its about nutritional, cultivation and isolation of bacteria, viruses and fungi

2.  Bacteria exhibit different modes of nutrition. They can
be:-
 Heterotrophic bacteria
 Autotrophic bacteria

3. Nutritional
habit of bacteria
Autotrophic heterotrophic
Photoautotrop
hic
-Green sulphur
bacteria
-Purple sulphur
bacteria
Chemoautotroph
ic
-Nitrifying
bacteria
-Iron bacteria
-Methanogens
Photoheterotrophi
c
Chemoheterotroph
ic
-parasitic
-saprophytic
-symbiotic

4.  Photosynthetic bacteria: use sunlight as source of energy
and electron donors are H2O, S and other sulphur
containing inorganic compounds.
 A) Green sulphur bacteria: contain bacterio chlorophyll
which is chemically similar to chlorophyll. Usually live in
anaerobic conditions.
CO2 + 12 H2S C6H12O6 + H2O + S
B) Purple sulphur bacteria : contain bacteriopurpurin. Here
the electron donors are some other sulphur containing
organic or inorganic compounds in place of H2S or
elemental S.

5. Chemoautotrophic bacteria
 Are without photosynthetic pigments.
a. Nitrifying bacteria: mainly found in soil.
- through their metabolic activities they convert
ammonia or ammonium ions into nitrates. The
formation of nitrates from ammonia or ammonium
ion is called nitrification.

6. b. Iron bacteria: found in iron containing rocks. Eg.
Ferrobacillus leptothrix.
c. Methanogens : produce methane and water from H2 and
CO2 eg. Hydrogenomonas.
H2 + CO2 CH4 + 2H2O + ENERGY

7. Heterotrophic bacteria
 Unable to synthesize their own metabolites and
depend on preformed organic compounds.
 They obtain carbon from organic compound made by
other organisms. Alcohols, fatty acids and other
organic substances serve as source of electron for
photosynthetic reaction.
 Eg. Purple non sulphur bacteria

8.  Chemo-heterotrophic bacteria:
- Take their food from other plants and animals.
- They obtain both energy and carbon sources from
organic compounds made by other organisms.
a) Parasitic bacteria: these can live in or upon
organisms, known as host, from which they
obtain their food and usually shelter.

9.  They may be Obligate parasite, grow only in living cells
and Facultative parasite (grow on dead material ).
b) Saprophytic bacteria: obtain their food from dead and
decaying organic matter of all living beings e.g, Bacillus
mycoides.
c) Symbiotic bacteria: symbiosis means a close ecological
relationship between two different species. This can be
mutually beneficial between the microbe and the host.
some bacteria live in symbiosis with other plants and
animals. Eg. Rhizobium legumenosarum.

10. Physical conditions required for growth of
bacteriaA) Temperature: the temperature that allows for most
rapid growth during a short period of time is known as
Optimum growth temp. A highest temp at which m.o
show growth is known as Maximum growth temp. and
the lowest temp at which m.o show growth is known as
Minimum growth temp.
Based on temp tolerance and its influence on growth ,
bacteria can be:-
1. Psychrophiles: able to grow at 0°C but have an optimum
temp of 15°C.
2. Mesophiles : grow within the range of 20-40 °C. All
bacteria that are pathogenic to humans and warm
blooded animals are mesophiles.

11. 3. Thermophiles : prefer high temp for growth (45-70°C).
 Some thermophiles which can grow in mesophilic range (
37-55°C) are called Facultative thermophiles. And those
which grow above 60°C are called true or obligate
thermophiles.

12. GROUP
GROWTH TEMP (°C)
EXAMPLES
MINIMUM OPTIMUM MAXIMUM
Obligate
psychrophiles
0-5°C 10-15 20
Vibrio
psychroerythrus
Facultative
psychrophiles
0-5°C 25-30 35
Pseudomonas
fluorescens
Mesophiles 15-20°C 35-40 45
Corynebacterium
diphtheriae
Facultative
thermophiles
35-40°C 50-55 65
Streptococcus
thermophilus
Obligate
thermophiles
50-55°C 65-70 80
Thermus
aquaticus

13. B) H- ion concentration:
• Bacteria are sensitive to variation in pH.
• Each bacteria has a pH range, above or below to
that bacteria cant survive.
• Optimum pH are at which bacteria grows best.
• Bacteria grows best at neutral or slightly alkaline
(pH-7.2-7.6.

14.  Depending on optimum pH value of m.o, they
can be classified as:-
1. Acidophiles- optimum pH range between 1-
6.5. eg. Lactobacillus acidophilus.
2. Neutrophiles – most bacteria grow in
narrow pH range of 6.5-7.5. eg. E-coli,
Salmonella typhi.
3. Alkalophiles – optimum pH range in between
7.5-14 . Eg, Vibrio cholerae (pH 9.0 )

15. C) Oxygen requirement
 Division is based on influence of oxygen into growth.
 Aerobic: require o2 for growth. Eg, E-coli
 Anaerobic: grow in absence of oxygen. Oxygen is toxic
to such cells and they cannot grow when incubated in
an air atmosphere.
 Some bacteria can tolerate low levels of oxygen are
called as tolerant anaerobes.
 Some bacteria cant tolerate even low levels of oxygen
and they may die after brief exposure to air, such
bacteria are called stringent bacteria. Eg, Clostridium
species.

