MORPHOLOGY AND CLASSIFICATION OF BACTERIA

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MORPHOLOGY AND
CLASSIFICATION OF BACTERIA

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Morphology and Classification of Bacteria
MICROBIOLOGY
MODULE
Microbiology
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1
MORPHOLOGY AND
CLASSIFICATION OF BACTERIA
1.1 INTRODUCTION
Microorganisms are a heterogeneous group of several distinct classes of living
beings. Based on the difference in cellular organization and biochemistry, the
kingdom protista has been divided into two groups namely prokaryotes and
eukaryotes. Bacteria and blue-green algae are prokaryotes, while fungi, other
algae, slime moulds and protozoa are eukaryotes. Bacteria are prokaryotic
microorganisms that do not contain chlorophyll. They are unicellular and do not
show true branching, except in higher bacteria like actinomycetales.
OBJECTIVES
After reading this lesson, you will be able to:
zdescribe the structure of Prokaryotic and Eukaryotic cell
zexplain the size of bacteria
zclassify bacteria based on the shape and arrangements
zdescribe the structure of bacterial cell wall
zdescribe the phases of Growth curve
zexplain the factors affecting the growth of bacteria
1.2 PROKARYOTES
The prokaryotic cells have the following characteristics such as
zNo organelles, all the action takes place in the cytosol or cytoplasmic
membrane

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zMost bacteria possess peptidoglycan, a unique polymer that makes its
synthesis a good target for antibiotics
zProtein synthesis takes place in the cytosol with structurally different
ribosome’s
Fig. 1.1: Prokaryote Cell
Fig. 1.2: Eukaryote Cell
Difference between Prokaryotic and Eukaryotic Cells
Character
Nucleus
Membrane-bound
organelles
Chromosome (DNA)
Prokaryotes
Absent. No nuclear
envelope
Absent
Single coiled chromosome
in cytoplasm ‘nucleoid’
region in association with
‘histone-like’ proteins
Eukaryotes
Present with nuclear
envelope and nucleolus
Present. Includes
mitochondria, chloroplasts
(plants), lysosomes
Multiple linear
chromosomes with histone
proteins

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1.3 BACTERIA
The major characteristics of Bacteria are based on their size, shape and
arrangements
1.3.1 Size
The unit of measurement used in bacteriology is the micron (micrometer)
1 micron (μ) or micrometer (μm) – one thousandth of a
millimeter
1 millimicron (mμ) or nanometer (nm) – one thousandth of a micron
or one millionth of a
millimeter
1 Angstrom unit (Å) – one tenth of a nanometer
The limit of resolution with the unaided eye is about 200 microns. Bacteria are
smaller which can be visualized only under magnification. Bacteria of medical
importance generally measure 0.2 – 1.5 μm in diameter and about 3-5 μm in
length.
Cell wall
Mitotic division
Ribosomes
Flagella
Cytoplasmic membrane
lipids
Mitochondria
Lysosomes
Golgi apparatus
Endoplasmic Reticulum
Eubacteria have a cell wall
of peptidoglycan Archaea
have cell walls of
pseudomurein
Absent
70S. Free in cytoplasm
when present consist of
protein flagellin
Eubacteria= Fatty acids
joined to glycerol by ester
linkageArchaea=
Hydrocarbons joined to
glycerol by ether linkage
Absent
Absent
Absent
Absent
No cell wall in animal
cellsPlant cell walls =
celluloseFungal cell walls =
chitin
Present
80S. Both free in cytoplasm
and attached to rough
E.R.70S in mitochondria
and chloroplasts
consist of 9+2 arrangement
of microtubules
Fatty acids joined to
glycerol by ester linkage
Present
Present
Present
Present

