BSCBO-

101

B. Sc. I YEAR Microbiology, Mycology

and Plant Pathology

DEPARTMENT OF BOTANY SCHOOL

OF SCIENCES UTTARAKHAND OPEN
UNIVERSITY
MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101
 BSCBO-
101

MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY

SCHOOL OF SCIENCES
DEPARTMENT OF BOTANY
UTTARAKHAND OPEN UNIVERSITY

Phone No. 05946-261122, 261123 Toll

free No. 18001804025 Fax No. 05946-
264232, E. mail [email protected]
htpp://uou.ac.in

UTTARAKHAND OPEN UNIVERSITY Page 1

MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101

Expert Committee Prof. J. C.

Ghildiyal Prof. G.S. Rajwar Retired Principal Principal Government PG
College Government PG College Karnprayag Augustmuni

Prof. Lalit Tewari Dr. Hemant Kandpal Department of Botany School of

Health Science DSB Campus, Uttarakhand Open University Kumaun
University, Nainital Haldwani

Dr. Pooja Juyal Department of

Botany School of Sciences
Uttarakhand Open University,
Haldwani

Board of Studies Late Prof. S. C.

Tewari Prof. Uma Palni Department of Botany Department of Botany HNB
Garhwal University, Retired, DSB Campus, Srinagar Kumoun University,
Nainital

Dr. R.S. Rawal Dr. H.C. Joshi Scientist, GB Pant National Institute of Department of
Environmental Science Himalayan Environment & Sustainable School of Sciences
Development, Almora Uttarakhand Open University,
Haldwa
ni

Dr. Pooja Juyal Department of

Botany School of Sciences
Uttarakhand Open University,
Haldwani

Programme
Coordinator

Dr. Pooja Juyal

Department of Botany
School of Sciences
Uttarakhand Open
University Haldwani,
Nainital

UTTARAKHAND OPEN UNIVERSITY Page 2

MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101
Unit Written By: Unit No.

1. Dr. Renu Negi 1 & 2 Associate Professor Department of Botany PDBH

Govt. P.G College, Kotdwar

2. Dr. Rajan Kumar Gupta 3 & 4 Associate Professor Department of Botany

PDBH Govt. P.G College, Kotdwar

3. Dr. Kapil Khulbe 5 Asst Professor, Department of Botany, DSB

Campus, Kumaun University, Nainital

4. Dr. Nisha Mishra 6 & 7 Retired Professor, Gorakhpur University

Gorakhpur-273009

5. Dr. Pooja Juyal 8 Department of Botany School of Sciences Uttarakhand

Open University Haldwani, Nainital

6. Dr. Reeta Sachan 9 Asst. Professor Department of Botany, Radhe Hari

Government PG College, Kashipur

7. Dr. Saurabh Guleri 10 & 11 Asst. Professor Department of Botany SGRR P.G
College, Pathri Bagh, Dehradun

8. Dr. Snehalata Bhandari 12 Asst. Professor BFIT, Technical Campus

Sudhowala, Dehradun

Course
Editor

UTTARAKHAND OPEN UNIVERSITY Page 3

MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101

Prof. Uma Palni Retired Professor,

Department of Botany DSB Campus,
Kumaun University Nainital

Title : Microbiology, Mycology and Plant Pathology ISBN No. : 978-

93-85740-57-2 Copyright : Uttarakhand Open University Edition :
2019

Published By: Uttarakhand Open University, Haldwani, Nainital-

263139
UTTARAKHAND OPEN UNIVERSITY Page 4
MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101

CONTENTS

BLOCK-1-GENERAL MICROBIOLOGY PAGE NO.

Unit-1-General account, distribution and classification of microorganisms, 7-40

Major microbes of food, water and soil

Unit-2-Isolation and Cultivation of Microorganisms, Instruments used in

Microbiological studies 41-68

Unit-3-Structure, Classification, Nutrition, Reproduction and Economic importance

of Bacteria 69-91

Unit-4-General account, Classification, Structure, Reproduction and Economic

importance of Viruses 92-106

BLOCK-2- FUNGI AND LICHENS PAGE NO.

Unit-5- Characters, Economic importance, Classification and General account of

major classes of Fungi 108-140

Unit-6- General account, Habit, Structure and Methods of reproduction in

Mastigomycotina, Zygomycotina, Ascomycotina 141-166

Unit-7-General account, Habit, Structure and Methods of Reproduction in

Basiodiomycotina, Deuteromycotina and Mycoplasma 167-194

Unit-8- Occurrence, General structure, Nutrition, Reproduction, Economic and

Ecological importance of Lichens 195-207

BLOCK-3- PLANT PATHOLOGY PAGE NO.

Unit-9-Infection, Disease resistance and General Symptoms 209-230

Unit-10-Symptoms, Morphology of the causal organism, Diseases cycle and

Control measures-I 231-246

Unit-11-Symptoms, Morphology of the causal organism, Diseases cycle and

Control measures-II 247-260

Unit-12-Plant Protection and Control measures of Plant Diseases 261-286

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MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101

BLOCK-1–GENERAL MICROBIOLOGY
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MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101

UNIT-1-GENERAL ACCOUNT, DISTRIBUTION AND

CLASSIFICATION OF MICROORGANISMS AND
MAJOR MICROBES OF FOOD, WATER AND SOIL
1.1- Objectives 1.2- Introduction
1.3-General account of
microorganism 1.4- Distribution
1.5- Classification 1.6-Major
microbes of:
1.6.1-Food 1.6.2-Water
1.6.3-Soil 1.7- Summary
1.8- Glossary 1.9- Self
assessment question
1.10- References 1.11-
Suggested Readings
1.12-Terminal Questions

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MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101
1.1
OBJECTIVES
Microbiology is the study of organisms invisible to our naked eye. This branch of
science explains the structure, nature, distribution, classification, occurrence,
physiology pathogenicity and application of microbes. This unit deals with the
introduction, general accounts, distribution, and classification of microbes and also
about the soil, water and food microbiology.
After reading this unit one is able
to:
• Know about micro-
organisms.
• Learn the variety of microorganisms which occur in the environment
surrounding us.
• Understand the existence of minute organisms and realize that these microscopic
organisms are living and perpetuate themselves by reproduction.
• Discuss the distinct group of microbes which differ in form and other characters but
resemble with each other in their small size and simple structure.
• Study the systematic position, and distribution of micro
organisms.

1.2
INTRODUCTION

Microbiology is the study of organisms too small to be clearly seen by the unaided
eye. Since objects less than about one millimeter in diameter cannot be clearly seen
and must be examined with a microscope, such living objects are collectively
referred as microorganisms or microbes. Therefore microbiology is defined as the
study of microorganisms. A variety of organisms like bacteria, protozoa, viruses,
fungi and algae are included in this category.

Regarding the place of microorganisms in the living organisms, satisfactory criteria

were unavailable until late 1940, when more definite observation of internal cell
structure was made possible with the aid of the powerful magnification provided by
electron microscope. Two cell types were discovered among these microorganisms.
In some organisms the cells contained nuclear substance which was not enclosed by
a nuclear membrane, while in others, a well-defined nucleus with a nuclear
membrane was present. These two patterns were called prokaryotic and eukaryotic
respectively. According to these special features of microorganisms, bacteria are
prokaryotic and fungi, algae and protozoa are eukaryotic. Viruses are left out of this
criteria as they are acellular organisms. Thus microbiology includes five major
branches namely, virology, bacteriology, phycology, mycology, and protozoology.
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MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101

1.3 GENERAL ACCOUNT OF

MICROORGANISMS

Antony–van Leeuwenhoek (1632-1723) was the first who studied in detail the
microbial content of a variety of natural substances under the microscope. The
various natural substances studied by Leeuwenhoek were water from rain barrels,
rivers, wells, sea, teeth scrapings and naturally fermented material like vinegar. His
observations were confirmed by others, but only in nineteenth century, the extent
and nature of microbial forms becames more apparent.
• Microbes are either unicellular or multicellular or non- cellular forms. Protozoa,
bacteria and some algae and fungi are unicellular forms and are made up of single
cells. While most of the algae and fungi are multicellular forms. Viruses lack a
cellular structure and hence they are non-cellular particles and occupy a border line
between living and non- living things.
• Based upon the presence or absence of nuclear membrane, microbes are of two
types namely Prokaryotes and Eukaryotes. Prokaryotes have incipient nucleus which
is suspended in the cytoplasm. This includes bacteria.
• The microbes such as protozoa, algae and fungi are eukaryote which contain a
nucleus, with a nuclear membrane and is well separated from the cytoplasm.

General characteristics of
Bacteria
1) Bacteria are small microscopic and least differentiated microorganisms. These are
believed to be amongst the first primitive organisms on the earth possessing typical
prokaryotic cell organization. 2) They are omnipresent, i.e. found in all possible
habitats. 3) They are unicellular and may live in association with others in colonies.
4) The size, shape and arrangement of bacterial cells vary and their size is about .5
micron to
3 micron. 5) They exhibit a variety of shapes e.g., spheres (coccus), rods
(bacillus), spirals (spirillum),
curved (vibrio)
etc.

Fig.1.1. Different shapes of

bacteria
6) They possess very rigid cell wall, which is not made up of cellulose, characteristic
of plant cell walls. It generally contains a peptidoglycan (murein), lipid and
lipopolysaccharides. The rigid cell wall determines the shape of bacterial cells. 7)
Nuclear material is not enclosed in a nuclear membrane. Nucleolus is absent. 8) An
extra chromosomal DNA called plasmid is usually present in the cytoplasm.

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MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101

Fig.1.2. A bacterium showing

appendages

9) Cell organelles includes 70s type ribosome and mesosome formed by

invagination of plasma membrane.Otherorganelles such as mitochondria,
lysosomes, Golgi body, endoplasmic reticulum,centriole etc. are absent.

Fig.1.3.Structure of a typical bacterium (E.

Coli)

10) Appendages like flagella and pili are present. Some pili are longer in some
bacteria and
are called as sex pili (Fig.1.2, 1.3). Motility is brought about by flagella. The Bacilli
and spirilla are motile and the cocci are non-motile. Thus the bacteria may motile or
non-motile. 11) When flagella are absent, the bacterium is called atrichous. In motile
bacteria, the number and position of flagella vary. The arrangement may be
monotrichous (a single polar flagellum), lophotrichous (a cluster of polar flagella),
amphitrichous (Flagella at both the ends either singly or in cluster), cephalotrichous
(two or more flagella at one end of the cell), peritrichous (cell surface evenly
surrounded by several flagella) (Fig.1.4). 12) The flagella are hair like or helical,
consists of a single minute filament which is made up of fibrils of flagellin protein.
Unlike hair a flagellum grows at its tip rather than at base. 13) Bacteria are either
Gram positive or Gram Negative. Gram positive bacteria retain violet colour on Gram
staining while Gram negative bacteria appear in red colour. This is because of the
difference in their cell walls. The cell wall of gram positive bacteria contains several
layers of peptidoglycan in addition to techoic acid and low quantity of lipoprotein and
lipid. In gram negative bacteria, cell wall has thin layer of peptidoglycan and high
amount of lipo protein and lipid. Techoic acid is absent in these bacteria.

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14) In some bacteria, shorter and thinner hair like appendages are present on the
surface of the cell wall. These structures are called pili or fimbrae. There function is
to adhere the cell to surfaces and sometimes help in transfer of genome to other
bacterial cells. These are called sex-pili.

Fig.1.4. Showing flagellation in

bacteria

15) A bacterial cell is protected by a cell envelope made up of a capsule, a cell wall
and a plasma membrane. The bacteria covered by a capsule are called capsulated
bacteria. While the bacteria which do not contain a capsule are called non-
capsulated bacteria. 16) Bacteria may be heterotrophic or autotrophic, Heterotrophic
may be parasitic, saprophytic or symbiotic. For nutrition, autotrophs use CO as the source
2

of carbon while heterotrophs use organic substances as the source of carbon. 17) Based
on temperature tolerance of bacteria they are of three types:-
• Mesophilic bacteria grow well in temperature between 25 C - 0

0
40 C.
• Thermophilic bacteria grow well above
0
40 C.
• Psychrophilic bacteria grow well in temperature less than 25 C. 18) On the basis
0

of availability of O bacteria may be aerobic or anaerobic or facultative

2

anaerobi
c.
 • Aerobic bacteria use oxygen for
respiration.
• Anaerobic bacteria use
CO .
2

• Facultative anaerobes use oxygen when it is available and use CO when oxygen is
2

not available. 19) Bacteria reproduce by binary fission, budding, fragmentation,

endospores, exospores and
conidiospores. 20) True sexual reproduction is lacking. However, genetic
recombination occurs by
conjugation, transformation and
transduction.

General Account of Algae 1) Algae are simple, chlorophyll bearing, and

unicellular or multicellular microorganisms.
Being chlorophyllous these are autotrophs. 2) Algae are heterogeneous groups.
They vary in size, habitat and reproductive processes. 3) Algae are ubiquitous and
abundantly present in sea water, fresh water, in damp soil, on
rocks, stones, barks of trees, on plants and
animals.

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4) Plant body of algae is called thallus which does not show differentiation into root,
stem,
leaf and true tissues. 5) Algae are aquatic or terrestrial. But most of them are
aquatic. They are either free living
or attached forms. 6) A few algae are parasites. Some algae are of specialized
habitats, e.g. parasites,
symbiotic cryophytes and thermophytes etc. 7) Algae are unicellular like
Chlamydomonasor multicellular like Spirogyra. Multicelluar algae may be in the form
of colonies like Volvox or in the form of filaments as Spirogyra. 8) The algae may be
prokaryotes or eukaryotes. All blue green algae are prokaryotes. 9) The cell consists
of a cell wall, a plasma membrane, cytoplasm and nucleus. The cytoplasm contains
mitochondria, plastids, ribosomes, Golgi complex, endoplasmic reticulum. 10) The
plastids in algae, contain pigments which are of three types –
(a) Chlorophylls: Five types chlorophyll (a, b, c, d and e) are found in different
algae.
Chlorophyll a is present in all the algae. (b) Carotenoids: These are the yellow
and orange pigments (namely carotenes and
xanthophylls) and are found in varied quantities in different algae. (c)
Biliproteins or phycobilins: These pigments include phycocyanin (blue in colour)
and phycoerythrin (red in colour) and presence of these pigments is the
characteristic feature of certain types of algae. 11) The pigments are present in
chloroplasts, which are of different shapes in different genera. The chloroplast
contains one or more spherical bodies called pyrenoids which are the centres of
starch formation. 12) Some algae are motile and possess flagella. 13) Reproduction
 in algae is of three types, namely, vegetative, asexual and sexual. Vegetative
reproduction takes place by fragmentation, fission, budding etc, asexual reproduction
by production of asexual spores (motile or non-motile). Asexual reproduction is most
common method of reproduction during favorable conditions. Algae reproduce
sexually during unfavorable conditions by producing gametes.

General Account of Fungi 1) Fungi are achlorophyllous, non-vascular

eukaryotic thallophytes. 2) They are non-green so heterotrophic microbes obtaining
their food in a soluble form by
uptake through plasma membrane. 3) Being heterotrophic, they live as
parasites, saprophytes or symbionts. 4) They are ubiquitous in distribution and occur
in any habitat where life is possible. 5) There are about 100,000 species of fungi. 6)
Plant body of fungi typically consists of branched filamentous hyphae which form a
network called mycelium. The hyphal structure is variously modified. 7) The
hyphae are aseptate, multinucleate in lower forms while septate and uni, bi or
multinucleate in higher forms. 8) Protoplasm remains surrounded by a distinct
cell wall made up of fungal cellulose
known as chitin. But in primitive slime moulds cell wall is
absent.

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9) Fungi are entirely devoid of chlorophyll but carotenoids are normally present.
Cytoplasm contains endoplasmic reticulum, mitochondria, Golgi bodies and many
non-living substances like reserve food. 10) In lower fungi the reproductive cells
(Asexual spores and gametes) are motile (uni or biflagellate). But the higher fungi
lack motile cells and show gradual reduction of sexuality. 11) Flagella are two types
(i) whiplash (acronematic) flagella are smooth and (2) Tinsel
(pentonematic) flagella with numerous minute hairs like structures on their surface.
12) Fungi are heterotrophic due to absence of chlorophyll. So they have to depend
for their
food on others. Therefore they may beof the following types: (a) Parasites obtain
their nutrition from other living plants or animals. Some of them live only
on living protoplasm and are called obligate parasites. Whereas others can also grow on
dead organic matter in absence of living host and are known as facultative saprophytes.
(b) Saprophytes obtain their nutrition from the dead decaying organic matter.
Among these, some saprophytes such as Mucor can obtain their nutrition only
from dead organic matter and are known as obligate saprophytes. On the other
hand some saprophytic fungi as Fusarium have the capacity to invade living
organisms and are known as facultative parasites. (c) Symbionts grow on other
living organisms and both are mutually benefited. Such association is known as
symbiosis, Lichens and mycorrhiza are common examples of this, in which fungal
partner shows mutualistic relationship with alga and roots of higher plants
respectively.
13) In unicellular fungi whole vegetative cell is transformed into a reproductive unit
such fungi are known as Holocarpic while in most of the fungi only a part of the
vegetative mycelium forms reproductive unit and rest remain vegetative. Such fungi
are known as Eucarpic.
14) Fungi reproduce by vegetative, asexual and sexual means. Vegetative by
fragmentation (e.g., Rhizopus, Alternaria etc.) fission (e.g., yeast) and budding (e.g.
yeast and Ustilago) Asexual reproduction occurs during favourable condition by the
formation of a variety of conidia and spores. Spores may be unicellular (e.g.
Aspergillus) or multicellular (e.g. Alternaria). They may be endogenous (developed
inside in pycnia or sporangia) or exogenous (developed outside on sporophores or
conidiophores). Some common asexual spores in lower fungi are motile known as
zoospores. (e.g., Phytophthora), Non motile known as aplanospores or conidia (e.g.
Mucor, Rhizopus). In Higher fungi these non- motilespores are called conidia, oidia
or chlamydospores. 15) Except for the class Deuteromycetes, sexual reproduction
occurs in all groups of fungi. It is completed in three phases (a) Plasmogamy (fusion
of protoplasm of two compatible gametes of sex cells) (b) karyogamy (fusion of two
nuclei from two gametes to form Dikaryon). (c) Meiosis (after karyogamy reduction
division takes place in diploid nucleus to form haploid stage). The sex organs if
present are called gamentangia which may form gametes. 16) The various methods
of sexual reproduction (by which the compatible nuclei are brought
together for plasmogamy) are as
follows:

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(a) Planogametic copulation (fusion of two naked motile gametes) May be either
Isogamy (fusing gametes morphologically similar) or Anisogamy (fusing gametes
are both morphologically and physiologically dissimilar) or Oogamy (fusion
between female gamete (egg) and male gamete (antherozoid). (b) Gametangial
contact (Male and female gametangia come in close contact with the
help of a fertilization tube). (c) Gametangial coupulation (Fusion of entire contents
of two compatible gametangia) and formation of zygote which develops into a resting
spore e.g., Mucor, Rhizopus. (d) Spermatization (sex organs are completely absent
and the sexual process is accomplished by minute spore like spermatia
(malegamete) and specialized receptive hyphae (female gamete) e.g., Puccinia. (e)
Somatogamy (sex organs are not at all formed but two vegetative cells or two
vegetative hyphae take over the sexual function and fuse together). e.g., Morchella
and Agaricus. 17) The optimum temperature for the growth of fungi is between 20 C 0

to 30 C. 18) Although light is not essential for growth, but for sporulation in many
0

species, some
light is necessary. 19) There are five basic types of life cycles in fungi as
asexual, haploid, haploid-dikaryotic,
haploid-diploid and
diploid.

