The process of growing microorganisms in culture by taking bacteria from the infection site (in vivo or environment) and grow them in artificial environment in the laboratory (in vitro).
Bacteria may require adequate nutrition, optimum pH, temperature and oxygen for growth and multiplication.
Suitable artificial media containing sources of carbon, nitrogen, hydrogen, oxygen, phosphorous and other elements such as sodium, potassium, magnesium, iron and growth factor (Vitamins) in very small amounts have been used for cultivation of microorganism.
When microorganisms are cultivated in the laboratory, a growth environment called a medium is used. The medium may be purely chemical (a chemically defined medium), or it may contain organic materials, or it may consist of living organisms such as fertilized eggs.
Microorganisms growing in or on such a medium form a culture.
A culture is considered a pure culture if only one type of organism is present and a mixed culture if populations of different organisms are present.
When first used, the culture medium should be sterile, meaning that no form of life is present before inoculation with the microorganism.
2. CULTIVATION
• The process of growing microorganisms in culture by
taking bacteria from the infection site (in vivo or
environment) and grow them in artificial environment
in the laboratory (in vitro).
2
3. MICROBIAL CULTIVATION
• Bacteria may require adequate nutrition, optimum pH,
temperature and oxygen for growth and
multiplication.
• Suitable artificial media containing sources of carbon,
nitrogen, hydrogen, oxygen, phosphorous and other
elements such as sodium, potassium, magnesium, iron
and growth factor (Vitamins) in very small amounts
have been used for cultivation of microorganism.
3
4. MICROBIAL CULTIVATION
• When microorganisms are cultivated in the laboratory,
a growth environment called a medium is used. The
medium may be purely chemical (a chemically defined
medium), or it may contain organic materials, or it may
consist of living organisms such as fertilized eggs.
• Microorganisms growing in or on such a medium form
a culture.
• A culture is considered a pure culture if only one type
of organism is present and a mixed culture if
populations of different organisms are present.
• When first used, the culture medium should be sterile,
meaning that no form of life is present before
inoculation with the microorganism.
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6. Definition: it is artificially prepared media which contains all
essential nutrient to provide for growth and multification of
particular microorganism.
There are two different kinds of media, defined media which
contain peptone, and undefined composition.
• An undefined medium (also known as a basal or complex
medium)
• Defined media (also known as chemically defined media or
synthetic media)
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7. • The majority of the commonly used culture media are commercially
available as dehydrated products. Which are reconstituted by the
addition of distilled water and then sterilised in the autoclave.
• Nutrient media
• This is an undefined medium because the amino acid source contains
variety of compounds with the exact composition being unknown.
Nutrient media contain all the elements that most bacteria need for
growth and are non-selective, so they are used for the general
cultivation and maintenance of bacteria kept in laboratory culture
collections.
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8. GENERAL CHARACTERISTICS OF CULTURE
MEDIA
Must give satisfactory and rapid growth from single culture
Should maintain pH during storage
Reasonably cheap and easily available
Maintain sterility throught expt.
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9. MEDIA FOR PURPOSE
1-Media for isolation
2-Selective or inhibitory media
3-Enrichment media
4-Media for maintenance
5-Media for determining nutritional requirements or ability to
use a substrate
6-Media for characterization
7-Media for screening
8-Media for microbiological assay of vitamins and amino acids
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10. USES OF MEDIA
Cultivation and isolation
Identification
Sterility test
Preservative efficacy test
Evaluation of disinfectant
To check the antimicrobial susceptabilty
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11. COMMON INGREDIENTS OF MEDIA
• Agar
• Peptone
• Yeast extract
• Meat Extract
• Water
• NaCl
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12. COMMON INGREDIENTS OF MEDIA
• Agar: Solidifying agent
• Peptone: Source of Nitrogen
• Yeast extract: as a source of vitamins.
• Meat Extract: Vitamins, corbohydrates.
• Water: uptake of nutrients(Micronutrients and
Macronutrients)
• NaCl: To maintain isotonicity
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13. Microbial Growth and Culture Media
Solid Media: Nutrient material that contains a solidifying agent
(plates, slants, deeps).
