daughter
cells) and
 heteroreplication (by which the information is
copied in mRNA sequences).
 Replication
(relation of bacteria’s structure and antibiotics)
 CYTOPLASM OF BACTERIA

 The cytoplasm of bacteria is a viscous watery

solution, or soft gel, containing a variety of organic
and inorganic solutes, and numerous small granules
called ribosomes.
 The variety of organelles seen in eukaryotic cells is
missing in bacteria because the cytoplasmic
membrane performs many complex functions carried
out by these organelles.
 The cytoplasm of bacteria also differs from that of the
eukaryotic organisms in not showing signs of internal
mobility.
 RIBOSOMES

 Ribosomes are complex globular structures

composed of several RNA molecules and many
associated proteins; they function as the active
centers for protein synthesis.
 Bacterial ribosomes are slightly smaller (10-20 nm)
than those of eukaryotic cells and they have a
sedimentation constant of 70S (Svedberg units),
being composed of a 30S (1 RNA molecule and 21
proteins, S1-S21) and a 50S subunit (2 RNA
molecules and 34 proteins, L1-L34).
Ribosomes
 Ribosomes
 They may be seen with the electron
microscope in number aprox. 20.000 per cell.
 They are strung together on strands of
messenger RNA (mRNA) to form polysomes
and it is at this site that the code of the mRNA
is translated into peptide sequences.
 INCLUSION GRANULES

 In many species of bacteria, round granules

are observed in the cytoplasm.
 These are not permanent or essential
structures, and may be absent under certain
conditions of growth.
 They appear to be aggregates of substances
concerned with cell metabolism, e.g. an excess
metabolite stored as a nutrient reserve.
INCLUSION GRANULES
(starch, glycogen, etc.)
 INCLUSION GRANULES
(exemples, functions)
 They consist of volutin (polyphosphate), lipid,
glycogen, starch or sulfur.
 Their demonstration may assist in the
identification of certain organisms (primary
taxonomic character);
 Ex.: the diphtheria bacillus (Corynebacterium
diphteriae) may be distinguished from related
bacilli found in the throat by its content of volutin
granules.
Corynebacterium diphteriae
(volutine granules - dark)
 MESOSOMES

 They are convoluted or multilaminated

membranous bodies visible in electron microscope.
 They develop by complex invagination of the
cytoplasmic membrane into the cytoplasm,
sometimes in relation to the nuclear body and often
from the sites of cross-wall formation during cell
division.
 Mesosomes are thought to be involved in the
mechanisms responsible for the compartmenting of
DNA at cell division and sporulation.
MESOSOMES
 CYTOPLASMIC MEMBRANE

 The cytoplasmic membrane is the physical

and metabolic barrier between the interior
and exterior of the bacterial cell.
 It is 5-10 nm thick, consists mainly of
lipoprotein and is visible in some ultrathin
sections examined with the electron
microscope (EM).
 Cell (Cytoplasmic) Membrane

 Flexible, phospholipid bilayer sheet

 Hydrophobic tails
 Hydrophillic heads
 May contain steroid- like molecules:
hopanoids to stabilize structure
Cell (Cytoplasmic) Membrane
 The lipid
molecules are
arranged in a
double layer
with their
hydrophilic
polar regions
externally
aligned and in
contact with a
layer of protein
at each surface.
Cytoplasmic membrane
Cytoplasmic membrane
 Cell (Cytoplasmic) Membrane

 Boundary between a cell and its environment

 Dynamic interface
 Changes with temperature, age, environment
Functions of the procaryotic plasma
membrane
 Osmotic or permeability barrier (separate
(s “outside”
from “inside”).
 Location of transport systems for specific solutes
(nutrients and ions)
 Mesosomes (internal invaginations of cytoplasmic
membrane).
 Energy generating functions, involving respiratory
and photosynthetic electron transport systems,
establishment of proton motive force, and
transmembranous, ATP-synthesizing ATPase
 Synthesis of membrane lipids (including
lipopolysaccharide in Gram-negative cells)
Functions of the procaryotic plasma
membrane
 Synthesis of murein (cell wall peptidoglycan)
 Assembly and secretion of extracytoplasmic
proteins
 Coordination of DNA replication and segregation
with septum formation and cell division
 Chemotaxis (both motility per se and sensing
functions)
 Location of specialized enzyme system
 CELL WALL

