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 Bacteria exhibit different modes of nutrition. They can
be:-
 Heterotrophic bacteria
 Autotrophic bacteria
Nutritional
habit of bacteria
Autotrophic heterotrophic
Photoautotrop
hic
-Green sulphur
bacteria
-Purple sulphur
bacteria...
 Photosynthetic bacteria: use sunlight as source of energy
and electron donors are H2O, S and other sulphur
containing in...
Chemoautotrophic bacteria
 Are without photosynthetic pigments.
a. Nitrifying bacteria: mainly found in soil.
- through t...
b. Iron bacteria: found in iron containing rocks. Eg.
Ferrobacillus leptothrix.
c. Methanogens : produce methane and water...
Heterotrophic bacteria
 Unable to synthesize their own metabolites and
depend on preformed organic compounds.
 They obta...
 Chemo-heterotrophic bacteria:
- Take their food from other plants and animals.
- They obtain both energy and carbon sour...
 They may be Obligate parasite, grow only in living cells
and Facultative parasite (grow on dead material ).
b) Saprophyt...
Physical conditions required for growth of
bacteriaA) Temperature: the temperature that allows for most
rapid growth durin...
3. Thermophiles : prefer high temp for growth (45-70°C).
 Some thermophiles which can grow in mesophilic range (
37-55°C)...
GROUP
GROWTH TEMP (°C)
EXAMPLES
MINIMUM OPTIMUM MAXIMUM
Obligate
psychrophiles
0-5°C 10-15 20
Vibrio
psychroerythrus
Facul...
B) H- ion concentration:
• Bacteria are sensitive to variation in pH.
• Each bacteria has a pH range, above or below to
th...
 Depending on optimum pH value of m.o, they
can be classified as:-
1. Acidophiles- optimum pH range between 1-
6.5. eg. L...
C) Oxygen requirement
 Division is based on influence of oxygen into growth.
 Aerobic: require o2 for growth. Eg, E-coli...
 Microaerophilic: grow best in low o2 levels but
cant tolerate leve of oxygen present in air
atmosphere. Eg, Campylobacte...
C) Moisture requirement
 Water is essential to bacterial protoplasm and
drying is lethal.
 Treponema is highly sensitive...
D) Light requirement
 Bacteria (except phototropic) grow well in dark.
 They are sensitive to UV light and other
radiati...
E) Osmotic effect
 Bacteria are more tolerant to osmotic variation
than most other cells due to strength of their cell
wa...
 Hyperosmotic environment: cell lose water and
undergoes plasmolysis (shrinkage of cells)
 Hypoosmotic environments : ce...
Halophiles
Salt loving organisms which require moderate to
large quantity of salt (NaCl).
Why do halophiles require sodium...
Responses to Salt
Chemical Requirement
1. Carbon sources
2. Nitrogen sources
3. Sulfur and phosphorus
4. Trace elements (e.g. copper, iron,
...
 Carbon
 Require for synthesising the cell components.
 Chemoheterotrophs use organic carbon sources
 Autotrophs use C...
 Sulfur
 Needed for synthesis of amino acids like
thiamine (B1) and biotin (a vitamin of B complex,
found in egg yolk, l...
 Trace elements
 Inorganic elements (mineral) required in small amounts
 Usually as enzyme cofactors
 Ex: iron, molybd...
Microbial Growth
 Microbial growth is defined as the increase in the
number of cells, which occurs by cell division
 Bin...
Binary Fission
Binary Fission
Phases of Growth
• Consider a population of organisms
introduced into a fresh, nutrient medium
• Such organisms display fo...
The Lag Phase
 The period between inoculation and beginning of
multiplication is the Lag Phase.
 Bacterial cell adjust i...
The Log Phase
 Exponential phase
 Organisms have adapted to a growth medium
 Cells divide steadily at constant rate
 G...
Synchronous growth: A
hypothetical situation in which
the number of cells in a
culture would increase in a
stair-step patt...
1) Cell division decreases to a point that new cells
are produced at same rate as old cell die.
2) The number of live cell...
Measuring Microbial Growth
Direct Methods Indirect Methods
 Plate counts
 Filtration
 MPN
 Direct count
 Turbidity
 ...
Direct method
1. Plate Count Method
 Most frequently used method.
 Inoculate plate with sample and count number of colon...
a) POUR PLATE:-
 Introduce 1.0 or 0.1 ml of inoculum into empty petri
dish.
 Add liquid nutrient medium, kept at 50°C.