The most common solidifier is agar, first used by Robert Koch.
Unique Properties of Agar:
Melts above 95oC.
Once melted, does not solidify until it reaches 40oC.
Cannot be degraded by most bacteria.
Polysaccharide made by red algae.
Originally used as food thickener (Angelina Hesse).
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14. TYPES OF CULTURE MEDIA
Based on the physical state
• Liquid medium: ( Absence of Agar)
• Without agar.
• for the proliferation of bacteria.
• e.g. Fluid Thioglycolate medium
• Solid medium:
• 1.5 to 2.5% agar.
• for the isolation and identification of bacteria
• e.g., slant, Petri dishes/plates.
• e.g. Nutrient agar
• Semisolid medium:
• 0.3-0.5% agar.
• for the observation of bacterial motility and preservation of bacteria.
• Eg. Nutrient Broth
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15. CLASSIFICATION OF M. O. ON THE BASIS OF
NUTRITIONAL REQUIREMENT
1.Source of energy: derive their energy from sunlight are called
Phototrophs
Obtained energy from chemical reaction called as chemotrophs ( E.coli)
2. Source of electron: bacteria reduced inorganic compounds as a electron
donars are called as lithotrophs.( e.g- Pseudomonas), organic
compound called as organotrophs.
3.source of corbon: autotrophs
4. source of Nitrogen
Source of sulphur, Phosphorus, mineral salt and growth
factor.
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16. TYPES OF MEDIA:
Bacteria infecting humans (commensals or
pathogens) are chemoorganoheterotrophs. When
culturing bacteria, it is very important to provide
similar environmental and nutritional conditions that
exist in its natural habitat. Hence, an artificial culture
medium must provide all the nutritional components
that a bacterium gets in its natural habitat. Most often,
a culture medium contains water, a source of carbon &
energy, source of nitrogen, trace elements and some
growth factors. Besides these, the pH of the medium
must be set accordingly. Some of the ingredients of
culture media include water, agar, peptone, casein
hydrolysate, meat extract, yeast extract and malt
extract.
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17. • 1) Classification based on consistency:
• Culture media are liquid, semi-solid or solid and biphasic.
• A) Liquid media: These are available for use in test-tubes, bottles or flasks.
Liquid media are sometimes referred as “broths” (e.g nutrient broth). In liquid
medium, bacteria grow uniformly producing general turbidity. Certain aerobic
bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as
thin film called ‘surface pellicle’ on the surface of undisturbed broth.
• . Sometimes the initial turbidity may be followed by clearing due to autolysis,
which is seen in penumococci. Long chains of Streptococci when grown in liquid
media tend to entangle and settle to the bottom forming granular deposits.
• Liquid media tend to be used when a large number of bacteria have to be
These are suitable to grow bacteria when the numbers in the inoculum is
suspected to be low. Inoculating in the liquid medium also helps to dilute any
inhibitors of bacterial growth. This is the practical approach in blood cultures.
Culturing in liquid medium can be used to obtain viable count (dilution
Properties of bacteria are not visible in liquid media and presence of more than
one type of bacteria can not be detected.
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18. • B) Solid media: Any liquid medium can be rendered by the addition
of certain solidifying agents. Agar agar (simply called agar) is the
most commonly used solidifying agent. It is an unbranched
polysaccharide obtained from the cell membranes of some species
red algae such as the genera Gelidium. Agar is composed of two
long-chain polysaccharides (70% agarose and 30% agarapectin). It
melts at 95oC (sol) and solidifies at 42oC (gel), doesn’t contribute
nutritive property, it is not hydrolyzed by most bacteria and is
free from growth promoting or growth retarding substances.
However, it may be a source of calcium & organic ions.
• Most commonly, it is used at concentration of 1-3% to make a solid
agar medium. New Zealand agar has more gelling capacity than the
Japanese agar. Agar is available as fibres (shreds) or as powders.