 The cell wall encases protoplast and lies immediately

external to the cytoplasmic membrane.
 It is 10-25 nm thick, strong and rigid.
 It supports the weak cytoplasmic membrane against
the high internal osmotic pressure (5 – 20 atms) of
the protoplasm and maintains the characteristic
shape of the bacterium in its coccal, bacillary,
filamentous or spiral form.
 The rigid cell wall is entirely absent in a few
unusual bacteria (e.g., mycoplasmas, genus
Mycoplasma).
Bacteria’s shapes/arrangement
 CELL WALL
 The integrity of the cell wall is essential to the
viability of the bacterium.
 If the wall is weakened or ruptured, the
protoplasm may swell from osmotic inflow of
water and burst the weak cytoplasmic
membrane (lysis).
Bacterial rigid cell wall is made up of peptidoglycan
and special structures

 Peptidoglycan (murein) is the principal

structural component of the cell wall.
 This compound is found in both Gram-positive
and Gram-negative bacteria, although is more
abundant in Gram-positive organisms.
Peptidoglican of Gram positive bacteria
Peptidoglican of Gram negative bacteria
Gram (+), (-) and acid fast bacterial
cell wall (comparison)
Gram (+) and Gram (-) bacterial cell
wall (comparison)
 Peptidoglycan
 Peptidoglycan polymers consist of repeating
dissacharides formed by:
- N-acetyl-glucosamine (NAG) and
- N-acetylmuramic acid (NAM).
 Peptidoglycan
 The N-acetylmuramic (NAM) acid links the
dissacharides to an oligopeptide chain
consisting of four aminoacids (usually L-
alanine, D-glutamic acid, either meso-
diaminopimelic acid – in Gram-negative
bacteria – or L-lysine – in Gram-positive
bacteria and D-alanine).
 Peptidoglycan
 Lysozyme hydrolyses peptidoglycan by
cleaving the glycosil bonds between N-acetyl-
muramic acid and the N-acetylglucosamine.
 Cell division
 Cell division occurs by the development, from the
periphery inwards, of a transverse cytoplasmic
membrane and a transverse cell wall, or cross wall.
 The cell wall plays an important part in cell
division.
 As the protoplast increases in mass, the cell wall is
elongated by the intercalation of newly synthesized
subunits into the various wall layers.
Division of cell
 Cell wall
 The cell wall can be demonstrated by special
staining methods, but most clearly by
electron microscope.
 It is not seen in conventionally stained smears
examined with the light microscope, but its
chemical structure is responsible for the
staining of bacteria in differential stainings.
 Cell wall
 Gram staining divides bacteria in gram-
positive (violet) and gram-negative (red);
 Ziehl-Neelsen staining divides bacteria in
acid-fast (red) and non-acid-fast (blue).
 Gram-staining
 Gram-staining, the most common criterion for
grouping medically important bacteria, is a simple
differential staining technique that employs crystal
violet (an aniline dye) as the primary stain and
fuchsin (a red dye) as a counter-stain.
 Gram-positive bacteria appear violet because they
retain the crystal violet and resist alcohol
decoloration.
 Gram-negative bacteria appear red because they are
decolorized completely by ethanol and they take up
fuchsin, the counter-stain.
 Gram staining
it is influenced by cell wall structure
 Staphylococcus
(Gram positive cocci arranged in
clusters)
Gram negative bacilli
 Acid fast bacilli
(ex. Mycobacterium tuberculosis)
 Gram-positive bacteria
 Gram-positive bacteria have a simpler, but
thicker cell wall, consisting primarily of tri-
dimensional peptidoglycan with teichoic acid
polymers (ribitol or glycerol phosphate
complexed with sugar residues) dispersed
throughout;
 some of this material (lipoteichoic acid) is
linked to lipids buried in the cell membrane.
Gram-positive bacteria
 Teichoic acids
 The teichoic acids are major surface antigens (see
immunology) in the Gram-positive species that
possess them.
 They bind magnesium ion and play a role in the
function of the cytoplasmic membrane’s enzymes.
 They bind autolytic enzymes, being thus involved in
the cell’s growth and division.
 They are receptors for bacteriophages (viruses of
bacteria).
Gram-positive bacteria
 Function of peptidoglycan
 Prevents osmotic lysis of cell protoplast and
confers rigidity and shape on cells
 Gram-negative bacteria
 Gram-negative bacteria have a cell wall that
is thinner than that of Gram-positive bacteria,
with bi-dimensional peptidoglycan and no
teichoic acids.
 An additional membrane, the outer
membrane, lies above the peptidoglycan layer.
 The outer membrane is much thicker than the
single peptidoglycan layer.
 The outer membrane
 The outer membrane is composed of:
 a bilipid layer,
 proteins and
 lipopolysaccharide (LPS, endotoxin).
Diagrams of the cell wall structure of Gram-negative
(left) and Gram-positive bacteria.
Key: peptidoglycan layer (yellow); protein (purple);
teichoic acid (green); phospholipid ( brown);
lipopolysaccharide (orange).
The outer membrane
 Cell wall of Gram (-) bacteria
 The bilipid layer is attached to the peptidoglycan by
lipoproteins that cross the periplasmic space
(Braun’s lipoproteins: anchors the outer membrane to
peptidoglycan – murein - sheet ).
 The proteins include porins, which form