16.  Microaerophilic: grow best in low o2 levels but
cant tolerate leve of oxygen present in air
atmosphere. Eg, Campylobacter jejuni.
 Facultative anaerobes: ordinarily aerobic but
can grow in absence of oxygen.
 Aerobic obtain energy through oxidation
 Anaerobes use hydrogen than oxygen

17. C) Moisture requirement
 Water is essential to bacterial protoplasm and
drying is lethal.
 Treponema is highly sensitive to drying.
 Spore may survive in dry state for several
decades.

18. D) Light requirement
 Bacteria (except phototropic) grow well in dark.
 They are sensitive to UV light and other
radiations.
 Cultures die if exposed to sunlight.

19. E) Osmotic effect
 Bacteria are more tolerant to osmotic variation
than most other cells due to strength of their cell
wall.
 Sudden exposure to hypertonic solution may cause
withdrawal of water and shrinkage of protoplasm.
 Sudden transfer from a concentrated solution to
a distilled water may leads to rupture of cells.

20.  Hyperosmotic environment: cell lose water and
undergoes plasmolysis (shrinkage of cells)
 Hypoosmotic environments : cell gain water
and swell and burst.

22. Halophiles
Salt loving organisms which require moderate to
large quantity of salt (NaCl).
Why do halophiles require sodium?
-cells need sodium to maintain high
intracellullar potassium concentration for
enzymatic function. Membrane transport
system actively transport sodium ions out of
the cells and concentrate potassium ions
inside.
- To maintain integrity of their cell wall.

23. Responses to Salt

24. Chemical Requirement
1. Carbon sources
2. Nitrogen sources
3. Sulfur and phosphorus
4. Trace elements (e.g. copper, iron,
zinc, and cobalt)
5. Vitamins (e.g. folic acid, vitamin B-12,
vitamin K)

25.  Carbon
 Require for synthesising the cell components.
 Chemoheterotrophs use organic carbon sources
 Autotrophs use CO2
 Nitrogen
 Major component of amino acids and proteins
 Most bacteria decompose proteins
 Some bacteria use NH4+ or NO3–
 A few bacteria use N2 in nitrogen fixation

26.  Sulfur
 Needed for synthesis of amino acids like
thiamine (B1) and biotin (a vitamin of B complex,
found in egg yolk, liver and yeast and involved in
synthesis of fatty acids and glucose).
 Some bacteria use SO4
2– or H2S
 Phosphorus
 PO4
3– is a source of phosphorus.
 Is an essential component of nucleotides, nucleic
acids etc.

27.  Trace elements
 Inorganic elements (mineral) required in small amounts
 Usually as enzyme cofactors
 Ex: iron, molybdenum, zinc
 Growth factors
 Some require organic compounds in minute quantities
for the growth, these are known as Growth factors.
 Some bacteria are capable of synthesizing their
entire requirement of vitamin from culture medium.
 Vitamins are added from outside in media for those
bacteria which are not capable of synthesize vitamins
from media.
 Eg. Staphylococcus aureus – Thiamine, Nicotinic
acid
Bacillus anthracis – Thiamine (Vit. B1)

28. Microbial Growth
 Microbial growth is defined as the increase in the
number of cells, which occurs by cell division
 Binary fission (equal cell division): A cell duplicates
its components and divides into two cells
 Septum: A partition that grows between two daughter
cells and they separate at this location
 Budding (unequal cell division): A small, new cell
develops from surface of existing cell and
subsequently separates from parent cell

29. Binary Fission

30. Binary Fission

31. Phases of Growth
• Consider a population of organisms
introduced into a fresh, nutrient medium
• Such organisms display four major phases
of growth in batch culture:
1. The lag phase
2. The logarithmic phase
3. The stationary phase
4. The death phase

33. The Lag Phase
 The period between inoculation and beginning of
multiplication is the Lag Phase.
 Bacterial cell adjust itself to adopt the new environment.
 Organisms do not increase significantly in number
 They are metabolically and physiologically active but are not
dividing.
 Grow in size, synthesize enzymes, and incorporate molecules
from medium
 Produce large quantities of energy in the form of ATP
 The length of the lag phase depends upon the nature of
medium.

34. The Log Phase
 Exponential phase
 Organisms have adapted to a growth medium
 Cells divide steadily at constant rate
 Growth occurs at an exponential (log) rate
 The organisms divide at their most rapid rate
 Time required for one bacterial division during this phase is known
as generation time. The number of bacteria present in each
generation period is almost twice than in previous period.
 Generation period depend on the type of species, nutrient in the
medium and physical conditions.