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19. • C) Semi-solid agar: Reducing the amount of agar to 0.2-0.5%
renders a medium semi-solid.
• Such media are fairly soft and are useful in demonstrating bacterial
motility and separating motile from non-motile strains (U-tube and
Cragie’s tube). Certain transport media such as Stuart’s and Amies
media are semi-solid in consistency. Hugh & Leifson’s oxidation
fermentationm test medium as well as mannitol motility medium are
also semi-solid.
• D) Biphasic media: Sometimes, a culture system comprises of both
liquid and solid medium in the same bottle. This is known as
biphasic medium (Castaneda system for blood culture). The
is added to the liquid medium and when subcultures are to be
the bottle is simply tilted to allow the liquid to flow over the solid
medium. This obviates the need for frequent opening of the culture
bottle to subculture.Besides agar, egg yolk and serum too can be
used to solidify culture media. While serum and egg yolk are
normally liquid, they can be rendered solid by coagulation using
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20. BACTERIAL GROWTH PATTERNS
• In liquid medium:
Superficial growth;
Turbidity/diffuse;
Precipitate growing;
(sediment)
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In solid medium:
Confluent growth / Smear;
Colony:
a cluster of microorganisms growing
on a solid medium. It is directly visible
and arises from a single cell.
26. COLONY- (CLONE)
• Colony- A bacterial population derived from one
bacterial cell. The cells within the colony have identical,
genus, species, genetic and phenotypic characteristics.
• Pure bacteria - derived from a single colony.
• Selection of a pure colony -most important for
bacterial identification
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27. TYPES OF CULTURE MEDIA
B. Depending on oxygen requirement
i. Aerobic media
e.g. Mac Conkey’s Broth
i. Anaerobic media
e.g. Roberson’s cooked medium
C. Depending on chemical composition
i. Simple or Basal media
ii. Synthetic or defined media
iii. Nonsynthetic or complex media
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28. TYPES OF CULTURE MEDIA
D. Depending on Functional type:
i. Enriched media
ii. Enrichment media
iii. Selective media
iv. Indicator media
v. Differential media
vi. Assay media
vii. Transport media
viii. Storage media
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29. SPECIAL MEDIA
I. ENRICHED MEDIA: (Liquid form)
Media is enriched by addition of substances such as
blood, serum and egg to basal medium to facilitate
growth of particular microorganism.
e.g. Blood Agar ( Streptococcus),
ChocolateAgar (Neisseria,haemophilus)
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30. BLOOD AGAR PLATE (BA)
• Nutrient agar with 5% sheep blood
• Differential – Identify hemolysis - Some bacteria secrete
enzymes that lyse red blood cells (hemolysins) such that a
clearing around the colony appears.
• b hemolysis- complete clearing (white hemolysis)
• a hemolysis – incomplete clearing (green hemolysis)
• g hemolysis- no hemolysis
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32. II. ENRICHEMENT MEDIA:
When a Specific substances is added in a liquid medium
which medium inhibits the growth of unwanted bacteria
and favours the growth of wanted bacteria.
e.g. Tetrathionate broth (inhibits the growth of E.Coli), allow
Salmonella
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33. MACCONKEY AGAR (MAC)
• Selective and differential medium.
• Selective - Gram positive bacteria are inhibited by the
presence of bile salts and crystal violet inhibitors in the
medium Most of gram negative bacteria will grow.
• Differentiate- Between Gram negative bacteria by their
ability to ferment lactose.
• Pink colonies- Bacteria that ferment lactose
(precipitation of some salts in media by acid production).
• Pale colonies- Non fermenters
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35. EOSINE METHYLENE BLUE (EMB),
• Differentiatial between lactose
fermenting and non fermenting
enteric bacteria
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36. TELLURITE GLYCINE AGAR (TGA)
• Selective- Tellurite glycine
and lithium inhibit most
bacteria
• Preferential growth of
Staphyloccoci coagulase
positive (Staphyloccocus
aureus)
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37. BLOOD AGAR PLATES ARE OFTEN USED TO DIAGNOSE INFECTION.
