Nep 1st Year Unit-3

Microbial diversity and technology/unit - 3
SYLLABUS: [Chapter No – 7]
MICROBIAL CULTURE AND PRESERVATION – Microbial cultures, Pure culture and axenic
cultures, subculturing, Preservation Methods – overlaying cultures with mineral oils,
lyophilisation. Microbial culture collections and their importance. A brief account on ITCC,
MTCC and ATCC.
MICROBIAL CULTURES
Microbial culture is a method of multiplying microbial organisms by letting them reproduce

in predetermined culture medium under controlled laboratory conditions.
A microbial culture medium is a mixture of substances that promotes and supports the
growth and differentiation of microorganisms. Culture media contain nutrients, energy
sources, growth-promoting factors, minerals, metals, buffer salts, and gelling agents (for
solid media). Culture medium is a gel or liquid designed to support the growth of
microorganisms or cells. There are different types of media for growing different types of
organisms or cells. One commonly used type of media is nutrient broth or agar.
The three main types of microbiological culture media are Liquid, semisolid and solid
media are routinely used for growth of micro-organisms. Culture methods involve taking
samples from the field and detecting the presence of microbe by culturing them.
PURE CULTURE:
In microbiology, a laboratory culture containing a single species of organism. A pure culture

is usually derived from a mixed culture (one containing many species) by transferring a small
sample into new, sterile growth medium in such a manner as to disperse the individual cells
across the medium surface or by thinning the sample manyfold before inoculating the new
medium. Both methods separate the individual cells so that, when they multiply, each will
form a discrete colony, which may then be used to inoculate more medium, with
the assurance that only one type of organism will be present. Isolation of a pure culture may
be enhanced by providing a mixed inoculum with a medium favouring the growth of one
organism to the exclusion of others.
. The top six methods used for obtaining pure culture of microorganisms.
1. Streak Plate Method:

This method is used most commonly to isolate pure cultures of bacteria. A small amount of
mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the
surface of the agar medium. The successive streaks “thin out” the inoculum sufficiently and
the micro-organisms are separated from each other.
1 | Afiya H. A., GSC - Hassan

These plates are incubated to allow the growth of colonies. The key principle of this method
is that, by streaking, a dilution gradient is established across the face of the Petri plate as
bacterial cells are deposited on the agar surface.
2. Pour Plate Method:

This method involves plating of diluted samples mixed with melted agar medium. The main
principle is to dilute the inoculum in successive tubes containing liquefied agar medium so
as to permit a thorough distribution of bacterial cells within the medium.
Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar
medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below
42°C). The bacteria and the melted medium are mixed well.
The contents of each tube are poured into separate Petri plates, allowed to solidify, and
then incubated. When bacterial colonies develop, one finds that isolated colonies develop
both within the agar medium (subsurface colonies) and on the medium (surface colonies).

These isolated colonies are then picked up by inoculation loop and streaked onto another
Petri plate to insure purity.
3. Spread Plate Method:

In this method, the mixed culture or microorganisms is not diluted in the melted agar
medium (unlike the pour plate method); it is rather diluted in a series of tubes containing
sterile liquid, usually, water or physiological saline.
A drop of so diluted liquid from each tube is placed on the center of an agar plate and
spread evenly over the surface by means of a sterilized bent-glass-rod. The medium is now
incubated. When the colonies develop on the agar medium plates, it is found that there are
some plates in which well-isolated colonies grow. The isolated colonies are picked up and
transferred onto fresh medium to ensure purity.
4. Serial dilution method:

As stated earlier, this method is commonly used to obtain pure cultures of those
microorganisms that have not yet been successfully cultivated on solid media and grow only
in liquid media.
A microorganism that predominates in a mixed culture can be isolated in pure form by a

series of dilutions. The aim of this dilution is to inoculate a series of tubes with a microbial
suspension so dilute that there are some tubes showing growth of only one individual
microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium,
containing 1,000 microorganisms i.e., 100 microorganisms/ml of the liquid medium.
If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we
would then have 100 microorganisms in 10 ml or 10 microorganisms/ml. If we add 1 ml of
this suspension to another 9 ml. Of fresh sterile liquid medium, each ml would now contain
a single microorganism.

5. Enrichment Culture Method:

Generally, it is used to isolate those microorganisms, which are present in relatively small
numbers or that have slow growth rates compared to the other species present in the mixed
culture.
The enrichment culture strategy provides a specially designed cultural environment by

incorporating a specific nutrient in the medium and by modifying the physical conditions of
the incubation. The medium of known composition and, specific condition of incubation
favors the growth of desired microorganisms but, is unsuitable for the growth of other types
of microorganisms.
AXENIC CULTURES:
A microbial culture that contains only one species, variety, or strain of

microorganism. Supplement Microorganisms can be grown under controlled laboratory
conditions in cultures where they are allowed to grow and reproduce.
The growth and maintenance of a single species in isolation, free from foreign or
contaminating species. Isolation in axenic culture is achieved usually by growing the species
in an environment that was sterilized previously and was thereby rid of contaminating
organisms.