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1.3.2 Microscopy
The morphological study of bacteria requires the use of microscopes. Microscopy
has come a long way since Leeuwenhoek first observed bacteria using hand-
ground lenses.
The types of microscope are
(i) Light or optical microscope
(ii) Phase contrast microscope
(iii) Dark field/ Dark ground microscope
(iv) Electron microscope
Light or optical microscope
They are of two types namely Simple and Compund Microscope
zSimple Microscope consists of a single lens. A hand lens is an example of
a simple Microscope.
zCompound Microscope consists of two or more lenses in series. The image
formed by the first lens is further magnified by another lens.
Bacteria may be examined under the compound microscope, either in the living
state or after fixation and staining. Examination of wet films or hanging drops
indicates the shape, arrangements, motility and approximately size of the cells.
But due to lack of contrast details cannot be appreciated.
Phase contrast microscope
This imposes the contrast and makes evident the structure within the cells that
differ in thickness or refractive index. The difference in the refractive index
between bacteria cells and the surrounding medium makes them clearly visible.
Retardation, by a fraction of a wavelength, of the rays of light that pass through
the object, compared to the rays passing through the surrounding medium,
produces phase difference between the two types of rays.
Dark field / Dark ground microscope
Another method of improving the contrast is the dark field microscope in which
reflected light is used instead of the transmitted light used in the ordinal
microscope. The contrast gives an illusion of increased resolution, so that very
slender organisms such as spirochete, not visible under ordinary illumination,
can be clearly seen under the dark field microscope.

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Electron Microscope
Beams of electron are used instead of beam of light, used in light microscope.
The object which is held in the path of beam scatters the electrons and produces
an image which is focused on a fluorescent viewing screen. Gas molecules
scatter electron, therefore it is necessary to examine the object in a vacuum.
INTEXT QUESTIONS 1.1
Match the following
Microscopes Properties:
1. Light microscope (a) reflected light
2. Phase contrast microscope (b) electron beam
3. Dark field microscope (c) light beam
4. Electron microscope (d) refractive index
1.3.3 Stained Preparations
Live bacteria do not show the structural detail under the light microscope due
to lack of contrast. Hence staining techniques are used to produce colour
contrast. Routine methods of staining of bacteria involve dying and fixing
smears – procedures that kill them. Bacteria have an affinity to basic dyes due
to acidic nature of their protoplasm. The commonly used staining techniques are
Simple Stains
Dyes such as methylene blue or basic fuchsin are used for simple staining. They
provide colour contrast, but impart the same colour to all bacteria.
Negative Staining
Bacteria are mixed with dyes such as Indian ink or nigrosin that provide a
uniformly coloured background against which the unstained bacteria stand out
in contrast. Very slender bacteria like spirochetes that cannot be demonstrated
by simple staining methods can be viewed by negative staining.
Impregnation Methods
Cells and structures too thin to be seen under ordinary microscope may be
rendered visible if they are impregnated with silver on the surface. These are
used for demonstration of spirochetes and bacterial flagella.

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Differential Stains
These stains impart different colours to different bacteria or bacterial structures,
the two most widely used differential stains are the Gram stain and Acid fast
stain. The gram stain was devised by histologist Christian Gram as a method of
staining bacteria in tissues.
Gram positive cells are simpler chemical structure with a acidic protoplasm. It
has a thick peptidoglycan layer. Teichoic acids are intertwined among the
peptidoglycan and the teichoic acids are the major surface antigen determinants
Gram negative cells are more complex, they are rich in lipids. The membrane
is bilayered as phospholipids, proteins and lipopolysaccharide. Lipopoly-
saccharides (LPS) are also known as endotoxin. Gram negative cells have a
peptidoglycan layer which is thin and formed by just one or two molecules. No
Teichoic acids are found in the cell wall of Gram negative bacteria. The Outer
membrane has Lipopolysaccharide channels with porins which transfer the
solutes across. Lipoprotein cross link outer membrane and peptidoglycan layer
Gram reaction may be related to the permeability of the bacterial cell wall and
cytoplasmic membrane to the dye-iodine complex, the Gram-negative, but not
the Gram-positive cells, permitting the outflow of the complex during
decolourisation. Gram staining is an essential procedure used in the identification
of bacteria and is frequently the only method required for studying their
morphology.
The acid fast stain was discovered by Ehrlich, who found that after staining with
aniline dyes, tubercle bacilli resist decolourisation with acids. The method as
modified by Ziehl and Neelsen, is in common use now.
INTEXT QUESTIONS 1.2
Match the following:
1. Simple stain (a) Silver
2. Negative stain (b) acids
3. Impregnation method (c) iodine complex
4. Acid fast stain (d) Methylene blue
5. Gram stain (e) Indian ink
1.4 SHAPE OF THE BACTERIA
Depending on their shape, bacteria are classified into several varieties
1. Cocci (from kokkos meaning berry) are spherical or oval cells

