Classification of Microbes

Dr.B.Reena Rajkumari
 Placing Bacteria
1735 Kingdoms Plantae and Animalia
1857 Bacteria and fungi put in the Kingdom Plantae –
“Flora”
1866 Kingdom Protista proposed for bacteria, protozoa,
algae, and fungi
1937 Prokaryote introduced for cells "without a nucleus"
1961 Prokaryote defined as cell in which nucleoplasm is
not surrounded by a nuclear membrane
1959 Kingdom Fungi
1968 Kingdom Prokaryotes proposed
1978 Two types of prokaryotic cells found

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 Taxonomy
• The science of classifying organisms
• Provides universal names for organisms
• Provides a reference for identifying organisms

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In 1970, Robert Whittaker developed a five-kingdom system of classification based
on ecology, evolution and structure.

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 The Three-Domain System

In 1980, Carl Woese proposed a new system based on ribosomal RNA (rRNA) analysis
of many different types of cells. Living things could be classified in three
superkingdoms or Domains: Archea, Eubacteria and Eukarya based on each group's
specific rRNA and other biochemical differences.
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 Living things classified in three
super kingdoms or Domains:
Archea, Eubacteria and Eukarya
based on each group's specific
rRNA and other biochemical
differences.

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 Classification of Prokaryotes
• Prokaryotic species: A population of cells with
similar characteristics
– Culture: Grown in laboratory media
– Clone: Population of cells derived from a single
cell
– Strain: Genetically different cells within a clone

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 Scientific Nomenclature
• Common names
– Vary with languages
– Vary with geography
• Binomial Nomenclature (genus + specific
epithet)
– Used worldwide
– Escherichia coli
– Homo sapiens

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 References
International Journal of Articles with evidence of
Systematic and Evolutionary new species or classification
Microbiology

Bergey’s Manual of Systematic Provides phylogenetic and

Bacteriology identification information on
bacteria and archaea

Approved Lists of Bacterial Lists species of known

Names prokaryotes
Based on published articles

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Phenotypic Classification Methods
Analytic Classification Methods
- Microscopic morphology
- Whole cell lipid analysis
- Macroscopic morphology
- Whole cell protein analysis
- Bio-typing

- Serotyping - Multi-locus locus enzyme electrophoresis

- Anti-biogram patterns
- Cell wall fatty acid analysis
- Phage typing

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Genotypic Classification of Bacteria

- Guanine and cytosine ratio

- DNA hybridization

- Nucleic acid sequence analysis

- Plasmid analysis
- Ribotyping

- Chromosomal DNA fragment

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Classification of Microbes

Dr.B.Reena Rajkumari
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Prokaryotic Form and Function

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 Structures common to all bacterial cells

• Cell membrane
• Cytoplasm
• Ribosomes
• One (or a few) chromosomes

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Structures found in most bacterial cells
• Cell wall
• Surface coating or glycocalyx

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 Structures found in some bacterial cells

• Flagella
• Pili
• Fimbriae
• Capsules
• Slime layers
• Inclusions
• Actin cytoskeleton
• Endospores

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Figure 4.1
Main groups of bacteria

Gram-positive cocci, bacilli and branching

I
bacteria

Gram-negative cocci, bacilli and comma-

II
shaped bacteria

III Spiral-shaped bacteria

IV Acid-fast bacteria

V Cell-wall-deficient bacteria

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 Bacterial classification
• Shape
• size
• Cell arrangement
• Stain
• Cell wall
• Flagella arrangement
• Motility
• Bacterial growth / Generation Time
• Capsule
• Endospore
• Pigment Production
• Oxygen requirement
• Nutrition
• Colony morphology
• Reproduction

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Bacterial Shapes, Arrangements, and Sizes

• Three general shapes

– Coccus- roughly spherical
– Bacillus- rod-shaped
• Coccobacillus- short and plump
• Vibrio- gently curved
– Spirillum- curviform or spiral-shaped
– Pleomorphism- when cells of a single species vary
to some extent in shape and size

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Size of different Microbes

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Meter Centimeter Millimeter Micrometer Nanometer Angstrom Picometer
100 m 10-2 m 10-3 m 10-6 m 10-9 m 10-10 m 10-12 m
0.000000001 0.0000000001 0.0000000000
1m 0.01 m 0.001 m 0.000001 m
m m 01 m