transmembrane channels involved in the

• transport of ions and

• hydrophilic compounds from the extracellular

compartment to the periplasm.

Porin
 Functions of porins
 Omp C and Omp F porins: proteins that form
pores or channels through outer membrane for
passage of hydrophilic molecules
 Omp A: provides receptor for some viruses
and bacteriocins (antibiotics produce by some
bacteria); stabilizes mating cells during
conjugation (see genetic chapter).
 Gram-negative bacteria are surrounded by two
membranes.
 The outer membrane functions as an efficient
permeability barrier because it contains
lipopolysaccharides (LPS) and porins.
Lipoproteins
 Lipopolysaccharide (LPS, endotoxin)

LPS is composed of a lipid portion (lipid A), a

polysaccharide rich core, and a
polysaccharide side chain.
 the lipid portion is heat-stable and
responsible for the biologic effects of
endotoxin;
 the polysaccharide rich core is antigen R of
gram-negative bacteria;
Lipopolysaccharide (LPS, endotoxin)
 the polysaccharide portion of LPS is antigenic
and is designed as the antigen O;
 antigen O determines electronegativity and
hydrophylia of the bacteria, thus confers smooth
culture characters, stability in suspensions and
antiphagocytic effects;
 antigen O has group specificity, being important
for the identification of bacteria.
1. LPS; 2 Braun lipoproteines; 3 Fosfolipide
4 outer membrane; 5 Peptidoglican layer
LPS
 The outer membrane
 The outer membrane confers several important
properties on Gram-negative bacteria:
- it protects the peptidoglycan from the effects of
lysozyme;
- it impedes the ingress of many antibiotics that are
thus rendered impotent;
- it alows the access of low molecular weight nutrients;
- components of the LPS, in particular the lipid A,
form endotoxin, which when released in the blood
stream, may give rise to the endotoxic shock.
The outer membrane
 The periplasmic space
 The periplasmic space is placed between the
outer membrane and the cytoplasmic
membrane.
 It contains the peptidoglycan, lipoproteins
and enzymes involved in:
- the extracellular digestion of gram-negative
bacteria or
- in the inactivation of antibiotics.
The periplasmic space
The periplasmic space
The periplasmic space
 Function of peptidoglycan
 Peptidoglycan prevents osmotic lysis and
confers rigidity and shape;
 outer membrane is permeability barrier;
 associated LPS and proteins have various
functions
 Functions of the cell wall
 The cell walls of bacteria deserve special attention for
several reasons:
1. They are an essential structure for viability, as
described above.
2. They are composed of unique components
(peptidoglycan) found nowhere else in nature.
3. They are one of the most important sites for attack
by antibiotics.
4. They provide ligands for adherence and receptor
sites for drugs or viruses.
5. They cause symptoms of disease in animals.
6. They provide for immunological distinction and
immunological variation among strains of bacteria.
 Acid - fast bacilli
 Acid-fast bacilli (mycobacteria, but also other
filamentous bacteria such as Nocardia) resist
destaining with acid-alcohol after staining with
carbol-fuchsin in Ziehl-Neelsen staining, so
they stain red.
Acid - fast bacilli (red)
 Acid - fast bacilli
 The basis for the staining is the presence of
unique fatty acids (e.g., mycolic acids) in the cell
wall.
 The high content of wax and lipids of the cell
wall confers the acid-fast bacilli resistance to
antimicrobial agents, heat, dryness, chemical
agents.
 They are facultative intracellular organisms and
they survive in macrophage cells.
Acid - fast bacilli (mycolic acids, arabinogalactan)
 Cell Wall-less Forms