General Account of Viruses 1) Viruses are exceptionally simple, filterable,

obligate, intracellular particles capable of
reproducing inside a living host. 2) These are extremely smaller in size (smaller
than bacteria) and it ranges from 20 nm to
 300 nm in diameter. 3) Inside a living, the viruses are active and they feed,
reproduce, grow and move. But when they live outside, they remain inactive and
behave as non- living things. They are also called living chemicals as they behave
like chemicals and can be crystallized. 4) Viruses differ fundamentally from cellular
organisms in that they contain only one type of nucleic acid either DNA or RNA. The
nucleic acid may be single or double stranded DNA or RNA and occur in either linear
or circular form. 5) Viruses do not contain cellular structures such as plasma
membrane, mitochondria, Golgi
complex, lysosomes, ribosomes etc. 6) Their basic structure consists of a protein
coat, (capsid) and nucleic acid. The smallest viruses known as virioids, consists of a
single strand of naked nucleic acid without protein coat. Capsid is made up of
several identical protein subunits known as capsomeres. These subunits are usually
arranged in the helical or polyhedral geometric forms which are specific for a
particular virus. 7) The capsomeres, forming the capsid (protein coat) of a virus, are
of two types: -Pentamer (made up of five identical monomers) and Hexamer(having
six monomers). Each monomer is connected with the neighbouring monomers on
either side with the help of bonds. Similarly capsomeres are also connected with
each other but the bonds between capsomeres are weaker.

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8) In complex forms (e.g., influenza and herpes virus and many plant viruses) the
virus particles are surrounded by on outer envelope. The envelope is membranous
and made up of protein, lipids and carbohydrates. Viruses with envelope are called
enveloped and those without envelope are said to be naked (e.g., TMV) (Fig.1.5).

Fig.1.5. Different structures in

Viruses
9) Viruses multiply by assembly line method. They do not divide. The cycle of
multiplication include
:
• attachment of virus to host
cell.
• penetration by genetic
material.
• production of virus components by the
cell.
• assembly of new virus components by the
cell.
• release from the host cell. 10) They lack enzymes for most metabolic
processes. 11) Viruses are also unique microorganisms as they lack machinery for
the synthesis of
proteins 12) They are obligate intracellular parasites of animals,
(protozoainsects,fish, birds, amphibians, mammals and humans) or plants
(angiosperms, gymnosperms, ferns, and fungi). 13) Many of the viruses have a close
biological relationship with an arthropod or other type
of vector on which they are dependent for their transmission from one host to the
other. 14) The viruses cause very serious diseases in crop plants, ornamental plants
and forests trees and many serious diseases in animals caused by viruses are well
known since the time immemorial.

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General Account of Protozoa 1) Protozoa are animal like organisms which

are motile unicellular, non-photosynthetic
eukaryotic microorganisms. 2) They normally obtain their food by ingesting other
organisms by a process called
phagocytosis. 3) These microorganisms are aquatic (fresh water or marine) or
terrestrial (in soil) but
majority of them are parasites in other animals including humans. 4) On the
basis of the type of movement, the microorganisms are divided into there types:
(a) Amoeboid protozoa: flagella are absent but a temporary projection of part of
cytoplasm called pseudopodium is present. (b) Flagellate protozoa: They have
either simple flagellum or a very complex flagellar
arrangement. (c) Ciliaryprotozoa: In some protozoa e.g. Paramecium, the surface
is covered with these
structures and cilia are shorter than flagella and have a co-ordinate motion. 5)
The cell is made up of a plasma membrane, cytoplasm and nucleus. The plasma
membrane may have outer protective coverings such as pellicles, shell, test or
torica.(Fig 1.6). 6) The cytoplasm is more or less homogenous substance consisting
of protein molecules. It is made up of an outer ectoplasm and an inner endoplasm.
Ectoplasm is gel like and endoplasm is more voluminous and fluid. 7) Some
protozoa secrete a resistant covering and from a cyst at certain times of their life
cycle. This protects organisms against adverse environments. It also functions as a
site for nuclear organization and serves as a means of transmission in parasitic
species. 8) The nucleus is typical eukaryotic. It has nuclear membrane, nucleoplasm
and chromosomes. Normally only one nucleus is present (e.g.,Amoeba). But in some
protozoa two similar nuclei are found while in others e.g. ciliate species to dissimilar
nuclei (one micro and another macro nucleus) are found. The macro nucleus is large
and controls the metabolic activities and regeneration process and smaller or micro
nucleus is responsible for reproductive activity. 9) On the basis of Nutrition the
protozoa are autotrophic, holozoic or parasitic. Amoeba uses pseudopodium for
gathering food, Paramecium uses cilia, and Suctorianus estentacles. The autotrophic
forms have chlorophyll for photosynthesis. e.g., Euglena. Some protozoa show
symbiotic relationship with other organisms.

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MICROBIOLOGY, MYCOLOGY AND PLANT PATHOLOGY BSCBO-101
 Fig.1.6.Various forms of
Protozoa

10) Asexual reproduction is some flagellate and ciliate species is associated

with cyst
formation. 11) Reproduction takes place by asexual and sexual
methods. Methods of asexual
reproduction are binary fission (e.g. Amoeba), multiple fission (e.g. Plasmodium),
and budding. Sexual reproduction takes place by conjugation (e.g. Paramecium).
And isogamy (e.g. Monocystis). 12) Protozoa play an important role in food chain
and food webs and are of particular importance in the ecological balance of many
communities. Some protozoa caused diseases in animals including humans which
are chronic and acute. In addition, these microorganisms have also become
important research tools for biologists and biochemists.

1.4 DISTRIBUTION OF MICROORGANISM

Microorganisms are cosmopolitan in distribution. They occur in every type of
habitat that can support life. This exceptional wide natural distribution is due to
physiological diversity exhibited by them. Following physiological characters
contribute to their survival in varied habitats:
(i) They can grow in inorganic environments without illumination. (Known as
chemolithotrophs). (ii) They have the capacity for rapid growth. (iii) They
have higher metabolic rates. (iv) They do not depend on the availability of
specific micronutrients in the environment.

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(v) Some of them (bacteria and cyanobacteria) can use nitrogen a capability not
known to
occur in any other
group.

Under suitable environmental conditions, the microbes frequently grow multiply and
produce spores, cysts and resting cells. We shall discuss the distribution of
microorganisms under following heads:
1. Microbes in soil 2. Microbes in
aquatic environment 3. Microbes
associated with plants 4.
Microbes in Air 5. Microbes in
Food 6. Microbes in Milk. 7.
Microbes of human body.

1. Microbes in Soil Man depends upon the soil for his food and the soil depends
upon the micro-organisms for its fertility. Agriculture would not be possible without
microorganisms in the soil. There are five major groups of microorganisms in the
soil. They are Bacteria, Fungi, Algae, Protozoa and viruses. One gram of soil has
about 200-500 billions of microorganisms.

Microbial Population in a fertile

soil
Type Number per gram Bacteria:
Direct count, 25 x 10 Dilution plate
8

15 x 10 Actinomycetes 7 x 10 Fungi
6 5

4 x 10 Algae 5 x 10 Protozoa 3 x
5 4

104

A. Bacteria: Bacteria are the larger group of soil microorganisms. The bacteria
which occur in soil are coci, bacilli and spiral forms. Among these, the bacilli are in
highest number and they swim actively in the soil solution. Some common soil
bacteria are the species of Pseudomonas, Arthrobacter, Achromobacter, Bacillus,
Clostridium, Micrococus, Flavobacterium, Chromobacterium and Mycobacterium.
Both autotrophic and heterotrophic bacteria occur in the soil. Different species of
Thiobacillus, Ferrobacillue, Nitrosomonas and Nitrobacterare also found.in the soil
as chemosynthetic autotrophic bacteria.
Environmental factors like temperature, moisture, pH and depth of the soil
affect the distribution of bacteria in the soil. Certain bacteria like Mycobacterium and
Pseudomonas are commonly found in the soil near the petroleum wells. These
bacteria are responsible for oxidizing ethane. Escherichia bacteria seldom occur in
the soil, many cellulolytic bacteria, such as species of Cytophaga and
Sporocytophaga are found in cellulose rich soil.

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B. Actinomycetes: A large number of actinomycetes are present in dry and warm

soil. They are particularly abundant in the soil rich in decomposed organic materials.
Species of Streptomyces, Micromonospora and Nocardia are some common
actinomycetes which occur in soil. They are responsible for the characteristic musty
or earthy smell of a freshly ploughed field and are capable of degrading many
complex chemical substances and thus play an important role, in soil environment.
C. Fungi: Several fungi are present in the soil and play an important role in the
improvement of soil nutrients in neutral and alkaline soils. Majority of soil fungi grow
in acidic soils with aerobic condition. Agricultural practices (crop rotation, use of
fertilizers and insecticide etc.) and the depth of the soil also influence the fungal
composition. Some important soil fungi are the species of Aspergillus, Botrytis,
Cephalosporium, Penicillium, Alternaria, Monilia, Fusarium, Verticillium, Mucor,
Rhizopus, Pythium, Chaetomium, and Rhizoctonia, Yeasts are not very common in
the soil except in Vineyard and orchard soils. Some fungi such as species of
Alternaria, Aspergillus, Cladosporim and Dematium are helpful in the preservation of
organic materials in the soil. Adding of organic matter to the soil stimulates soil
fangal flora. It is to be noted that the mycelium of fungi play an important role in
binding the soil particles because of their interwoven hyphae.
Some phytopathogenic fungi also live in soil, often as saprophytes e.g.
Spongospora of myxomycetes that cause powdery scab of potato tuber;
Phytophthora and Alternaria species cause late blight of potato and early blight of
potato respectively.
D. Algae: The algae are widely distributed in the soil even in deserts. Many algae
occur on the surface of moist soils where sufficient light is available. The growth of
algae is beneficial for soil conservation and in improving soil structure. In paddy
fields, blue green algae play a significant role in nitrogen fixation.
The most commonly occurring algae isolated from the soil are the members of
cyanophyceae and chlorophyceae e.g. Nostoc, Cylindrospermum, Anabaena and
Chlorella, Chlorococcus and Scytonema. In addition to these, certain diatoms are
also frequently occur in the soil.
E. Protozoa: Protozoans are present in great abundance in the upper layer of the
soil and their number has a direct effect on bacterial population, because they ingest
bacteria. Depending upon the condition of the soil, protozoans may exist in
vegetative or cystform. Protozoans present in the soil belong to the class
Mastigophora(species of Bodo, Cercobodo, Cercomonas Monas, Spiromonasetc)
class sarcodina(Amoeba, Biomyxa, Nuclearia, Trinema) and class ciliata are
(Colpoda, Gastrostyla, oxytrichaetc).
F. Viruses: Viruses are present in very small number in the soil. Bacteriophages
ingest bacteria and actinomycetes and some viruses infect the fungi present in the
soil.

2. Microorganisms in aquatic environment Water is unique environment for

micro-organisms. A large number of micro-organisms are carried from air and soil
through various sources before water reaches reservoirs like rivers, lakes, ocean etc.
Aquatic habitats are only marginally aerobic. Microorganisms occurs in all

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depths. Drifting microbial life of aquatic environment is called Plankton. There are
various types of microorganisms in the aquatic environment .Some major examples
are as follows:

I. Bacteria: Bacteria are heterotrophic organisms of water, which may live in close
association with the algal flora of water. They develop well both in fresh and marine
water, near the submerged vegetation and just above the mud layer. Anaerobic
bacteria and few fungi also grow at the bottom sediments where are algae are
absent. Pigmented and non-pigmented bacteria like Pseudomonas,
Chromobacterium, Achromobacter, Flavobacterium and Micrococus occur frequently
in unpolluted water Escherichia coli, Streptococcus faecal is, Proteus vulgaris and
Clostridium perfringens, the characteristic intestinal tract inhabiting bacteria are also
found in polluted water. Species of certain bacteria like Pseudomonas vibrio,
Favobacterium and Achromobacter may be present in surface layers of sea water.
These marine bacteria have important roles in nitrogen, sulphur, Phosphorus and
carbon cycles within the sea. II. Fungi: Certain fungi like Saprolegnia,
Manoblepharis and Chytrids occur in well aerated waters. These are saprophytes
that grow on dead algae and small animals mainly in fresh water habitats. They are
important decomposers in aquatic environments. Some water moulds are parasitic
on the gills of fish. The fungi which occur in sea water include the species of
Chytridium, Patersonia and Ophiobolus. III. Algae: Many planktonic and benthic
species of algae occur in different habitats of water both fresh and marine. On the
basis of their habitat in fresh and marine water, algae are divided into following three
categories: (a) Epipelic Algae: Algae growing on deposit sediment in water. e.g.:-
Oscillatoria,
Navicula. (b) Epipsammic Algae: Algae attached among the coatings of
bacteria on sand grains.
e.g. – Fragilaria, Chaetophora, etc. (Fresh water) and Raphoneis, Amphora,
Rivularia
etc. (Marine
forms)
(c) Plankotonic Algae: Free floating algae. e.g., Anabaena, Pandorina,
Chlamydomonas etc. (Fresh water) and Rhizosolenia, Coratium, Chaetoceros,
Peridiumetc (Marine forms). IV. Protozoa: The Protozoans are generally found in
water films surrounding the soil particles. Protozoans like Uroglenopsis, along with
algae Eudorina and Volvox produce an undesirable odour to water and pollute the
fresh water ecosystem. Aquatic protozoans, in planktonic forms are very commonly
distributed in fresh and marine water. These Protozoans are Ciliates, Flagellates and
heliozoans etc.

3. Micro-organism in Association with

plants
Plant parts leaves, stems, flowers, fruits, seeds and roots are literally covered
with micro- organisms of various kinds. Some of the common associations of soil-
micro organisms with plant are –
(A) Rhizosphere – Microbes influencing root and its surrounding. These
are
(a) Legume Root Nodule e.g.,
Rhizobium

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(b) Associative Nitrogen fixative. e.g., Azotobacter and

Azospirillum.
(B) Mycorrhizae- These are fungus- root
association.
The roots of about 80% of all kinds of vascular plants are normally involved in
symbiotic association with mycorrhizae. Generally three types of mycorrhizal
association have been found:
I Ectomycorhizal: In this fungi grow as external sheath around the tip of the root
with limited inter cellular penetration of the fungus into the cortical region of root. This
type of association is common in oak, birch, beech and coniferous trees.
II Endomycorrhizal: In this, fungal hyphae penetrate the outer cortical cells of the
plant root where they grow intracellularly and form coils, swellings, or minute
branches. These are characterized by two intracellular structures called Vesicles and
arbuscules. That's why these are called vesicular – arbuscular mycorrhizae (VAM).
This is found in wheat, corn, beans, tomatoes, apples, oranges and many
commercial crops and grasses.
III Ectendomycorrhizal Association: This type of association has more persistant
Intracellular infections of cortical cells found predominantly in the family
Orchidaceae.
(C) Actinorrhizae: Actinomycete association with plant roots is called
actinorrhizae. They are formed by the association of frankia strains. Frankia
strains are capable of nitrogen fixation and are important in the life of plants.
(D) Tripartile Association: When a plant develops relationship with two different
types of micro-organisms it is called Tripartile association. Following types of
associations are examples of tripartite association:
(i) Endomycorrhizae plus rhizobia including Rhizobium and
Bradyrhizobium.
(ii) Endomycorrhizae and
actinorrhizae.
(iii) Ectomycorrhizae and
actinorrhizae.
(iv) Ectendomycorrhizae and
actinorrhizae.

4. Microbes in
Air
Air as a matter of fact, it not a suitable medium for the growth of the micro-
organisms and studies indicate that higher the altitude, one might expect to find
fewer micro-organisms. Air itself does not support growth of the microorganisms but
they are either borne on dust particles, in moisture droplets expelled by men during
talking, coughing and sneezing. Micro- organisms are more numerous in the air
during dry weather than in wet weather because rain washes them out of the air.
A variety of microorganisms are found in air over populated land areas. These
include spores of Bacillus and Clostridium, ascospores of Yeasts, Conidia of moulds,
cysts of protozoans, unicellular algae, Non spore forming bacteria (Micrococcus
luteus), non- pathogenic bacteria, gram negative rods (Chromobacterium) etc. The
spores of many pathogenic fungi (e.g., rusts) causing crop diseases, plant pollens,
seeds (minute) are transmitted from one place of another through air currents.

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A number of human diseases are air borne and are transmitted by infectious
dust e.g.
Diphtheria(Corynebacteriumdiphtheria);Tuberculosis(Mycobacteriumtuberculosis),
whooping cough (Bordetella pertussis), children's influenza (Haemophilies
influenzale) etc.