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2. Bacilli (from baculus meaning rod) are rod shaped cells
3. Vibrios are comma shaped curved rods and derive their name from their
characteristics vibratory motility.
4. Spirilla are rigid spiral forms.
5. Spirochetes (from speira meaning coil and chaite meaning hair) are flexuous
spiral forms
6. Actinomycetes are branching filamentous bacteria, so called because of a
fancied resemblance to the radiating rays of the sun when seen in tissue
lesions (from actis meaning ray and mykes meaning fungus)
7. Mycoplasmas are bacteria that are cell wall deficient and hence do not
possess a stable morphology. They occur as round or oval bodies and as
interlacing filaments.
Fig. 1.3: Shapes of bacteria.

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INTEXT QUESTION 1.3
Match the following:
1. Bacilli (a) coma
2. Cocci (b) flexous spiral form
3. Vibrio (c) rigid spiral form
4. Sprillum (d) rod shaped
5. Spirochetes (e) spherical shaped
Bacteria sometime show characteristic cellular arrangement or grouping.
According to the plane of cellular division, cocci may be arranged in pairs
(diplococci), chains (streptococci), groups of four (tetrads) or eight (sarcina),
or grape like clusters (staphylococci).
Fig. 1.4: Arrangement of Cocci.
INTEXT QUESTIONS 1.4
Match the following:
1. Diplococci (a) groups of four
2. Streptococci (b) groups of eight
3. Tetrads (c) occurs in pairs
4. Sarcina (d) grape like clusters
5. Staphylococci (e) occurs in chains

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Fig. 1.5
1.5 BACTERIAL STRUCTURE
The outer layer or cell envelope consists of two components, a rigid cell wall
and beneath it a cytoplasmic or plasma membrane. The cell envelope encloses
the protoplasm, comprising the cytoplasm, cytoplasmic inclusions such as
ribosomes and mesosomes, granules, vacuoles and the nuclear body.
Cell wall
Beneath the external structures is the cell wall. It is very rigid & gives shape to
the cell. Its main function is to prevent the cell from expanding & eventually
bursting due to water uptake. Cell Wall constitutes a significant portion of the
dry weight of the cell and it is essential for bacterial growth & division. The cell
wall cannot be seen by direct light microscopy and does not stain with simple

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stains. It may be demonstrated by microdissection, reaction with specific
antibodies, mechanical rupture of the cell, differential staining procedures or by
electron microscopy.
Chemically the cell wall is composed of peptidoglycan. Mucopeptide
(peptidoglycan or murien) formed by N acetyl glucosamine & N acetyl muramic
acid alternating in chains, cross linked by peptide chains. Embedded in it are
polyalcohol called Teichoic acids. Some are linked to Lipids & called
Lipoteichoic acid. Lipotechoic acid link peptidoglycan to cytoplasmic membrane
and the peptidoglycan gives rigidity.
The functions of Teichoic acid are
zgives negative charge
zmajor antigenic determinant
ztransport ions
zanchoring
zexternal permeability barrier
Characteristics Gram Positive Gram Negative
Thickness Thicker Thinner
Variety of amino acids Few Several
Lipids Absent Present
Teichoic acid Present absent
Outer Membrane
Outer membrane is found only in Gram-negative bacteria, it functions as an
initial barrier to the environment and is composed of lipopolysaccharide (LPS)
and phospholipids
Lipopolysaccharide (LPS)
The LPS present on the cell walls of Gram-negative bacteria account for their
endotoxic activity and antigen specificity.
A bacterium is referred as a protoplast when it is without cell wall. Cell wall
may be lost due to the action of lysozyme enzyme, which destroys peptidoglycan.
This cell is easily lysed and it is metabolically active but unable to reproduce.
A bacterium with a damaged cell wall is referred as spheroplasts. It is caused
by the action of toxic chemical or an antibiotic, they show a variety of forms
and they are able to change into their normal form when the toxic agent is
removed, i.e. when grown on a culture media