1/1,000,000,0 1/10,000,000, 1/1,000,000,0

1/100 m 1/1,000 m 1/1,000,000 m
00 m 000 m 00,000 m

hundreth of a thousandth of millionth of a billionth of a ten billionth of trillionth of a

meter a meter meter meter a meter meter

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 Arrangement, or Grouping
• Cocci- greatest variety in
arrangement
– Single
– Pairs (diplococci)
– Tetrads
– Irregular clusters
(staphylococci and
micrococci)
– Chains (streptococci)
– Cubical packet (sarcina)
• Bacilli- less varied
– Single
– Pairs (diplobacilli)
– Chain (streptobacilli)
– Row of cells oriented
side by side (palisades)
• Spirilla
– Occasionally found in
short chains
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Arrangement of cells dependent on pattern of division
and how cells remain attached after division:

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 The Cell Envelope: The Boundary layer of
Bacteria

• Majority of bacteria have a cell envelope

• Lies outside of the cytoplasm
• Composed of two or three basic layers
– Cell membrane
– Cell wall
– In some bacteria, the outer membrane

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Differences in Cell Envelope Structure
• The differences between gram-positive and
gram-negative bacteria lie in the cell envelope
• Gram-positive
– Two layers
– Cell wall and cytoplasmic membrane
• Gram-negative
– Three layers
– Outer membrane, cell wall, and cytoplasmic
membrane

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 Structure of the Cell Wall
• Helps determine the shape of a bacterium
• Provides strong structural support
• Most are rigid because of peptidoglycan
content

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 Structure of the Cell Wall, cont.
• Keeps cells from rupturing because of changes in
pressure due to osmosis
• Target of many antibiotics- disrupt the cell wall,
and cells have little protection from lysis
• Gram-positive cell wall
– A thick (20 to 80 nm), homogeneous sheath of
petidoglycan
– Contains tightly bound acidic polysaccharides
• Gram-Negative Cell Wall
– Single, thin (1 to 3 nm) sheet of peptidoglycan
– Periplasmic space surrounds the peptidoglycan
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 • Macromolecule composed of
a repeating framework of
long glycan chains
• cross-linked by short peptide
fragments provides strong,
flexible support
• keep bacteria from bursting
or collapsing because of
changes in osmotic pressure

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Bacterial cell wall

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 Comparison of Bacterial cell wall

Acid-fast bacteria (Mycobacteria) have a thick peptidoglycan cell wall, which is

surrounded by a wax like coat of mycolic acid and glycolipids that is impermeable
to many substances and imparts resistance to many disinfectants and antibiotics.

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Mycobacterial cell wall – Mycolic acid

Mycobacterial cell wall:

1-outer lipids,
2-mycolic acid,
3-polysaccharides(arabino-galactan),
4-peptidoglycan,
5-plasma membrane,
6-lipoarabinomannan (LAM),
7-phosphatidylinositol mannoside,
8-cell wall skeleton

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 Nontypical Cell Walls
• Some aren’t characterized as either gram-
positive or gram-negative
• For example, Mycobacterium and Nocardia-
unique types of lipids (acid-fast)
• Archaea - unusual and chemically distinct cell
walls
• Some don’t have a cell wall at all -
Mycoplasmas- lack cell wall entirely

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 Mycoplasmas and Other Cell-Wall-
Deficient Bacteria
• Mycoplasma cell membrane is stabilized by
sterols and is resistant to lysis
– Very small bacteria (0.1 to 0.5 µm)
– Range in shape from filamentous to coccus
– Not obligate parasites
– Can be grown on artificial media
– Found in many habitats
– Important medical species: Mycoplasma pneumoniae

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 Mycoplasma pneumoniae

Mycoplasma fermentans

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Hoechst staining mycoplasma
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• Some bacteria lose their cell wall during part
of their life cycle
– L-forms
– Arise naturally from a mutation in the wall-
forming genes
– Can be induced artificially by treatment with a
chemical that disrupts the cell wall
• When this occurs with gram-positive cells, the cell
becomes a protoplast
• With gram-negative cells, the cell becomes a
spheroplast