SUBCULTURING:
Sub-culturing is a procedure of transferring of microorganism into fresh nutritive medium

from its stock culture. It includes transfer of culture from slant to slant, slant to plate, plate
to plate, plate to slant, solid medium to broth, and broth to solid media.
Subculturing prolongs the lifespan of the cells or microorganisms, allowing for long-term
maintenance and observation of the culture.
PRESERVATION METHODS:
Once a microorganism has been isolated and grown in pure culture, it becomes necessary to
maintain the viability and purity of the microorganism by keeping the pure culture free from
contamination. Normally in laboratories, the pure cultures are transferred periodically onto
or into a fresh medium (sub-culturing) to allow continuous growth and viability of
microorganisms. The transfer is always subject to aseptic conditions to avoid contamination.
Since repeated sub-culturing is time-consuming, it becomes difficult to maintain a large

number of pure cultures successfully for a long time. In addition, there is a risk of genetic

changes as well as contamination. Therefore, it is now being replaced by some modern

methods that do not need frequent sub-culturing.
These methods include refrigeration, paraffin method, cryopreservation, and lyophilization

(freeze-drying).
PARAFFIN METHOD/ PRESERVATION BY OVERLAYING CULTURES WITH MINERAL OIL
This is a simple and most economical method of maintaining pure cultures of bacteria and
fungi. In this method, sterile liquid paraffin is poured over the slant (slope) of culture and
stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and
prevents dehydration of the medium. This condition helps microorganisms or pure culture
to remain in a dormant state and, therefore, the culture can be preserved from months to
years (varies with species).
The advantage of this method is that we can remove some of the growth under the oil with
a transfer needle, inoculate a fresh medium, and still preserve the original culture. The
simplicity of the method makes it attractive, but changes in the characteristics of a strain
can still occur.
Agar slant cultures:
 All microbiology laboratories preserve micro-organisms on agar slant.

 The agar slants are inoculated and incubated until good growth appears.
 They are then covered with sterile mineral oil to a depth of 1 cm above the tip of slant
surface.
 The slants are incubated for 24hr or more and are then stored in a refrigerator.
 These cultures are periodically transferred to fresh media.
 Transfers are made by removing a loop full of the growth, touching the loop to the glass
surface to drain off excess oil, inoculating a fresh medium and then preserving the initial
stock culture.
 Time intervals at which the transfers are made which varies with the origin and
condition of growth.

 This is a simple and most economical method of preserving bacteria and fungi where
they remain viable for several years at room temperature.
LYOPHILIZATION (FREEZE-DRYING)
Freeze drying is a process where water and other solvents are removed from a frozen
product via sublimation (means to transform solid to vapour).
Sublimation occurs when frozen liquid goes directly to a gaseous state without entering a
liquid phase.
It is recommended to use slow rates of cooling, as this will result in the formation of vertical
ice crystal structures, thus allowing for more efficient water sublimation from the frozen
product. Freeze-dried products are hygroscopic and must be protected from moisture
during storage. Under these conditions, the microbial cells are dehydrated and their
metabolic activities are stopped; as a result, the microbes go into a dormant state and retain
viability for years. Lyophilized or freeze-dried pure cultures are then sealed and stored in the
dark at 4°C in refrigerators.
Freeze-drying method is the most frequently used technique by culture collection centers.
Many species of bacteria preserved by this method have remained viable and unchanged in
their characteristics for more than 30 years.
The Lyophilisation Process

 In this process the microbial suspension is placed in small vials.

 A thin film is frozen over the inside surface of the vial by rotating it in mixture of dry ice
(solid carbon dioxide) and alcohol, or acetone at a temperature of −78oC .
 The vials are immediately connected to a high vacuum line. This dries the organism
while still frozen.
 Finally, the ampules are sealed off in a vacuum with small flame.
 These cultures can be stored for several years at 40°C.
 This method is also employed for preservation of toxins, sera, enzymes and other
biological material.
 To revive microbial cultures it is merely necessary to break open the vial aseptically, add
a suitable stale medium, and after incubation make further transfers.
 The process permits the maintenance of longer number of culture without variation in
characteristics of the culture and greatly reduces the danger of contamination.
Advantage of Lyophilization
1. Only minimal storage space is required; hundreds of lyophilized cultures can be stored
in a small area.
2. Small vials can be sent conveniently through the mail to other microbiology
laboratories when packaged in a special sealed mailing container.
3. Lyophilized cultures can be revived by opening the vials, adding the liquid medium,
and transferring the rehydrated culture to a suitable growth medium.
MICROBIAL CULTURE COLLECTIONS AND THEIR IMPORTANCE
Microbes constitute the largest biomass on the earth and comprise of three domains of life
(bacteria, archaea and eukaryotes). Almost 90 % of this diversity is still unexplored.
These microbes play an integral and unique role in the functioning of the ecosystems in
maintaining a sustainable biosphere and productivity.
They are responsible for nutrient recycling and detoxification. They act as biological control
agents, biocatalysts and produce a wide variety of products that have pharmaceutical and
industrial applications. They are also thought to be solutions for the food and energy crisis
that the world may face in future.
We also understand today that they would play fundamental role in improving the quality of
life, alleviate poverty, malnutrition, etc.
Microbial Culture collections are at the very heart of efforts to conserve biological diversity.
 Their primary aim is to preserve their strain holdings while providing pure cultures
and genetic materials required for biotechnology applications, teaching, research