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Cytoplasmic membrane
Cytoplasmic membrane is present immediately beneath the cell wall, found in
both Gram positive & negative bacteria and it is a thin layer lining the inner
surface of cell wall and separating it from cytoplasm. It acts as a semipermeable
membrane controlling the flow of metabolites to and from the protoplasm.
Cytoplasm
The cytoplasm is a Colloidal system containing a variety of organic and
inorganic solutes containing 80% Water and 20% Salts, Proteins. They are rich
in ribosomes, DNA & fluid. DNA is circular and haploid. They are highly coiled
with intermixed polyamines & support proteins. Plasmids are extra circular
DNA.
Fig. 1.6
Ribosomes
They are the centers of protein synthesis. They are slightly smaller than the
ribosomes of eukaryotic cells
Mesosomes
They are vesicular, convoluted tubules formed by invagination of plasma
membrane into the cytoplasm. They are principal sites of respiratory enzymes
and help with cell reproduction
Cytoplasmic Inclusions
The Inclusion bodies are aggregates of polymers produced when there is excess
of nutrients in the environment and they are the storage reserve for granules,
1 μm

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phosphates and other substances. Volutin granules are polymetaphosphates
which are reserves of energy and phosphate for cell metabolism and they are also
known as metachromatic granules.
Nucleus
The Nucleus is not distinct and has no nuclear membrane or nucleolus and the
genetic material consist of DNA. The cytoplasmic carriers of genetic information
are termed plasmids or episomes.
Capsule
Capsule is the outer most layer of the bacteria (extra cellular). It is a condensed
well defined layer closely surrounding the cell. They are usually polysaccharide
and if polysaccharide envelops the whole bacterium it is capsule and their
production depends on growth conditions. They are secreted by the cell into the
external environment and are highly impermeable. When it forms a loose mesh
work of fibrils extending outward from the cell they are described as glycocalyx
and when masses of polymer that formed appear to be totally detached from the
cell and if the cells are seen entrapped in it are described as slime layer.
The Capsule protects against complement and is antiphagocytic. The Slime layer
& glycocalyx helps in adherence of bacteria either to themselves forming
colonial masses or to surfaces in their environment and they resists phagocytosis
and desiccation of bacteria.
Flagella
Flagella are long hair like helical filaments extending from cytoplasmic
membrane to exterior of the cell. Flagellin is highly antigenic and functions in
cell motility. The location of the flagella depends on bacterial species as polar
situated at one or both ends which swims in back and forth fashion and lateral
at along the sides.
The parts of flagella are the filament, hook and the basal body. Filament is
external to cell wall and is connected to the hook at cell surface, the hook & basal
body are embedded in the cell envelope. Hook & filament is composed of protein
subunits called as flagellin. Flagellin is synthesized within the cell and passes
through the hollow centre of flagella. The arrangement of flagella may be
described as
(i) Monotrichous – single flagella on one side
(ii) Lophotrichous – tuft of flagella on one side
(iii) Amphitrichous – single or tuft on both sides
(iv) Peritrichous – surrounded by lateral flagella

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Structure Flagella Type Example
Monotrichous Vibrio cholerae
Lophotrichous Bartonella
bacillifornis
Amphitrichous Spirillum serpens
Peritrichous Escherichia coli
Fig. 1.7: Flagella.
Various types of mobility is observed because of the presence of the flagella as
Serpentine motility is seen with Salmonella, Darting motility with Vibrio and
Tumbling motility with Listeria monocytogenes
Pili / Fimbriae
Hair-like proteinaceous structures that extend from the cell membrane to
external environment are pili which are otherwise known as fimbriae. They are
thinner, shorter and more numerous than flagella and they do not function in
motility. The fimbriae is composed of a subunit called pilin.
There are two types pili namely Non-sex pili (Common pili) eg. fimbriae or type
IV and the sex pili. The fimbriae are antigenic and mediate their adhesion which
inhibits phagocytosis. The sex pili help in conjugation.
INTEXT QUESTIONS 1.5
Match the following:
1. Monotrichous (a) single or tuft on both sides
2. Lophotrichous (b) surrounded by lateral flagella
3. Amphitrichous (c) single flagella on one side
4. Peritrichous (d) tuft of flagella on one side
Spore
Some bacteria have the ability to form highly resistant resting stage called
spores, which helps them to overcome adverse environmental conditions that are
unfavorable for vegetative growth of cell. They are not a reproductive form and