 A few bacteria are able to live or exist without a cell

wall.
 There are two groups of bacteria that lack the
protective cell wall peptidoglycan structure, the
Mycoplasma species, one of which causes atypical
pneumonia and some genitourinary tract infections
and the L-forms, which originate from Gram-positive
or Gram-negative bacteria and are so designated
because of their discovery and description at the
Lister Institute, London.
 Cell Wall-less Forms
 The mycoplasmas and L-forms are all Gram-
negative and insensitive to penicillin and are
bounded by a surface membrane structure. L-
forms arising "spontaneously" in cultures or
isolated from infections are structurally related to
protoplasts and spheroplasts;
 all three forms (protoplasts, spheroplasts, and L-
forms) revert infrequently and only under special
conditions.
 Cell Wall-less Forms
 The mycoplasmas are a group of bacteria that lack
a cell wall.
 Mycoplasmas have sterol-like molecules
incorporated into their membranes and they are
usually inhabitants of osmotically-protected
environments.
 Mycoplasma pneumoniae is the cause of primary
atypical bacterial pneumonia, known in the
vernacular as "walking pneumonia".
 Cell Wall-less Forms
 For obvious reasons, penicillin is ineffective in
treatment of this type of pneumonia.
 Sometimes, under the pressure of antibiotic therapy,
pathogenic streptococci can revert to cell wall-less
forms (called spheroplasts) and persist or survive in
osmotically-protected tissues.
 When the antibiotic is withdrawn from therapy the
organisms may regrow their cell walls and reinfect
unprotected tissues.
 CAPSULES, MICROCAPSULES AND
LOOSE SLIME
 Many bacteria, including several pathogenic
species, are surrounded by a discrete covering
layer of a relatively firm gelatinous material
that lies outside and immediately in contact
with the cell wall (glicocalix).
 Microcapsule
 When this layer, in the wet state, is wide
enough (0.2 µ m or more) to be resolved with
the light microscope, it is called a capsule.
 When it is narrower, and detectable only by
indirect, serological means, or by electron
microscopy, it may be termed a microcapsule.
 Microcapsule
 The capsular gel consists largely of water and it
has only a small content of solids (e.g. 2%).
 In most species, the solid material is a complex
polysaccharide, though in some species its main
constituent is polypeptide or protein.
 Extracellular polymer is synthesized by enzymes
located at the surface of the bacterial cell.
 Loose slime
 Loose slime, or free slime, is an amorphous,
viscid colloidal material that is secreted
extracellularly by some non-capsulate bacteria
and also, outside their capsules, by many
capsulate bacteria.
 In capsulate bacteria the slime is generally
similar in chemical composition and antigenic
character to the capsular substance.
 Slime forming bacteria. Capsular slime
surrounding bacterial cell is clearly visible
 Capsule’s functions
 It is probable that the principal action of
capsules and microcapsules is to protect the
cell wall against attack by various kinds of
antibacterial agents, e.g. bacteriophages,
colicines, complement, lysozyme and other
lytic enzymes, that otherwise would more
readily damage and destroy it.
 Capsule’s functions
 In the case of certain capsulate pathogenic organisms
(e.g. pneumococ-cus, pyogenic streptococci, anthrax
bacillus) good evidence has been obtained to show
that the capsule protects the bacteria against
ingestion by the phagocytes of the host.
 The capsule is thus an important agent determining
virulence, and non-capsulate mutants of these bacteria
are found to be non-virulent.
 The capsule is also involved in the adherence of
bacteria to different surfaces, including host’s cells.
 Capsule’s functions
 The capsular substance is usually antigenic
and the capsular antigens play a very
important part in determining the antigenic
specificity of bacteria.
 When capsulate pneumococci are treated with
type-specific antiserum, the sharpness of
outline of the capsule is greatly enhanced. This
is referred to as the " capsule-swelling
reaction ".
 The capsule is colorless in usual stainings.
20th
"Capsule-swelling reaction"
 FLAGELLA