and other purposes, since their major task is to collect, preserve and make microbial
strains accessible to the public.
 To provide identification services according to the expertise of the culture collection
about different kinds of microorganisms.
 Some culture collections also offer microbiological services to academic institutions
and industry, and conduct studies related to systematics, thus contributing to the
wealth of knowledge and leading to the discovery of new taxa.
 Culture collections and gene banks therefore play a significant role, not only in the
development of biotechnology-based industries and in education, but more
importantly, in the conservation of microbial strains which constitute part of a
country's heritage.
 The use of microorganisms and cell cultures to solve agricultural, food, health,
energy, and environmental problems is now rapidly increasing and with this, there is
an ever-growing need for the preservation and storage of newly isolated strains,
genetically engineered strains, strains with plasmids and other useful
microorganisms.
A BRIEF ACCOUNT ON ITCC, MTCC AND ATCC
Culture collections are centres that provide authentic examples of organisms, often
microorganism cultures that can be grown or maintained in the laboratory. They normally
have a public service role and often provide other biological resources and services.
ITCC (THE INDIAN TYPE CULTURE COLLECTION)
The Indian Type Culture Collection (ITCC), was established in 1936 in the then Division of
Mycology and Plant Pathology, Indian Agricultural Research Institute[IARI], New Delhi with a
view to furnish the knowledge on living fungi.
It is known for National Repository for maintenance, supply and identification of fungal
cultures. Research Institutes of ICAR, CSIR, ICMR, State Agricultural Universities (SAUs),
traditional universities, national and multinational companies dealing with fungal cultures
are the regular clients and beneficiaries of the dedicated services provided by the ITCC. The
beneficiaries of ITCC are not only from India but also from other countries like Iran, Iraq,
Pakistan, Bangladesh, Nepal, Sri Lanka, Germany,France, United Kingdom and U.S.A.
The main objectives of ITCC are to act as a repository, to supply authentic fungal cultures
and identification of fungi as well as to provide related services to farmers, technocrats and
scientists working in research Institutions, Universities and Industries for teaching,
demonstration and investigational purposes to mycologists and plant pathologists
throughout the country.
SERVICES:

Identification of fungal and bacterial cultures

Identification of different groups of fungi (Zygomycetes, Ascomycetes and
Basidiomycetes) and fungi like organisms (Oomycetes) and plant pathogenic bacteria
are done on payment basis received from researchers and students of different parts
of the country along with the prescribed request form. The report is sent after
identification.
Supply of fungal cultures
Authentic fungal and bacterial cultures (Phytopathogenic and selected bacterial
biocontrol agents) are supplied as an active cultures on agar slants and are
dispatched along with the report by surface mail within the country while, by air mail
outside the country after three to four weeks of receiving the request in the
prescribed format.
Deposition of cultures
Fungal and bacterialcultures which are morphologically and molecularly identified
and are of Indian origin having some special features (producers of enzymes, toxins,
antibiotics) and newly reported taxa (including new genera, new species and new
records) will be deposited at ITCC to ensure ex-situ conservation of microbial
diversity.
MTCC (MICROBIAL TYPE CULTURE COLLECTION)
The Microbial Type Culture Collection and Gene Bank (MTCC), a national facility
established in 1986 is funded jointly by the Department of Biotechnology (DBT)
and the Council of Scientific and Industrial Rese arch (CSIR), Government of
India.
The MTCC, housed at the Institute of Microbial Technology (IMTECH),

Chandigarh, has established itself as a distinguished culture collection centre
for microbial resources in India.
The main objectives of this national facility are to act as a depository, to supply
authentic microbial cultures and to provide related services to the scientists
working in research institutions, universities and industries.
The MTCC scientists are actively engaged in research programmes on microbial

diversity, taxonomy and related areas and have so far described about 100
novel microbial taxa, including bacteria, actinomycetes and yeasts.
The main objectives of MTCC are as follows:
 ex-situ conservation of microbial resources of India

 To provide authentic microbial cultures to industries as well as academic

and research institutes
 To provide identification, freeze drying and other microbiology related

services
 To act as a depository of patent cultures, and
 To take up research activities on microbial diversity, taxonomy,

phylogeny and related areas in microbiology.
The MTCC scientists are actively engaged in research programmes on microbial

diversity, taxonomy and related areas and have so far described about 100
novel microbial taxa, including bacteria, actinomycetes and yeasts.
ATCC (American Type Culture Collection)
ATCC or the American Type Culture Collection is a nonprofit organization which collects,
stores, and distributes standard reference microorganisms, cell lines and other materials for
research and development.
ATCC headquarters and production facilities are based in Manassas, Virginia.
Established in 1925 to serve as a national center for depositing and distributing

microbiological specimens, ATCC has since grown to distribute in over 150 countries. It is
now the largest general culture collection in the world.
SERVICES
 ATCC provides specialized services as a biological resource center. Individuals and

groups can employ a safe deposit service for their own cell cultures, providing a
secure back-up for valuable biomaterials if required.
 ATCC also is able to retain secure samples of patented materials and distribute them
according to instructions and approval of the patent holder
 ATCC also provides biological repository management services to institutions,
agencies and companies wishing to outsource the handling of their own culture
collections.

SYLLABUS (CHAPTER - 8)
Viruses: general structure and classification of viruses; ICTV system of classification.