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Structure
Flagella
Pili
Sex pilus
Common pili or fimbriae
Capsules (includes “slime
layers” and glycocalyx)
Cell wall
Gram-positive bacteria
Gram-negative bacteria
Plasma membrane
Ribosomes
Inclusions
Chromosome
Plasmid
Functions(s)
Swimming movement
Stabilizes mating bacteria
during DNA transfer by
conjugation
Attachment to surfaces;
protection against
phagotrophic engulfment
Attachment to surfaces;
protection against phagocytic
engulfment, occasionally
killing or digestion;
protection against
desiccation
confers rigidity and shape on
cells
confers rigidity and shape;
outer membrane is
permeability barrier;
associated LPS and proteins
have various functions
Permeability barrier;
transport of solutes; energy
generation; location of
numerous enzyme systems
Sites of translation (protein
synthesis)
Often reserves of nutrients;
additional specialized
functions
Genetic material of cell
Extrachromosomal genetic
material
Predominant chemical
composition
Protein
Protein
Protein
Usually polysaccharide;
occasionally polypeptide
Peptidoglycan (murein)
complexed with teichoic
acids
Peptidoglycan (murein)
surrounded by phospholipid
protein-lipopolysaccharide
“outer membrane”
Phospholipid and protein
RNA and protein
Highly variable;
carbohydrate, lipid, protein
or inorganic
DNA
DNA
Characteristics of Bacteria Cell Structures

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not a storage granule. These spores are resistant to bactericidal agents and
adverse physical conditions. Each spore can give rise to only one endospore
which play a role in heat resistance. Spores consists of three layers namely core,
cortex and spore coat
Fig. 1.8: Spere
1.6 GROWTH AND MULTIPLICATION OF BACTERIA
Bacteria divide by binary fission and cell divides to form two daughter cells.
Nuclear division precedes cell division and therefore, in a growing population,
many cells having two nuclear bodies can be seen. Bacterial growth may be
considered as two levels, increase in the size of individual cells and increase in
number of cells. Growth in numbers can be studied by bacterial counts that of
total and viable counts. The total count gives the number of cells either living
or not and the viable count measures the number of living cells that are capable
of multiplication.
1.6.1 Bacterial Growth Curve
When bacteria is grown in a suitable liquid medium and incubated its growth
follows a definite process. If bacterial counts are carried out at intervals after
innoculation and plotted in relation to time, a growth curve is obtained. The
curve shows the following phase
(i) Lag phase
Immediately following innoculation there is no appreciable increase in number,
though there may be an increase in the size of the cells. This initial period is the
time required for adaptation to the new environment and this lag phase varies
with species, nature of culture medium and temperature.

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(ii) Log or exponential phase
Following the lag phase, the cell starts dividing and their numbers increase
exponentially with time.
(iii) Stationary phase
After a period of exponential growth, cell division stops due to depletion of
nutrient and accumulation of toxic products. The viable count remains stationary
as an equilibrium exists between the dying cells and the newly formed cells.
(iv) Phase of decline
This is the phase when the population decreased due to cell death.
Number of
bacteria
Time
Decline
phase
Stationary
phase
Log
phase
Lag
phase
Fig. 1.8: The growth curve of bacteria showing different phases
The various stages of bacterial growth curve are associated with morphological
and physiological alterations of the cells. The maximum cell size is obtained
towards the end of the lag phase. In the log phase, cells are smaller and stained
uniformily. In the stationary phase, cells are frequently gram variable and show
irregular staining due to the presence of intracellular storage granules. Sporulation
occurs at this stage. Also, many bacteria produce secondary metabolic products
such as exotoxins and antibiotics. Involution forms are common in the phase of
decline.
1.7 FACTORS THAT AFFECT THE GROWTH OF
BACTERIA
Many factors affect the generation time of the organism like temperature,
oxygen, carbon dioxide, light, pH, moisture, salt concentration.
Nutrition
The principal constituents of the cells are water, proteins, polysaccharides,
lipids, nucleic acid and mucopeptides. For growth and multiplication of bacteria,
the minimum nutritional requirement is water, a source of carbon, nitrogen and
some inorganic salts.

