Microbiology (BT 13102)

Unit II
Taxonomy:
• The science of classification mainly of living forms is called taxonomy.
• The objective of taxonomy is to classify living organism i.e. to establish the relationships
between one group of organism and another and to differentiate between them.
Classification: Orderly arrangement of units under study into groups of larger units.
Scientific Nomenclature: As common names are rarely specific and are often misleading, a system
of scientific names which is called scientific nomenclature was developed in 8 th century by Carolus
Linnaeus
• This system gives every organism two names usually derived from Latin or Greek.
• These are genus and specific epithet (species) and both names are underlined/italicized.
• Genus name is always capitalized and in always a noun and the species name is written in
lowercase and is adjective.
• As this system gives two names to each organism the system is called Binomial
nomenclature
• Homo sapiens
• Noun – Genus – man Adj – wise
• Rules for assigning names to newly classified bacteria and for assigning bacteria to taxa are
established by International Committee on systematic Bacteriology are published in
bacteriological code.
• According to this Bacterial code scientific names are to be taken from Latin (Genus can be
taken from Greek)

Taxonomic Hierarchy : All organisms can be grouped into a series of subdivision that make up
the taxonomic hierarchy
• Species: Basic unit of classification and Group of closely related organisms that breed
among themselves
• Genus: Consists of species that differ from each other in certain ways but are related by
descent
• Family: Group of similar genera
• Order: Group of similar families
• Class: Group of similar orders
• Division: Group of similar classes
• Kingdom: Group of similar divisions
Phylogenetic Hierarchy
• Grouping organism according to common properties implicit that a group of organism
evolved from common ancestor.
• Each species retains some of characteristics of the ancestor.
• Some of information used to classify and to determines evolutionary relationships in higher
organism comes from fossils but as fossil evidence is not available for majority of
prokaryotes their phylogeny must be based on other kinds of evidences. …….??
• Charles Darwin theory of evolution explains that the many similarities among organism are
a result of their descent from common ancestors. The arrangement of organism into
taxonomic categories called taxa reflects degree of relatedness among organism.
• The most fundamental taxonomic or phylogenetic division is that between prokaryote and
eukaryote.
• One theory is that eukaryote cells evolved from prokaryote cells living inside one
another (as endosymbionts) (oldest living fossil) are remains of organism that lived
more than 3.5 billion years ago and they are prokaryotes.
• Eukaryotic cells evolved recently about 1.4 billion years ago.
Classification Systems:
• Until 18th century the classification of living organism placed all organism into one of two
kingdoms, plants and animal.
• In microbiology we study some organism that are predominantly plants live and others are
animal like and some that share characteristics common to both plants and animals.
• Since there organism that do not fall naturally in either plant or animal kingdom. It was
proposed that new kingdom be established to include those organism which typically neither
are plant or animals.
• Earliest proposal was made by German Zoologist E H Haeckel – 1866.
• He suggested that a third kingdom Protista be formed to include those unicellular micro
organisms that neither plant like/ nor animal like.
• These organism the protists include bacteria, algal, fungi and protozoa.
• Bacteria – Lower Protist
• Fungi, algal and protozoa – Higher Protist
• Kingdom Protista left some questions unanswered ……of what criteria can be used to
distinguish bacteria from yeast. ???
• No satisfactory observations were available till 1940 and after electron microscopy, the
absence of membrane bound internal structure in one group of protest (bacteria) and
presence of membrane found structure in others (fungi, algae and protozoa) it was decided
that these organism can be classified into eukaryote and prokaryote organism.
• Bacteria – Prokaryotic organism
• Fungi, protozoa and algae – (Eukaryotic), Plant and animals are also eukaryotes.
• Viruses are not included in this scheme.
 In 1969 Robert H. Whittaker founded the five kingdom system of biological
classification, which shows prokaryotes to be the ancestors to all eukaryotes.
In this system all prokaryotes are included in kingdom prokaryotes (Monera).
The four eukaryotic kingdoms are distinguished according to nutritional requirements, patterns of
development, tissue differentiation and possession of 9+2 flagella.
• Simple eukaryote mostly unicellular are grouped as Protista.
• They have flagella at sometime during their life cycle. This kingdom includes moulds, slime
moulds, protozoa, protozoa and primitive eukaryote algae.
Fungi include the unicellular yeast, multicellular moulds and macroscopic species as mushrooms.
• Fungus absorbs dissolved organic matter through its plasma membrane to obtain row
materials for vital functions.
• Most fungi lack flagella and develop from spores or from fragment of hyphae.
The kingdom planta (plants) includes some algae and all mosses, ferns, conifers and flowering
plants.
• All members are multi-cellular and to obtain nutrition, plants uses photosynthesis.
Animalia:
• The kingdom of Animalia (animals) includes sponges, worms, insects animals with
backbones vertebrates.
• Animal obtain nutrients and energy by ingesting organic matter.