35. Synchronous growth: A
hypothetical situation in which
the number of cells in a
culture would increase in a
stair-step pattern, dividing
together at the same rate
Nonsynchronous growth: A
natural situation in which an
actual culture has cell dividing
at one rate and other cells
dividing at a slightly slower
rate

36. 1) Cell division decreases to a point that new cells
are produced at same rate as old cell die.
2) The number of live cells stays constant.
Decline (Death) Phase
1) Condition in the medium become less and less
supportive for cell division
2) Cell lose their ability to divide and thus die
3) Number of live cells decreases at a logarithmic
rate.
4) it may be due to nutritional exhaustion or
autolytic enzymes.
Stationary Phase

37. Measuring Microbial Growth
Direct Methods Indirect Methods
 Plate counts
 Filtration
 MPN
 Direct count
 Turbidity
 Metabolic activity
 Dry weight

38. Direct method
1. Plate Count Method
 Most frequently used method.
 Inoculate plate with sample and count number of colonies.
 Assumptions:- 1. each colony originate from a single
bacterial cell.
2. original inoculums is homogenous.
3. no cell aggregates are present.
 ADV:- 1. measures viable cells.
 DISADV :- 1. take 24hrs or more for visible colonies to
appear.
2. only count between 25-250 colonies are accurate.
3. must perform serial dilutions to get appropriate
numbers per plate.

39. a) POUR PLATE:-
 Introduce 1.0 or 0.1 ml of inoculum into empty petri
dish.
 Add liquid nutrient medium, kept at 50°C.
 Gently mix, allow to solidify and incubate at 37°C.
 DISADV:- 1. not useful for heat sensitive bacteria.
b) SPREAD PLATE:-
 Introduce 0.1ml of inoculum onto surface of petri-dish
containing solid medium.
 Spread with sterile glass rod.
 Colonies grow on surface of medium.

40. Figure 6.17
Plate Counts

41. Filtration
 Used to measure small quantities of bacteria. Eg.
Faecal bacteria in lake or ocean water.
 A large sample (> 100ml) is filtered through a millipore
filter.
 The microbe are retained in the filter disc and the disc
is placed on a culture medium in a petri plate.
 The plates are incubated and the colonies are counted
on the filter disc.

42. The Most Probable Number (MPN) Method
 Used mainly to grow
bacteria that will not
grow on solid medium.
 Dilute the sample
repeatedly and inoculate
several broth tubes for
each dilution.
 Count number of positive
tubes in each set.

43. Direct Counts
 Another way to measure bacterial growth are:
1. Direct microscopic count
2. Counting chamber method
1. Direct microscopic count:-
1. Also called Breed Method.
2. Known volume of suspension (0.1ml) is spread uniformly over the
glass slide with specific area (1 sq. cm.)
3. The smear is the fixed, stained and examined under lens and cells
are counted.
4. It is not possible to examine entire area so a few areas are
observed and an average is taken.
5. Total cells per square cm is then calculated by determining number
of microscopic fields per sq cm.

44. Petroff – Hausser counting
In Petroff-Hausser counting chamber, bacterial suspension
is introduced onto chamber with a calibrated pipette.
Microorganisms are counted in specific calibrated areas.
Number per unit volume is calculated using an appropriate
formula
Total no. of bacterial = Number of cells counted x dilution
cells.mm3 area counted x depth of fluid

45. The Petroff-Hausser Counting Chamber

46. Countable number of colonies

47. Indirect methods
Turbidimetric method:
 As the bacteria multiply in media, it becomes turbid.
 Spectrophotometer is used to determine the percentage
transmission or absorbance.
 The instrument works on principle which states that light
absorbance is directly proportional to the turbidity of the
medium.
 Adv is no incubation time is required.
 Diasadv is discrimination between live and dead bacteria is not
possible.
 It is not possible to measure cultures grown in colored media.

48. Indirect Methods
A Spectrophotometer: This instrument can be used to measure bacterial
growth by measuring the amount of light that passes through a suspension of
cells

49. Turbidity

50. Turbidity
The less light transmitted, the more bacteria in sample.

51. Metabolic Activity
 Measurement of specific chemical change by
metabolic activity of microbes can be
correlated with the microbial growth.
Assuming the amount of a certain metabolic
product, ex acids, CO2 produced = direct
proportion of no of bacteria present

52. Dry Weight
 Simple and direct method.
 Culture suspension is centrifuged and pellet is
repeatedly washed to remove all foreign particles.
 The residue is then dried and weighed.
 Weight of dried culture = direct proportion of no of
bacteria present
 Disadv – does not distinguish between live and dead
cells.

53. Bacteriological media
Media is an artificially prepared mixture for
various nutrients for growth and multiplication of
m.o.
Common ingredients of media:-
1. water: tap or distilled water may be used for the
preparation of culture media by dissolving various
organic and inorganic compounds.
2. Peptones : complex mixture of partially digested
proteins obtained from meat, casein, fibrin, soya etc.
They mainly supply nitrogenous material and also act as
buffer. Stored in tightly closed container as it is
hygroscopic and become sticky when come in contact
with air.