5. Microorganisms in Food Microbes are in direct competition with the human for
the nutrients present in food. So foods are ideal culture media for microbes, and
different food items become contaminated with microbes, which are present in soil,
the bodies of plants and animals, water, air, equipments during processing or
preparation. Microorganisms that occur in foods may be divided into following
categories:- I- Beneficial organisms which bring about desirable fermentations e.g.,
those which are
used in the preparation of cheese, vinegar etc. II- Harmful organisms which are
responsible for decay of substances rich in organic matter,
and undesirable fermentations. III- Pathogenic organisms causing dreadful
diseases and food poisoning by their toxic
secretions. IV- Microorganisms themselves form food e.g., single cell
proteins and Mushrooms.
The effect depends upon the type and numbers of microbes and also on the nature
of food i.e., whether cooked, preserved or processed. Sometimes specific microbes
are added to food to get a desired effect. e.g., Poipickled cabbage (Lactobacillus
plantarum).
Food rich in proteins (meat, eggs, etc.), those with carbohydrates (vegetables and
fruits) are spoiled by different types of micro-organisms by the process of
putrefaction and decomposition (e.g. Pseudomonas, Micrococcus and Bacillus).

6. Microbes in
Milk
A number of micro-organisms thrive in milk (which is one of the nature's most
preferred food), and its products. Milk inside the udders is free from bacterial
contamination, but as it comes through the teat of the udder, it is contaminated as
bacteria are always present at the teat canals of the udder. Micrococci and
streptococci constitute the teat microflora.
Milk can be easily contaminated by pathogenic organisms to the milk in
various ways. Since bacteria are present everywhere including hay, feeds and
ground. The important direct possible ways of contamination are –
(i) Milking utensils (ii) Hay and other feeds, (iii) Hands of milkers, (iv) The udder of
cow and buffalo and (v) the skin of animal. Various types of micro-organisms are
found in milk. The presence and absence of these organisms depend on sanitary
quality and conditions of production. The important microorganisms in milk are:
I. Bacteria: Bacteria form the major section of microbes that grow well in milk. They
may produce beneficial or desirable effects and detrimental or undesirable effects.
The study of these bacteria in relation to milk and milk products is known as dairy
bacteriology.

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(a) Beneficial
Effects
Microorganisms are deliberately added to milk to produce fermented milk
products, to create new pleasing food flavours and odours. Yogurt is produced
by adding two bacteria: Streptococcus thermophilus and Lactobacillus bulgaricus
in the ratio of 1:1. Streptococcus produces acid and Lactobacillus produce
aroma components. Cheese, which has been thought to be developed 5000
B.C. is one of the oldest human foods. About 2000 varieties of cheese are
produced all over the world. Cheeses are classified on the basis of texture or
hardness as:-
Cheese Bacteria Used I. Softcheese -
Streptococcus lactis and
(ripened) S.cremoris II. Soft cheese -
Streptococcus lactis
(unripened) S. cremoris
S. thermophilus Lactobacillus bulgaricus III Semi soft -
Streptococcus lactis and Brevibacteriumlineus
S. cremoris IV. Hard - S. lactis, S.cremoris and
Lactobacilluscasei
S.durans, S. thermophilus V. Very Hard - S. lactis, S.
cremoris, S. tshermophilusand
Lactobacillus
bulgaricus

(b) Detrimental or undesirable Effect Spoilage occurs when

microorganisms degrade the carbohydrates, proteins and fats of milk. Examples
of common types of spoilage in dairy products and associated bacteria are
following:-
Spoilage type Bacteria involved Souring - Lactobacillus sp. and
Streptococcus sp. Sweet curdling - Bacillus sp., Proteus sp.,
Micrococcus sp. Gas production - Clostridium sp. and coliform
bacteira Red rot - Serratiasp. Grey rot - Clostridiumsp.
II Yeasts: They are found in milk and milk products. They act on lactose and
produce acid and carbon dioxide. Some produce gassy fermentation and some are
lipolytic in action. These contaminate milk through feed and soil. e.g.,Torulacremoris,
Torulalactis.
III. Moulds: They contaminate and grow in large number on the surface of butter,
cream, khoa and cheese. The colour may be black, grey, blue or white. They
produce undesirable odour also. Some are lipolytic and some are proteolytic e.g.
Penicillium sp, Cladosporium and Gleotrichum.

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IV Bacteriophages: Bacteriophages present in milk kill the bacteria in the starters

and interfere in the process of fermentation which is essential to produce certain milk
products such as butter and cheese.
V Viruses and Protozoa: These are not common organisms present in milk
products but
they occur in some rare
conditions.

7. Microbes of Human
body
The human foetus in uterus is free from bacteria and other microorganisms.
Within hours after birth, it begins to acquire a normal microbiota within the first week
or two. After that a variety of microorganisms become associated with the human
body. Thousands of microbes are present around us and many inhabit human body
in natural course. Most of the microbes of the human body are commensals, as they
do not harm the host. They obtain their nourishment from secretion and excretory
wastes of the human body. Some microbes act as scavengers by ingesting excretory
wastes or are beneficial to the host. e.g., – certain intestinal bacteria synthesize
vitamin E and K, whereas others protect the host from the pathogenic microbes.
I. Microbes of
skin
The human skin always remains in contact with bacteria present in the air, but
most of them are unable to grow since the skin secretes some bactericidal
substances.Staphylococcus, Streptococcus, Propioni bacterium, moulds, yeasts and
some pathogenic bacteria live on the surface of the skin. Some dermatophytic fungi
viz. Epidermophyton, Microsporum and Trichophyton may colonize in the skin and
produce athlete's foot and ringworm diseases.

II. Microbes of the mouth

cavity
 Continuous presence of soluble nutrients and abundance of moisture in the
mouth cavity provides a suitable environment for the growth of microorganisms.
Some common microbes are Staphylococcus aureus, S. epidermidis, S. mitis,
Lactobacilli, Actinomycetes, Bacteroidesoralis, Candida albicans,
Treponemadenticala, Mycobacteria, Entamoeba sp. and Trichomonasetc.

III. Microbes of Gastro – Intestinal

tract
Several micro-organisms such as staphylococcus aureus, S. epidermidis,
Haemophilus influenzae and Neisseria inhabit the pharynx. Stomach contains very
few microorganisms because of its acidic pH. When the stomach functions normally,
it is devoid of microbes due to the presence of gastric juices. Many gram + ve
facultative bacteria and fungus Candida albicansare found in the duodenum.
Similarly a large number of micro-organisms are found in the large intestine (colon).
They include gram (-)ve bacilli gram (+)vebacilli, Enterobacter, Escherichia coli,
Proteus and Candida albicans. In addition to these certain Protozoans
Trichomonashominis, Entamoeba hartmsanni, are also present.

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IV. Microbes of the mucous membrane of

the eye
Staphylococcus albus, Comybacterium xerosis and mycoplasmas are usually
associated with the mucous membrane of the eye.

V. Microbes in Respiratory
tract
We inhale a large number of adsorbed micro-organisms along with dust-
particles. Most of them are trapped in the nasal cavity. Some Staphylococci, aerobic
Corynebacteria, besides other cocciand bacilli inhabit the nasal cavity.

VI. Microbes of Genito-

urinarytract
The upper genitourinary tract consisting of kidneys, ureters and urinary
bladder is usually free of microorganisms. In both male and female, a few bacteria as
Staphylococcus epidermidis, Streptococcus faecalis and Corynebacterium sp. are
usually present in the distal portion of urethra. In the adult female genital tract, the
major microorganisms are the acid tolerant Lactobacillus sp., Bacteroides sp.,
aerobic corynebacteria, Peptostreptococus sp. and Enterococci, Mycobacterium
smegmatis and mycoplasmas.
1.5
CLASSIFICATION
In the early 19 century the traditional classification of living organisms was given by
th

Aristotle. He classified the living organisms into two kingdoms Plantae and Animalia.
Plantae includes algae, fungi and Bacteria & other plants. Other kingdom Animalia
includes all animals including protozoa.
A three kingdom classification was given by E. Haeckel in 1866. Heincludeda
the third kingdom Protista and classified living organisms as follows:
1- Protista (Algae, fungi, bacteria and
Protozoa) 2- Plantae (excluding unicellular
algae and fungi) 3- Animalia (excluding
protozoa.)
Later, Copeland (1956) suggested a four kingdom system of classification of living
organisms. He placed bacteria and blue-green algae in the fourth Kingdom Monera
and fungi in the third kingdom Protista.
Whittaker in 1969 proposed a five kingdom system of classification of living
organisms. His classification is as follows:
1- Monera (Bacteria and Cyanobacteria) 2- Protista
(unicellular algae, slime molds and protozoa) 3-
Fungi 4- Plantae (Eukaryotic multicellular plants) 5-
Animalia (excluding protozoa)

Hawker and Linton in 1971 classified microorganisms into 3

heads: I. Viruses: Specific group of sub-cellular obligate
parasites.

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II. Prokaryote: organisms with prokaryotic organization. This

included-
(a) Bacteria (unicellular forms without a definite nucleus). (b) Higher Bacteria
(filamentous actinomycetes to filterable mycoplasmas). (c) Rickettsiae (Parasitic
bacteria having the appearance of tiny rod shaped or spherical
resembling bacteria and viruses). (d) Cyanobacteria
(photosynthetic forms of prokaryotic organization) III. Eukaryote :
organisms with eukaryotic organization which includes-
(a) Algae (unicellular eukaryotic algae belonging to Chlorophyceae,
Chrysophyceae and
Euglenophyceae). (b) Fungi (moulds) (fungi with unicellular to multicellular
hyphae with cottony growth
belongs to Phycomycetes, Ascomycetes, Basidiomycetes, and deuteromycetes).
(c) Slime moulds (organisms with slimy mass of naked motile protoplasm called
 plasmodium). (d) Protozoa (unicellular animals without chlorophyll. and are
divided into sarcodina,
sporozoa, ciliophora and mastigophora. (e) Nematodes
(multicellular thread like cylindrical worms).
Recently microorganisms are classified into two major
groups:
(A) Acellular Infectious
agents (B) Cellular
Infectious agents

(A) Acellular Microorganisms: These are called acellular organisms as they do not
possess three minimum characteristics of a cell- Presence of a membrane, genetic
material and metabolic machinery.
These are of following
types:
I. Viruses: Ultramicroscopic obligate parasites made up of nucleic acid, lack
cytoplasm
and cell organelles and multiply only within the living host cell. A Provisional
committee or Nomenclature of viruses (P.C.N.V.), headed by system proposed by
Lwoff, Horne and Tournier (1962). According this system all viruses are grouped
into a single phylum –Vira which is divided into subphyla, classes, orders, suborders
and families. On the basis of following characters:
(a) Type of nucleic acid DNA or RNA. (b) Symmetric helical Cuboidal or biral. (c)
Presence or absence of envelope around capsid. (d) Diameter of helical nucleo
capsids and no. of capsomeres present in cuboidal viruses.

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II Viroids:
These are small naked RNA molecules. These were first discovered by Diener
(1971). They replicate autonomously. Viroid RNA is usually single stranded,
circular and is of very low molecular weight viroids cause various plant disease
such as potato spindle tuber (by PSTV), citrus exocortis, chrysanthemum stunt,
cucumber pale fruit etc.
III Prions: These are rod-shaped proteinaceous infectious particles without nucleic
acid; Prions were named by S.B. Prusiner (1984) who was awarded Nobel Prize in
Medicine in 1997. Prions cause a variety of mammalian neurodegenerative
diseases. These are generally fatal and are referred to as transmissible spongiform
encephalopathies (TSES).These include diseases like scraple, disease of sheep;
CreutzfeldtJakob disease (CJD) and Kuru – Human diseases. Prions also cause
madcow disease of bovino spongiform encephalopathy (BSE).
IV Virusoides: They are also called satellite RNA. They are encapsulated by plant
viruses packaged together with a viral genome. They cannot replicate independently
and do so with the help of an associated virus.
(B) Cellular Micro-organisms: These microorganisms are cellular as their cells
possess a membrane, genetic material and metabolic machinery. These are two
types on the basis of presence or absence of nuclear membrane.

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1. Prokaryotic microbes & 2. Eukaryotic

microbes.

1. Prokaryotic
microbes
These are unicellular microorganisms. These show prokaryotic cell organization.
Prokaryotic micro-organisms are again subdivided into two-
(A) Archebacteria & (B) Eubacteria
(A) Archebacteria: These are the primitive type of bacteria which lack muramic acid
in their cell walls; the membrane lipids have ether linked aliphatic branched chains.
These possess distinctive RNA polymerase enzymes. Their ribosome is also of
different composition in shape. These bacteria are categorized into (a) methanogenic
bacteria (b) extreme halophiles and (c) thermoacidophiles.
(B) Eubacteria (True bacteria):This comprises a vast majority of bacteria. The
Peptidoglycan cell wall contains muramic acid. The membrane lipids have ester
linked straight chained fatty acids.
These are divided into following
groups –
(i) Spirochetes: These are gram-negative, chemohetrotrophic bacteria, and are
distinguished by their structure and motility. They lack external rotating flagella but
still can move through very viscous solutions, exhibiting creeping or crawling
movement when in contact with a solid surface. Their unique style of motility is due
to the presence of an axial filament. Two or more than a hundred prokaryotic flagella
called periplasmic flagella, extend from both ends of the cyinder and often overlap
one another. Spirochetes are anaerobic, facultatively anaerobic or aerobic. Some
pathogenic forms are: Treponemapallidum, Borrelia burgdorferi, Leptospira and
causing syphilis, lyme disease and Leptospirosis respectively.

(ii) Rickettsias: These belong to order Rickettsiales. These are a group of Gram-
negative intracellular parasitic bacteria. These resemble viruses in their very small
size and intracellular existence. They differ from viruses in having both DNA and
RNA, a plasma membrane, ribosome, enzymes etc. They are intermediate between
bacteria and viruses. Some of the important pathogenic forms are:
Rickettsia prowazeki causes typhus fever; Rickettsia
rickettsii causes rockey mountain spotted fever;
Rickettsia orientalis causes scrub typhus; and
Rickettsia burnetti causes Q fever

(iii) Mycoplasma
(Mollecutes)
Mycoplasmas are prokaryotes without cell wall and are placed in the class
molecules (Mollis = Soft; cutis = skin). They are pleomorphic and occur in any shape
such as spherical, pear shaped, branched and helical filaments. They are non-motile
but can glide along liquid covered surfaces. They are usually facultative anaerobes
but a few are obligate anaerobes. Their genome is one of the smallest (about 5-10 x
10 daltons) found in prokaryotes. They live
8

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as saprophytes or parasites or pathogens of plants, animals, man or insects. The

common examples of mycoplasmaare:
Mycoplasma pneumoniae (causing mycoplasmal pneumonia);
Mycoplasmamycoides and gallisepticum causing contagious bovine
pleuropneumonid in cattles, and in chicken respectively. Mycoplasma urealyticum
causes genital infection.
(iv)Cyanobacteria (Blue green
algae)
Cyanobacteria are blue green algae or blue green bacteria. They form a
connecting link between bacteria and green plants. They are prokaryotic. They are
photoautotrophs as they contain chlorophyll which is located in thyllakoids. Their
photosynthetic system closely resembles that of eukaryotes, because they possess
phycobili proteins as accessory pigments like red algae. Cyanobacteria vary greatly
in shape and appearance. They may be unicellular and exists in colonies of various
shapes or multicellular and form filaments called trichomes.
Cyanobacteria are not toxic to man. The cytoplasm contains phycobillins and
carboxysomes. They have heterocysts which are specialized cells for nitrogen
fixation and akinetes for spore formation. Common examples are: Anabaena,
Nostoc, Chlorococcus, Oscillatoria, Stigonemaetc.
(V)
Actinomycetes:
Actinomycetes include fungus like bacteria. (Actis = rays; mykes =
fungus)
These are aerobic, gram positive bacteria that form branching usually non-
fragmenting hyphae and bear asexual spores. Most actinomycetes are non-motile.
Motility is confined to flagellated spores only. They are mainly soil inhabitants and
can degrade a variety of organic compounds.
Actinomycetes are chemoorganotrophs, as they rely on organic compound for
their energy. These organisms are of great economic importance as they produce
about 85% of know antibiotics.
The important actinomycetes are the following: Streptomyces(produces
streptomycin): Erythromycine & Chloramphenical, tetracycline;
Micromonospora(produces gentamycin) .Some actinomycetes fix atmospheric
nitrogen and live symbiotically (e.g. Frankiasp). A few actinomycets are pathogens of
human, animals and plants e.g. Thermoactinomyces vulgaris whichcauses a
respiratory disease called Farmer‘s lung in humans.

2. Eukaryotic
Microbes
These are they microorganism with eukaryotic organization. Such organisms
are divided into three parts: I Unicellular Eukaryotic Microbes, II Multicellular
Eukaryotic Microbes (Fungi) & III Helminthes.
I. Unicellular Eukaryotic
Microbes
These are solitary or colonial unicellular microbes with eukaryotic
organization. They are usually aerobic forms, and can be motile or non- motile.
Motility is due to cilia, flagella or pseudopodia. They are subdivided into following
types:

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(a) Photosynthetic Protists (Unicellular

algae) :
It includes unicellular photo synthetic organisms belonging to different groups
Chlorophyceae, Euglenophyceae, Xanthophyceae, Pyrrophyceae, Bacillariophyceae
and Chrysophyceae. Some major group of this are

1- Dianoflagellates: These belong to Chrysophyceae. They are unicellular with

cellulose cell wall. They possess two flagella. They reproduce only asexually. Sexual
reproduction is unknown. Some of them e.g. Gonyaulaxsecretes a toxin which kills
marine animals like fish known as 'red tide'. Some Dianoflagellates are
phosphorescent and make the sea Scarface glow in the dark. 2- Diatoms: These
belong to bacillariophyceae. They lack flagella. Diatoms are diploid and reproduce
asexually as well as sexually. Diatoms are nearly indestructible because of
deposition of silica in their cell walls. They leave behind large amounts of cell wall
deposits called diatomaceous earth. 3- Euglenoids: These are Euglena like protists.
They lead plant like and animal like lives. These are free living forms found in fresh
water ponds and ditches on damp soil. They do not possess a cell wall. The
flagellum is duplicated before the cell divides. e.g. Euglena.
(b)Consumer – Decomposer
protists:
These resemble fungi in appearance and life style but are more closely
related to protists in cellular organization, reproduction and life cycle. These include:
I. Acellular Slime moulds
(Myxomycota)
They are wall less mass of multinucleate protoplasm e.g. Physarum. It slowly
streams or glides over decaying leaves or logs. This strand of protoplasm is called
plasmodium. Feeding is by phagocytosis.

II. Cellular Slime moulds(Acrasiomycota) These are numerous individual

amoeboid cells which aggregate together and move like a mass of protoplasm. The
individual cells are not fused. This is called pseudo plasmodium. e.g.Dictyostelium.