...
Figure 6.17
Plate Counts
Filtration
 Used to measure small quantities of bacteria. Eg.
Faecal bacteria in lake or ocean water.
 A large sample (>...
The Most Probable Number (MPN) Method
 Used mainly to grow
bacteria that will not
grow on solid medium.
 Dilute the samp...
Direct Counts
 Another way to measure bacterial growth are:
1. Direct microscopic count
2. Counting chamber method
1. Dir...
Petroff – Hausser counting
In Petroff-Hausser counting chamber, bacterial suspension
is introduced onto chamber with a cal...
The Petroff-Hausser Counting Chamber
Countable number of colonies
Indirect methods
Turbidimetric method:
 As the bacteria multiply in media, it becomes turbid.
 Spectrophotometer is used...
Indirect Methods
A Spectrophotometer: This instrument can be used to measure bacterial
growth by measuring the amount of l...
Turbidity
Turbidity
The less light transmitted, the more bacteria in sample.
Metabolic Activity
 Measurement of specific chemical change by
metabolic activity of microbes can be
correlated with the ...
Dry Weight
 Simple and direct method.
 Culture suspension is centrifuged and pellet is
repeatedly washed to remove all f...
Bacteriological media
Media is an artificially prepared mixture for
various nutrients for growth and multiplication of
m....
4. Yeast extract: prepared from cells of yeast such as
saccharomyces. It contain carbohydrates, amino acids,
growth factor...
Properties of Agar
1. Act as good solidifying agent
2. No nutritional value in media
3. Bacteriologically inert
4. Stable ...
Culture media Depending on
physical state
Depending on
oxygen requirement
Depending on
chemical
composition
Depending on
f...
1. Depending on physical state:-
1. Solid media (1.5-2.5% agar) . Eg, Nutrient agar
2. Semisolid media (0.2-0.5% agar) . E...
3. Depending on chemical composition:-
1. Simple
2. Synthetic
3. Complex / non-synthetic
4. Depending on function:
1. Enri...
Simple media: include peptone, water and nutrient
broths which form the basis of most media used
in the study of common b...
Special media
 Enrichment media: when a specific substance is
added in a liquid medium which inhibits the growth of
unwan...
 Physical conditions of a culture media may be adjusted
and make as selective for growth of specific m.o.
 Eg, McConkey’...
Transport media: delicate m.o like Neisseria
gonorrhoeae which may not survive the time taken
for transporting the specim...
 Assay media: these media have specific composition
and are used for the assay of antibiotics, amino acids
and vitamins.
...
Storage media: help in preservation and storage of
bacteria for a considerable long period.
Eg, Robertson’s cooked meat ...
ISOLATION OF BACTERIA
 Pure culture consist of a population of only one species of
m.o.
 The isolation of one kind of m....
Streak plate method
 Most widely used method.
 Prepared by streaking a small amount of mixed
cultures over the surface o...
The Streak Plate Method
POUR PLATE MEHOD
 In this , the mixed culture is diluted directly in tubes of
liquid agar medium.
 The medium is maintai...
Disadv:-
1. M.o trapped beneath the surface of the medium when
it solidify. Hence, surface as well as subsurface colonies
...
SPREAD PLATE METHOD
 In this method, the mixed culture is diluted in the
culture medium.
 A sample is removed from each ...
 Adv:-
1. Simple method
2. Only surface colonies are formed
3. Used for counting the m.o present in the inoulum
4. M.o ar...
Isolation of bacteria
 The microbial population in our environment is very
large and complex. For example a single sneeze...
1. Physical method of selection
a. Incubation temperature: To select the
psychrophilic, mesophilic and thermophilic bacter...
2. To select alkali tolerated organism, a high pH medium
can be used. Eg, to select the cholera causing
bacterium, vibrio ...
2. chemical method of selection:-
1. By the use of particular type of substrate: selection of
particular bacteria by use o...
2.Use of dilute media: certain aquatic bacteria such as
Caulobactor species, are capable of growing with very
low levels o...
3. Use of inhibitory or toxic chemicals: the addition of
low levels of certain chemicals such as dyes, bile salts,
antibio...
3. Biological methods of selection:
A disease producing species occurring in a mixed
culture can often be selected by taki...
FUNGI
Includes yeast, molds and mushrooms.
Donot contain chlorophyll, hence they are saprophytic
i.e, obtain food from d...
The fungus hat causes disease belongs to a specific
group known as Fungi imperfecti.