ON THE RIGHT IS A POSITIVE STREPTOCOCCUS CULTURE; ON THE
LEFT A POSITIVE STAPHYLOCOCCUS
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38. III. SELECTIVE MEDIA: ( Solid form)
favor the growth of particular microorganism and inhibit
the growth of others..
e.g. MacConkeys agar ( E.Coli),
Jensen Agar medium
( Mycobacterium Tuberculosis)
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39. IV. INDICATOR MEDIA:.
e.g. Wilson and Blair
S. Typhi Reduces sulphite to sulphide in the presence of
glucose and the colonies have black metallic shine.
39
40. V. DIFFERENTIAL MEDIA:
Used to distinguish between different types of bacteria based
on some observable characteristics.
e.g. Blood agar medium( Hemolytic)
MacConkeys medium ( Lactose fermenter & non-
lactose fermentor)
These media provide environments in which different bacteria can be
distinguished from one another. For instance, violet red bile
agar is used to distinguish coliform bacteria such as Escherichia
coli from noncoliform organisms. The coliform bacteria appear
as bright pink colonies in this media, while noncoliforms
appear a light pink or clear.
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41. • Certain media are both selective and differential. For instance,
MacConkey agar differentiates lactose-fermenting bacteria from
nonlactose-fermenting bacteria while inhibiting the growth of
Gram-positive bacteria. Since lactose-fermenting bacteria are
often involved in water pollution, they can be distinguished by
adding samples of water to MacConkey agar and waiting for
growth to appear.
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42. PURE CULTURE OF BACTERIA
Definition: culture started from a single cell
Purpose: to isolate and maintain a single type of
bacteria
Methods: Sterilization -- removing all contaminants
Aseptic technique --handling cultures and
materials
Growth medium -- providing for cell
nutrition
Techniques: Streak plate (twice) -- most common
Spread plate (primarily used for counting)
Goal: to dilute bacteria
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43. • In order to work with microorganisms in the laboratory, it is
desirable to obtain them in pure cultures. Pure cultures of
bacteria can be obtained by spreading bacteria out and
permitting the individual cells to form masses of growth called
colonies. One can then pick a sample from the colony and be
assured that it contains only one kind of bacteria. Cultivating
these bacteria on a separate medium will yield a pure culture.
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44. PURE CULTURE TECHNIQUES
• Streak plate method
• Spread plate techniques
• Pour plate techniques
i. tube dilution
ii. Serial dilution
• Micromanipulator
• Roll tube technique
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45. ADVANTAGES OF PURE
CULTURE TECHNIQUES
• To isolate microorganism from heterogeneous or
mixed population
• To study morphology of bacteria
• To study role played by specific microorganisms.
• For identification of species
• Cultivation and Multiplication
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46. STREAK PLATE METHOD
• Principle: By spreading a large amount of bacteria over the
large surface area of a plate, the amount of bacteria is
diluted until individual cells are spread on the surface of the
plate. From these individual cells a single colony arises. All
of the cells in this colony are genetically identical
• In order to identify bacteria, it is necessary to obtain a pure
culture. This is one by using the streak-plate method.
Bacterial cells are spread over the surface of an agar plate in
a continuous dilution, so that the cells will be separated
from each other. When the plate is incubated, those
individual cells will grow into colonies that originated from a
single cell.
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48. Contamination of a streak plate resulting from leaving the plate open too long
48
49. Contamination of a streak plate resulting from leaving the plate open too long
49
50. STREAK PLATES
• Allow for the growth of isolated colonies on the surface of the agar.
• Used to isolate clones of a particular bacterial species/strain.
• An isolated colony, one that is not touching any other colonies, is
assumed to be a pure culture.
• May observe colony morphology that can be used to help identify the
bacterial species.
• Colonies of the same organism may grow differently on different
media, e.g. the shape, color, growth pattern of the colony may differ
on other types of media.