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Bacteria can be classified nutritionally, based on their energy requirement and
on their ability to synthesise essential metabolites. Bacteria which derive their
energy from sunlight are called phototrophs, those who obtain energy from
chemical reactions are called chemotrophs. Bacteria which can synthesise all
their organic compounds are called autotrophs and those that are unable to
synthesise their own metabolites are heterotrophs.
Some bacteria require certain organic compounds in minute quantities. These
are know as growth factors or bacterial vitamins. Growth factors are called
essential when growth does not occur in their absence, or they are necessary for
it.
Oxygen
Depending on the influence of oxygen on growth and viability, bacteria are
divided into aerobes and anaerobes.
Aerobic bacteria require oxygen for growth. They may be obligate aerobes like
cholera, vibrio, which will grow only in the presence of oxygen or facultative
anaerobes which are ordinarily aerobic but can grow in the absence of oxygen.
Most bacterial of medical importance are facultative anaerobes. Anaerobic
bacteria, such as clostridia, grow in the absence of oxygen and the obligate
anaerobes may even die on exposure to oxygen. Microaerophilic bacteria are
those that grow best in the presence of low oxygen tension.
Carbon Dioxide
All bacteria require small amounts of carbon dioxide for growth. This
requirement is usually met by the carbon dioxide present in the atmosphere.
Some bacteria like Brucella abortus require much higher levels of carbon
dioxide.
Temperature
Bacteria vary in their requirement of temperature for growth. The temperature
at which growth occurs best is known as the optimum temperature. Bacteria
which grow best at temperatures of 25-40°C are called mesophilic. Psychrophilic
bacteria are those that grow best at temperatures below 20°C. Another group of
non pathogenic bacteria, thermophiles, grow best at high temperatures, 55-80°C.
The lowest temperature that kills a bacterium under standard conditions in a
given time is known as thermal death point.

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Moisture and Drying
Water is an essential ingredient of bacterial protoplasm and hence drying is lethal
to cells. The effect of drying varies in different species.
Light
Bacteria except phototrophic species grow well in the dark. They are sensitive
to ultraviolet light and other radiations. Cultures die if exposed to light.
H-ion concentration
Bacteria are sensitive to variations in pH. Each species has a pH range, above
or below which it cannot survive and an optimum pH at which it grows best.
Majority of pathogenic bacteria grow best at neutral or slightly alkaline pH (7.2
– 7.6)
Osmotic Effect
Bacteria are more tolerant to osmotic variation than most other cells due to the
mechanical strength of their cell wall. Sudden exposure to hypertonic solutions
may cause osmotic withdrawal of water and shrinkage of protoplasm called
plasmolysis.
WHAT YOU HAVE LEARNT
zBacteria are prokaryotic microorganism that do not contain chlorophyll
zThey are unicellular and do not exhibit true branching.
zThe morphological study of bacteria requires the use of microscope like
optical or light microscope, phase control microscope, dark/field microscope,
electron microscope
zStaining techniques like simple stain, negative stain, impregnation stain,
differential stains are used to exhibit structure of bacteria
zBacteria are classified based on the shape as cocci, bacilli, vibrio, Spirilla.
And based on arrangements they are classified as diplococci, streptococci,
tetrads, sarcina, staphylococci
zBacterial cell has cell wall, inner protoplasm and other components
zBacterial growth phase has a lag phase, log phase, stationary phase and a
decline phase

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TERMINAL QUESTIONS
1. Describe the structure of cell wall
2. Classify bacteria based on shaped and arrangement with examples
3. Explain the factors affecting the growth of the bacteria
4. Describe growth curve
ANSWERS TO INTEXT QUESTIONS
1.1
1. (c) 2. (d) 3. (a) 4. (b)
1.2
1. (d) 2. (e) 3. (a) 4. (b) 5. (c)
1.3
1. (d) 2. (e) 3. (a) 4. (c) 5. (b)
1.4
1. (c) 2. (e) 3. (a) 4. (b) 5. (d)
1.5
1. (c) 2. (d) 3. (a) 4. (b)