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Stains.
As microbes are small in size and relative transparent, bacteria must be
stained to be visible under the light microscope. Staining characteristics may
be used in classification. The major types of staining reactions are the
following:
a. The simple stain uses a single dye (e.g., methylene blue, basic fuchsin,
and crystal violet) to colour the cells.
b. The gram stain is a differential staining procedure in which the purple
stain is retained in the thick peptidoglycan layer of the gram-positive
bacteria, whereas the colour is removed by an alcohol rinse from the
thinner gram-negative walls. The gram-negative cells are then stained red
by the counterstain safranin
c. The acid-fast stain uses a procedure to stain cells that have an outer
layer of a waxy lipid (acid-fast, stained red). This waxy layer prevents
removal of the stain by an acid rinse. The stain is removed from cells that
lack the waxy layer, which are then counterstained blue (non–acid-fast).
Bacteria of the genus Mycobacterium are acid-fast bacilli (AFB).
d. The endospore stain
e. The capsule stain
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Flagella

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 Flagella
• Some bacteria posses Flagella
(singular : Flagellum)
• They are long filamentous
appendages that contain globular
protein flagellin arranged in several
chains .
• It intertwines and forms a helix
• It is attached to a hook and a basal
body which anchors the flagellum
to the cell wall and plasma
membrane
• basal body -stack of rings firmly
anchored in cell wall
• rotates 360o

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 Flagellar arrangement

Monotrichous- single flagellum Peritrichous- dispersed randomly

on one pole over the structure of the cell
Lophotrichous- small bunches or Amphitrichous - single flagellum
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Attachment of Flagella

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 Movement by flagella

There are many schemes for flagellation in bacteria, of which peritrichous flagella and a single polar
(mono-trichous) flagellum are two types. a) In the case of peri-trichous flagella, such as those found
in Escherichia coli, counter-clockwise (CCW) flagellar rotation results in the formation of a helical
bundle that propels the cell forward in one direction in a smooth-swimming motion (a 'run'). By
contrast, the presence of clockwise (CW) rotation causes unbundling of the helical bundle, allowing
the bacterium to randomly reorient its direction (a 'tumble').b) In the case of a single polar
flagellum, CCW rotation propels the cell forward in a run, whereas CW rotation propels the cell
backward with a concomitant random reorientation.
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• Chemo taxis (from chemo- + taxis) is the movement of an organism in response to a
chemical stimulus.
• Somatic cells, bacteria and other organisms direct their movements according to
certain chemicals in their environment.
• This is important for bacteria to find food (e.g., glucose) by swimming toward the
highest concentration of food molecules

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• The early days of the development of microscopy (Leeuwenhoek),
• Description of chemotaxis was first made by T W. Engelmann (1881) and W.F. Pfeffer (1884) in
bacteria and H.S. Jennings (1906) in ciliates.
• The Nobel Prize laureate I. Metchnikoff also contributed to the study of the field with
investigations of the process as an initial step of phagocytosis.
• The significance of chemotaxis in biology and clinical pathology was widely accepted in the
1930s.
• The most important aspects in quality control of chemotaxis assays were described by H. Harris
in the 1950s.
• The pioneering works of J. Adler represented a significant turning point in understanding the
whole process of intracellular signal transduction of bacteria.
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swarming

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Type of Motility

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 Brownian motion or Movement
• The Brownian motion, first observed in 1827 by the English botanist Robert
Brown.
• He was investigating a suspension of microscopic pollen particles in solution
• He observed movement even in pollen samples that had been dead for
more than 100 years.
• This is due to random molecular bombardment of tiny bacterial cells by the
molecules of the solvent:

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Axial Filaments

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• These non-emergent flagella, or endoflagella (endo- meaning 'internal') ,
• These internal flagella form a bundle or axial filament.
• The filaments contain a core of flagellin (FlaB) and in some species also contain a sheath
of a second type of flagellin (FlaA).
• The sheathed filaments are thicker (about 25 nanometres in diameter).
• Found in spirochetes
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 Motility Medium
Soft Agar Deep Test for motility
• This special media relies on the ability
of motile bacteria to move through a
tube of semisolid medium.
• The growth of motile bacteria in such a
tube will produce turbidity throughout
the solid medium, whereas non-motile
organisms will grow only along the line
of inoculation.
• The media we will use has 0.35% agar
instead of the usual 1.5%
• Motile organisms (such as E. coli) will
exhibit growth radiating from the stab
inoculation line.
Non motile organisms (such as
Staphylococci or Streptococci) will
exhibit growth only along the stab
inoculation line