Structure and multiplication of TMV, SARS – COV2, and Bacteriophage(T2). Cultivation of
viruses, Vaccines and types.
Virus is an infective agent that typically consists of a nucleic acid molecule in a protein coat,
is too small to be seen by light microscopy, and is able to multiply only within the living cells
of a host.
Viruses (Latin ‘Venum – poisonous fluid’) are simplest forms of life. They are not cells, but
their study has provided a great deal of information about cells. Study of viruses is a branch
of biology called Virology. Viruses are cellular parasites. They are smaller than bacteria and
have a much more simplified organization.
The largest viruses are about 300nm in size, whereas the smallest known viruses are about
20nm
 Russian Botanist Iwanowsky (1892) was first to give clear cut evidence of virus. He
demonstrated their occurrence in tobacco leaves suffering from mosaic disease.
 W. M. Stanley, an American microbiologist crystallized tobacco mosaic virus (TMV)
after isolating from infected tobacco leaf juice. He thus showed that viruses are not
like typical cells. Stanley was awarded Nobel Prize for this work.
GENERAL CHARACTERS:
(a) They are non-cellular, self-replicating agents.
(b) They can grow and multiply intracellularly as an obligate parasite (i.e., grow only in
living host) or remain inert outside the host.
(c) Depending on the symmetry, they are of three types: cubical, helical and complex.
(d) The viruses consist of two parts: the centrally placed nucleic acid, covered by
protein coat.
(e) The nucleic acid is either DNA or RNA, but both do not remain together.
(f) The nucleic acid may be single or double stranded.
(g) The outer coverings i.e., shell or capsid is made up of protein units, called
capsomeres; except some animal viruses which are with additional polysaccharides.
(h) They have no machinery of their own for protein synthesis and thereby they use host
machinery for the synthesis of protein.
(i) During replication their nucleic acid directs the host cell to make different parts of
virus and when these parts assemble together they form a complete infectious
particle, the virion.
(j) They are transmitted very easily from one organism to another organism.
GENERAL STRUCTURE
There are predominantly two kinds of shapes found amongst viruses: rods, or filaments, and
spheres. The rod shape is due to the linear array of the nucleic acid and the protein subunits
making up the capsid. The sphere shape is actually a 20-sided polygon (icosahedron).
Virus is made of three components capsid, envelope and nucleic acid.
Capsid - The capsid is the protein shell that encloses the nucleic acid; with its enclosed
nucleic acid, it is called the nucleocapsid. This shell is composed of protein organized in
subunits known as capsomers. They are closely associated with the nucleic acid and reflect
its configuration, either a rod-shaped helix or a polygon-shaped sphere.
The capsid has three functions:
1) It protects the nucleic acid from digestion by enzymes,
2) Contains special sites on its surface that allow the virion to attach to a host cell,
and
3) Provides proteins that enable the virion to penetrate the host cell membrane and,
in some cases, to inject the infectious nucleic acid into the cell's cytoplasm.

Envelope - Many types of virus have a glycoprotein envelope surrounding the nucleocapsid.
The envelope is composed of two lipid layers interspersed with protein molecules
(lipoprotein bilayer. Many viruses also develop spikes made of glycoprotein on their
envelopes that help them to attach to specific cell surfaces.
Nucleic Acid – DNA or RNA is found at the core of the virus.
CLASSIFICATION OF VIRUSES:
Viruses can be classified primarily on their phenotypic characteristics, core content,

chemical composition, capsid structure, size, shape and genome structure.
I. Classification based on the presence of nucleic acid
DNA virus
The virus, having DNA as its genetic material. There are two different types of DNA virus
Single-stranded (ss) DNA virus: e.g. Picornaviruses, Parvovirus, etc.
Double-stranded (ds) DNA virus: e.g. Adenovirus, Herpes virus, etc.
RNA virus
The virus, having RNA as its genetic material. There are two different types of RNA virus
Double-stranded (ds) RNA virus: e.g. Reovirus, etc.
Single-stranded (ss) RNA virus: It is further classified into two Positive sense RNA (+RNA)
and negative sense RNA (-RNA).
Poliovirus, Hepatitis A, Rabies virus, Influenza virus are examples of single-stranded RNA
virus.
II. Classification based on the structure or symmetry
1. Rod or Spiral shaped or helical symmetry virus. E.g. TMV
2. Cubical or icosahedral symmetry shaped virus. E.g. Reovirus, Picornavirus
3. Complex virus. E.g.Bacteriophage
III. Classification based on the host range
Based on the type of host, there are three different types of viruses:
Animal viruses: These viruses infect by invading the cells of animals, including humans.
Prominent examples of animal viruses include the influenza virus, mumps virus, rabies virus,
poliovirus, Herpes virus, etc.
Plant viruses: These viruses infect plants by invading the plant cells. Well-known examples
of plant virus include the potato virus, tobacco mosaic virus, beet yellow virus, and turnip
yellow virus, cauliflower mosaic virus, etc.

Bacteriophage: The virus which infects bacterial cells is known as bacteriophage. There are
many varieties of bacteriophages, such as DNA virus, λ page, etc.
STRUCTURE AND MULTIPLICATION OF TMV
 TMV is a simple rod-shaped helical virus.
 TMV consisting of centrally located single- stranded RNA (5.6%) enveloped by a

protein coat (94.4%).
 The rod is considered to be 3,000 Å in length and about 180 Å in diameter.
 The protein coat is technically called ‘capsid’.
 R. Franklin estimated 2,130 sub-units, namely, capsomeres in a complete helical rod

and 49 capsomeres on every three turns of the helix; thus there would be about 130
turns per rod of TMV.
 The diameter of RNA helix is about 80 Å and the RNA molecule lies about 50 Å
inward from the outer-most surface of the rod. The central core of the rod is about
40 Å in diameter.
 Each capsomere is a grape like structure containing about 158 amino acids and
having a molecular weight of 17,000 dalton as determined by Knight.
 The ssRNA is little more in length (about 3300 Å) slightly protruding from one end of
the rod. The RNA molecule consists of about 7300 nucleotides; the molecular weight
of the RNA molecule being about 25,000 dalton.

MULTIPLICATION OF TMV
 Plant viruses like TMV penetrate and enter the host cells in toto and their replication
completes within such infected host cells.
 Inside the host cell, the protein coat dissociates and viral nucleic acid becomes free
in the cell cytoplasm.
 Although the sites for different steps of the viral multiplication and formation of new
viruses have not yet been determined with absolute certainty, the studies suggest ha
alter becoming free in the cell cytoplasm the viral-RNA moves into the nucleus
(possibly into the nucleolus).
 The viral-RNA first induces the formation of specific enzymes called ‘RNA
polymerases’ the single-stranded viral-RNA synthesizes an additional RNA strand
called replicative RNA.
 This RNA strand is complementary to the viral genome and serves as ‘template’ for
producing new RNA single strands which is the copies of the parental viral-RNA.