In immunocompetent humans these fun...
Cultivation of fungi:
 Grows on usual bacteriological culture media at temp 20-
30 °C.
 Grows very slowly in comparison ...
Three types of media which are suitable for the growth
of fungi are:
1. Natural media- pieces or infusions of fruits, vege...
One of the best known and oldest media for the growth
of fungi is Sabouarud’s Dextrose Agar (SDA) which
includes maltose a...
Fungi reproduction
 A large no. of fungi get reproduced both asexually and
sexually.
 Asexual reproduction:
 Most commo...
 Flagellum is much more complex.
 Base of flagellum enters the cell and gets attached to the
nucleus by a structure term...
Several types of spores:
Conidiospores
Blastospores
Chlamydospores
Sporangiospores
Arthrospores
 Sexual reproduction:-
 It is characterised by the strategical union of 2
compatible nuclei ; and the entire phenomenon ...
VIRUSES
 Are infective agents that typically consists of a nucleic
acid molecule in a protein coat and is too small to be...
 Non-living characters:
 They don't have protoplasm
 They don't have enzyme system
 They don't have to respire
 They ...
STRUCTURE OF VIRUSES
 A virion is a complete, fully developed viral particle
composed of nucleic acid surrounded by a coa...
Nucleic Acid
Spike
Projections
Protein
Capsid
Lipid Envelope
Virion
Associated
Polymerase
 Nucleic acid:
 Contain single kind of nucleic acid either DNA or RNA,
which is the genetic material.
 The % of nucleic...
 In some viruses, the capsid is covered by an envelope
which usually consists of some combination of lipids,
proteins and...
EFFECT OF PHYSICAL AND CHEMICAL AGENTS ON
VIRUSES
 Most human pathogenic viruses are inactivated after the
exposure of 60...
 Viruses that contain lipid are inactivated by organic
solvents such as chloroform and ether.
 Isopropyl alcohol is less...
LIFE CYCLE OF BACTERIOPHAGE
 Bacteriophage exhibit 2 different type of life cycle:
1. Lytic cycle: there is intracellular...
LYTIC CYCLE
Steps in Viral Replication
A. Attachment. This is the first step in viral replication.
Surface proteins of the virus inter...
C. Uncoating (disassembly). A complex process. This process
makes the nucleic acid available for transcription to permit
m...
E. Assembly and Release. The process of virion assembly
involves bringing together newly formed viral nucleic
acid and the...
LYSOGENIC CYCLE
 Following entry into the host cell, phage nucleic acid
become integrated with the bacterial chromosomes....
Cultivation of virus
 Virus are obligate parasite and cannot grow on inanimate
culture media.
 Viruses can be cultivated...
Methods for Cultivation of Virus
 Generally three methods are
employed for the virus
cultivation
1. Inoculation of virus ...
1. Laboratory animals:
 Laboratory animals play an essential
role in studies of viral pathogenesis
 Live animals such as...
 The different routes of inoculation in
mice are:
 intracerebral
 subcutaneous
 intraperitoneal
 or intranasal
 Afte...
 Due to cost and risk to handlers, monkeys find less
application in virology. The poliomyelitis after
intracerebral or in...
Inoculation of Virus in Embryonated Eggs
 Goodpasture and Burnet in 1931 first used
the embryonated hen’s egg for the
cul...
 Chicken, duck, and turkey eggs are the most common
choices for inoculation
 The egg used for cultivation must be steril...
Inoculation of Virus
 The egg must be injected through
the shell, usually by drilling a hole
or making a small window
 T...
 Viruses multiplying in embryos may or
may not cause effects visible to the
naked eye
 The signs of viral growth include...
Parts of Embryonated Egg
 The air sac is important to the
developing embryo for
respiration and for pressure
adjustments
...
 The yolk sac is the source of nourishment for the
developing Embryo
 As the embryo develops, the yolk sac decreases in ...
Routes of Viral Inoculation
 An embryonated egg offers
various sites for the
cultivation of viruses
 The different sites...
 The chosen route of inoculation and age of the embryo
are determined by the given virus selectivity for a
certain membra...
Advantage of embryonated eggs
1. Eggs are much simpler to handle than animals
2. Most economical and easily available
3. A...
Cell Cultures
 Prior to the advent of cell culture, animal viruses could
be propagated only on whole animals or embryonat...
There are mainly three type of cultures:
1. Organ culture
2. Explant culture
3. Cell culture
Organ culture:-
Useful for the isolation of some viruses which appear to be
highly specialized parasites of certain organs...