 Motile bacteria possess filamentous appendages

known as flagella, which act as organs of
locomotion.
 The flagellum is a long, thin filament, twisted spirally
in a wave form.
 It is about 0.02 µ m thick and is usual several times
the length of the bacterial cell.
 It originates in the bacterial protoplasm and is
extruded through the cell wall.
 According to the species, there may be one to several
(e.g. 1-20) flagella per cell.
FLAGELLA
 The arrangement of flagella
 The arrangement of flagella may be
monotrichus – a single flagellum at one end of
the bacterium, amphitrichous – two flagella,
each at one end of the bacterium, lofotrichous
– a group of flagella at one end of the
bacterium, or peritrichous, when they
originate over the surface of the cell.
 The presence, number and position of the
flagella are primary taxonomic
 Flagella
 Flagella consist largely or entirely of a
protein, flagellin, and are driven by the rotary
action of a swivel-like basal hook.
 They can be demonstrated easily and clearly
with the electron microscope, ussually
appearing as simple fibrils without internal
differentiation.
 Flagella
 They are invisible by the light microscope, but
may be shown by the use of special staining
methods (e.g., silver staining), and in special
circumstances by dark-ground illumination.
Because of the difficulties of these methods,
the presence of flagella is commonly inferred
from the observation of motility.
Lofotrichous flagella
Bacterial Flagellum Structure
 Motility
 Motility may be observed either
microscopically or by noting the occurence of
spreading growth in semi-solid agar medium.
 On microscopical observation of wet films,
motile bacteria are seen swimming in different
directions across the field, with a darting,
wriggling or tumbling movement.
Semisolid agar
 Motility
 Among the spirochete, motility appears to be a
function of the cell body, since flagella do not occur.
 The most characteristic movement is a fast spiral
rotation on the long axis with slow progression in the
axial line; movements of flexion and lashing
movements may be observed.
 Some spirochetes possess an axial filament and others
a band of fibrils wound around their surface from
pole to pole.
Spirochete’s flagella
 Spirochete’s flagella
 Treponema and Borrelia have numerous thin
axial filaments, disposed around the
protoplast, while Leptospira has only two
thicker filaments, forming an axistil,
surrounded by the protoplast.
 It has been suggested that these structures may
contribute to motility, either through being
themselves contractile or by acting as
stiffeners for recoil against the contractile
protoplast.
 Motility
 Motility may be beneficial in increasing the
rate of uptake of nutrient solutes by
continuously changing the environmental fluid
in contact with the bacterial cell surface.
 Random movement and dispersion through the
environment may be beneficial ensuring that at
least some cells of the strain reach every
locality suitable for colonization.
 Motility
 Bacteria tend to migrate towards regions
where there is a higher concentration of
nutrient solutes (a process known as
chemotaxis) and away from regions containing
higher concentrations of disinfectant
substances (negative chemotaxis).
 Motility
 It might be supposed that the power of active
locomotion would assist pathogenic bacteria in
penetrating through viscid mucous secretions
and epithelial barriers, and in spreading
throughout the body fluids and tissue, but it
must be noted that many non-motile pathogens
(e.g. brucellae and streptococci) are no less
invasive than motile ones.
PROTOPLASTS, SPHEROPLASTS AND
L-FORMS
 Weakening, removal or defective formation of the
cell wall is involved in the production of the various
abnormal forms called spheroplasts, protoplasts
and L-forms.
 Complete removal of the bacterial cell wall of a
Gram-positive bacterium results in the formation of
protoplasts, which are constituted by the cytoplasmic
membrane and the bacterial contents.
 Protoplasts require an isotonic medium in order to
assume a spherical configuration; they cannot
maintain integrity when placed in a hypo- or
hypertonic medium.
PROTOPLAST
 SPHEROPLASTS
 The complexity of the Gram-negative cell wall results
in innate resistance to enzymatic destruction of the
cell wall. Gram-negative cells with damaged cell
walls become spheroplasts (i.e., they assume a
spherical shape) even in a nonisosmotic medium (i.e.,
they are resistant to differences in osmotic pressure
between the extracellular and intracellular
compartments).
 Spheroplasts commonly revert to normal bacterial
morphology when transferred to culture medium
lacking the cell wall inhibitor.
 L-FORMS
 L-forms (wall-less organisms) may emerge during
antibiotic therapy. These aberrant organisms can
cause persistent infection, resisting the effects of
antibiotics whose mechanism of action involves
interference with cell wall formation.
 They differ from the parent bacteria in lacking a rigid