 The new viral-RNAs are released from the nucleus into die cytoplasm and serve as
messenger-RNAs (mRNAs).
 Each mRNA, in cooperation with ribosomes and t-RNA of the host cell directs the
synthesis of protein subunits.
 After the desired protein sub-units (capsomeres) have been produced, the new viral
nucleic acid is considered to organize the protein subunit around it resulting in the
formation of complete virus particle, the virion.
 No ‘lysis’ of the host cell, as seen in case of virulent bacteriophages, takes place. The
host ells remain alive and viruses move from one cell to the other causing systemic
infection. When transmitted by some means the viruses infect other healthy plants.
SARS COV-2
(Severe acute respiratory syndrome coronavirus 2)
The word “coronam” in latin means “crown” crown-like appearance under an electon
microcope due to spike glycoprotein on the envelope.
Corona virusbelongs to coronaviridae family causes diseases in mammals and birds. In

humans it causes respiratory infections ranging from the common cold to more severe
diseases such as Middle East respiratory syndrome (SARS) and Cov 19.
It is an enveloped virus made of linear ssRNA (+sense) with a spike proteins, nucleoproteins
and membrane proteins etc.
Structure of SARS COV-2
The structure of sars cov 2 is made of spike proteins, nucleoproteins, membrane proteins,
envelope and a genome.
Spike proteins: it is made up of glycoprotiens, it helps in binding with the host receptors
and facilitates entry.
Nucleoproteins: proteins bond to RNA genome forms nucleocapsid.
Membrane proteins: it is the central organizer of Cov assembly which determines shape of
viral envelope.

Envelope: these envelope proteins interacts with membrane protein to form viral envelope
Genome: ssRNA (+ sense strand) 30kb in length.
MULTIPLICATION OF SARS COV2:
The life cycle of SARS COV2 virus inculdes 6 steps:
1. Virus Entry
2. Translation of Viral Replication Machinery
3. Replication
4. Translation of Viral Structure Proteins
5. Virion Assembly
6. Release of Virus
Virus entry:
Binding of the spike proteins to the [ACE]2 receptors of host that causes some
conformational change allowing the entry of the virus inside into cytoplasm by endocytosis.
Translation of Viral Replication Machinery and Replication:

As the genome is ssRNA (+ sense strand = which means it is ready for translation and
contains the 5 prime cap and a polyA tail and is recognized by eukaryotic ribosomes)
On translation it forms proteins like replicase then many proteins are synthesized for a
succesfull life cycle of a virus translation and transcription must takes place.
The replication – transcription complex [RCT] is formed which involves many proteins
Translation of Viral Structure Proteins and Virion Assembly:
The part of the genome is broken down to form subgenomic RNA that specifically codes for
specific parts of the virus (nucleoprotein, spike, envelope etc) which are translated by
ribosomes that are bound to the endoplasmic reticulum (ER).
The ER forms double membrane vesicles (DMVs) in which the viral RNA is replicated and
shielded from the host’s innate immune system.
Nsp3 creates pores through which viral RNA leaves the DMVs for virion assembly. The
nucleocapsid proteins (N) remain in the cytoplasm and are assembled from genomic RNA.
They fuse with the virion precursor which is then transported from the ER through the Golgi
apparatus to the cell surface via small vesicles.

Release of Virus:
Virions are then released from the infected cell through exocytosis and search another host
cell.
BACTERIOPHAGE
Bacteriophage (In greek, phagein –to eat, bacteria – cater) A phage is a virus that attacks
bacteria. Phages are among the best known of viruses because of the ease of studying
bacterial systems.
Bacteriophages are viruses that infect bacteria. They are abbreviated as phages.
F. Twort (1915) and F. d ‘Herelle (1917) independently discovered bacteriophages.
Bacteriophages are highly host specific
STRUCTURE: Many phages are among the most structurally complex viruses known. The T2
phage is a typical one common group of phages. T2 Phage is a cellular.
Its body is divided into two parts- head and tail; the head is hexagonal and the tail is rod-
shaped, a protein shell surrounds the body.
Inside the shell, there is a’ double-strand DNA in its head. The body of the virus, called a
capsid, is a complex structure with a head containing DNA and a tail apparatus that attaches
to receptors on the surface of the bacterial host.
At the upper end of the trail, there is a collar, and at the lower end, there is a base plate,
some spikes and attachment fibers. In Viruses, there is no nucleus, cell membrane,
cytoplasm, and other organelles.

MULTIPLICATION OF T2 BACTERIOPHAGE (LYTIC CYCLE)
Step-1: Adsorption/ attachment
Attachment sites on the bacteriophage adsorb to receptor sites on the host bacterium.
Most bacteriophages adsorb to the bacterial cell wall, although some are able to adsorb to
flagella or pili. Specific strains of bacteriophages can only adsorb to specific strain of host
bacteria. This is known as viral specificity.
Step-2: Penetration
In the case of bacteriophages that adsorb to the bacterial cell wall, a bacteriophage enzyme
"drills" a hole in the bacterial wall and the bacteriophage injects its genome into the
bacterial cytoplasm. Some bacteriophages accomplish this by contracting a sheath which
drives a hollow tube into the bacterium. This begins the eclipse period. The genomes of
bacteriophages which adsorb to flagella or pili enter through these hollow organelles. In
either case, only the phage genome enters the bacterium so there is no uncoating stage.
Step-3: replication (DNA and protein synthesis)
Enzymes coded by the bacteriophage genome shut down the bacterium's macromolecular
(protein, rna, dna) synthesis. The bacteriophage replicates its genome and uses the
bacterium's metabolic machinery to synthesize bacteriophage enzymes and bacteriophage
structural components
Step -4: Maturation (assembly)