III. Water mould

(Oomycota):
They consist of finely branched filaments called hyphae. Their cell walls are
made up of glucanc and cellulose. Very small amount of chitin is found in rare cases
They reproduce sexually by a large egg cell and a small antheridium, asexual
reproduction by biflagellate zoospores. They are saprophytes (e.g. Saprolegnia) or
parasites (e.g. Phytophthora infestans). Another significant feature of this group is
presence of hydroxyproline amino acid in the wall protein. (c) Protozoan Protists:
Protozoa are animal like protists that can be defined as motile eukaryotic
unicellular microorganisms. They resemble multicellular animals in their morphology
and physiology.

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Some of them secrete a resistant Covering and form cyst which protects organisms
against adverse environments. These organisms move by pseudopodium, flagella or
cilia, and reproduce asexually and sexually. They play an important role in food
chains and food webs. Many of them are parasitic in humans and animals.

I. Multicellular Eukaryotic
Fungi:
This group includes the organisms which are placed in the kingdom fungi by
Whittaker. They exist primarily in the form of filamentous hyphae. They lack
chlorophyll, and reproduce asexually, sexually or by both methods. They are
important decomposers and play an important role in recycling of minerals, thus are
of great importance to humans in both beneficial and harmful ways. They are further
divided into four groups (1) Phycomycetes (2) Ascomycetes (3) Basidiomycetes and
(4) Deuteromycetes.
(3). Helminthes: The kingdom Animalia has only one group of microscopic
organisms the helminthes. Tape worm, flukes and round worms are collectively
called helminthes. There are two types of parasitic helminthes based upon
morphological form. They are the flatworms, belonging to phylum Platyhelminthes
with a very thin segmented body and round worms belonging to phylum
Aschelminthes with an elongate, cylindrical, unsegmented body plan.
All helminthes are multicellular animals. They absorb nutrients through their
body wall while living in the host intestine; most worms complete their life cycle on
two hosts, and have the capacity to regenerate. Some pathogenic helminthes are:
Ascarislumbricoides (Round worm); Necatoramericanus (Hook worm); Fasciola
hepatica (Sheep liver fluke); Taeniasolium (Pork tape worm) and Taeniasaginata
(Beef tap worm) etc.

1.6 MAJOR
MICROBES
1.6.1 Major Microbes of
food
Food is an indispensable item for all living organisms. All food items are
associated with microbes in one form or other. Even naturally occurring food such as
fruits and vegetables contain microorganisms. Major microbial flora of common food
are listed below:

Types of food Microbial flora

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1. Milk I. Biochemical Types - Streptococcus lactis, S. cremoris

II. Acid producers –Lactobacilli, Microbacteria, Coliform, Micrococcus III. Gas
produces –Coliforms, Clostridium, Torulacremoris IV. Proteolytic –Bacillus,
Pseudomonas, Proteus, Streptococcus V. Lipolytic –Pseudomonas, Achromobacter,
Candida, PenicssSSillium VI. Mesophilic- Bacillus coagulans VII. Thermophilic –
Bacillus stearothermophilus 2. Dairy Products Lactobacillus, Leuconostoc,
Micrococcus, Streptococcus,
Geotrichum 3. Raw milk Alcaligene, Bacillus, E.coil, Lactobacillus, Leuconostoc and
Streptococcus 4. Fruits and vegetables Bacillus, Pseudomonas, Salmonella,
Corynebacterium, Erwinia, E.coli, Aspergillus. Botrytis, fusarium, Trichothecium,
saccharomyces, moniliaandRhizopus 5. Egg & Egg Products Pseudomonas
fluorescens, P.ovalis, Salmonella, Proteus
thamnidium, moulds and yeasts. 6. Meat Clostridium, Enterobacteria, Micrococcus,
Streptococcus faecalis, Proteus, Pseudomonas, Staphylococcus, Aspergillus,
Candida. 7. Fish Pseudomonas, Chromobacterium, Micrococcus, Flavobacterium,
Corynebacterium, Sarcina, Serratia, Bacillus, E.coli, Clostridium. 8. Bread
Saccharomyces cerevisiae, Enterobacter. Lactobacillus brevis, Clostridium, Bacillus
polymyxa, Serratiamarceseens (red or bloody bread), Rhizopusnigricans,
Penicillium, Aspergillums, Mucor (Bread mould). 9. Poultry Pseudomonas, Proteus,
Chromobacter. E.coli, Salmonella,
Bacillus. 10. Shellfish Bacillus, Alcaligenes, Proteus,
Coliforms. 11. Fermented Food Streptococcus actis, lactobacillus, Clostidium,
Leuconostoc,
Acetobacter, Saccharomyces,Pediococcus 12. Beef
Cladosporium, Sporotrichum 13. Seafood Pseudomonas and Vibrio 14. Pickles
Brettanomyces, Debaryomyces, Leuconostocmesenteroides.

Besides these, some microorganisms are foods intoxicating. The food

intoxication results due to ingestion of exotoxins secreted by microbes in food. The
intoxication symptoms appear immediately after consuming contaminated food
because it does not require growth of disease causing microorganisms. Some of the
major food intoxicating diseases, their causal organisms and foods involved are
listed below:

(A) Major Food

intoxications

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Disease Microbe Food involved

Food poisoning Bacillus cereus Meats, potatoes, Cereals.
Rice products, pudding.
Botulism Clostridium botulinum Fish, meats and low acid
canned food. Perfringens food poisoning Clostridium perfringens Animal and Poultry
Products. Staphylococcal food Poisoning Staphylococuus aureus Meat, dairy Products,
Poultry, custard and starch containing food. (B) Major Food inflations
Campylobacteriosis Campylobacter jejuni Raw milk, raw chicken and
poultry products.
Listeriosis Listeria monocytogenes Poorly processed dairy
products. Salmonellosis
Salmonella typhimurium Poultry, eggs, dairy products,
meat. Shigellosis Shigellasonnei, S. flexneri Insanitary cooked food, fish,
potatoes and salads.
Yersiniosis Yersinia enterocalitica Milk and meat product.

E.coli enteritis Cheese and raw vegetable

1.6.2 Major Microbes of

Water
Water is unique physical environment, andfavours the existence of many
types of micro organisms that are not common in soil. Micro organisms occur in all
depths. The surface film and bottom sediments have a high concentration of
microorganisms. Drifting microbial life of aquatic environment is called plankton,
composed of phytoplankton and Zooplankton. The bottom region of the body of
water harbours largest number and kinds of microorganisms called benthic
microorganisms. The movement of water by wind, tide and currents affect the
distribution of microorganisms.
(a) Major microbes of Ponds and
Lakes
Lakes and ponds of temperate region show thermal stratification which influences
the microbial population in different seasons. Common fresh water micro
organisms are Pseudomonas, flavobacterium, Aeromonas, and Acaligenes,
Clostridium, Thiothrix and Thiobacillus. Besides this both, Cyanobacteria and
many algae contribute to massive water blooms. (b) Major Microbes of sea
Diatoms, Cyanobacteria, Silicoflaellates, Dinoflagellates,etc.Chlamydomonas
are major phytoplanktons. Many microorganisms, particularly algae and
cyanobacteria cause a condition called Red sea and Red tides.Brown, amber
or greenish yellow colourationis also due to abundance of micro organisms.

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Common marine forms are vibrio, Actinobacter, Pseudomonas,

Flavobacterium, Staphylococcus, several sps of Phycomycetes,
Deuteromycetes and Myxomycetes and a number of protozoa and species of
fungi also occur in sea water.
(c) Microbes of Domestic
water
The domestic water is obtained from rivers, streams, ponds, dams, lakes,
wells and bore wells. The micro organisms of domestic water include viruses,
bacteria, algae, protozoa and fungi. Some of the microbes are listed below:
Bacteria : Streptococcus faecalis, S. bovis, S. equines,
Pseudomonas,
Alginomonas, Xanthomonas, Escheriachia coli,
Enterobacter, Aerobacter, Salmonella, Bacillus,
Micrococcus, Shigella, Proteus, KlebsiellaSerratia
Viruses : Poliovirus
Protozoa : Entamaebahistolytica and Giardia
Fungi : Achlyaamericana, Dictyuchuspisci, Pythiumundulatum,
Saprolegni
a
Algae : Anabaena, Microcyrtis, Nostoc, Oscillatoria, Oedogonium,
Spirulin
a

(d) Microbes of Sewage or Waste

Water
Major microbes include coliform bacteria and micro organisms other than
coliform bacteria. Major Microbes other than Coliform bacteria are:-
Bacteria:Streptococcusfaecalis, S. faecium, S. bovis and S. equines; some
slime forming bacteria; Sphaerotilus and Gallionella (Iron bacteria);
Thiobacillus (Sulphur bacteria).
Algae: Microcystis, Spirulina etc. produce nuisance characteristics and
produce toxic
substance
also.
Viruses:Polio virus (enter through the human and animal intestinal
tracts).
(e) Pathogenic water borne
microbes
Some of the bacterial viruses and protozoan pathogens are able to survive in
water and infect humans. Some of the water borne diseases is listed below:
Microorganism Disease Vibrio cholera - Cholera Camphylobacter -
Gastroenteritis and Diarrohea Salmonella typhi - Typhoid Leptospira -
Jaundice, Haemorrhagic effects. Giardia lamblia - Diarrohea Cryptosporidium
- Acute enteroicolitis Naegleriafowleri - Primary amoebic meningo encephalitis
(PAM)
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1.6.3 Major Microbes of

Soil
There are five major groups of micro organisms in the soil. They are bacteria,
fungi, Algae, Protozoa and Viruses.
I. Bacteria: These are the largest group of soil microbes. Practically all kind of
bacteria
may be found in Soil. Major bacteria of soil are categorized as : 1. True bacteria: Of
all the micro organisms‘ true bacteria are the most abundant in
soils. Most commonly isolated bacteria from soils are – (a) Gram negative
bacilli:Pseudomonas, Agrobacterium,Achrobacter, Rhizobium,
Flavobacterium. (b) Gram positive non Sporing bacilli: Corynebacterium,
Arthrobacter, cellulomonas : (c) Gram positive cocci : Micrococcus, Sarcina (d)
Gram Positive Spore formingbacilli : Bacillus (aerobic) Clostridium (anaerobic) 2.
Nitrogen fixing bacteria: (a) Symbiotic bacteria: Live in the roots of legumes
e.g.Rhizobiumsps., (Rhizobium leguminosarum on peas, lentils etc, R. japonicum
on soyabeans, R. phaseoli on beans, R. trifoli on Red, White and other clovers
etc). (b) Non- symbiotic bacteria live freely in soils-Azobacter (Aerobic),
Clostridium
(Anaerobic) (c) Sulphuroxidizer:Thiobacillusthiooxidans,
Desulfovibriodesulfuricans (d) ProteolyticBacteria:Clostridiumhystolyticum,
Proteus vulgais, Pseudomonas
fluorescens, Bacillus cereus. (e) Bacteria involved in nitrification:Nitrosomonas,
Nitrosococcus, Nitrosospira, and
Nitrosocysti
s.
Besides above mentioned bacteria, some soil inhabiting bacteria are pathogenic and
cause plant diseases. e.g. Agrobacterium causes galls, Erwinoa sp. causes soft rot
as well as dry necrosis in carrots, potato and cucumber.
II. Actinomycetes: Many actinimycetes are present in the soil, e.g. Streptomyces,
Nocardia, Micromonospora. The filamentous actinomycete Streptomyces produces
an odour causing compound called geosmin. Streptomyces species are responsible
for potato scab disease. III. Cyanobacteria and other Algae : A number of
cyanobacteria are abundant lyoccurin moist soils, e.g. Anabaena, Nostoc,
Cylindrospermum microcystis, Oscillatoria etc. They fix molecular nitrogen present in
the air and improve the fertility of soil. These are used as biofertilizers. Besides
cyanophyceae, other algal genera are also present in soil. These are Chlorella,
Chlorococcum, Cladophora, Botrydiopsis, Bumilleria. Navicula, Pinnularia, Fragilaria
etc. IV. Fungi: Mucor, Rhizopus, Pythium,Penicillum, Aspergillus, Alternaria,
Hormodendron, cladosporium and yeasts are common in vineyard and orchard soils.
Some soil fungi are good soil binders. e.g., Aspergillus,Cladosporium, Rhizopus and
Penicillium. Some are pathogenic and cause diseases. e.g., Alternariasolani causing
early blight of potato.
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V. Protozoa: Many types of Protozoa are found in the soil. Some major soil
protozoans are
:

Mastigophora- Bodo, Cercomonas, Monas, Euglena, Spiromonas,

Spongomonas, oikomonas Sarcodina- Amoeba, Biomyxa, Harmanella,
Lecythium, Nucbaria,
Trinema, Naegleria Cilliata- Colpidium, Calpoda, Gastrostyla, Halteria,
Oxytricha,
Pleutotricha, Vorticella,
Uroleptus
Protozoa do not serve any major function in the soil. They engulf bacteria and
maintain some equilibrium of the bacterial flora of soil.
VI. Viruses: Bacterial Viruses (Bacteriophages), as well as plant and animal viruses,
are present in soil. Viruses transmit genetic material from one bacterium to another
through transduction. The bacteriophages have some effects on the ecology of the
bacteria also.

The Rhizosphere
microbes:
Rhizosphere is the area of soil around the root system. Based on the intimacy
of microbial association with root system, the rhizosphere is divided into two regions,
namely endorhizosphere and exorhizosphere. Microorganisms colonize the
rhizosphere to utilize them as food. Certain fungi form symbiotic association with root
to form mycorrhiza. Some examples of rhizospheremicroorganisms are as follows:-
Fungi –Aspergillus flavus, A. niger, A. fumigates, A. terreus; Cladosporium
herbarum; Fusarium oxysporum, F. solani.
Bacteria –Pseudomonas, Rhizobium, Bacillus, Agrobacterium, Micrococcus,
Azobacter, Mycobacterium

Microorganisms as Bio
fertilizers
The nutrients of biological origin added to the soil to enrich the soil fertility are
called biofertilizers or microbial fertilizers. The organisms used as biofertlizers
include:
Rhizobium, Azospirillum, Azotobacter, Azotococcus, Anabaena, Nostoc,
Plectonema and Tolypothrix.
Phosphate Solubilizing microbes: Bacillusmegaterium, Xantnomonas,
Pseudomonas, Aspergillus, and Pencilliumdigitatum.
 The spores of VAM fungi like Glomus, Gigaspora, A.caulospora, Sclerocystis and
Endogoneare used as VAM biofertilizers.

1.7 SUMMARY
In this unit you have learnt
that–

• Microbiology is the study of

microorganisms.
• Microorganisms are invisible creatures, too small to beseenwiththe
naked eye.

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• Microbes are widely distributed and are omnipresent. Theycan be isolated from air,
water, soil, in living plants and animals and dead organic substanses.
• Microbes areeitherUnicellularormulticellularornon cellular forms, and are
prokaryotes or eukaryotes.
• Microorganisms include viruses, Bacteria, Algae, Fungi and Protozoa. Non cellular
microbes are viruses. Unicellular microbes have single cells e.g., Protozoa, Bacteria,
someAlgae and some fungi. Multicellular microbes have many cells e.g.,
FungiandAlgae.
• Microbes have the capacity for rapid growth. They do not depend on the availability
of specific micronutrients in theenvironment.
• Some of the microbes (bacteria and cyanobacteria) can
fixatmosphericnitrogen.
• Soil is an excellent natural medium formicroorganisms. Man depends upon the soil
for his food. The soil depends uponthemicro organisms for its fertility.
• Food is an indispensable item for alllivingmicro organisms. All food items are
associatedwithmicroorganismsinoneform or other.
• In aquatic environment micro organisms occur from water surface to greater
depths. The various water sources as ponds, pools, Lakes, rivers and oceans show
a great diversity in theirmicroflora.

1.8
GLOSSARY
Actinomycetes: Gram Positive Bacteria that are characterized by the formation of
branching filaments. Adenoviruses: A group of DNA viruses, causing infection of
the upper respiratory tract. Antibiotic: A substance of microbial origin that has
antimicrobial activity and it kills the other micrograms. Archaebacteria: A group of
bacteria that includes primitive type of bacteria in which cell wall lack muramic acid.
Bacillus: A rod shaped bacterium. Bacteriophage: A virus whose host is a
bacterium and replicates within bacterial cell. Biofertilizers: The nutrients of
biological origin added to the soil to enrich the soil fertility are called biofertilizers.
Botulism: A neuroparalytic disease due to an exotoxin produced by the bacterium
clostridium botulinum in improperly canned or preserved food.
Chemoorganotraphs: organisms relying on organic compounds for their energy
source. Ectomycorrhiza: Amycorrhiza in which the fungal hyphae grow only
intercellularly, never entering the cell wall of the host cell. Endomycorrhiza:
Amycorrhiza in which the fungal hyphae penetrate the cell wall of the host cell.
Epipelic Algae: Algae growing on deposit sediment in water. Epipsammic Algae:
Algae attached among the coatings of bacteria on sand. Eubacteria: True bacteria
in which cell wall contain muramic acid. Mycorrhiza: A symbiotic association
between fungus and plant roots. Prions: Proteinaceous rod shaped infectious
particles without nucleic acid.

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Symbionts: Two organisms living together with mutual benefits. VAM:

Vesicular Arbuscular Mycorrhizal fungi. These are also called endomycorrhiza.
Virioids: Small naked RNA molecule. Yoghurt: Fermented liquor made from
milk.

1.9 SELF ASSESSMENT QUESTIONS 1.9.1

Choose the correct answer from the given
below:
(a) Which one of the following isarchaebacteria?
(i) Blue– green (ii) Rickettsias (iii) green sulphar
(iv) Methanogens

(b)Which of the following water is free from bacteria? (i) deep

well water (ii) sea water (iii) Water of hot spring (iv) rain
water as it falls down

(c) Bacteria which in association with legume roots fix atmospheric nitrogen are
called:
(i)Azobacter (ii) Paseudomonas (iii)Rhizobium (iv)
E.coli

1.9.2 Fill in the

blanks:
a) The Characteristic earthy smell of freshly ploughed field is due to a
compound
called ................ produced by
streptomyces.
b) Actinomycete association with plant root is
called.................
c) Botulism is caused by.........................
1.9.3 Answer the following in one word or in one
sentence.
a) Name the microorganism which causes red rot of
milk.
b) Name a symbiotic
bacterium.
c) What name is given to a virus which infects
bacteria?
1.9.4 Write true or false
(T/F).
a) Prions are single stranded RNA containing cellular infections
agents.
b) Flagellated cells are absent in
cyanobacteria.
c) Viruses contain both RNA and
DNA.
1.9.5 Explain the following
–
i) Associative nitrogen fixation.
ii) How do microorganisms contribute to body
odour?
iii) Compare prokaryotic and eukaryotic
microorganisms.