54. 4. Yeast extract: prepared from cells of yeast such as
saccharomyces. It contain carbohydrates, amino acids,
growth factors and inorganic salts. Is used mainly as
source of vitamins.
5. Meat extract: prepared from fresh meat by hot water
extraction method. It contain gelatin, peptones, amino
acids, purines, minerals, carbohydrates and growth
factors etc.
6. Agar: a long chain polysachcharide obtained from
seaweed algae. Algae which yields agar are called
Agarophytes. Agar is a mixture of 2 polysachcharides
such as aggarose (70%) and agaropectin (30%).

55. Properties of Agar
1. Act as good solidifying agent
2. No nutritional value in media
3. Bacteriologically inert
4. Stable or firm at different temperature used
for incubation
5. Melts at 95-98°C and remains liquid upto 40-
42°C
6. Easily available and economical

56. Culture media Depending on
physical state
Depending on
oxygen requirement
Depending on
chemical
composition
Depending on
functional type

57. 1. Depending on physical state:-
1. Solid media (1.5-2.5% agar) . Eg, Nutrient agar
2. Semisolid media (0.2-0.5% agar) . Eg, Nutrient
broth containing 0.5% agar.
3. Liquid media(absence of agar). Eg, Fluid
thioglycollate broth.
2. Depending on oxygen requirement:-
1. Aerobic media. Ex. MacConkey’s broth
2. Anaerobic media. Eg. Robertson’s cooked meat
medium.

58. 3. Depending on chemical composition:-
1. Simple
2. Synthetic
3. Complex / non-synthetic
4. Depending on function:
1. Enrichment media
2. Selective media
3. Indicator media
4. Transport media
5. Assay media
6. Differential media

59. Simple media: include peptone, water and nutrient
broths which form the basis of most media used
in the study of common bacteria.
Complex media: usually contain complex materials
of biological origin such as blood or milk or beef
extract. The exact chemical composition of this
media is not known but it provide all the growth
factors for the cultivation of unknown bacteria.
Synthetic media: prepared from pure chemical
substances and the exact composition of the
medium is known.

60. Special media
 Enrichment media: when a specific substance is
added in a liquid medium which inhibits the growth of
unwanted bacteria and favors the growth of wanted
bacteria.
Eg, Tetrathionate broth, slenite F broth. These media
inhibits coliforms (eg, E-coli) and allow growth of
pathogenic cultures (salmonella species) in feces.
 Selective media: like enrichment media but is in solid
form.
 When a substance is added to solid medium which
inhibits the growth of unwanted bacteria and supports
the growth of required bacteria in the form of colonies
is known as selective medium.

61.  Physical conditions of a culture media may be adjusted
and make as selective for growth of specific m.o.
 Eg, McConkey’s media (E-coli) . It contains sodium
taurocholate which selectively allow the growth of
Gram –ve bacteria by inhibiting gram+ve bacteria.
Indicator media: these media contain indicator
which changes color when a bacterial species grow in
them. Eg. Wilson and Blair medium, Salmonella typhi
reduces sulphite to sulphide in the presence of
glucose and the colonies have a black metallic shine.

62. Transport media: delicate m.o like Neisseria
gonorrhoeae which may not survive the time taken
for transporting the specimen to the lab or may
be overgrown by non-pathogens and pathogens.
 Special media are devised for such type of
delicate m.o which are called transport media. Eg,
Stuart’s transport medium.

63.  Assay media: these media have specific composition
and are used for the assay of antibiotics, amino acids
and vitamins.
 Media containing specific components are also used for
testing the disinfectants.
 Differential media: certain reagents or supplements,
when incorporated into culture media, may allow
differentiation of various kinds of bacteria.
 Example, if a mixture of bacteria is inoculated onto a
blood containing agar media, some bacteria may
haemolyse the RBC, others do not. Thus, one can
distinguish between hemolytic and non-hemolytic
bacteria on the same media.

64. Storage media: help in preservation and storage of
bacteria for a considerable long period.
Eg, Robertson’s cooked meat medium, blood agar
slants etc.

65. ISOLATION OF BACTERIA
 Pure culture consist of a population of only one species of
m.o.
 The isolation of one kind of m.o. from a mixture of many
different kinds is called the pure culture techniques.
 Different methods used are:-
1. Streak plate method
2. Pour plate method
3. Spread plate method

66. Streak plate method
 Most widely used method.
 Prepared by streaking a small amount of mixed
cultures over the surface of solid medium in petri plate
with a platinum or nichrome wire.
 The purpose of streaking is to thin out the inoculum
successively so that microbes get separated.
 In the beginning, microbes are crowded and colonies
develop closely but as the streaking proceeds cells get
separated as the needle contain less cells.
 Hence at the last streak, few and clearly separated
colonies are developed.