The phage parts assemble around the genomes
Step – 5: Release (lysis)
Usually, a bacteriophage-coded lysozyme breaks down the bacterial peptidoglycan causing

osmotic lysis and release of the intact bacteriophages.
CULTIVATION OF VIRUSES:
As viruses are intracellular obligatory parasites, they always need living cells for their
growth. They cannot be grown on any artificial media.
There are three methods employed for the cultivation of animal viruses
1. Animal inoculation
2. Embryonated eggs or chick embryo method.
3. Tissue culture or cell culture.
Animal Inoculation – Susceptible experimental animals like Mice, Monkey, Rabbits, and
Guinea Pigs etc. are used for the cultivation of viruses. Virus sample to be cultivated should
injected into the experimental animal. It is important to select specific host animal for
particular viruses. Route of inoculation of viral sample in the host cell also play important
role in cultivation of viruses. Other factors such as age and immunity of host animal also
affect the growth of viruses in the host.
Eg. Mice are the most widely employed animals in virology. The growth of the virus in
inoculated animals may be indicated by death, disease or visible lesion. Disadvantages of
animal inoculation are that immunity may interfere with viral growth.

Embryonate Eggs or Chick embryo method: Good pasture (1931) was the first who used
hen’s embryonated egg for the cultivation of viruses. Embryonated egg provides several
sites for the cultivation of viruses. Viz
1. Chorio- allantoic membrane
2. Allantoic cavity
3. Amniotic cavity
4. Yolk sac
5. Embryo Different site is used for growth of different viruses.
Tissue Culture: Steinhardt and colleagues (1913), was the first who used bits of tissue or
organ for the cultivation of viruses. Now advance techniques are develop in Tissue culture.
Three types of tissue cultures are available.
 Organ culture: - Small bits of organs are used for the cultivation of virus.
 Explants culture: - Fragments of minced tissue can be grown as ‘explants’ embedded

in plasma clots.
 Cell culture: - This is most common method for viral cultivation and growth of
viruses. Different types of cell cultures are used for different viruses

VACCINE AND ITS TYPES:
A vaccine is a suspension of weakened, killed, or fragmented microorganisms or toxins or

other biological preparation, such as those consisting of antibodies, lymphocytes, or mRNA,
that is administered primarily to prevent disease.
TYPES: There are several basic types of vaccines. Some vaccines are described here.
1. Attenuated whole-agent vaccines use living but attenuated (weakened) microbes.

Examples of attenuated vaccines are the Sabin polio vaccine and those used against
measles, mumps and rubella (MMR). The widely used vaccine against the tuberculosis
bacillus and certain of the newly introduced, orally administered typhoid vaccines contain
attenuated bacteria.

2. Inactivated whole-agent vaccines use microbes that have been killed. Inactivated virus
vaccines used in humans include those against rabies, influenza and polio (the Salk polio
vaccine). Inactivated bacterial vaccines include those for pneumococcal pneumonia, cholera,
pertussis (whooping cough) and typhoid.
3. Toxoids which are inactivated toxins, are vaccines directed at the toxins produced by a
pathogen. Examples: Vaccines against tetanus and diphtheria.
4. Subunit vaccines use only those antigenic fragments of a microorganism that best
stimulate an immune response. Subunit vaccines that are produced by genetic modification
techniques, meaning that other microbes are programmed to produce the desired antigenic
fraction, are called recombinant vaccines.
For example, the vaccine against the hepatitis В virus consists of a portion of the viral
protein coat that is produced by genetically modified yeast.
5. Conjugated vaccines Conjugate’ means ‘connected’ or ‘joined’. With some bacteria, to

get protection from a vaccine you need to train the immune system to respond to
polysaccharides (complex sugars on the surface of bacteria) rather than proteins. It
developed in recent years to deal with the poor immune response of children. The
polysaccharides are combined with porteins such as diphtheria toxoid. This approach has
led to the very successful vaccine for Haemophilic influenza type b, which gives significant
protection.
6. Nucleic acid vaccines or DNA vaccines are among the newest and most promising
vaccines, although they have not yet resulted in any commercial vaccine for humans. Ex-
periments with animals show that plasmids of “naked” DNA injected into muscle results in
the production of the protein encoded in the DNA.
ICTV SYSTEM OF CLASSIFICATION:
The International Committee on Taxonomy of Viruses (ICTV) is a committee which

authorizes and organizes the taxonomic classification of viruses.
They have developed a universal taxonomic scheme for viruses and aim to describe all the
viruses of living organisms.
Members of the committee are considered to be world experts on viruses. The committee
operates authoritative database (ICTVdB) containing taxonomic information for 1,950 virus
species, as of 2005. It is open to the public and is searchable by several different means.
The official objectives of the ICTV are:

1. To develop an internationally agreed upon taxonomy for viruses.

2. To develop internationally agreed upon names for virus taxa, including species and
subviral agents.
3. To communicate taxonomic decisions to all users of virus names, in particular the
international community of virologists, by publications and via the Internet.
4. To maintain an index of virus names.
5. To maintain an ICTV database on the Internet, this records the data that characterize
each named viral taxon, together with the common names of each taxon in all major
languages.
 Proposals for new names, name changes, and the establishment and taxonomic
placement of taxa are handled by the Executive Committee of the ICTV in the form
of proposals.
 All relevant ICTV subcommittees and study groups are consulted prior to a decision
being made.
 The name of a taxon has no status until it has been approved by ICTV, and names will
only be accepted if they are linked to approved hierarchical taxa.
 If no suitable name is proposed for a taxon, the taxon may be approved and the
name be left undecided until the adoption of an acceptable international name,
when one is proposed to and accepted by ICTV.
 Names must not convey a meaning for the taxon which would seem to either
exclude viruses which are rightfully members of that taxa, exclude members which
might one day belong to that taxa, or include viruses which are members of different
taxa.