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Answers
Keys:
1.9.1- (a)-(iv), (b)-(iv), (c)-
(iii)
1.9.2- (a)-Geosmin, (b)-Actinorrhizae, (c)- Clostridium
botulinum
1.9.3-(a)-Serratiamarcescens, (b)-Rhizobium, (c)-
Bacteriophage
1.9.4-(a)-False, (b)- True, (c)-
False

1.10
REFERENCES
• Atlas, R.M. Principle of
Microbiology.
• Bhagat Singh and Renu Singh, 2011. Microbiology for Medical Sciences. I.K.
International Publishing House Pvt. Ltd. New Delhi.
• Casida, L.E. 1968, Industrial Microbiology, John Wiley and sons, New
York.
• Clifton, A. 1958, Introduction to the Bacteria, MC Graw, Hill Book Co. New
York.
• Doelle, H.W. and C.G., Heden 1986. Applied Microbiology, Kluwer Academic
Press, London.
• Dubey, R.C. & D.K. Maheshwari. A text book of Microbiology. S.Chand& Co. New
Delhi.
• Frazier, W.C. & D.C. Westhoff. 1988. Food Microbiology. Tata McGraw
Hill.
• Kaushik, P. 1996, Introductory Microbiology. Emkay Publ.
Delhi.
• Mandahar, C.L. 1978, Introduction to Plant viruses. S. Chand & Co. Ltd.,
Delhi.
• Mukherjee, K.G., and Ved Pal Singh, 1997. Frontiers in Applied Microbiology,
Rastogi Publ. Meerut.
• Norris, J.R. and D.W. Ribbons 1970. Methods in Microbiology. Academic Press,
London.
• Pelezar, M.J. Chan, ECS and Kreig, N.R. 1993, Microbiology, concept and
Applications. M.C. Graw Hill, New York.
• Power, C.B. and H.F. Daginawala, 1970. Methods in Applied Microbiology, Rastogi
Publ. Meerut.
• Prescott, I. J., J.P.M. Harley & A.D. Klein, Microbiology. Tata MC Graw
Hill.
• Robinson, R.K., 1990. Dairy Microbiology, Elsevier Applied Sciences,
London.

1.11 SUGGESTED
READINGS
• Ananthanarayan&Panicker, 1997. Text Book of Microbiology Orient
Longman.
• Bhagat Singh and Renu Singh, 2011.Microbiology for Medical Sciences I.K.
International Publishing House Pvt. Ltd. New Delhi.
• Dubey, R.C. & D.K. Maheshwari. A Text Book of Microbiology. S. Chand & Co.
Delhi.
• Kaushik, P. 1996. Introductory Microbiology.Emkay Publ.
Delhi.
• Mandahar, C.L. 1978. Introduction to Plant viruses. S. Chand & Co. Ltd.,
Delhi.

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• Subbarao, N.S. 1994. Soil Micro organisms& Plant Growth. Oxford & IBH Pvt. Ltd.
New Delhi.

1.12 TERMINAL
QUESTIONS
1. Give a brief account of Salient features of main groups of
microorganisms.
2. Write short notes on the following:
(i) Archebacteria (ii) Viroids and Prions (iii) Mycoplasma
(iv) Cyanobacteria (v) Actinomycetes (vi) Spirochete.
3. Discuss the different approaches to classification of
microorganisms.
4. What are the applications of the following
microorganisms?
(a) Rhizobium (b) Bacillusthuringiensis
5. List various types of microbial food spoilage and name the organisms
responsible in each
instance.
6. Discuss the distribution of microorganisms in
soil.
7. Differentiate the following:
(i) Prokaryotes and Eukaryotes
 (ii) Ectomycorrhiza and
Endomycorrhiza
(iii) Autotrophs and
Heterotrophs
(iv) Archaebacteria and
Eubacteria

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UNIT 2- ISOLATION AND CULTIVATION OF

MICROORGANISMS, INSTRUMENTS USED IN
MICROBIOLOGICAL STUDIES
2.1- Objectives
2.2- Introduction
2.3- Sterilization
2.4- Preparation of culture
Media
2.5- Dispensingthe
medium
2.6- Some important
culturemedia
2. 7- Methods of obtaining pure
cultures.
2.8- Methods of isolation
2.9- Cultivation of viruses
2.10- Culture
techniques
2.11- Instruments used in microbiological
studies.
2.12-
Summary.
2.13-
Glossary
2.14- Self assessment
questions.
2.15-
References
2.16- Suggested
Readings
2.17 Terminal
Questions.

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2.1 OBJECTIVES
It is well known that microorganisms occur in natural environments; and are
contaminated and mixed with several other forms of life. To know more about them
and to study them individually, they have to be separated from mixed forms and
need to be cultured under artificial conditions.This unit deals with Isolation of
microorganisms and their growth in the laboratoryconditions and also various culture
techniques and instruments used formicrobiological studies. After reading this
unit,one will be ableto:

• Get an idea of the cultivation of

microorganisms.
• Know about different culture media and their
preparation.
• Understand the methods of isolationof
microorganisms.
• Obtain pure
cultures.
• Know about thecultivation and culture techniques
ofviruses.
• Study the different microbiological
instruments.

2.2 INTRODUCTION
You know that microorganisms occur innatural environment; they
arecontaminated and mixed with several other forms of life. In order to
understandmore about them we have to separate and study them individually for this
purpose we have to isolate microorganisms and culture them under artificial
conditions. The technique of growing microorganisms in an artificial medium is
known ascultivation.
Cultivation of microorganisms is done in the laboratory and requires favorable
environmental conditions such as nutrient sources of energy, appropriate
temperature, oxygen and pH etc. Since various types of microorganisms grow
together in a suitable environment, a number of isolation techniques are used to
obtain pure culture of just one species of a microorganism.

2.3 STERILIZATION
In microbiology Sterilization is an important term which needs making
anything free of any form of life. For detail study of a microorganism, one needs a
pure culture of an organism. It ia obtained by taking utmost care to avoid
contamination through the atmosphere, glassware, media or other instruments used
in the culture technique. The growth of unwanted microbes in the culture is called
contamination and these unwanted microbes are called contaminants.
A number of precautionary measures are taken to prevent contamination
during sterilization and creating a germ free condition is called aseptic condition.
There are following three methods of sterilization:
(A)
Physical
(B)
Chemical

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(C)
Gaseous
(A) Physical Methods : The
frequently used physical methods are
I. Sterilization by heat-
(a) Dry heat
sterilization
(b) Wet heat
sterilization
II. Sterilization by filtration III.
Sterilization by ultra violet radiations IV.
Sterilization by ionizing radiations.
I. Sterilization by heat-Heat is most commonly applied in the microbiological
laboratories for sterilization. (a) Dry heat sterilization: Direct heating of the
instruments in a flame is an easy way of sterilization. Inoculating needles,
scissors, forceps, scalpels etc. are commonly sterilized by direct heat while the
neck and mouth of specimen tubes, flasks and culture tubes are also passed
through flame till they become sterilized. The process of sterilizing the articles
with flame is called flaming.
Another method of dry heat sterilization is to keep thoroughly washed and dried
glass wares such as Petri dishes, beakers, flasks, culture tubes etc. inside
thermostatically controlled electric oven. Complete sterilization is accomplished
by maintaining a temperature of 160 C for not less than 4 hrs. inside the oven.
0

(b) Wet heat sterilization: Wet heat(steam) is more efficient method and is
preferred in sterilizing the media used for culturing micro-organisms. The common
ways by which Wet heat is employed in the laboratory are: Boiling, Pasteurization,
Tyndallization and autoclaving. i. Boiling: It is a common method of sterilization. All
the instruments used for cultivation are kept in a container filled with distilled water
and allowed to boil for at least 15 minutes. If the articles are not to be used
 immediately, these should be stored in a sterile container. ii. Pasteurization: Many
substances such as milk are treated with uncontrolled heating at temperatures well
below boiling. This process is called pasteurization. Milk, beer and many other
beverages are usually pasteurized. This process does not sterilize a beverage, but
it does kill any pathogen present. Milk can be pasteurized in two ways (i) in the
older method; the milk is heated at 63 C for 30 minute. (ii) Flash pasteurization
0

consists of quick heating to about 72 C for 15 seconds followed by rapid cooling. iii.
0

Tyndallization: Sometimes a heat sensitive material is sterilized by fractional steam

sterilization, called tyndallization. The container with the material to be sterilized is
heated at 90-100 C for 30minutes on each of three consecutive days and incubated
0

at 37 C in between. The first heating will destroy all microbes except bacterial
0

endospores. Most of these germinate when incubated at 37 C and are killed by the
0

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second heating. Any remaining spores are destroyed by the second incubation and
third heat treatments, many materials specially the liquid media for microbial
cultures. iv. Autoclaving: Steam under pressure is more efficient method of
sterilization this technique and is known as autoclaving. Autoclave is a cylindrical
metallic vessel with double walls. There are various types of autoclaves in use.

1) Simple autoclave: In which the body is made up of gun metal and it is

cylindrical in appearance and closed at one end by hinged door. A gasket seal is
provided between the door and cylinder. It can withstand high temperature. A
perforated metal tray is provided within the barrel which is used for keeping those
articles which are to be sterilized. The water present below the perforated tray is
boiled by an electric heater to produce the steam.

2) Steam jacketed autoclave: It is a modified form of simple autoclave. In simple

autoclave much of heat is wasted from the surface of barrel. To check this, a
steam jacket is provided around the barrel in large autoclave. Inside the
autoclave steam pressure is increased and the temperature increases
proportionately. Usually autoclaving is done at 15 lb. pressure for 15 minutes.
The autoclave is used to sterilize most of the solid and liquid media required for
microbial cultures.

II. Sterilization by filtration : It is a best way to sterilize the solutions of heat

sensitive materials. This method simply removes the microbes instead of directly
destroying them. There are two types of filters.
(a) Depth filters: These consist of fibrous or granular materials that have been
bonded into a thick layer filled with twisting channels of small diameter. The
solution containing microorganisms is sucked through this layer under vacuum
and microbial cells are removed by physical screening, Depth filters are made of
diatomaceous earth, unglazed porcelain (chamberlain filters), asbestos filters etc.
(b) Membrane filters: These filters have replaced depth filters. Such filters are
circular porous membranes, made of cellulose acetate, cellulose nitrate,
polycarbonate, polyvinylidene fluoride or other synthetic materials. Membranes
with pores about 0.2, μm in diameter are used to remove vegetative cells from
 solutions. The solution is forced through the filler with a vacuum or with pressure
from a syringe or nitrogen gasbottle and collected in previously sterilized
containers. Membrane filters remove microorganisms like a sieve with minute
pores. (c) Air sterilization by filtration: Air can also sterilize by filtration.
Surgical masks and cotton plugs on culture vessels are two common examples
that let air in but keep microorganisms out. Laminar flaw biological safety
cabinets fitted with high efficiency particulate air (HEPA) filters remove 99.97%
particles from the air.

III. Radiation: Sunlight is the major source of radiation on the earth. It includes
visible light, ultraviolet radiation (UV), Infrared rays and radio waves. At the surface
of the earth, very little UV

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radiation is found. The ozone layer presents between 25 and 30 miles above the
earth‘s surface absorbs somewhat larger UV rays. This elimination of UV is crucial
because it is quite damaging to living system. UV rays kill all kinds of
microorganisms due to its shortwave length and high energy does not penetrate
glass, dirt films, water and other substances very effectively. Many forms of
electromagnetic radiation are very harmful to microorganisms. As the wavelength of
electromagnetic radiation decreases, the energy of radiations increases e.g. gamma
rays and x-rays are much more energetic than the visible light of infrared rays.
Electromagnetic radiation acts like a stream of energy packets called photons. Each
photon possesses a quantum of energy whose value depends upon the wavelength
of the radiation.

IV. The ionizing radiations, because of very short wavelength or high energy cause
atoms to lose electrons ionize. Two major ionizing radiations are:
(i) X-rays produced artificially (ii) Gamma rays– Which are emitted during
ionizing radioisotope decay. Low levels of ionizing radiations cause mutations
and may indirectly results in death while higher levels are directly lethal. Ionizing
radiation is an excellent sterilizing agent and penetrates deep into objects.
Gamma radiation has also been used to pasteurize meat and other food.

(B) Chemical Methods:

It is a quick method of sterilizing instruments, glass apparatus or any other
article used in culture technique. The chemicals usually act as disinfectants because
they cannot readily destroy bacterial endopores. There are a number of chemicals
known for their property as:
(a) Disinfectant or germicidal (germ killing) e.g. Lysol, Cresol,
etc. (b) Antiseptic (microbial growth stopping) e.g. Ethyl
andisopropyl alcohols. (c) Sanitizer (reducing microbial
population to sap limit) e.g. Silver nitrate, mercuric chloride and
some other forms of mercury.
(C) Gaseous Methods: Many heat sensitive materials such as disposable plastic
syringes, petridishes, catheters, heart lung machine components etc. are now a days
sterilized with ethylene oxide gas. It is both microbicidal and sporicidal. It kills by
combining with cell proteins. It is very effective sterilizing agent as it rapidly
penetrates packing materials, even plastic wraps.
Betapropiolactone (BPL) is occasionally used to sterilize vaccines and sera. It
also destroys microorganisms more rapidly than ethylene oxide but does not
penetrate materials well and may be carcinogenic. Because of this, BPL has not
been used extensively.

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2.4 PREPARATION OF CULTURE

MEDIA
The organisms are grown on suitable culture media. A culture medium is a
nutrient preparation which provides a balanced mixture of the required nutrients at
concentrations that will permit good growth of microorganisms.
The culture media are generally of following
types:
I. Living culture media. II. Natural
culture medium III. Synthetic
culture medium. IV. Complex
media or non-Synthetic.

Culture media are variously

classified as:
(A) On the basis of composition
:
1- Living culture media : Such media require living cells, tissues or callus to be
parasitized by the microorganisms
to be cultured. Chick embryo is commonly used for cultivation of certain viruses.
2- Natural or Empirical culture Media : The empirical or natural media mostly
contain either one or many in gradient. In such medium, the exact composition is not
defined. Natural media are convenient and inexpensive. However, these are not
ideal media for many organisms. Milk, Skim milk, wine diluted blood, vegetable
juices are some of the natural media. 3- Synthetic medium: Synthetic medium is
prepared by mixing many components in a particular ratio. In this the exact chemical
composition of the medium is known. Such media contain highly pure organic and
inorganic compounds. Nutrient Agar is synthetic medium. 4. Complex Media:
Complex media are those, in which chemical composition is not well defined. These
media are non- synthetic. These media are useful for culturing a variety of
microorganisms. Peptone, yeast extract, meat extract, Beef extract etc. used in
complex media. (B) On the basis of Physical state :
1- Liquid media: These are defined as water based solutions that do not solidity
at temperature above freezing and flow freely when the Container is tilted. These
media are made by dissolving various solutes in distilled water. The liquid media
are termed broths, milks or infusions. 2- Semisolid Media: The media which
exhibit a clot-like consistency at room temperature are called semi soiled media.
They do not flow freely. They contain a solidifying agent agar or gelatin that
thickens them but does not produce a firm substrate. These are used to
determine the motility of bacteria and to localize a reaction at a specific site. 3-
Solid Media: The media which provides firm surface on which cells can form
discrete colonies are called solid media. These are useful in isolating and sub
culturing bacteria and fungi. They are of two types.

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(i) Liquefiable solid media: These are also called reversible solid media, They
contain a solidifying thermoplastic agent. The most widely used agent is agar-
agar. Agar is solid at room temperature and is flexible and mouldable. It has the
property to hold moisture and nutrients. It is a non-digestible nutrient for the vas-
majority of microorganisms. (ii) Non-liquefiable solid media: These are not
thermoplastic. They includes materials such as rice grains (used to grow fungi),
cooked meat and potato slices. These media start out solid and remain solid after
heat sterilization.
Potato dextrose agar
(PDA)
This is used for growing fungi and is prepared in the laboratory. The
ingredients are :
Potatoes peeled and sliced –
200gm. Dextrose – 20 gm. Agar
– 15 gm. Distilled water– 1000
ml.
Potatoes sliced are first steam cooked in 500 ml water and agar is mixed in
other 500 ml. water. Now both are mixed together; filtered and dextrose is added.
(C) On the basis of function (Functional
Types)
1. General Purpose media: Those media which support the growth of many
microorganisms, are called general purpose
media.
These are non- synthetic and contain a number of nutrients that could support the
growth
of both pathogens and non-
pathogens.
 2. Enriched Media: The specially fortified media are called enriched media.
These contain complex organic substances such as blood, serum, hemoglobin or
special growth factors like vitamins, amino acids which are the requirments of
some microorganisms to grow. Bacteria that require growth factors and complex
nutrients are called as fastidious (e.g.: Streptococcus pneumonia). 3. Selective
and Differential Media: These media are meant for special microbial groups.
These help in the preliminary identification of a genus or even a species, in a
single step. (a) Selective Media: These contain one or more substances that
inhibits the growth of certain microbe/ microbes but not others e.g. Use of dyes
like basic fuchsin and crystal violet favours the growth of gram negative bacteria.
Some selective media contain strong inhibitory substances. e.g.: Tellurie is used
to isolate oral streptococci from saliva. Some nutrients are used in the media
specifically, e.g. cellulose for cellulose digesting
bacteria. (b) Differential Media: These media are used for the growth of several
types of microorganisms but are used to differentiate different groups of
microbes. These are also used for in tentative identification of microorganisms on
the basis of their biological variations. Variations maybe in colony size and the
colour, the colour changes of media and the formation of bubbles and
precipitation properties. These

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variations are due to the type of agents added and the way the cells react to
them. Blood agar is a differential as well as enriched medium. It distinguishes
between hemolytic and non- hemolytic bacteria. Hemolytic bacteria produce
clear zones around their colonies as a result of red blood cell destruction.
(D) Some Miscellaneous
Media:
1- Reducing Medium: A reducing medium contains a substance cystine that
absorbs oxygen reducing its availability. These media are useful in growing
anaerobic bacteria and also in determining oxygen requirement.
2- Carbohydrate fermentation Medium: These contain sugars that can be
fermente
d.
3- Transport Media: These are used to maintain and preserve specimens for a
period of time prior to clinical examination. These are also used to sustain delicate
species that die rapidly if not kept under stable condition.
4. Assay Media: These are used to test the effectiveness of antimicrobial drugs and
also to assess the effect of antiseptics, cosmetics and preservatives on the growth of
microorganisms.
5. Enumeration Media: These are used by industrial and environmental
microbiologists to count the numbers of organisms in milk, water, food, soil and other
samples.