67. The Streak Plate Method

68. POUR PLATE MEHOD
 In this , the mixed culture is diluted directly in tubes of
liquid agar medium.
 The medium is maintained in a liquid state at temp.
45°C to allow thorough distribution of the inoculum.
 Inoculated medium is transferred into petri plate,
allowed to solidify and incubated.
 In serial dilution technique, the original inoculum
may be diluted by water so that the concentration of
microbes gradually become less.

69. Disadv:-
1. M.o trapped beneath the surface of the medium when
it solidify. Hence, surface as well as subsurface colonies
are developed and it is very difficult to isolate and
count the sub surface colonies.
2. Tedious method, time consuming and requires skilled
hands.
3. M.o are subjected to hot shock as the temp is about
45°C.
4. Method is unsuitable for isolating psychrophiles
bacteria.

71. SPREAD PLATE METHOD
 In this method, the mixed culture is diluted in the
culture medium.
 A sample is removed from each dilution tube (0.1ml)
and placed onto the surface of agar plate.
 Spread the culture and incubate the plates and observe
isolated colonies after 24 hrs.

73.  Adv:-
1. Simple method
2. Only surface colonies are formed
3. Used for counting the m.o present in the inoulum
4. M.o are not exposed to higher temperature.

74. Isolation of bacteria
 The microbial population in our environment is very
large and complex. For example a single sneeze may
disperse from 10,000 to 1,00,000 bacteria. So to study
the characteristics of one species, that species must be
isolated using selective methods.
 To isolate a particular bacteria from mixed culture
there are different type of selective methods such as:-
1. Physical method
2. Chemical method
3. Biological method

75. 1. Physical method of selection
a. Incubation temperature: To select the
psychrophilic, mesophilic and thermophilic bacteria,
cultures are incubated at different temp.
For example, for psychrophiles the incubation temp
is 0°C to 5°C, and for thermophiles the incubation
temp is equal to 60°C.
b. pH of media:
1. To select the acid tolerant bacteria, a low pH medium
can be used. Eg, to select the lactobacilli present in
cheddar cheese, the pH of the medium is maintained
at 5.35 with an acetic acid/ acetate buffer. Other m.o
cant grow at such low pH.

76. 2. To select alkali tolerated organism, a high pH medium
can be used. Eg, to select the cholera causing
bacterium, vibrio cholera from stool sample, we can
use a medium with high pH of 8.5. Most of the
intestinal bacteria are unable to grow at this pH.
c. Heat treatment: To select the endospore forming
bacteria, a mixed culture can be heated to 80°C for 10
minutes before being used to inoculate culture
media. Vegetative cells are killed at this temp but the
endospore forming bacteria survive and
subsequently germinate at this temp.

77. 2. chemical method of selection:-
1. By the use of particular type of substrate: selection of
particular bacteria by use of a single substrate i.e,
single carbon or nitrogen.
For example, isolation of a soil bacteria which utilizes a
very complex organic compound α-conidendrin (a
constituent of wood), if isolated in nutrient agar
media, chance of finding is very limited. So a liquid
enrichment medium is prepared in which α-
conidendrin is a source of carbon. Under these
conditions only α-conidendrin utilizing bacteria will
be able to grow well and can be isolated.

78. 2.Use of dilute media: certain aquatic bacteria such as
Caulobactor species, are capable of growing with very
low levels of carbon and nitrogen sources. Consequently,
one way to isolate such bacteria is to inoculate a mixed
culture in a very dilute medium. For example, a broth
containing only 0.01% peptone. The medium must have
low enough levels of nutrients that other kinds of
organisms will not be able to grow well in it.

79. 3. Use of inhibitory or toxic chemicals: the addition of
low levels of certain chemicals such as dyes, bile salts,
antibiotics etc to culture media can be useful for the
selection of certain kind of bacteria.
1. Many Gram-ve bacteria can grow in the presence of low
concentration of various dyes that inhibit the growth of
Gram+ve bacteria.
2. Intestinal bacteria can grow in the presence of bile salts
such as sodium deoxycholate whereas non-intestinal
bacteria are usually inhibited.

80. 3. Biological methods of selection:
A disease producing species occurring in a mixed
culture can often be selected by taking advantage of its
pathogenic properties.
Eg, a sputum sample containing Streptococcus
pneumoniae is ordinarily contaminated by many other
bacterial species. However, laboratory mice is injected
into the mice, the pathogens will multiply extensively.
Nonpathogenic bacteria present in the sample will be
either inhibited or killed by the defense mechanism of
the animal. In a sense, the animal serves as the
selective medium.

81. FUNGI
Includes yeast, molds and mushrooms.
Donot contain chlorophyll, hence they are saprophytic
i.e, obtain food from dead organic matter or parasitic
ie, they obtain nourishment from living organisms.
Most fungi are not pathogenic in nature.