SYLLABUS: CHAPTER – 9
Viroids – general characters and structure of Pstvd, Prions – general characters and prion
diseases. Economic importance of viruses.
___________________________________________________________________________
VIROIDS
GENERAL CHARACTERS:
 Viroids are obligate intracellular parasites.
 Viroids are small single-stranded, circular RNAs that are infectious pathogens. They
donot have capsid (protein)
 Until 1970 viruses were considered as the smallest infectious agents.
 viroids were discovered by T.O. Diener for the first time in 1971 on potato spindle tuber.
 Till now 33 viroids are known. Most viroids cause plant diseases and most commo example
is potato spindle tuber viroid.
 the human disease known to be caused by viroid is hepatitis - D
STRUCTURE OF VIROIDS:
Structure of viroid was first shown directly by electron microscope; Viroid’s are small,
circular, single stranded RNA molecules. They consist a short stretch of highly
complementary circular single stranded RNA without protein coat. Viroid’s are 240 to 380
nucleotides long and all of them have dumb-bell structures
H. J. Cross (1979) sequenced the nucleotide sequence of the Potato spindle Tuber Virus
(PSTV). It consists of 359 ribonucleotides and is characterized by numerous intermolecular
base pairings that lends ability in the structure. Structurally, it has been divided into file
structural/functional domains

These are:
1. Conserved central domain.
2. Pathogenic domain
3. Variable domain
4. Left terminal domain, and
5. Right terminal domain.
The structural domains are related to specific functions. The conserved central domain,
mainly the upper strand is involved with cleavage and ligation of RNA. The left and right
domain still waits for the function. Members of the avsunviroid group are different from the
structure described above. They lack a conserved central Domain (CCR) and possess a
ribozyme activity (a ribozyme is a catalytic RNA molecule, in the case RNA cleavage in the
ribozymic activity).
POTATO SPINDLE TUBER VIROID DISEASE:
The main host is potatoes but the disease also affects tomatoes and solanaceous members.
Symptoms: Causes foliage to be spindly, very upright, with overlapping leaflets and
sometimes with upward rolling of terminal leaflets. Plants will be stunted. Tubers may show
the following deformities; small, elongated, cylindrical, spindle or dumb-ell-shaped, with
prominent eyes evenly distributed over the tuber, and cracking. Sprouting is slower than in
healthy tubers.
Transmission and dispersal
The disease is mechanically transmitted by contact between healthy and diseased plants,
tractor wheels, tools, etc. Within potato plants, it is found most readily in the upper leaves
and tubers. Transmission in true seed of potato depends upon the cultivar. Long Range
dispersal mainly occurs through the movement of infected tubers and true seed. PSTVd can
also be spread by aphids.

Control:
There are no chemical or biological controls available to control PSTVd within infected
plants. Therefore control is essentially through the use of healthy virus-free seeds or
planting material, and good crop sanitation practices. If infection does occur, destruction of
infected plants and strict hygiene measures to prevent infection of subsequent crops are the
only courses of action.
PRIONS
Prions are protenaceous molecules without nucleic acid association. Prions are smaller than
viruses and can only be seen through an electron microscope when they have aggregated
and formed a cluster. The term "prion" is derived from proteinacious infectious particles
and refers to the pathogen that causes transmissible spongiform encephalopathies (TSEs).
It was first discovered by Stanley B Prisoner, and was awarded Nobel Prize in medicine.
Prions can survive heat, radiations and chemical treatment that normally inactivate viruses.
This small infectious particle is a disease-causing form of a protein called cellular prion
protein (PrPc). PrPc is mainly found on the surface of cells in the central nervous system
(CNS),
These include scrapie disease in sheep, bovine spongiform encephalopath (BSE or mad cow
disease) in cattle, Creutzfeldt-Jakob Disease (CJD) and Kuru in humans.
PRION DISEASES:
Prion diseases (also called transmissible spongiform encephalopathy) are very rare: All most
all known prion diseases are neurologic diseases. Creutz Feldt-Jacob disease, make up about
85% of the cases. There are about 1 to 1.5 cases per one million people per year.

HUMAN PRION DISEASES:

Creutzfeldt-Jacob Disease (Bovine Spongiform Encephalopathy):
A person can get the disease by eating of BSE (Bovine spongiform encephalopathy) infected
beef having a blood transfusion, from a person with the disease, injection of human growth
hormone extracted from bodies with the disease, or through infected surgical instruments.
Patients suffer from Ataxia or disequilibrium (a patient cannot stand or walk well because
he cannot maintain his equilibrium. This is because of a disease in cerebellum), dementia or
loss of mentality (progressive loss of cognitive functions) usually die after one year.
Kuru Disease:
This disease is seen people living in New Guinea who eat the brains of dead people. Patients
suffer from ataxia, dementia and inability in moving their eyes. Patients usually die within
two years.
Gerstmann-Straussler-Scheinker Syndrome:
Very rare syndrome, presents with ataxia and dementia Animal prion diseases.
ANIMAL PRION DISEASES
 Bovine Spongiform Encephalopathy (BSE)