2.5 DISPENSING THE MEDIUM

 Dispensing is the process in which the medium is poured into the sterilized
flasks, culture tubes and Petri dishes. The unsterilized medium is usually poured into
the flasks and culture tube by semiautomatic syringe, funnel and automatic filter. The
liquid medium (Broth) is dispensed into culture tube or flasks which are plugged with
nonabsorbent cotton wool plugs. The pouring of sterilized medium is usually carried
out in Petri dishes which are already sterilized in a special sterilized inoculation
chambers.

2.6 SOME IMPORTANT CULTURE MEDIUM

Some important media which are generally used for culturing the
variousmicro- organisms are given below.
(A) For Bacteria
I. Nutrientagar: Beef extract - 3.0 gm.
Peptone -5.0 gm. Agar - 15.0 gm. Distilled
Water - 1,000 ml.
Heat unit/ agar and peptone dissolve. Adjust PH to 6.6
to 7.0.
II. Asparagin Mannitol agar
K HPO ----------------------1.0 gm.
2 4

KNO -------------------------0.5 gm.

3

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MgSo 4 7H2O----------------0.2 gm. Fe cl 3

6H2O------------------in traces.
Nacl---------------------------0.1 gm.
Asparagine-------------------0.5
gm. Mannitol---------------------1.0
gm. Agar--------------------------
15.0 gm. distilled
water---------------1,000ml.

After the agar and salts have been dissolved add the mannital and adjust the
PH to 7.4.
Mac Conkey‘s agar medium is a typical selective medium. It made up of the
following components –
Peptone - 20 gm. Lactose - 5gm.
Neutralredsolution - 10 ml. ( 1%)
Nacl - 5gm. Bile salt - 1.5 gm. Agar
- 13.5 gm. Crystal violet - 0.001gm.
Distilled water - 1.000 ml.
It is used for the culture and isolation of gram negative lactose fermenting
bacteria.
 • Blood Agar is the most commonly used differential medium. The blood agar
medium consists of the following components ;
Infusion from beef heart - 500gm.
Tryptose - 10gm.
Nacl - 5gm.
Agar - 15gm.
Distilled water - 1000ml.
If mixtures of bacteria are inoculated into this medium, the hemolytic and non-
hemolytic bacteria can be identified.
(B) For fungi: I. Potato dextrose agar
(PDA) Potato and sliced potato
-------------200gm.
Dextrose--------------------------------20 gm.
Agar-------------------------------------15 gm.
Distilled water -------------------------
1,000ml.

II. Czapek- Dox agar : Sucrose

--------------------------30.0 gm.

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Sodium nitrate ------------------2.0 gm.

Potassium chloride -------------0.5gm
Magnesiumsulphate--------- 0.5 gm.
Ferrous sulphate ----------------0.01
gm. Dipotassium phosphate--------
1.0 gm. Agar ------------------------------
15gm. Distilled water --------------------
1,000ml.

III. Rose- Bengal agar (Cooke’s

medium) Glucose
-----------------------------10.00 gm
Peptone -------------------------------5.00
gm. Dipotassium phosphate -----------
1.00 gm. Magneciumsulphate
---------------0.50gm. Streptomycin
-----------------------30.00gm. Agar
---------------------------------15.00 gm.
Rose- Bengal----------------------- 0.035
gm. Distilled water ---------------------
1,000ml.
 The antibiotic is sterilized separately and added aseptically to the sterilized
medium. The medium is specially recommended for isolation of fungi in the
presence of large numbers of bacteria,
IV. Smith and Dawson’s
medium : Glucose
--------------------- 10.0 gm.
NaNo ----------------------1.0 gm. KH
2 2

Po --------------------1.0 gm. Agar

4

-------------------------15.0gm. Rose
Bengal ----------------0.067 gm.
Soil extract------------------ 1,000
ml.
The soil extract is prepared by autoclaving500 gmof loam soil in 1200 ml. water
for one
hour. The extract is then filtered through
paper.
(C) For Actinomycetes:
Ken knight and Munaier‘s
medium Dextrose
---------------------1.00 gm. KH 2

Po --------------------0.10 gm. NaNo ----------------------

4 3

0.10 gm. Kcl---------------------------

0.10gm. MgSo4 7H O--------------0.10 gm.
2

Agar -------------------------15.0gm.
Distilled water---------------1,000
ml.

(D) For specialized

fungus : (i) For Yeasts
Malt extract agar

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Malt extract----------------------30.0
gm. Peptone---------------------------
5.0 gm.
Agar------------------------------20.0
gm. Distilled water------------------
1,000 ml.

(ii) For Aspergillus niger : Raulins

Medium Sugar ------------------- 70.0 gm
Tartaric Acid----------- 4.0 gm.
Ammonium nitrate ------4.0 gm.
Potassium carbonate ------- 0.6 gm.
 Ammonium phosphate-----0.6 gm.
Magnesium carbonate-------o.4 gm.
Ammonium sulphate--------- 0.25 gm.
Zinc sulphate (crystal)-------- 0.07 gm.
Ferrous sulphate(crystal)------0.07 gm.
Potassium silicate -------------0.07 gm.
Distilled water ----------------
upto1,000ml.

2.7 METHODS OF OBTAINING PURE

CULTURE
Pure Culture: A culture containing only one type of microorganism is called a pure
culture. The microbes are found distributed everywhere in nature and hence they
occur only as a mixed form. When culture media are inoculated with substances
such as soil, water or excreta, many kind of organisms develop simultaneously, and
it results the growth of a mixed culture of microbes. For the study of a single
microorganism a pure culture of that organism is required.
Any technical procedure for obtaining pure culture is dependent upon the
isolation of a single viable microbe which is allowed to multiply in a suitable culture
medium. The first reliable method of isolation of pure culture was devised by Robert
Koch in 1881 and his theory is known as ̳Koch‘s Postulated‘
Koch’s
Postulates
1- The pathogen or organism must be constantly associated with the symptoms
of the
disease in all diseased plants examined. 2- The pathogen must be isolated and
grown in pure culture on nutrient media. 3- The pathogen from pure culture must
be isolated and inoculated on healthy plants of some species on which the
disease appears. andit must produce the same symptom of disease on
inoculated plants. 4- The pathogenmust be again isolated in pure culture and its
culture and characteristics must be resemble with previous culture. So that the
isolated pathogen is identified and confirmed for the disease.

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If nutrient agar isinoculated with fluid and is then solidified and kept underfavorable
temperature, many of the microbes that have been introduced are able to multiply
and formdistinct colonies. If the colonies are not closely crowded, a pure culture may
be obtained by touching a colony with the tip of a sterile needle and inoculating it in a
fresh culture medium.
2.8 METHODS OF ISOLATION
The isolation of one kind of microorganisms from a mixture is called pure
culture technique. The petridishes or flasks with sterilized needles are inoculated
with an organism and are placed in culture chamber for its growth. Before inoculation
both the hand and inoculation instruments etc. are sterilized by wiping them with
cotton wool soaked in alcohol.
Some important methods used for obtaining pure culture are as
follows:
I. Pour Plate Method : In this method the mixed culture is diluted in sterile medium
and the diluted mixture is added to culture tubes containing melted agar medium.
The contents of the tubes are then poured into a sterile petridish and allowed to
solidify. The plates are then incubated. The cells of different of microorganisms
develop into colonies and cells from individual colony are picked up for further
culture.

II. Streak Plate method : Streaking is the most widely used method of isolation. This
method is most suitable for bacterial and fungal cultures. In streak plate method, the
mixed culture is taken on a sterile wire loop (inoculum) and is drawn back and forth
on a solid agar medium in a petridish. The successive streaks thin out the culture
sufficiently. In this method, isolated individual cells are deposited on some region of
the plate. The needle is flamed and allowed to cool after each streaking. Several
such streaks are made on the medium. The streaking is done in some definite
pattern (Fig.2.1). Fig.2.1 Different ways of streaking on agar plate

Streaking method needs a lot of care not to break the surface of the medium
during streaking incubation each cell grows into a colony.

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III. Serial Dilution Method : The dilution of sample in successive stages is called
serial dilution. This method is most suitable for the bacteria and fungi which cannot
be easily isolated by streaking method. The mixture of microorganisms is diluted
serially in culture tubes of sterile medium until the last tube contains only a single
organism. In this method, the dilution factor increases in a regular fashion e.g.: 1/10,
1/100, 1/1000 etc. In this method 1ml. of sample is mixed to 9ml. of sterile water in
culture tube. This gives a tenfold dilution and this dilution factor is represented as
1/10 or 10 , Now from this 10 fold dilution, 1 ml. of sample is taken and is added to 9
-1

ml. of sterile water taken in a second culture tubes Now the second tube contains a
100 fold dilution and the dilution factor is represented as 1/100 or 10-2
 Fig.2.2 Serial dilution
technique

Similarly from tube two, 1ml. of sample is taken and is added to 9 ml. of sterile
water taken in a third culture tube. Now the third tube contain a 1000 fold dilution
factor is represented as 1/1000 or 10 . In same way, culture tubes 4 and 5 are
-3

prepared. Tube 5 provides 10 dilution and tube 5 provides 10 dilution. A 6 Tube

-4 -5 th

is prepared as a control containing 10 ml. of sterile water only.

Then from each tube, 1 ml. of diluted sample is taken and is added to an agar
plate (a petridish containing 10 to 15 ml. of melted agar medium). The 6 agar
plates are incubated at 25-30 C for 24 hours. A luxuriant growth of most of the
0

bacteria is obtained. By the serial dilution, the chance of dominant organism in

pure condition in the culture is increased. Ultimately, a little amount of the
suspension is pipette out and spread over the medium of petridish(Fig.2.2).
IV- Spread Plate Method:

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It is a modification pour plate method. In this method, the mixed culture is

serially diluted in sterile distilled water. A small amount of diluted mixture is then
poured on the surface of the agar plate and it is spread evenly using a sterile bent
glass rod. The isolated cells grow into colonies.

V- Single cell method:

In this method, a suspension of microbes is placed on the cavity slide.
Thereafter, a single cell is removed with the help of sterile micropipette with the aid
of a microscope. The cell is then transferred to sterile culture. The colony obtained,
originated from single cell.

VI- Enrichment Culture Method:

In this method, a particular nutrient, which favours the growth of the desired
bacterium, is added to the medium. When the mixed culture is placed in this
enriched medium, the desired bacterium will grow dominantly.

VII- Selective culture

method:
In this method a selective medium is taken which contains a chemical, which
suppresses undesirable species. e.g.: Crysisal violet inhibits gram positive bacteria,
when crystal violet is added to the medium, the medium will select the gram negative
bacteria.

VIII- Differential Culture

Method:
 In this method the specific chemicals are used in the medium and different
microorganisms are isolated on the basis of their colour, e.g.: in eosin- methylene
blue agar medium, E. coli will produce colonies with a brilliant green metallic colour
and Acrobactor acrogens will produce pink colonies with dark centres.
After obtaining the pure culture of a desired microbe, it may be grown and
maintained as a pure culture. This pure culture can be maintained by transferring the
organisms from one to another culture tube. This process is called subculturing.

2.9 CULTIVATION OF
VIRUSES
Viruses are unable to reproduce independently, So, they cannot be cultured
like other microorganisms. These are cultured indifferent ways depending upon the
type of living host which they require for multiplication.
I. Cultivation of plant viruses: Plant viruses can be cultivated in various ways.
Plant tissue cultures, cultures of separated cells or cultures of protoplast may be
used for cultivating plant viruses, Viruses can also be grown in whole plants.
Leaves of a healthy plant are mechanically inoculated when rubbed with a
mixture of viruses and an abrasive such as carborudum orcelite. When the cell
walls are broken by the abrasive, the viruses come in direct contact with plasma
membrane and infect the exposed host cells. Alocalized necrotic lesion often
develops

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due to the rapid death of cells in the infected area. Even when lesions do not
appear, the infected plant may show other symptoms such as change in colour or
leaf shape. Some plant Viruses are transmitted only if a diseased part is grafted
into a healthy plant.

II. Cultivation of Animal Viruses : In the past animal viruses were cultivated by
inoculating suitable host animals or embryonated eggs, usually six to eight days
after laying. Before inoculation the shell surface of egg is disinfected with iodine
and penetrated with a small sterile drill. After inoculation, the hole is sealed with
gelatin and the egg incubated. Because viruses reproduce only in a certain parts
of the embryos, so they must be injected into the proper region. The virus
infection produces a local tissue lesion called pock appearance which varies and
is characteristic of the virus.
Now a days, animal viruses are grown in tissue culture on monolayer of animal
cells. This technique evolved with the development of growth media for animal
cells and by the discovery of antibiotics for the preventions of bacterial and fungal
contamination.
 III. Cultivation of Bacteriophages : Bacteriophages are cultivated in either broth
or agar cultures of young, actively growing bacterial cells. The number of host
cells destroyed is so large that turbid bacterial cultures may clear rapidly as a
result of cellysis. For preparing agar culture the bacteriophage sample is mixed
with cool, liquid agar and a suitable bacterial culture. This mixture is then quickly
poured into a petridish containing a bottom layer of sterile layer. Wherever a
virion comes to rest in the top agar, the virus infects an adjacent cell and
reproduces eventually. Bacteriallysis result in a plaque in the opaque layer. The
appearance of plaque is characteristic of the phage being cultivated.

2.10 CULTURE
TECHNIQUES
Microbiologists use five basic techniques (also called five I‘s) to culture,
manipulate, examine and characterize microorganisms. These are:
(i) Inoculation (ii) Isolation (iii) Incubation (iv) Inspection (v) Identification These
techniques are used by all microbiologists, whether a beginner laboratory student or
researcher is attempting to isolate a useful bacterium from the soil or a clinical
microbiologist trying to find out the cause of patient‘s infection. These techniques
thus, help in handling and maintaining microorganisms as discrete entities.
(i) Inoculation: The process of transfer of inoculum (a sample containing
microorganisms) into a container of nutrient medium, which provides an
environment in which they grow is called inoculation. Inoculum may be obtained
from soil, water sewage, foods, air and inanimate objects. For determining the
cause of an infectious

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disease, inoculum is obtained from body fluid (blood, cerebrospinal fluid),

discharges (Sputum, urine, faeces) or diseased tissue.
The culture vessels (culture tubes, conical flasks, or petridishes) containing
appropriate culture media are inoculated using tools such as loops, needles,
pipettes, etc. For a properly controlled experiment, sterilization of the glassware,
equipment and culture media is necessary. This means that the inoculation must
start with a sterile medium. All inoculating tools and culture vessels should be
sterile. Measures are also taken to prevent the entry of undesirable microbes
while inoculating. This procedure is carried out in special rooms fitted with UV
lamps. Now a days for inoculation, special laminar flow (biological safety
cabinets), fitted with HEPA fitters are used.
For inoculation in culture tubes agar slants are
prepared.
Preparation of
Agarslants:
 Liquified agar medium is poured into culture tubes. The culture tubes are
plugged with cotton wool and sterilized in autoclave. The sterilized tubes are taken
out and placed in a slanting position and then allowed to cool. The sloppy surface
provides maximum area of the agar medium in the culture tube for the growth of
microorganisms (Fig.2.3).

Fig.2.3 Culture tube with agar

slant

Transfer of the Inoculum: The inoculation is done in inoculation chamber,

completely sterilized with ultraviolet light. The hand should also be cleaned and
sterilized with rectified spirit. For culture tube inoculation, the tube containing
inoculum with sterilized agar medium slant is held in one hand and the inoculating
needle in the other. The cotton plugs of the tubes are taken out with the help of
fingers in front of the flame of spirit lamp. The inoculum is picked out with the help of
needle and then inserted within the agar surface of the tube. Plugging of the tube is
immediately done to avoid contamination. For petridish inoculation, the lid is
removed to a minimum and inoculation is done in the centre of the dish. The
inoculated tubes or petridishes are finally incubated at desired temperature.
(ii) Isolation: Isolation is separation of the pathogen from host tissue or
from mixed
culture or its inoculation in culture media. To achieve proper isolation, a small
number of cells is inoculated into a relatively large volume or an expansive area
of medium. It generally requires (i) a medium with firm surface (ii) a petri plate (a
clear flat dish with a cover) and (iii) an inoculating loop. There are several ways
of isolation to obtain pure culture: as I. Pour plate method II. Streak plate
method III. Serial Dilution method. IV. Spread plate method V. Single cell
method. VI. Enrichment culture method VII. Selective Culture method. VIII.
Differential culture method. All these methods of isolation are already discussed
in detail in earlier chapter.

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(iii)Incubation: Once a culture vessel containing medium is inoculated, it is

placed in a temperature controlled chamber or incubator to facilitate
multiplication. This is called incubation. Usually they are incubated at 20 to 40 C 0

in Laboratory. Incubators can also control the content of atmospheric gases like
oxygen and carbon dioxide that may be required for the growth of microbes.
During the incubation period, the microbe multiplies and produces some visible
manifestation of growth.
 (iv) Inspection: It is essential to inspect a culture macroscopically during various
stages of inoculations. Colonies of microorganisms are readily visible especially
the colonies of bacteria and fungi appears. Colonies are actually large masses of
clinging cells. The colonies exhibit distinctions in size, shape, colour and texture.
Colony development on agar surface helps the microbiologists in identifying
microorganisms. Once a microorganism is isolated, it is a standard practice to
make a second level. Cultural called a subculture. This is done by removing a
small sample from one well isolated colony and transferring it into a separate
container of media. Because a colony consists of only one type of
microorganism, it yields a pure colour or auxenic culture for further testing and
identification. Cultural inspection is also done by using a microscope. This gives
information of many characteristics of cell microbiology including size, shape and
details of internal and external structures.