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Hanging Drop method

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Generation Time

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 Inclusions
• Inclusions- also known as inclusion bodies
– Some bacteria lay down nutrients in these inclusions
during periods of nutrient abundance
– Serve as a storehouse when nutrients become
depleted
– Some enclose condensed, energy-rich organic
substances
– Some aquatic bacterial inclusions include gas vesicles
to provide buoyancy and flotation
– Are not enclosed by membranes
– Example- sulfur granules of photosynthetic bacteria &
Polyphosphate granules of Corynebacterium
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 Metachromatic granules

• Polyphosphate granules of Corynebacterium and Mycobacterium are called

metachromatic granules because they stain a contrasting color in methylene
blue
• Green coloured, rod shaped bacteria that are arranged at angles to each other
resembling English letter 'L', 'V' or Chinese letter pattern along with bluish
black metachromatic granules at the poles seen
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 Magnetosomes
• Magnetotactic bacteria contain
granules with iron oxide- give
magnetic properties to the cell
• Present in several gram-negative
bacteria (Aquaspirillum
magnetotacticum for example)
• Act like magnents
• Have been demonstrated to
decompose hydrogen peroxide
• In vitro so it has been suggested
that magnetosomes may protect
cells from hydrogen peroxide
accumulation

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 Endospore-Forming Bacteria
• These bacteria have a two-phase life cycle
– Phase One- Vegetative cell
• Metabolically active and growing
• Can be induced by the environment to undergo spore
formation (sporulation)

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 Phase Two: Endospore
• Stimulus for sporulation- the depletion of nutrients
• Vegetative cell undergoes a conversion to a
sporangium
• Sporangium transforms in to an endospore
• Hardiest of all life forms
– Withstand extremes in heat, drying, freezing, radiation,
and chemicals
– Heat resistance- high content of calcium and dipicolinic
acid
– Some viable endospores have been found that were more
than 250 million years old

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 Different Types of spore formation

Bacillus subtilis and Acetonema longum form endospores inside the mother cell,
Myxococcus xanthus differentiates from a rod-shaped vegetative cell to a spherical
spore, and Streptomyces coelicolor aerial hyphae simultaneously form multiple
septa before sporulation. Green, inner membrane; red, peptidoglycan; blue, outer
membrane; gray, spore coat; P, prespore; M, mature spore.

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 Differences between endospores and vegetative cells

Property Vegetative cells Endospores

Typical Gram-positive murein Thick spore coat, cortex, and
Surface coats cell wall polymer; crystalline S- unique peptidoglycan core wall;
layer no S-layer
Microscopic appearance Nonrefractile Refractile

Calcium dipicolinic acid Absent Present in core

Cytoplasmic water activity High Very low

Enzymatic activity Present Absent

Macromolecular synthesis Present Absent

Heat resistance Low High

Resistance to chemicals and
Low High
acids
Radiation resistance Low High

Sensitivity to lysozyme Some sensitive; some resistant Resistant

Sensitivity to dyes and staining Sensitive Resistant

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• Germination
– Breaking of dormancy
– In the presence of water and a specific germination
agent
– Quite rapid (1 ½ hours)
– The agent stimulates the formation of hydrolytic
enzymes, digest the cortex and expose the core to
water
• Medical Significance
– Several bacterial pathogens
• Bacillus anthracis
• Clostridium tetani
• Clostridium perfingens
• Clostridium botulinum
– Resist ordinary cleaning methods

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 Akinete
• An akinete is a thick-walled dormant cell derived
from the enlargement of a vegetative cell.
• It serves as a survival structure.
• It is a resting cell of cyanobacteria and unicellular
and filamentous green algae.
• They are thick-walled cells, produced by most
strains of Nostocales and Stigonematales.
• They were first described before the discovery
of bacterial endospores (Carter 1856).
• Development of akinetes from a vegetative cell
involves:
– increase in size
– gradual disappearance of gas vacuoles
– increase in cytoplasmic density, number of
ribosomes & cyanophycin granules
• The akinetes are filled with food reserves, and
have a normal cell wall surrounded with 3 layer
coat
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 Capsule

Diagram of bacterial mucoid-like structures:

1. capsule; 2. slime layer; 3. biofilm

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 Capsule / Slime layer
• Capsules are usually polymers of simple sugars (polysaccharides)
• The capsule of Bacillus anthracis is made of polyglutamic acid and
Glycoprotein in Y.pestis
• Most capsules are hydrophilic (“water-loving”)
• Help the bacterium avoid desiccation (dehydration) by preventing water
loss.
• Capsules can protect a bacterial cell from ingestion
• Prevent destruction by white blood cells (phagocytosis).
• A capsular layer of extracellular polysaccharide material can enclose many
bacteria into a biofilm and serves many functions
• A capsule is closely associated with cells , organized layer and does not
wash off easily, while a slime layer is unorganized layer more diffuse and is
easily washed away.
• Capsulated bacteria form mucoid colonies on solid media
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 What Is a Biofilm?
• Glycocalyx are instrumental in the formation of biofilms.
• A biofilm is a living ecosystem made of millions of bacterial cells, their wastes and
other extracellular products.

Oral Biofilm & Plaque

• The slime layer of Streptococcus mutans allows this bacteria and others to
accumulate on tooth enamel (causes of cavities).
• Other bacteria in the mouth become trapped in the slime and form a biofilm,
eventually building up as plaque.

Medical Impact of Biofilms

• Biofilms also have other serious medical implications.

• Persistent biofilms containing pathogenic bacteria can be problematic when they
accumulate on damaged tissues and internal medical devices, such as catheters and
pacemakers.

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• Biofilm formation involves attachment, colonization, and growth of
microorganisms (Forsythe, 2000).
• Different nutrients in aquatic environments absorbed on surfaces to form
conditioning film with different physicochemical properties.
• Physicochemical properties of a surface determine how bacteria attach.
• Biofilm forms when bacteria adhere to conditioned surfaces in aquatic
environments and excrete an exo-polysaccharide (EPS), glue-like substance
that can anchor them to all kinds of material - such as metals, plastics, soil
particles , medical implant materials, and tissue.
• A biofilm can be formed by a single bacterial species. In nature, many species of
bacteria, fungi, algae, protozoa, and debris form biofilms

Advantages of biofilm growth mode

1) Protection from antimicrobial agents;
2) Increased availability of nutrients for growth;
3) Increased binding of water molecules, reducing the possibility of
dehydration;
4) Proximity to progeny and other bacteria, facilitating plasmid transfer

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Biofilm in Dental Plaque

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Microbial
Biofilm
Formation:

Biofilm growth cycle. Initially, free-swimming cells settle on a surface via reversible attachment
(a), which triggers the production of extracellular polymeric substances (EPS) for surface adhesion
(b). Biofilm proliferates within the EPS matrix (c) and exhibits typical biofilm morphology on
maturation (d) in a microorganism such as Pseudomonas. During biofilm dispersal (e), individual
free-swimming cells are released from mature biofilms to colonize new surfaces, which completes
the biofilm growth cycle.
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 • Diagrammatic representation of cyclic steps
involved in the formation of an active
biofilm.
• Cells initially attach by physico–chemical
interactions or extracellular matrix protein
secretion to form a cell monolayer, in which
cells express pili and have twitching motility
and/or the ability to undergo chemotaxis.
• Cells proliferate in the monolayer and other
microbes attach to form an active biofilm,
the development and distortion of which is
influenced by environmental factors such as
hydrodynamic and mechanical stress.
• Cells in the mature biofilm are motile and
undergo chemotaxis, which leads to
spreading of biomass and an increased rate
of horizontal gene transfer.
• As cells die, active bioconversion and/or
biodegradation leads to solute transfer to or
from the bulk liquid, which results in
eventual biofilm detachment.
• The processes of formation and detachment
of cells are repeated in a cycle, thereby
enabling further development of similar
biofilms, which can subsequently attain new
dimensions as a result of environmental
influences.
• The approximate time period for which each
B.Reena Rajkumari of the phases persist is shown on the left.
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 What is necessary for biofilm analysis?
A universal toolbox for biofilm investigation focusing on process relevant
parameters are required. These include
(i) Spatial distribution of cells on the substratum,
(ii) Distribution of metabolically-active cells throughout the biofilm,
(iii) Analysis of the EPS matrix,
(iv) Biofilm thick-ness,
(v) Requirement of oxygen, growth substrates,
(vi) Biotransformation substrates and their respective con-centration profiles in
the biofilm, and
(vii) Response to process relevant stress (e.g., solvents, shear forces).