 Chronic Wasting Disease (CWD)
 Scrapie
 Transmissible mink encephalopathy
 Feline spongiform encephalopathy
 Ungulate spongiform encephalopathy
ECONOMIC IMPORTANCE OF VIRUSES
Viruses have both harmful and beneficial roles in human life. Actually no virus directly
involves in human welfare, in most cases scientists use them as beneficiary agents in
different fields.
Harmful Effects of Viruses
 A vast number of viruses cause human and animal diseases. Some viruses are
epidemic which spreads rapidly too many people and some viruses are pandemic
which spread diseases worldwide. COVID-19 (corona virus disease) is very life
threatening disease that already has taken the lives of nearly few lakhs people
worldwide. In 2003, the severe acute respiratory syndrome (SARS) also took the lives
of nearly 800 people worldwide.
 Besides, a vast number of viruses cause plant diseases as rice tungro baciliform virus
(RTBV), tobacco mosaic virus (TMV), tomato/papaya ring spot virus, tomato leaf curl,
potato leaf roll virus, etc.
 Viruses are also used as biological warfare and weapons in many countries. Viruses
are tiny but they have the ability to produce diseases which can cause death and
damage to huge populations in epidemics and pandemics.
BENEFICIAL EFFECT OF VIRUSES
 Viruses are utilised in the production of vaccines, used to develop immunity against
viral infection.
 There are many industrial uses of viruses. Viruses are used in preparation of sera and
vaccines to be used against diseases like rabies, polio, hepatitis B, papillomavirus,
etc.
 The multiplication of viruses in bactereial cells is also utilized in the production of

antibodies. It is used in various pharmaceutical products such as proteins, vaccine,
antigens and antibodies.
 Viruses are also used in biological studies. They have gained a prominent position in
world because of their value as biological research tools.
 They are broadly used in research in the field of genetic engineering, molecular
biology and medicine due to their capacity to fast reproduction and plainness
structure.
 Viruses are used in bacteriophage therapy. Bacteriophages have been researched for
their use in therapy.
 There are many uses of viruses in medicine. In this case, it is being used as vectors or
carriers that take the required material for treatment of a disease to various target
cells.
 In the life science, it plays an important role to understand the basic mechanisms of
molecular genetics. It is also used in genetic engineering for the production of
cloning.
 It is used for gene therapy. In this case, viral genes are replaced by the human gene.
 Viruses are used in biological control by human in eradicating pests like insects (by
NPV) and in controlling the population of organisms such as rabbits by inducing viral
infection.
 The viruses also played a central role in the early evolution, before the diversification
of bacterial, archaea and eukaryotes, at the time of the last universal common
ancestor of life on earth. Viruses are still one of the largest reservoirs of unexplored
genetic diversity on earth. By holding both the living and nonliving characters,
viruses got the importance in determining the origin of life.
 Viruses are also used in aquatic environment to recycling carbon. About one million
of viruses are found in one teaspoon of seawater. Among them, majority are
bacteriophages. They are not harmful to animals and plants and are used as
scavengers to eradicate the bacteria present in the polluted water.

Nep 1st Year Unit-3

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Nep 1st Year Unit-3

Uploaded by

Copyright:

Available Formats

Microbial diversity and technology/unit - 3

Microbial culture is a method of multiplying microbial organisms by letting them reproduce

In microbiology, a laboratory culture containing a single species of organism. A pure culture

1. Streak Plate Method:

1 | Afiya H. A., GSC - Hassan

2. Pour Plate Method:

2 | Afiya H. A., GSC - Hassan

3. Spread Plate Method:

4. Serial dilution method:

A microorganism that predominates in a mixed culture can be isolated in pure form by a

3 | Afiya H. A., GSC - Hassan

5. Enrichment Culture Method:

The enrichment culture strategy provides a specially designed cultural environment by

A microbial culture that contains only one species, variety, or strain of

4 | Afiya H. A., GSC - Hassan

Sub-culturing is a procedure of transferring of microorganism into fresh nutritive medium

Since repeated sub-culturing is time-consuming, it becomes difficult to maintain a large

5 | Afiya H. A., GSC - Hassan

changes as well as contamination. Therefore, it is now being replaced by some modern

These methods include refrigeration, paraffin method, cryopreservation, and lyophilization

PARAFFIN METHOD/ PRESERVATION BY OVERLAYING CULTURES WITH MINERAL OIL

Agar slant cultures:

 All microbiology laboratories preserve micro-organisms on agar slant.

6 | Afiya H. A., GSC - Hassan

The Lyophilisation Process

7 | Afiya H. A., GSC - Hassan

MICROBIAL CULTURE COLLECTIONS AND THEIR IMPORTANCE

8 | Afiya H. A., GSC - Hassan

A BRIEF ACCOUNT ON ITCC, MTCC AND ATCC

ITCC (THE INDIAN TYPE CULTURE COLLECTION)

9 | Afiya H. A., GSC - Hassan

Identification of fungal and bacterial cultures

The MTCC, housed at the Institute of Microbial Technology (IMTECH),

The MTCC scientists are actively engaged in research programmes on microbial

The main objectives of MTCC are as follows:

 ex-situ conservation of microbial resources of India

10 | Afiya H. A., GSC - Hassan

 To provide authentic microbial cultures to industries as well as academic

 To provide identification, freeze drying and other microbiology related

 To act as a depository of patent cultures, and

 To take up research activities on microbial diversity, taxonomy,

The MTCC scientists are actively engaged in research programmes on microbial

ATCC (American Type Culture Collection)

ATCC headquarters and production facilities are based in Manassas, Virginia.

Established in 1925 to serve as a national center for depositing and distributing

 ATCC provides specialized services as a biological resource center. Individuals and

11 | Afiya H. A., GSC - Hassan

Viruses: general structure and classification of viruses; ICTV system of classification.

(a) They are non-cellular, self-replicating agents.

(f) The nucleic acid may be single or double stranded.

Virus is made of three components capsid, envelope and nucleic acid.

The capsid has three functions:

1) It protects the nucleic acid from digestion by enzymes,

13 | Afiya H. A., GSC - Hassan

Nucleic Acid – DNA or RNA is found at the core of the virus.

Viruses can be classified primarily on their phenotypic characteristics, core content,

I. Classification based on the presence of nucleic acid

III. Classification based on the host range

14 | Afiya H. A., GSC - Hassan

STRUCTURE AND MULTIPLICATION OF TMV

 TMV is a simple rod-shaped helical virus.

 TMV consisting of centrally located single- stranded RNA (5.6%) enveloped by a