(v) Identification: The microorganisms isolated are identified by a combination of

microscopic and macroscopic appearance. These are useful in differentiating
simple prokaryotic cells from larger complex eucaryotic cells. Although,
appearance is of no use in the identification of bacteria because of similar
morphologies. For their identification other techniques that characterize their
cellular metabolism are used. These involve biochemical tests that can determine
fundamental chemical characteristics such as nutrient requirements, products
releasing during growth, temperature and gas requirements and methods of
deriving energy. By compiling macroscopic and microscopic characters and
results of biochemical tests, a profile is prepared which is then used in the final
identification of a microbe. Thus, microorganisms are identified in terms of their
macroscopic morphology, their microscopic characters, and their biochemical
reactions and genetic characteristics.
Maintenance of
culture:
Culture of microorganism is maintained for further studies. Many research
laboratories require a line of stock cultures. The stock cultures are continuously
maintained species that represent ―living Catalogues‖. The largest culture collection
is ̳American Type culture collection located in Rockville, Maryland, U.S.A. It
maintains frozen and freeze-dried fungal, bacterial, viral and algal cultures. Disposal
of cultures:
Some cultures are potential health hazard. Therefore, they require immediate
and proper disposal. Microbial cultures are disposed off in two ways: (1) Steam
sterilization by

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autoclave, and (ii) incineration (burning). Both are effective methods for destroying
microorganisms.

2.11 INSTRUMENTS USED IN MICROBIOLOGICAL

STUDIES
 Following are some of the basic requirements and equipments for culturing
microorganism under artificial conditions:
I. Culture vessels: Culture media are contained in culture vessels usually culture
tubes, conical flasks and petridishes. These are made of high quality corning
glass. The petri dishes are special culture dishes named after their inventor Julies
Richard Petri. Petri developed these dishes in 1887. They consists of two round
halves, top half overlapping the bottom.

Fig.2.4 Culture vessels; A. Culture tube B. Conical flask. C. Petri disc. D.

Culture Bottles

Culture tubes are of two size, the smaller are without rim and Large with rim. Almost
all sizes of flasks ranging from 50 ml to 1000 ml are used for microbial culture.
These are used to contain culture media before and after the sterilization and also
for culture the pathogen in liquid or semisolid medium (Fig.2.4). II. Plugs: Tubes and
flasks containing media or culture are always plugged with cotton wool so that, any
air which enters inside is filtered from all contaminating microorganisms. A plug
should project inside the tube about an inch and should have tuft outside the tube,
by which it can be taken out. The plug should fit accurately and tightly, but not so
tightly that it cannot be extracted when gripped between any two fingersof one hand.
The plug should also retain its shape, so that after withdrawl, it can readily be a
reinstered. III. Inoculating tools: For inoculation, tools like inoculating needles,
inoculating loops, syringes, etc. are used. Inoculating needles/ loops are made up of
plantinum wire/ nichrome wire fixed into a metal or glass rod at one end. In
inoculating needles the wire is straight whereas in inoculating loops, the free end of
the wire is bent in the form of a loop (Fig.2.5).

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 Fig.2. 5 Inoculating tools: A. Inoculation needle, B. Inoculation loop, C.
Syringe

IV Sterilization
Equipments:
Following equipments are used in sterilization of glass ware and culture
media: (i) Oven for dry sterilization (ii) Autoclave for steam sterilization
(iii) Filter sterilization Equipment (iv) Sterile rooms or inoculation
chambers (v) Laminar flow biological softy cabinets

(i) Oven for dry

sterilization:
Dry heat is mainly used to sterilize glass ware or other heat stable materials.
The objects are wrapped in aluminium foil and exposed to a temperature of
170 C for 90 minutes in an oven. It is an electrically operated device. It
0

consists of a big chamber with insulated walls and is fitted with electrical
hearers to raise the temperature level and a thermostat to maintain
temperature at a desired level (Fig.2.6).

Fig.2.6. Hot air

oven

(ii) Autoclave: Autoclave is the instrument used to sterilize culture media, glass
ware and other tools by high pressure steam, which developed inside the
sterilizing chamber by heating water. Steam pressure increases inside the
chamber with increasing heating time. The body of autoclave is made up of a
thick double walled cylinder. Inside the

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 cylinder at its bottom is fitted an electric immersion rood. The mouth of the
cylinder is fitted with a heavy tightly fitting lid. A pressure gauge is attached to
the lid to monitor pressure inside the cylinder. An outlet valve is also attached
to the lid. Laboratory autoclaves are commonly operated at a steam pressure
of 15lbm above atmospheric pressure which corresponds to a temperature of
2

120 C. Even bacterial spores that survive several hours of boiling are rapidly
0

killed at 120 C. The temperature at 15lb pressure for 15minutesis sufficient to

0

kill any organism, and to achieve complete sterility. If an autoclave is not

available, pressure cookers can be used for purpose of sterilization (Fig.2.7).

Fig.2.7
Autoclave

(iii)Filter Sterilization
Equipment :
Solutions of heat labile materials are sterilized by filtration through filters
capable of retaining microorganisms. The most commonly used filters are:
1. Membrane filters (Millipore filters): These consist of porous discs of
cellulose
esters.

2. Seitz filters: these consist of discs of asbestos cellulose

mixture.
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3. Sintered glass filters: These are prepared by fusing together fine

glass
fragment
s.

4. Candle filters: These are made up of unglazed

ceramic.

Fig.2.8 A to D Various types of

filters

The filter is appropriately mounted on a funnel like structure. The mounting is

inserted into a receiving flask. The complete assembly is sterilized by heat. The
solution to be sterilized is poured into the filter. Its passage through filter is
accelerated either by suction on the receiving flask or by pressure on the unfiltered
liquid (Fig.2.8. A-D).
(iv) Sterile Rooms (Inoculation chambers) : Inoculation is done in sterile rooms
or inoculation chambers. These are fitted with ultraviolet bulbs / lamps which emit
UV light in wave length range 260 to 270 mm. They are useful for killing microbes
in air and on object surfaces.

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(v) Laminar flaw Biological Safety

Cabinets :
The laminar flaw cabinets are available in different sizes. They can be placed
where needed and thus, eliminated the necessity for a separate room.
Laminar flow biological safety cabinets are filled with high efficiency
particulate air (HEPA) fitters which remove 99.97% of 0.3 μmparticles. These
are one of the most important air filtration systems. Laminar flaw cabinets
force air through HEPA fitters, then project a vertical curtain of sterile air
across the cabinet opening to protect a researcher from microorganisms
being handled within the cabinet and to prevent room contamination
(Fig.2.9).

Fig.2.9, A schematic diagram showing the air flow pattern in a biological

safety cabinet

V. Incubators: Majority of fungi grow reasonably well at room temperature, however

in order to induce maximum rate of growth and in some cases, to promote the
formation of certain type of spores and fruiting structures, higher or lower
temperature is essential. Incubator is the instrument, used for such purpose. It is an
electrical instrument similar to hot air ovens in construction and operation. The range
of temperature varies from room temperature generally between 20 C to 50 or 60 C.
0 0

The cultures of microbes are incubated at suitable temperature in these chambers

(Fig.2.10).
 Fig.2.10, An incubator for maintaining
cultures

VI. Colony
Counters:
This device is used to count the colonies of microorganisms developing on a culture
plate. These are generally of 2types - A. Quebec colony counter :

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In quebec colony counter, there is a platform which is marked with cross ruling (small
squares). An illuminator is there below the platform to illuminate the colonies and
above the platform is present a magnifying lens. This lens magnifies the colonies
and thus helps in counting. For counting the colonies, the culture plate is mounted on
the platform and illuminated from below. The colonies are counted easily against
back ground of small squares (Fig 2.11. A).

B. Electrical colony counter : The electrical colony counters are fined with an
electrode for marking the location of each colony, when a colony is touched by the
electrode; it is automatically recorded in the counter (Fig.2.11. B).

Fig.2.11. Colony counters A. Quebec colony counter B. Electrical

colony counter

C. Counting Chambers : To determine microbial members through direct counting,

counting chambers are used. These are easy, inexpensive and relatively quick. It
also gives information about the size and morphology of the organisms, being
counted. Petroff Hausser counting chambers are used for counting bacteria.
Haemocytometers are used for counting large eukaryotic microorganisms. Large
microorganisms such as protozoa, algae and yeasts (non filamentous forms) can be
directly counted with electric counters like coulter counter. It gives accurate
results with larger cells and is extensively used to count red and white blood cells. It
is not useful in counting bacteria because of small debris particles, formation of
filaments etc (Fig.2.12).

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Fig.2.12 Coulter
counter

2.12
SUMMARY
In this unit you have learnt
that –

• Microorganisms occur in natural environments; they are contaminated and

mixed with several other form of life.
• To study microorganism individually we have to separate them from mixed
forms and culture or grow them under artificial conditions.
• As they are widely distributed and undesirable microbes may enter into an
experiment and cause misleading results. To overcome these problem different
methods like sterilization, pure culture etc is done.
• In the isolation of microorganism for detailed study, one must take care to avoid
contaminations, for this various sterilization methods are used to produce aseptic
condition for microbial culture.
 • The microorganisms are grown on suitable culture media. A culture medium is a
nutrient preparation which provides a balanced mixture of the required nutrients
at concentrations that will permit good growth of microorganism.
• Various types of culture media are prepared for different microorganisms,
nutritional requirements of microbes vary from a few simple inorganic compounds
to complex organic compounds. So culture media used for culturing microbes are
extremely varied in nutrient component and consistency.
• Microorganisms usually grow in complex, mixed, population containing several
types. Isolation of single species or single microorganism is done to obtain pure
culture or auxenic culture various ways like spread plate method streak method,
Dilution methods, pour plate method, etc. are used to get pure culture or single
individual.
• For culturing obligate parasites (that cannot grow on artificial media) e.g.
viruses, live cell culture or host animals are required.

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• For all the culture process, to grow, to examine and to characterize

microorganisms five basic techniques are used by all microbiologists. These are:
(1) Inoculation (ii) Incubation (iii) Isolation (iv) Inspection and (v) Identification.
• Culture vessels, inoculation tools, sterilization equipments, inoculation
chambers, Laminate flow, incubators, colony counters and counting chambers
are the basic equipments used in culture techniques.

2.13
GLOSSARY
Acclimated micro-organism: Any microorganism that is able to adapt to
environmental
changes, such as change in
temperature.
Actinomycetes: A group of micro-organisms apparently intermediate between
bacteria and
fungi, and classified as
either.
Agar: A gelatin like material obtained from red algae and used to prepare culture
media on
which microbes are
grown.
Antimicrobial: A chemical that either destroys or inhibits the growth of microscopic
and
submicroscopic
organisms.
Auxenic culture: Pure culture i.e. a microorganism of a single species, growing in a
medium
free of other
microorganisms.
Bacterial filter: A special type of filter through which bacterial cells
cannot pass.
Bacteriophage: A virus that infects specific bacteria and usually
kills them.
Bacteriostatic: A chemical or physical agent that prevents bacterial growth and
their
multiplication without
killing.
Culture: to grow microorganisms artificially on a prepared food
material.
Culture medium: The prepared food material on which micro-organisms are
cultured.
Disinfectant: A physical or chemical agent that frees a plant, organ or tissue from
infection.
Incubation period: The Period between penetration of a host by a pathogen and its
first
appearance of symptoms on the
host.
Inoculate: To bring a pathogen into contact with ahost
plant.
Inoculation: Transfer of a pathogen into a host or culture
media.
Inoculum: The pathogen or causal part which causes disease when brought into
contact with
the
host.
Isolate: A single spore or culture derived from a mixture of microbes
culture.
Isolation: Separation of a pathogen or microbe from the host and its culture on a
nutrient
 mediu
m.

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Mechanical Inoculation: Inoculation of plant with a virus through transfer of sap

from a
virus infected plant to a healthy
plant.
Secondary Inoculum: Inoculum produced by Infections that took place during the
same
growing
season.
Serum: The watery portion of the blood remaining after
coagulation.
Sterilization: The elimination of pathogen or microorganisms from any surface, or
making
free from
microbes.
Virus: A submicroscopic obligate parasite consisting of nucleic acid and
protein.

2.14 SELF ASSESSMENT

QUESTIONS
2.14.1 Choose the correct answer from the given
below:
(a) Agar agar is obtained from: (i) Blue green algae (ii)
Green algae (iii) Brown algae (iv) Red algae

(b) Special culture dishes were invented by: (i) Robert Kotch
(ii) Julius Richard Petri (iii) Coulter (iv) None of the above

(c) The instrument in which wet heat sterilization is done to sterilize

objects is: (i) Autoclave (ii) Laminar flow (iii) Hotair oven (iv) Inoculation
chamber

2.14.2 Fill in the

blanks:
(a) A medium of known composition is called....................... medium. (b) A Pure
culture is a population of cells derived from....................... (c) One of the most
important air filtration system filled with high efficiency particulate
air (HEPA)
is...................................

2.14.3 Answer the following in one word or in one

sentence:
(a) How are heat labile materials sterilized? (b) Name the electrical counter with the
help of which largemicroorganisms such as
protozoa, algae and yeasts can be directly counted. (c) Heat sensitive
materials e.g. disposable plastic, syringes, petri dishes, catheters etc.
are sterilized with which
chemical?
2.14.4 Write True or False
(T/F):
(a) Cultivation of microorganism on a solid agar medium in a culture tube kept in a
slanting
position is called agarslant
culture.

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(b) The process of transfer of a sample containing micro organism into a

container of
nutrient medium is called
incubation.
(c) Selective and differential media are extensively used in isolation and
identification of
microbe
s.

2.14.5 Explain the

following –
(a) Types of culture media.
(b) Methods of sterilization for cultivation.
(c) Instruments used in cultivation of
microbes.
Answers
Keys:
Q.1- (a)-(iv), (b)-(ii), (c)-
(i)
Q.2- (a)-Synthetic, (b)-single cell, (c)- Laminar flaw
cabinets
Q.3- (a)-Filter sterilization, (b)-Coulter counter, (c)-Ethyline
oxide
Q.4-(a)-True, (b)-False, (c)-
True

2.15
REFERENCES
• Ananthanarayan&Panicker, 1997, Text Book of Microbiology oriental Long
man publishers.
• Bhagat Singh and Renu Singh, 2011. Microbiology for Medical
sciences. I.K. International Publishing House Pvt. Ltd., New Delhi.
• Baron, E.J., Peterson, L.R. &Finegold, S.M. Bailey &Scolt's
Diagnostic Microbiology, 1990, Mosby Publishers.
• Casida, L.E. 1968, Industrial Microbiology, John wiley and sons, New
York.
• Doelle, H.W. and C.G. Heden 1986. Applied Microbiology, Kluwer Academic
Press, London.
• Kamat, M.N. 1953. Practical Pathology. Prakash Publishing,
Poona.
• Kaushik. P. 1996, Introductory Microbiology. Emkay Publ.
Delhi.
• Mukherjee , K.G. and Ved Pal Singh, 1997. Frontiers in Applied
Microbiology, Rastogi Publ. Meerut.
• Norris, J.R. and D.W. Ribbons 1970. Methods in Microbiology. Academic
Press, London.
• Power, C.B. and H.F. Daginawala 1996. General Microbiology 2 vols. Himalaya
Pub. House, New Delhi.
• Prescott, I.J., J.P.M. Harley & A.D. Klein, Microbiology. Tata MC Graw
Hill.
• Robinson, R.K. 1990, Dairy Microiology. Elsevier Applied Sciences,
London.
• Ross, F.C. 1983. Introductory Microbiology. Charles E. Merril Publ. Co.
Columbus. Ohio.
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2.16. SUGGESTED
READINGS
1. Alexander, M. 1971. Microbial Ecology, John willeg&
sons.
2. Brock, T.D. & M.T. Madigen. Biology of Micro-organisms prentice Hall.
3. Bridge, E.A. Modern Microbiology. WMC Brown Publisher, oxford
England.
4. Bhagat Singh and Renu Singh, 2011.Microbiology for medical sciences.
I.K.
International Publishing House Pvt. Ltd. New
Delhi.
5. Dubey, R.C. & D.K. Maheshwari, A text Book of Microbiology. S. Chand &
Co.
Delhi
.
6. Kaushik, P. 1996. Introductory Microbiology.EmkayPubl,
Delhi.

2.17. TERMINAL
QUESTIONS
1. Give an account of the cultivation of micro
organisms.
2. Write short notes on the following –
(i)Synthetic media (ii) Non synthetic media
(iii)Autoclave (iv) Laminar Flow biological safety chamber
(v) Koch‘s postulates (vi) Dry heat sterilization.
3. Describe briefly the Basic requirements and apparatus for culturing
microorganism.
4. Discuss the different types of culture media used for successful culturing of
microbes.
5. Define sterilization with reference to microbiology Discuss different
sterilization
 method
s.
6. Describe in brief the five basic techniques of culturing microorganisms
under
artificial
conditions.
7. Differentiate the following –
i) Pure culture and Mixed culture ii)
Selective media and Differential media iii)
Inoculation and contamination iv)
Pasteurisation and Tyndallisation

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UNIT-3- STRUCTURE, CLASSIFICATION,

NUTRITION, REPRODUCTION AND ECONOMIC
IMPORTANCE OF BACTERIA
3.1-
Objectives
3.2-
Introduction
3-3-Structure of
Bacteria
3.4-
Classification
3.5-
Nutrition
3.6-
Reproduction
3.7-Economic
Importance
3.8-
Summary
3.9-
Glossary
3.10-Self assessment
question
3.11-
References
3.12-Suggested
Readings
3.13-Terminal
Questions

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3.1
OBJECTIVES
After reading this unit student will be
able:
 • To study the structure of
bacteria.
• To understand the characteristic features of
bacteria.
• To know different shapes of
bacteria.
• To classify the
bacteria.
• To analyze the nutrition and economic importance of
bacteria.