cell wall and, in consequence, regular size and shape,
but they are nevertheless viable and capable of
growing and multiplying on a suitable nutrient
medium.
 Spore
 Some species, notably those of the genera
Bacillus and Clostridium, develop a highly
resistant structure or endospore, whereby the
organism can survive in a dormant state
through a long period of starvation or other
adverse environmental condition.
 Sporulation versus germination
 The process does not involve multiplication:
 in sporulation, each vegetative cell forms only one
spore, and in subsequent
 germination each spore gives rise to a single
vegetative cell.
 Certain specific antigens develop in the spore that
are not found in the vegetative cells.
 Spores are involved in the transmission of certain
diseases.
 Sporulation
 Sporulation occurs as a response to starvation or, at
least, the exhaustion of a limiting substance.
 In certain species, sporulation may be induced by
depletion of nutrients necessary for vegetative
growth;
 at the same time, the process requires a continued
supply of other minerals (potassium, magnesium,
manganese and calcium salts), and favorable
conditions of moisture, temperature, pH, oxygen
tension, etc.
 Sporulation
 The spore is formed inside the parent vegetative cell
(hence the name “endospore”).
 It develops from a portion of the protoplasm near one
end of the cell (“the forespore”), incorporates part of
the nuclear material (equivalent to one genome) of
the cell and aquires a thick covering layer, the
“cortex”, and a thin, but tough, outer “spore coat”
consisting of several layers.
 Spores of some species have an additional, apparently
rather loose covering known as the “exosporium”.
Spore’s structure
Sporulation
Sporulation
 Shape, dispozition
 The appearance of the mature spore varies
according to the species, being spherical, ovoid
or elongated, occupying a terminal,
subterminal or central position, and being
narrower than the cell, or broader and bulging it.
 Finally, the remainder of the parent cell
disintegrates and the spore is freed.
Clostridium spp.
C. tetani (culture smear)
Bacillus spp. (culture smear)
Bacillus anthracis (the lack of spore
in vivo – capsule)
Bacillus (culture smear- special
staining for spore)
Spore’s dispozition
 Application
 Spores are much more resistant than the
vegetative forms to injurious chemical and
physical influences, including exposure to
disinfectants, drying and heating.
 Thus, application of moist heat at 100-120°C
for a period of 10-20 minutes may be needed
to kill spores, whereas heating at 60°C suffices
to kill vegetative cells; spores can be used in
the control of sterilization.
 Spore = resistance
 Spores may remain viable for many years, either in the dry state
or in moist conditions unfavorable to growth.
 The marked resistance of spores has been attributed to several
factors in which they differ from vegetative forms:
- the impermeability of their cortex and outer coat,
- their high content of calcium and dipicolinic acid,
- their low content of water and their
- very low metabolic and enzymatic-activity.
 Germination
 Germination of the spore occurs when the
external conditions become favorable to
growth by access to moisture and nutrients.
 It is irreversible and involves rapid degradative
changes.
 Germination
 The spore successively loses its heat resistance
and its dipicolinic acid; it loses calcium, it
becomes permeable to dyes and its refractility
changes.
 In the process of germination, the spore swells,
its cortex disintegrates, its coat is broken and a
single vegetative cell emerges.
Germination
 Germination
 When mature, the spore resists coloration by
simple stains, appearing as a clear space
within the stained cell protoplasm.
 Spores are slightly acid-fast and may be
stained differentially using special staining.
 Spore
 The presence, shape and position of the
spores are primary taxonomic characters of
bacteria.
Spore’s dispozition
 Bacterial reproduction

 Among bacteria, multiplication takes place by simple

binary fission.
 The cell grows in size, usually elongating to twice its
original length, and the protoplasm becomes divided
into two approximately equal parts by the ingrowth of
a transverse septum from the plasma membrane and
cell wall.
 In some species, the cell wall septum, or cross-wall,
splits in two and the daughter cells separate almost
immediately.
 Bacterial reproduction
 In others, the cell wall of the daughter cells remain
continuous for some time after cell division and the
organisms grow adhering in pairs, clusters, chains or
filaments.
 If cross-wall splitting is thus delayed in an organism in
which the cross-walls of successive cell divisions are all
formed in parallel planes, the cells will be grouped in
chains.
 If it is delayed in an organism that forms successive
cross-walls in different planes, e.g. ones at right angles to
each other, the cells will be grouped in pairs, forming
either cubic or irregular clusters.
Cells’ division