Three kingdom classification

In 1978, Carl Richard Woese proposed three kingdom system of classification.
• The grouping of all bacteria in to prokaryote in five kingdom system has been based on
microscopic observation.
• Woese observation/proposal was based on modern techniques in molecular biology and
biochemistry that revealed that there are two types of prokaryotic cells.
• This discovery was based on the fact that after comparing sequence of nucleotides in rRNA
of different kinds of cells.
• It is clear that there are three distinctly different cells groups.
(1) Eukaryotes
(2) Eubacteria (True Bacteria)
(3) Archaebacteria
Woese believed that archaebacteria and eubacteria while similar in appearance should form
their own separate branches on evolutionary tree and all eukaryotes (divided into Protista, fungi,
plants and animalia in Whittaker system should be grouped third kingdom.
Archaea bacteria differ from Eubacteria
a) Cell Wall don’t contain peptidoglycan.
b) They live in extreme environment
c) They carry out unusual metabolic process.
(i) Methanogens – Strict anaerobe that produce CH4 from CO2 and H2
(ii) Extreme Halophiles – Required high concentration of salt for survive
(iii) Thermoacidophilic – Grow in hot & acidic environment.

Goals of Classification:
a) Stability: Classification that are subject to frequent radical changes leads to confusion. So every
attempt should be made to device classification that need minor changes as new information
becomes available.
b) Predictability: By knowing characteristic of one member of group it should be possible to assume
that other members of same group have similar properties

Methods of Classification:
1. Intuitive Method:
A scientist / microbiologist who is familiar with properties of organism he is studying for years
decides that organism represent one or more species or generation.
The disadvantage is that characteristic of an organism that seem important for one man may not be
so important for other and different taxonomist may arrive at different classification
2. Numerical Taxonomy:
In this method, a scientist may determine many characteristic (100-200) for each strain giving each
characteristic equal weightage. Then using computer percentage similarity of each strain to every
other strain is determined.

This approach was given by Michael Adanson and is known as Adansonian or Numerical
Taxonomy.
• The basis of the approach is the assumption that either each phenotypic character is given
equal weightage then it should be possible to express numerically the taxonomic distances
between organism in term of number of characters that are shared relative to the total
number of character examined.
• The significance of this method is greatly influenced by number of character studied.
• To obtain a high degree of significance, a large number of characters have to be examined.
3. Genetic Relatedness:
a) G+C
• Most reliable method based on degree of genetic relatedness bacteria organism (based on
DNA – the genetic material).
%G + C – Similar – same / similar species
%G + C – different – not similar species.

But organism that are completely different / unrelated may have similar %G+C value……????
b) DNA Homology:
 • Double stranded DNA molecule of two organism are heated to convert them to single
strands, single strand of one organism are then mixed with those from other organism and
allowed to cool.
• If organism are related heteroduplexes will form.
c) ) Ribosomal RNA Homology and Ribosomal RNA Oligonucleotide Cataloging
• Ribosome are small granular which manufacture proteins are composed of protein and
RNA. Ribosome RNA (rRNA) is coded by only small fraction of DNA molecule – rRNA
cistron.
• In all bacteria the nucleotide sequence of these rRNA genes has been found to be highly
conserved i.e. during evolution the nucleotide sequence has changed more slowly than that
of building of DNA molecule.

Classification Characteristics:
a) Morphological
• Cell shape, size, structure
• Cell array
• Occurrence of special structure
Staining reaction, motility
b) Chemical Characteristics
Compound: LPS in cell wall G –ve
Teichoic acid – G +V
DNA/RNA – virus
c) Cultural Characteristics:
 Only inorganic:
• Organic compound
• Complex natural substance (Peptone, yeast, blood)
• Living host (rickettesia)
 Condition for growth- Temperature, Oxygen and light
 Grows in specific manner- sediment, pellicle...., colonies
d) Metabolic Characteristics
• Obtain energy:
• Utilize various organic/inorganic compound
• Processes/Enzymes
e) Genetic Characteristics:
• DNA – feature that are constant and characteristic for each organism.
• DNA base composition – G+C 23 - 71%
• Plasmid DNA – Circular DNA molecule that are capable of autonomous replication with in
bacteria cell and this presence can confer special characteristic on the cells that posses
them.
f) Pathogenecity Characteristics:
• Ability to cause disease.
• Various organisms are responsible for disease .
g) Ecological Characteristics:
 Habitat and distribution and interaction
• Marine – fresh
• Soil
• Air
• Widely distributed – Not limited to particular environment.