3.2-
INTRODUCTION

―Bacteria, the Hidden Heroes of the earth are very much

powerful‖
Bacteria Bacteria are microscopic unicellular prokaryotic organisms characterized
by the lack of a membrane-bound nucleus and membrane-bound organelles. Once
considered a part of the plant kingdom, bacteria were eventually placed in a
separate kingdom, Monera. Bacteria fall into one of the two groups, Archaebacteria
(ancient forms thought to have evolved separately from other bacteria) and
Eubacteria. A recently proposed system classifies the Archaebacteria, or Archaea,
and the Eubacteria, or Bacteria, as major groupings (sometimes called domains)
above the kingdom level.
Bacteria were the only form of life on earth which exist for 2 billion years. They were
first observed by Antony van Leeuwenhoek in the 17th century; bacteriology as an
applied science began to develop in the late 19th century, as a result of research in
medicine and in fermentation processes, especially by Louis Pasteur and Robert
Koch.
These microorganisms remarkably adaptable to diverse environmental conditions.
They are found in the bodies of all living organisms and on all parts of the earth—in
land, terrains and ocean depths, in arctic ice and glaciers, in hot springs, and even in
the stratosphere. Our understanding of bacteria and their metabolic processes has
been expanded by the discovery of species that can live only deep below the earth's
surface and by species that thrive without sunlight in the high temperature and
pressure near hydrothermal vents on the ocean floor. There are more bacteria, as
separate individuals, than any other type of organism; there can be as many as 2.5
billion bacteria in one gram of fertile soil.

3.3 STRUCTURE OF
BACTERIA
Bacteria display a wide diversity of shapes and sizes. Their cells are about one-tenth
the size of eukaryotic cells and are typically 0.5–5.0 micrometers in length (Fig.3.1).
However, a few species- for example, Thiomargarita namibieniensis and
Epulopiscium fishelsoni - are up to half a millimetre long and are visible to the
unaided eye; E. fishelsoni reaches 0.7 mm. Among the smallest bacteria are
members of the genus Mycoplasma, which measure

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only 0.3 micrometres, as small as the largest viruses. Some bacteria may be even
smaller, but these ultra microbacteria are not well-studied.

Fig.3.1: The Bacterial

cell

Most bacterial species are either spherical, called cocci (sing. coccus, from Greek
kókkos, grain, seed), or rod-shaped, called bacilli (sing. bacillus, from Latin baculus,
stick). Elongation is associated with swimming. Some bacteria, called vibrio, are
shaped like slightly curved rods or comma-shaped; others can be spiral-shaped,
called spirilla, or tightly coiled, called spirochaetes. A small number of species even
have tetrahedral or cuboidal shapes. More recently, bacteria were discovered deep
under Earth's crust that grows as branching filamentous types with a star-shaped
cross-section. The large surface area to volume ratio of this morphology may give
these bacteria an advantage in nutrient-poor environments. This wide variety of
shapes is determined by the bacterial cell wall and cytoskeleton, and is important
because it can influence the ability of bacteria to acquire nutrients, attach to
surfaces, swim through liquids and escape predators.
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Fig.3.2: A biofilm of thermophilic bacteria in the outflow of Mickey hot Springs,

Oregon, approximately 20 mm thick

Many bacterial species exist simply as single cells, others associate in characteristic
patterns: Neisseria form diploids (pairs), Streptococcus form chains, and
Staphylococcus group together in "bunch of grapes" clusters. Bacteria can also be
elongated to form filaments, for example the Actinobacteria. Filamentous bacteria
are often surrounded by a sheath that contains many individual cells. Certain types,
such as species of the genus Nocardia, even form complex, branched filaments,
similar in appearance to fungal mycelia.

Fig.3.3- The range of sizes shown by prokaryotes, relative to those of other

organisms and biomolecules

Bacteria often attach to surfaces and form dense aggregations called biofilms or
bacterial mats (Fig.3.2). These films can range from a few micrometers in thickness
to upto half a meter in depth, and may contain multiple species of bacteria, protists
and archae. Bacteria living in biofilms display a complex arrangement of cells and
extracellular components, forming secondary structures such as microcolonies,
through which there are networks of

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channels to enable better diffusion of nutrients. In natural environments, such as soil

or the surfaces of plants, the majority of bacteria are bound to surfaces in biofilms.
Biofilms are also important in medicine, as these structures are often present during
chronic bacterial infections or in infections of implanted medical devices, and
bacteria protected within biofilms are much harder to kill than individual isolated
bacteria (Fig.3.3).
Even more complex morphological changes are sometimes possible. For example,
when starved of amino acids, Myxobacteria detect surrounding cells in a process
known as quorum sensing, migrate toward each other, and aggregate to form fruiting
bodies up to 500 micrometres long and containing approximately 100,000 bacterial
cells. In these fruiting bodies, the bacteria perform separate tasks; this type of
cooperation is a simple type of multicellular organisation. For example, many cells
migrate to the top of these fruiting bodies and differentiate into a specialised dormant
state called myxospores, which are more resistant to drying and other adverse
environmental conditions than are ordinary cells.

The bacterial
surface
Cell Wall: The cell envelop is composed of the plasma membrane and cell wall. As
in other organisms, the bacterial cell wall provides structural integrity to the cell. In
prokaryotes, the primary function of the cell wall is to protect the cell from internal
turgor pressure caused by the much higher concentrations of proteins and other
molecules inside the cell compared to its external environment. The bacterial cell
wall differs from that of all other organisms by the presence of peptidoglycans which
is located immediately outside of the cytoplasmic membrane. Peptidoglycan is made
up of a polysaccharide backbone consisting of alternating N-Acetylmuramic acid
(NAM) and N-acetylglucosamine (NAG) residues in equal amounts. Peptidoglycan is
responsible for the rigidity of the bacterial cell wall and for the determination of cell
shape. It is relatively porous and is not considered to be a permeability barrier for
small substrates. While all bacterial cell walls (with a few exceptions e.g.
extracellular parasites such as Mycoplasma) contain peptidoglycan, not all cell walls
have the same structures. The cell wall is required for bacterial survival, but several
antibiotics stop bacterial infections by interfering with cell wall synthesis. There are
two main types of bacterial cell walls, those of gram-positive bacteria and those of
gram-negative bacteria, which are differentiated by their Gram-staining
characteristics. For both these types of bacteria, particles of approximately 2 nm can
pass through the peptidoglycan. If the bacterial cell wall is entirely removed, it is
called a protoplast but when it is partially removed, it is called a spheroplast. β-
Lactam antibiotics such as penicillin inhibit the formation of peptidoglycan cross-links
in the bacterial cell wall. The enzyme lysozyme, found in human tears, also digests
the cell wall of bacteria and is the body's main defence against eye infections in
human beings.

The gram-positive cell

wall
Gram-positive cell walls are thick and the peptidoglycan layer constitutes almost
95% of the cell wall in some gram-positive bacteria and as little as 5-10% of the cell
wall in gram- negative bacteria. The cell wall of some gram-positive bacteria can be
completely dissolved by lysozyme. In other gram-positive bacteria, such as
Staphylococcus aureus, the walls are

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resistant to the action of lysozyme. The matrix substances in the walls of gram-
positive bacteria may be polysaccharides or teichoic acids. The latter are very
widespread, and found only in gram-positive bacteria. There are two main types of
teichoic acid: ribitol teichoic acids and glycerol teichoic acids. The latter one is more
commonly found. These acids are polymers of ribitol phosphate and glycerol
phosphate, respectively, and only located on the surface of many gram-positive
bacteria. However, the exact function of teichoic acid is not fully understood. A major
component of the gram-positive cell wall is lipoteichoic acid. One of its purposes is
providing an antigenic function. The lipid element is to be found in the membrane
where its adhesive properties assist in its anchoring to the membrane (Fig.3.4).
 Fig.3.4: Cell Wall composition of
Bacteria

The Gram-negative cell

wall
Gram-negative cell walls are thin and unlike the gram-positive cell walls, they contain
a thin peptidoglycan layer adjacent to the cytoplasmic membrane. The chemical
structure of the outer membrane's lipopolysaccharides is often unique to specific
bacterial sub-species and is responsible for many of the antigenic properties of these
strains. Lipopolysaccharides, also called endotoxins, are composed of
polysaccharides and lipid A which are responsible for much of the toxicity of gram-
negative bacteria. It consists of characteristic lipopolysaccarides embedded in the
membrane (Fig.3.4).

Plasma Membrane: The plasma membrane or bacterial cytoplasmic membrane is

composed of a phospholipid bilayer and thus has all of the general functions of a
cell

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membrane such as acting as a permeability barrier for most molecules and serving
as the location for the transport of molecules into the cell. In addition to these
functions, prokaryotic membranes also function in energy conservation as the
location about which a proton motive force is generated. Unlike eukaryotes, bacterial
membranes (with some exceptions e.g. Mycoplasma and methanotrophs) generally
do not contain sterols. However, many microbes do contain structurally related
compounds called hopanoids which likely fulfil the same function. Unlike eukaryotes,
bacteria can have a wide variety of fatty acids within their membranes. Along with
typical saturated and unsaturated fatty acids, bacteria can contain fatty acids with
additional methyl, hydroxy or even cyclic groups. The relative proportions of these
fatty acids can be modulated by the bacterium to maintain the optimum fluidity of the
membrane.

Fig.3.5. - Structure of bacterial plasma

membrane
As a phoshpholipid bilayer, the lipid portion of the outer membrane is impermeable to
charged molecules. However, channels called porins are present in the outer
membrane that allow for passive transport of many ions, sugars and amino acids
across the outer membrane. These molecules are therefore present in the periplasm,
the region between the cytoplasmic and outer membranes. The periplasm contains
the peptidoglycan layer and many proteins responsible for substrate binding or
hydrolysis and reception of extracellular signals. The periplasm is thought to exist in
a gel-like state rather than a liquid due to the high concentration of proteins and
peptidoglycan found within it. Because of its location between the cytoplasmic and
outer membranes, signals received and substrates bound are available and
transported across the cytoplasmic membrane using transport and signalling
proteins imbedded there (Fig.3.5).
Flagella: Many kinds of bacteria have slender, rigid, helical flagella (singular,
flagellum) composed of the protein flagellin. These flagella range from 3 to 12
micrometers in length and are very thin—only 10 to 20 nanometers thick. They are
anchored in the cell wall and help to spin or pulling the bacteria through the water
like a propeller. The number and position of flagella vary in bacteria. The
arrangement may be monotrichous (a single polar flagellum), lophotrichous (a cluster
of polar flagella), amphitrichous (flagella at both the ends either singly or in cluster),
cephalotrichous (two or more flagella at one end of bacterial cell),

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peritrichous (cell surface evenly surrounded by several lateral flagella) or atrichous

(cells devoid of flagella).
A flagellum consists of three basic parts – the basal body, hook and filament. The
basal body attaches the flagellum to the cell wall and plasma membrane. It is
composed of a small central rod inserted into a series of rings. In gram positive
bacteria, only a distal (inner) pair of rings is present while in gram negative bacteria
two pairs of rings (the proximal and distal) are connected by a central rod. The hook
of the flagellum is present outside the cell wall and connects filament to the basal
body. It consists of different proteins. The hook of gram positive bacteria is slightly
longer than the gram negative bacteria. The outermost long part of flagellum is called
filament. It is made up of globular proteins called flagellin which are arranged in
several chains and form a helix around a hollow core (Fig.3.6).
 Fig.3.6- Structure of Bacterial
flagella

Pili (Fimbriae): These are other hair like structures that occur on the cells of some
bacteria. They are shorter than bacterial flagella. They are .2 – 20 mμ long and
about 7.5 to 10 nanometers thick (Fig.3.7). Pili help the bacterial cells attach to
appropriate substrates and exchange

Fig.3.7- Bacteria showing structure

of pili

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genetic information. Pili originate from cytoplasm. According to the function, pili are
of two types, (a) common pili which act to adhere the cell to the surface, (b) sex pili
which join the other bacterial cell for transfer of genome. These structures also affect
the metabolic activity of the bacterial cell. They are also equipped with antigen
properties as they act as thermolabile non-specific agglutinogen.

The Cell
Interior
The most fundamental characteristic of bacterial cells is their prokaryotic
organization. Bacterial cells lack the extensive functional compartmentalization seen
within eukaryotic cells.

Internal membranes: Many bacteria possess invaginated regions of the plasma

membrane that function in respiration or photosynthesis. These are called
mesosomes. These structures form the site for respiratory activity. The cytoplasmic
membrane is also the site of many metabolic activities, e.g. the organic and
inorganic nutrients are transported by permeases through plasma membrane. It
consists of enzymes of biosynthetic pathways that synthesize different components
of cell wall, such as peptidoglycogen, techoic acid, phospholipids and
polysaccharides.
The cytoplasm of bacterial cell is thick and semi-transparent. It lacks cytoskeleton
and cytoplasmic streaming compartmentation of cell organelles is absent in bacterial
cell. Concentrated deposition of certain substances is dectectable in the cytoplasm
of some bacteria. The volutin granules are found in some bacteria which serve as
reserve source of phosphate. Another reserve carbon and energy source called
polybeta-hydroxybutyrate is also found in aerobic bacteria. In some bacteria that live
in aqatic habitats, bright refractile bodies which are hallow and have regular shape
with more or less conical ends are observed by electron microscopy. These are gas
vacuoles which provide buoyancy.

Nucleoid region: Bacteria lack nuclei and do not possess the complex
chromosomes characteristic of eukaryotes. Instead, their genes are encoded within a
single double-stranded ring of DNA that is crammed into one region of the cell known
as the nucleoid region. Many bacterial cells also possess small, independently
replicating circles of DNA called plasmids. Plasmids contain only a few genes,
usually not essential for the cell‘s survival. They are best thought of as an excised
portion of the bacterial chromosome.

Ribosomes: Bacterial ribosomes are smaller than those of eukaryotes and differ in
protein and RNA content. Antibiotics such as tetracycline and chloramphenicol can
be used to observe the difference—they bind to the bacterial ribosomes and block
protein synthesis, but they do not bind to eukaryotic ribosomes. The ribosomes have
two sub units, a larger 50 s sub unit and a smaller 30 s sub unit. Each is composed
of proteins and ribosomal RNA.

3.4-
CLASSIFICATION
Classification is done to describe the diversity of bacterial species by naming and
grouping organisms based on similarities. Bacteria can be classified on the basis of
cell structure, cellular metabolism or on differences in cell components such as DNA,
fatty acids, pigments, antigens and quinones. While these schemes allowed the
identification and

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classification of bacterial strains, it was unclear whether these differences

represented variation between distinct species or between strains of the same
species. This uncertainty was due to the lack of distinctive structures in most
bacteria, as well as lateral gene transfer between unrelated species. Due to lateral
gene transfer, some closely related bacteria can have very different morphologies
and metabolisms. To overcome this uncertainty, modern bacterial classification
emphasizes molecular systematics, using genetic techniques such as
guanine/cytosine ratio determination, genome-genome hybridization, as well as
sequencing genes that have not undergone extensive lateral gene transfer, such as
the rRNA gene. Classification of bacteria is determined by publication in the
International Journal of Systematic Bacteriology, and Bergey's Manual of Systematic
Bacteriology. The International Committee on Systematics Bacteriology (ICSB)
maintains international rules for the naming of bacteria and taxonomic categories
and for the ranking of them in the International Code of Nomenclature of Bacteria.
The term "bacteria" was traditionally applied to all microscopic, single-cell
prokaryotes. However, molecular systematic showed prokaryotic life to consist of two
separate domains, originally called Eubacteria and Archaebacteria, (now called
Bacteria and Archaea) that evolved independently from an ancient common
ancestor. These two domains, along with Eukarya, are the basis of the three domain
system, which is currently the most widely used classification system in microbiology.
However, due to the relatively recent introduction of molecular systematic and a
rapid increase in the number of genome sequences that are available, bacterial
classification remains a changing and expanding field. For example, a few biologists
argue that the Archaea and Eukaryotes evolved from Gram-positive bacteria.
Identification of bacteria in the laboratory is particularly relevant in medicine, where
the correct treatment is determined by the bacterial species causing an infection.
Consequently, the need to identify human pathogens was a major impetus for the
development of techniques to identify bacteria.

Fig.3.8- Phylogenetic tree showing the diversity of bacteria, compared

to other organisms. Eukaryotes are colored red, archae green and
bacteria blue

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The Gram stain, developed in 1884 by Hans Christian Gram, characterises bacteria
based on the structural characteristics of their cell walls. The thick layers of
peptidoglycan in the "Gram-positive" cell wall stain purple, while the thin "Gram-
negative" cell wall appears pink. By combining morphology and Gram-staining, most
bacteria can be classified as belonging to one of four groups (Gram-positive cocci,
Gram-positive bacilli, Gram-negative cocci and Gram-negative bacilli). Some
organisms are best identified by stains other than the Gram stain, particularly
mycobacteria or Nocardia, which show acid-fastness on Ziehl-Neelsen or similar
stains. Other organisms may need to be identified by their growth in special media,
or by other techniques, such as serology.
Culture techniques are designed to promote the growth and identify particular
bacteria, while restricting the growth of the other bacteria in the sample. Often these
techniques are designed for specific specimens; for example, a sputum sample will
be treated to identify organisms that cause pneumonia, while stool specimens are
cultured on selective media to identify organisms that cause diarrhoea, while
preventing growth of non-pathogenic bacteria. Specimens that are normally sterile,
such as blood, urine or spinal fluid, are cultured under conditions designed to grow
all possible organisms. Once a pathogenic organism has been isolated, it can be
further characterised by its morphology, growth patterns, pattern of hemolysis, and
staining.
As with bacterial classification, identification of bacteria is increasingly using
molecular methods. Diagnostics using such DNA-based tools, such as polymerase
chain reaction, are increasingly popular due to their specificity and speed, compared
to culture-based methods. These methods also allow the detection and identification
of "viable but nonculturable" cells that are metabolically active but non-dividing.
However, even using these improved methods, the total number of bacterial species
is not known and cannot even be estimated with any certainty. Following present
classification, there are a little less than 9,300 known species of prokaryotes, which
includes bacteria and archaea; but attempts to estimate the true number of bacterial
diversity have ranged from 10 to 10 total species – and even these diverse estimates
7 9

may be off by many orders of magnitude (Fig.3.8).

3.5-
NUTRITION
Bacteria exhibit an extremely wide variety of metabolic types. The distribution of
metabolic traits within a group of bacteria has traditionally been used to define their
taxonomy, but these traits often do not correspond with modern genetic
classifications. Bacterial metabolism is classified into nutritional group on the basis of
three major criteria: the kind of energy used for growth, the source of carbon, and the
electron donors used for growth. An additional criterion of respiratory
microorganisms is the electron acceptors used for aerobic or anaerobic respiration.
Carbon metabolism in bacteria is either heterotrophic, where organic carbon
compounds are used as carbon sources, or autotrophic, meaning that cellular carbon
is obtained by fixing carbon dioxide.

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