Cell culture:-
 very popular and useful technique routinely used for
cultivation of viruses.
 Tissues are dissociated in...
Diploid cell strains are derived from primary cell
cultures established from a particular type of tissues,
such as lung o...
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
Nutrition, cultivation and isolation of bacteria
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Nutrition, cultivation and isolation of bacteria

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

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Nutrition, cultivation and isolation of bacteria

  1. 1.  Bacteria exhibit different modes of nutrition. They can be:-  Heterotrophic bacteria  Autotrophic bacteria
  2. 2. 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
  3. 3.  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.
  4. 4. 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.
  5. 5. 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
  6. 6. 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
  7. 7.  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.
  8. 8.  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.
  9. 9. 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.
  10. 10. 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.
  11. 11. 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
  12. 12. 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.
  13. 13.  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 )
  14. 14. 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.
  15. 15.  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
  16. 16. 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.
  17. 17. 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.
  18. 18. 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.
  19. 19.  Hyperosmotic environment: cell lose water and undergoes plasmolysis (shrinkage of cells)  Hypoosmotic environments : cell gain water and swell and burst.
  20. 20. 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.
  21. 21. Responses to Salt
  22. 22. 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)
  23. 23.  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
  24. 24.  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.
  25. 25.  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)
  26. 26. 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
  27. 27. Binary Fission
  28. 28. Binary Fission
  29. 29. 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
  30. 30. 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.
  31. 31. 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.
  32. 32. 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
  33. 33. 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
  34. 34. Measuring Microbial Growth Direct Methods Indirect Methods  Plate counts  Filtration  MPN  Direct count  Turbidity  Metabolic activity  Dry weight
  35. 35. 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.
  36. 36. 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.
  37. 37. Figure 6.17 Plate Counts
  38. 38. 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.
  39. 39. 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.
  40. 40. 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.
  41. 41. 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
  42. 42. The Petroff-Hausser Counting Chamber
  43. 43. Countable number of colonies
  44. 44. 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.
  45. 45. 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
  46. 46. Turbidity
  47. 47. Turbidity The less light transmitted, the more bacteria in sample.
  48. 48. 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
  49. 49. 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.
  50. 50. 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.
  51. 51. 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%).
  52. 52. 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
  53. 53. Culture media Depending on physical state Depending on oxygen requirement Depending on chemical composition Depending on functional type
  54. 54. 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.
  55. 55. 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
  56. 56. 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.
  57. 57. 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.
  58. 58.  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.
  59. 59. 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.
  60. 60.  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.
  61. 61. Storage media: help in preservation and storage of bacteria for a considerable long period. Eg, Robertson’s cooked meat medium, blood agar slants etc.
  62. 62. 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
  63. 63. 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.
  64. 64. The Streak Plate Method
  65. 65. 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.
  66. 66. 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.
  67. 67. 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.
  68. 68.  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.
  69. 69. 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
  70. 70. 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.
  71. 71. 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.
  72. 72. 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.
  73. 73. 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.
  74. 74. 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.
  75. 75. 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.
  76. 76. 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.
  77. 77. 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.”
  78. 78. 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.
  79. 79. 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)
  80. 80. 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.
  81. 81. 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.
  82. 82.  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.
  83. 83. Several types of spores: Conidiospores Blastospores Chlamydospores Sporangiospores Arthrospores
  84. 84.  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.
  85. 85. 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.
  86. 86.  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.
  87. 87. 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
  88. 88. Nucleic Acid Spike Projections Protein Capsid Lipid Envelope Virion Associated Polymerase
  89. 89.  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.
  90. 90.  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.
  91. 91. 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.
  92. 92.  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.
  93. 93. 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.
  94. 94. LYTIC CYCLE
  95. 95. 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.
  96. 96. 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.
  97. 97. 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.
  98. 98. 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.
  99. 99. 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
  100. 100. 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
  101. 101. 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
  102. 102.  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
  103. 103.  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.
  104. 104. 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
  105. 105.  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
  106. 106. 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
  107. 107.  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
  108. 108. 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
  109. 109.  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
  110. 110. 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
  111. 111.  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
  112. 112. 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.
  113. 113. 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
  114. 114. There are mainly three type of cultures: 1. Organ culture 2. Explant culture 3. Cell culture
  115. 115. 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.
  116. 116. 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.
  117. 117. 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).

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