• Colony Morphology Characteristics
• Colony color
• Type of hemolysis (if grown on Sheep Blood Agar)
• Form
• Elevation
• Margin
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52. POUR PLATE METHOD
• Another method of separating bacteria is the pour plate
method. With the pour plate method, the bacteria are mixed
with melted agar until evenly distributed and separated
throughout the liquid. The melted agar is then poured into an
empty plate and allowed to solidify. After incubation, discrete
bacterial colonies can then be found growing both on the agar
and in the agar.
• In this method, the mixed culture is diluted directly in tubes of
liquid (cooled ) agar medium.
• Inoculum is maintained in liquid state at temp 45oC
• Transfer into Petri plate
• Allow to solidify
• And incubate
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55. ADVANTAGES
• Surface and subsurface colonies are developed
• Counting of colonies is possible
55
56. DISADVANTAGES
• Tedious, time consuming and requires skill
• MO subjected to hot shock b’cos liquid medium is maintained
at at temp 45oC
• Unsuitable for isolating psychrophile bacteria( damaged by the
melted agar)
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57. Two processes for isolating bacteria from a mixed culture. (a) The streak plate technique. (b) The
pour plate technique.
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58. SERIAL DILUTION METHOD
As stated earlier, this method is commonly used to
obtain pure cultures of those microorganisms that
have not yet been successfully cultivated on solid
media and grow only in liquid media. A microorganism
that predominates in a mixed culture can be isolated
in pure form by a series of dilutions
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59. If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then
have 100 microorganisms in 10 ml or 10 microorganisms/ ml. If we add 1 ml of this suspension to
another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism. If this
tube shows any microbial growth, there is a very high probability that this growth has resulted from
the introduction of a single microorganism in the medium and represents the pure culture of that
microorganism.
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62. SPREAD PLATE TECHNIQUES
• Used for isolation of diluted mix population of
microorganism so that individual colonies can be
isolated.
• In this techniques microorganism are spread over the
solidified agar with a sterile glass rod or spreader.
• The theory behind this techniques is that as that as the
petridish is rotated at some stage single cell will be
deposited from a clump of cell. Some of these cells will
be separated from each other by a distance sufficient
to allow the colonies to develop on agar medium.
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65. • The inoculum is subjected to serial dilution in a sterile liquid medium,
and a large number of tubes of sterile liquid medium are inoculated
with aliquots of each successive dilution. The aim of this dilution is to
inoculate a series of tubes with a microbial suspension so dilute that
there are some tubes showing growth of only one individual microbe.
For convenience, suppose we have a culture containing 10 ml of
liquid medium, containing 1,000 microorganisms i.e., 100
microorganisms/ml of the liquid medium
65
66. ADVANTAGES
• Simple method
• Used to isolate the microorganism from any sample like:
Milk,food,soil etc.
• Embedded colonies are developed hence microbial count is possible.
• Microorganism are not exposed to higher temperature.
• Disadvantages:
• Chances of contamination
• If the sample is not spread properly or evenly then isolation is
possible.
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67. MICROMANUPULATOR
Device that can pick up single cell from a
colony of mixed culture. These are used in
conjunction with microscope to pick up a
single bacterial cell from hanging drop
techniques.
The single microbial cell is gently sucked into
the micropipette and transferred on to a large
drop of sterile medium on another cover slip.
67
68. ADVANTAGES
• Method makes one reasonably sure of the pure culture coming from
single cell.
• However device has to be used with skill and precision.
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69. 5. ROLL TUBE TECHNIQUES
• Used for isolation of stringent anaerobes
• In this method stoppered anaerobic culture tube is used for
isolation which has been coated with a prereduced agar medium
containing oxygen free nitrogen.
• When stopper is removed the tube is kept anaerobic by
continuously flushing oxygen free CO2 from a gas canula.
• Inoculation is done with a transfer loop held against the agar
surface as the tube is being rotated by a motor.
• Inoculation starts from the bottom and draws the loop gradually
upward.
• After inoculation the tube is restoppered and and incubated
anaerobically to get well isolated colonies.
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