82. The fungus hat causes disease belongs to a specific
group known as Fungi imperfecti.
In immunocompetent humans these fungi usually
cause minor infections of the hair, nails, mucous
membranes or skin.
Fungus can be defined as “ those m.o that are
invariably nucleated, spore- bearing and do not posses
chlorophyll, generally reproduce both asexually and
sexually and have somatic structural features that are
essentially surrounded by cell walls comprising of
polysaccharides or chitin.”

83. Cultivation of fungi:
 Grows on usual bacteriological culture media at temp 20-
30 °C.
 Grows very slowly in comparison to bacteria.
 When fungi is to be isolated, it is better to adopt a
medium which supports their growth but is not optimal
for the growth of bacteria.
 Acid (pH=5.6) media that incorporates a relatively high
concentration of sugar are tolerated by molds but are
inhibitory to many bacteria.

84. Three types of media which are suitable for the growth
of fungi are:
1. Natural media- pieces or infusions of fruits, vegetables,
cereal grains or animal tissues.
2. Culture media prepared from peptones, plant extracts,
agar and other components of variable compositions.
3. Synthetic / chemical media.
Natural medium generally used is agar and glucose
which may be combined and added.
Another medium for cultivation of yeast and molds
contain infusion of potato with agar and glucose
(PDA medium)

85. One of the best known and oldest media for the growth
of fungi is Sabouarud’s Dextrose Agar (SDA) which
includes maltose and peptone as its main ingredients.
It has partial selective action because of its high sucrose
content and low pH.
Chloramphenicol is incorporated in the culture medium
to prevent contamination by bacteria.
The fungal growth can be identified by its color,
morphology on visual examination.
By the addition of some antibiotic and by preventing the
contamination, pre culture of the fungus is isolated.

86. Fungi reproduction
 A large no. of fungi get reproduced both asexually and
sexually.
 Asexual reproduction:
 Most common procedure is usually accomplished by help of
spores.
 Salient features are:-
 Produced by fragmentation of aerial hyphae
 Progeny genetically identical to parent
 Simplest form of available fungal spore is known as Zoospore,
which possess no rigid cell wall and is propelled by flagella.

87.  Flagellum is much more complex.
 Base of flagellum enters the cell and gets attached to the
nucleus by a structure termed as Rhizoplast.
 Sporangium designates the asexual reproductive structures. In
its early stage it is found to be loaded with nuclei and
protoplasm.
 Cleavage takes place whereby numerous sections invariably
developed into corresponding uninucleate zoospores.
 Finally, following a motile phase, the resulting zoospores
encysts, loosing its flagellum and rests quietly just prior to
germination.

88. Several types of spores:
Conidiospores
Blastospores
Chlamydospores
Sporangiospores
Arthrospores

90.  Sexual reproduction:-
 It is characterised by the strategical union of 2
compatible nuclei ; and the entire phenomenon may be
distinctly divided into three phases:-
1. Phase-I : the union of gametangia (i.e, sex organs) brings
the nuclei into close proximity within the same protoplast. It
is referred as Plasmogamy.
2. Phase-II : known as Karyogamy, which takes place with
fusion of 2 nuclei. Above two process takes place in
immediate sequence in lower fungi whereas in higher fungi
they do occur at altogether different time periods.
3. Phase-III : known as meiosis, takes care of nuclear fusion
whereby actual number of chromosomes is distinctly and
significantly reduced to its original haploid state.

92. VIRUSES
 Are infective agents that typically consists of a nucleic
acid molecule in a protein coat and is too small to be
seen by light microscope.
 It is able to multiply only within the living cells of host.
 Living characteristics:
 They are nucleoproteins and contain either DNA or RNA
surrounded by a protein coat.
 They replicate, although inside the living cell.
 Capable of synthesising protein for thin layer coat.
 They cause disease like bacteria and fungi.

93.  Non-living characters:
 They don't have protoplasm
 They don't have enzyme system
 They don't have to respire
 They are perfect obligate parasites which means they
depend upon specific host cells for their reproduction
and development.
 The virus particle attach to the host cell by adsorption
with the help of tail fiber and dissolve the cell wall of
the host cell.

94. STRUCTURE OF VIRUSES
 A virion is a complete, fully developed viral particle
composed of nucleic acid surrounded by a coat that
protects it from the environment and serves as a
vehicle of transmission from one host cell to another.
 Viruses are not cellular and therefore do not contain
nucleus, cytoplasm or cell membrane.
 Structure of virus include:
 Nucleic acid
 Capsid and envelope

95. Nucleic Acid
Spike
Projections
Protein
Capsid
Lipid Envelope
Virion
Associated
Polymerase

96.  Nucleic acid:
 Contain single kind of nucleic acid either DNA or RNA,
which is the genetic material.
 The % of nucleic acid in relation to protein is about 1%
for the influenza viruses and about 50% for certain
bacteriophages.
 Capsid and envelope:
 The nucleic acid of virus is surrounded by a protein coat
called Capsid.
 Each capsid is composed of protein subunits called
capsomers.