A wide array of methods for micro scale investigations has been developed
ranging from traditional colony forming unit (CFU) counting to intrinsically-
tagged fluorescent proteins or reporter gene-coupled epifluorescence.

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Scanning electron microscopy (SEM) ,environmental scanning electron microscopy (ESEM), Confocal laser scanning microscopy (CLSM) , Atomic force microscopy
(AFM) , Optical coherence tomography (OCT B.Reena Rajkumari 128
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 Colony Morphology
• Colony morphology is based on the
• size,
• colour,
• shape, and
• texture of colonies
that are grown in pure culture on an agar plate.
• Each colony originates from a colony-forming unit (CFU),
consisting of a single cell or group of adherent cells

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Various colonies of bacteria and pigment production on solid cultivation
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Dry, Chalky Nocardia Colonies On NYC Agar
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 Pigment Production in Microbes
• Bacteria are pigmented or coloured.
• Pigmented bacteria are also known as chromo bacteria.
• Bacterial pigments are water soluble or insoluble;
• water soluble pigments are diffused in the growth medium.
• Chemically, bacterial pigments are pyrrole, phenazine, carotenoid,
xanthophyll and quinine or Quinone derivatives.
• The pigment molecules are synthesized in cell wall or periplasmic space.
• We can visualize pigmentation in bacteria in specific growth medium or by
staining bacterial cells with a dye to observe under microscope.
• It has been proved that only aerobic and facultative aerobic bacteria are
pigmented because, molecular oxygen is essential for pigmentation.
Therefore, anaerobic bacteria are non-pigmented.
• Pigment synthesis is also dependent on light, pH, temperature and media
constituents like indicator dyes.
• They display all the colours from rainbow including light or dark tinges and
unusual colours like black, white, brown, golden, silver and fluorescent
green, yellow or blue.
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 Pigments
Purple: Spirillum rubrum
Violet: Chromobacterium violacein
Indigo: Janthinobacterium lividum
Blue: Streptomyces coelicolor (actinorhodin edible)
Green: Chlorobium tepidum
Yellow: Xanthomonas campestris (xanthomonadins)
Orange: Sarcina aurentiaca
Red: Serratia marcescens (prodigiosin)
Brown: Rhizobium etli
Black: Prevotela melaninogenica
Golden: Staphylococcus aureus
Silver: Actinomyces sp.
White: Staphylococcus epidermidis
Cream: Proteus vulgaris
Pink: Micrococcus roseus
Maroon: Rugamonas rubra
Fluorescent blue/green: Pseudomonas aeruginosa (Pyocyanin)
Fluorescent yellow: Pseudomonas fluorescens (Pyoverdin/fluorescein)
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 Pigments merit and demerits
In pathogenesis:
• Resistance to phagocytosis
• Heat resistance and acid stability
• In vitro antibody formation enhancers
• Antitumor properties
Industrial applications:
• In paint formulations
• Alternatives to colour additives of plant origin
• In textile dyeing
• Food colorants
• Source of vitamin A
• In therapeutics
• Indicators of oil spill
• Biosensors and markers of water, soil and air pollution

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Colony counter and Lens

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 Temperature Adaptation Groups
Psychrophiles
• optimum temperature 15oC
• capable of growth at 0 - 20oC
• This group includes those responsible for the spoilage of food in
refrigerators.
Mesophiles
• optimum temperature 37oC
• Range 10o - 40oC (45)
• All major human and animal pathogens are mesophiles.
• Some are thermoduric, meaning that they can withstand slightly
higher temperature ranges
Thermophiles
• optimum temperature 60oC
• capable of growth at 40 - 70oC
Hyperthermophiles
• Archaea that grow optimally above 80°C
• found in seafloor hot-water vents
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Effects of NaCl concentration on
different bacteria

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 Water
• Water is essential to all living cells as the primary solvent in which chemical
activities take place and as a reactant in metabolic reactions such as
hydrolysis.

• Some microorganisms can produce resting stages such as endospore and

cysts when in the absence of water, but all must have water in order to be
metabolically active.

• Osmotic pressure is the pressure exterted on a cell by the movement of

water toward the highest concentration of dissolved solutes across a semi-
permiable membrane .

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