Differences between Prokaryotes and Eukaryotes

Group Prokaryote Eukaryote

Group (e.g.,) Bacteria Alga, fungi, protozoa, plant and

animals

Size 1-2 by 1-4μm or less Greater than 5μm in width or

diameter

Genetic System Nucleoid, chromatin body/Nuclear material Nucleus, mitochondria,

chloroplast

Structure Not bounded by nuclear membrane Bounded

One circular chromosome More than 1 chromosome
Chromosome don’t contain histones Histone-Pr
Nucleolus absent Present nucleolus

Sexuality Zygote nature is merozygotic (partial Zygote is diploi

diploid)
Group Prokaryote Eukaryote

Cytoplasmic nature and Structure

Stream Ab Pr

G. Vacuole Pr Ab

Mesosome Pr Ab

Ribosome 70S 80S

Mitochondria, chloroplast Golgi Ab Pr

Cytoplasmic Membrane Sterols Ab Pr

Cell wall (Murein/peptidoglycan) Pr Ab

Pseudopodia Ab Pr

G+C 28-73% About 40%

 Staining is a technique used to enhance contrast in samples, generally at
the microscopic level. Stains and dyes are frequently used .

Microorganisms cannot be studied properly because they

are transparent, Colourless and therefore difficult to see,
when suspended in an aqueous medium.

Several stains and staining procedures are there to study the

properties of various microorganisms and their differentiation
into specific groups /genera/ species.

Chemical substances commonly used to stain bacteria are known as Dyes

Chemically a dye (stain) is as an organic compound containing a benzene ring plus a chromophore
and auxochrome group

 Fixed, stained preparations are most frequently used for the observations for the
morphological characteristics of bacteria
 Cells are made more clearly visible after they coloured
 Differences between cells of different species and within the same species can be
demonstrated by use of appropriate staining solutions (Differential or selective staining)..

Types of staining

Differential staining
Simple staining

Requires more than one dye separated

Use of a single stain
by a decolorizing agents
Visualisation of morphological
shape and arrangement
Identification Visualisation of structures
e.g., methylene blue, crystal
violet, carbol fuchsin

Gram Capsule Spore

Acid fast
stain stain stain
stain
Simple Staining:
Elucidate the morphology and arrangement of bacterial cells
Positively charged stain colors the negatively charged cells, making them stand out against the light
background.
Negative Staining:
• Requires an acidic dye such as India Ink or Nigrosin.
• Since the surface of most bacterial cells is negatively charged, the cell surface repels
the stain.
• The background is coloured, but the bacterial cells are not.
• Bacteria will show up as clear spots against a dark background
Advantage:
• Bacteria are not heat fixed so they don't shrink.
• Capsulated bacteria difficult to stain can be observed by this technique.
Grams Staining:
Gram staining method, named after the Danish bacteriologist who originally devised it in 1882, Hans
Christian Gram, is one of the most important staining techniques in microbiology
Used to distinguish and classify bacterial species into two large groups: Gram-positive
bacteria and Gram-negative bacteria
• It differentiates bacteria by the chemical properties of their cell walls. Gram-positive cells
have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal
violet.
• Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash
out on addition of ethanol. They are stained pink or red by
the counterstain, commonly safranin or fuchsine.
Gram Positive:
• Gram –ve bacterial cell wall is thin, complex, multi-layered structure and contains high lipid
content, in addition to protein and mucopeptides.
• Higher amount of lipid is readily dissolved by alcohol , resulting in the formation of large
pores in the cell wall which do not close appreciably on dehydration of cell wall proteins thus
facilitating the leakage of Crystal violet iodine (CV-I) Complex results in decolraization of
bacteria which later takes the counter stain safranin appears red.
Gram Positive:
• Gram +ve bacterial cell walls are thick because of their different composition (lower lipid
content), become dehydrated during treatment with alcohol.
• Pore size decreases, permeability is reduced and the CV-I complex cannot be extracted.
Therefore these cells remain purple violet.
• Endospore Staining:
• Demonstrates spore structure in bacteria as well as free spores
• Some bacteria are capable of changing into dormant structures that are metabolically
inactive and do not grow or reproduce.
• Since these structures are formed inside the cells, hence called endospore. Ferdinand Cohn
discovered existence of endospores in bacteria.
• These are remarkably resistant to heat, radiation, chemicals.
• Heat resistance of spores has been linked to their high content of calcium and dipicolinic
acid.
• Spores are differentially stained by using dyes that penetrate the spore wall
• An aqueous primary stain (Malachite green) is applied and steamed to enhance penetration
of the impermeable spore coats.
• Once stained the endospore do not readily decolorize and appear green within red cells
Acid Fast Staining:
Differential stain
• Bacteria are classified as acid fast if they retain the primary stain (carbol fuchsin) after
washing with strong acid and appear red, as non acid fast if they loose their color after
washing with acid and counter stain by methylene blue.
• Property of acid fastness appears due to presence of high content of lipid called mycolic
acid in cell wall.
Capsule Staining:
• Some bacterial cells surrounded by a mucilaginous substances forming a viscous coat
around the cell known as capsule.
• Composed of polysaccharide, may composed of other materials e.g., B. anthracis has a
capsule of poly D- glutamic acid.
• Done by Negative attaining.