97.  In some viruses, the capsid is covered by an envelope
which usually consists of some combination of lipids,
proteins and carbohydrates.
 Depending on the viruses, envelopes may or may not be
covered by spikes, which are carbohydrate protein
complexes that projects from the surface of the
envelope.
 Some viruses attach to host cells by means of spikes.
This is one of the characteristics for the identification of
viruses.
 The capsid of a nacked virus protects the nucleic acid
from nuclear enzymes in biological fluids and promote
the virus attachment to susceptible host cells.

98. EFFECT OF PHYSICAL AND CHEMICAL AGENTS ON
VIRUSES
 Most human pathogenic viruses are inactivated after the
exposure of 60°C for 30 minutes.
 Viruses are stable at low temp and routinely stored at -40°C
to -70°C.
 Some are rapidly inactivated by drying.
 Heat is the most suitable method for virus disinfection.
 UV light inactivates viruses by damaging their nucleic acid
and has been used to prepare viral vaccines.

99.  Viruses that contain lipid are inactivated by organic
solvents such as chloroform and ether.
 Isopropyl alcohol is less active against non-
enveloped viruses.
 The most generally active agents are chlorine, iodine,
aldehydes, ascorbic acid and ozone.

100. LIFE CYCLE OF BACTERIOPHAGE
 Bacteriophage exhibit 2 different type of life cycle:
1. Lytic cycle: there is intracellular multiplication of
phages followed by lysis and release of progeny virions.
2. Lysogenic cycle: the phage DNA becomes integrated
with the bacterial genome, replicate synchronously
without any cell lysis.

102. LYTIC CYCLE

103. Steps in Viral Replication
A. Attachment. This is the first step in viral replication.
Surface proteins of the virus interact with specific receptors
on the target cell surface. These may be specialized proteins
that are more widely distributed on tissues throughout the
body. The presence of a virus-specific receptor is necessary
but not sufficient for viruses to infect cells and complete the
replicative cycle.
B. Penetration. Enveloped viruses (e.g., HIV, influenza virus)
penetrate cells through fusion of the viral envelope with the
host cell membrane. Non-enveloped viruses penetrate cells by
translocation of the virion across the host cell membrane or
receptor mediated endocytosis of the virion.

104. C. Uncoating (disassembly). A complex process. This process
makes the nucleic acid available for transcription to permit
multiplication of the virus.
D. Transcription and Translation. After infection and
penetration of part of viral genome produces early mRNA
molecules, which are translated into a set of early proteins.
 These serve to switch off host cell macromoleculare synthesis,
degrade the host DNA and starts to make components of viral DNA.
 The viral DNA replicates and also starts to produce batch of late
mRNA molecules, which produce the protein for phage coat.
 The late messages are translated into subunits of the capsid
structure, which condense to form phage head, tail and tail fiber.

105. E. Assembly and Release. The process of virion assembly
involves bringing together newly formed viral nucleic
acid and the structural proteins to form the
nucleocapsid of the virus.
 The release of progeny particles takes place by sudden
explosion.
 Lysozyme synthesized within the cell causes the bacterial cell
wall to break down and the newly produced bacteriophage are
released from the host cell.
 Each cycle of phage reproduction may require 20-60 min and
in a single phage infection around 200 or more progeny are
produced.

106. LYSOGENIC CYCLE
 Following entry into the host cell, phage nucleic acid
become integrated with the bacterial chromosomes.
Integrated nuclei known as prophage.
 The prophage behaves like segment of the host
chromosomes and replicate synchronously in bacterial cell.
This is called lysogeny and the bacterium carrying
prophage is called lysogenic bacterium.
 Everytime the host cells machinery replicates the bacterial
chromosome, it also replicates the prophage DNA.
 However a rare spontaneous event or the action of UV light
or certain chemical can lead to excision of the phage DNA
and to initiation of the lytic cycle.

108. Cultivation of virus
 Virus are obligate parasite and cannot grow on inanimate
culture media.
 Viruses can be cultivated within suitable hosts, such as a
living cell
 The primary purposes of viral cultivation are:
1. To isolate and identify viruses in clinical specimens
2. To prepare viruses for vaccines
3. And to do detailed research on viral structure,
multiplication cycles, genetics, and effects on host cells
 Viruses not only need living cells to grow in but also they
are specific about the type of cell they infect and grow in

109. Methods for Cultivation of Virus
 Generally three methods are
employed for the virus
cultivation
1. Inoculation of virus into
animals
2. Inoculation of virus into
embryonated eggs
3. Tissue culture

110. 1. Laboratory animals:
 Laboratory animals play an essential
role in studies of viral pathogenesis
 Live animals such as monkeys, rabbits,
guinea pigs are widely used for
cultivating virus
 Mice are the most widely employed
animals in virology

111.  The different routes of inoculation in
mice are:
 intracerebral
 subcutaneous
 intraperitoneal
 or intranasal
 After the animal is inoculated with the
virus suspension, the animal is:
 observed for signs of disease
 visible lesions
 or is killed so that infected tissues can
be examined for virus

112.  Due to cost and risk to handlers, monkeys find less
application in virology. The poliomyelitis after
intracerebral or intraspinal inoculation in monkeys
causes typical paralytic disease.
 The growth of virus in inoculated animals may be
indicated by death, disease or visible lesions.

113. Inoculation of Virus in Embryonated Eggs
 Goodpasture and Burnet in 1931 first used
the embryonated hen’s egg for the
cultivation of virus
 The process of cultivation of viruses in
embryonated eggs depends on the type of
egg being used
 Eggs provide a suitable means for:
 the primary isolation and identification of
viruses
 the maintenance of stock cultures
 and the production of vaccines

114.  Chicken, duck, and turkey eggs are the most common
choices for inoculation
 The egg used for cultivation must be sterile and the
shell should be intact and healthy
 Rigorous sterile techniques must be used to prevent
contamination by bacteria and fungi from the air and
the outer surface of the shell
Inoculation of Virus

115. Inoculation of Virus
 The egg must be injected through
the shell, usually by drilling a hole
or making a small window
 The viral suspension or suspected
virus- containing fluid is injected
into the fluid of the egg
 The exact tissue that is inoculated
is guided by the type of virus
being cultivated and the goals of
the experiment

116.  Viruses multiplying in embryos may or
may not cause effects visible to the
naked eye
 The signs of viral growth include:
 Death of the embryo
 Defects in embryonic development
 and localized areas of damage in the
membranes, resulting in discrete opaque
spots called pocks
 Certain viruses can also be detected by:
 their ability to agglutinate red blood cells
 or by their reaction with an antibody of
known specificity
Detection of Viral Growth

117. Parts of Embryonated Egg
 The air sac is important to the
developing embryo for
respiration and for pressure
adjustments
 The shell and shell membrane
function both as a barrier and as
an exchange system for gases and
liquid molecules
 The chorioallantoic sac and its
contents (allantoic fluid) remove
waste products produced by the
developing embryo
 This Membrane and its contents
increases in size as the embryo
grows

118.  The yolk sac is the source of nourishment for the
developing Embryo
 As the embryo develops, the yolk sac decreases in size
until it is completely absorbed into the digestive system
of the mature embryo
 The amnion is a thin membrane that encloses the
embryo and Protects it from physical damage
 It also serves as an exchange system and is best seen in
the younger embryos

119. Routes of Viral Inoculation
 An embryonated egg offers
various sites for the
cultivation of viruses
 The different sites of viral
inoculation in embryonated
eggs are:
1. Chorioallantoic
membrane(CAM)
2. Amniotic Cavity
3. Allantoic Cavity
4. Yolk sac

120.  The chosen route of inoculation and age of the embryo
are determined by the given virus selectivity for a
certain membrane or developmental stage of the
embryo
 For example Infectious bronchitis virus is propagated in
the yolk sac of a 5-6 day old embryo
 whereas Rous-sarcoma virus is inoculated on the
chorioallantoic membrane of a 9-11 day old embryo and
will produce pocks (lesions) 5-10 days post-infection
Routes of Viral Inoculation

121. Advantage of embryonated eggs
1. Eggs are much simpler to handle than animals
2. Most economical and easily available
3. Are clean and bacteriologically sterile.
4. Don’t need feeding and caging
5. Donot have immune mechanism like animals to
counteract virus infection.
6. Chick embryo offer several sites for cultivation of viruses.

122. Cell Cultures
 Prior to the advent of cell culture, animal viruses could
be propagated only on whole animals or embryonated
chicken eggs
 Cell cultures have replaced embryonated eggs as the
preferred type of growth medium for many viruses
 Cell culture consists of cells grown in culture media in
the laboratory
 These cultures can be propagated and handled like
bacterial cultures; they are more convenient to work
with than whole animals or embryonated eggs

123. There are mainly three type of cultures:
1. Organ culture
2. Explant culture
3. Cell culture

124. Organ culture:-
Useful for the isolation of some viruses which appear to be
highly specialized parasites of certain organs e.g.
tracheal ring organ culture is employed for the isolation
of coronavirus.
Explant culture:-
Minced tissue may be grown as explantembedded in
plasma clots. This is not useful in virology.

125. Cell culture:-
 very popular and useful technique routinely used for
cultivation of viruses.
 Tissues are dissociated into component cells by the cells are
washed, counted and suspended in a growth medium
distributed in petri plates, test tubes or bottles.
 The cells adhere in glass surface and grow out to form a
monolayer sheet.
 Cultures made directly from live tissues are primary cell
cultures. Primary cell lines are normal cells freshly taken
from the body and cultured. They are capable of only limited
growth in culture. They are useful for isolation and
cultivation of viruses for vaccine production.

126. Diploid cell strains are derived from primary cell
cultures established from a particular type of tissues,
such as lung or kidney, which is embryonic in origin.
They are of single type and can undergo 50-100
divisions before dying. They posses the normal diploid
karyotype (set of chromosomes).