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CULTURE MEDIA
Microbiological Culture
Method of cultivating microbial organisms by letting
them reproduce in predetermined culture media under
controlled laboratory conditions
Culture Media
• Study of microorganisms depends on its ability to grow in
the laboratory
• Growth of microorganisms is possible only if suitable culture
media is available
• A culture medium is defined as a solid or liquid preparation
used for the growth, transport and storage of
microorganisms
• The effective culture medium must contain all the nutrients
required for the growth of the microorganism.
How many types of growth media?
• There are two major types of growth media:
• Cell culture: which use specific cell types
derived from plants or animals
• Microbiological culture: which are used for
growing microorganisms, such as bacteria or
yeast.
Nutrient Requirements
• Nutritional requirements vary greatly among the
microorganisms
• In common all microorganisms need sources of energy,
nitrogen, carbon, water, phosphorus, sulfur, and
various minerals
• The exact composition of a satisfactory medium will
rely on the species to be identified and cultivated
Reasons for Culturing
• Bacteria have to be grown (cultured) for them to be identified
and subsequent clinical diagnosis
• Culturing on solid media is another convenient way of
separating bacteria in mixture
• Bacteria have to be cultured in order to obtain antigens from
developing serological assay for vaccines
• Certain genetic studies and manipulations of the cells also
need that bacteria be cultured invitro
History of culture media
• Louis Pasteur used simple broths made up of urine or
meat extracts
• Robert Koch realized the importance of solid media
and used potato pieces to grow bacteria
• It was on the suggestion of Fannie Eilshemius, wife of
Walther Hesse (who was an assistant to Robert Koch)
that agar was used to solidify culture media
History of culture media
• Before the use of agar, attempts were made to
use gelatin as solidifying agent. Gelatin had
some inherent problems….
• It existed as liquid at normal incubating
temperatures (35-37incubating temperatures
(35-37oC)
• Digested by certain bacteria
Agar
• Used for preparing solid medium
• Polysaccharide extract obtained from seaweed
• 2% agar is employed in solid medium
• Agar is an ideal solidifying agent as it ia:
• No nutritive value
• Bacteriologically inert i.e, no affected on the growth of
bacteria
• Remains solid at 370C
• Melts at 980C
• It is transparent
Composition of culture media
• Provide similar environmental and nutritional conditions
that exist in its natural habitat
• An artificial culture medium must provide all the nutritional
components that a bacterium gets in its natural habitat
• A culture medium contains water, a source of carbon &
energy, source of nitrogen, trace elements and some growth
factors
• The pH of the medium must be set accordingly
Classification of Media
• Bacterial culture media can be classified based on
1.Consistancy
2.Nutritional component
3.Functional use
Classification of Culture Media
I. Based on the consistency
a) solid medium
b) liquid medium
c) semi solid medium
II. Based on the ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
III. Based on functional use or application
1) Basal media
2) Special media
• Enriched media
• Selective media
• Enrichment media
• Differential media
• Transport media
• Indicator media
• Anaerobic media
Classification based on consistency
A) Solid media:
• An agar plate is a Petri dish that contains a growth medium (typically
agar agar plus nutrients)used to culture microorganisms.
• Agar is the most commonly used solidifying agent
• Used for separating mixed bacteria
• Bacteria can be identified basing on colony character
• Used for the isolation of bacteria as pure culture
• Colony morphology, pigmentation, hemolysis can be appreciated.
• Eg: Nutrient agar, Blood agar
B) Liquid media:
• Contains no agar
• Used for profuse growth, inoculum preparation
• Eg: Nutrient broth
• Available for use in test-tubes, bottles or flasks
• Referred to as broths (e.g nutrient broth)
• Bacteria grow uniformly producing general turbidity
• Mixed organisms cannot be separated
C) Semi-solid agar:
• Contains 0.5% agar
• fairly soft and useful in demonstrating bacterial motility and
separating motile from non-motile strains
• Eg: SIM
Different Types of Media
• Basal/Simple media: Basal media are basically
simple media that supports most non-
fastidious bacteria
• Ex: Peptone water, nutrient broth and nutrient
agar
• NB consists of peptone, yeast extract, NaCl
NB + 2% agar = Nutrient agar
• Complex media
• Media other than basal media
• They have added ingredients
• Provide special nutrients
• specially prepared for research purposes
• composition of every component is well known.
• Synthetic or defined media
• Media prepared from pure chemical substances and its
exact composition is known
• Eg: peptone water – 1% peptone + 0.5% NaCl in water
• Enriched media
• Enriched media are media that have been supplemented with highly
nutritious materials such as blood, serum, egg yolk or yeast extract
for the purpose of cultivating fastidious organisms.
• Addition of extra nutrients to basal medium makes them enriched
media
• Used to grow bacteria that are exacting in their nutritional needs
• Ex: Chocolate agar, Blood agar
• Enrichment media
• Liquid media used to isolate pathogens from a mixed culture
• Media is incorporated with inhibitory substances to suppress the
unwanted organism
• Eg: Selenite F Broth –for the isolation of Salmonella, Shigella
• Alkaline Peptone Water – for Vibriocholerae
• Enriched media contains the nutrients required to support the
growth of a wide variety of organisms including some
fastidious ones
• Used to grow as many different types of organisms as are
present in the specimen
• Enrichment media promotes the growth of a particular
organism by providing it with essential nutrients and rarely
contains certain inhibitory substances to prevent the growth
of normal competitors
• Ex: Selenite F Broth –favors the growth of Salmonella and also
prevent the growth of normal competitors like E.coli.
• E.coli does not die in the medium but they donot flourish like
salmonella does
Blood agar and Chocolate agar
• Selective media enrichment media
• Selective media allows the growth of certain type of organisms,
while inhibiting the growth of other organisms.
• Designed to inhibit unwanted commensal or contaminating bacteria and help
to recover pathogen from a mixture of bacteria
• This selectivity is achieved in several ways. For example, organisms that have
the ability to utilize a given sugar are screened easily by making that particular
sugar the only carbon source in the medium for the growth of the
microorganism.
• Like wise Inhibitory substance is added to solid media
• Any agar media can be made selective by addition of certain inhibitory agents
(antibiotics, dyes, chemicals, alteration of pH or a combination of these) that
don’t affect the pathogen.
• Examples of Selective media
• Thayer Martin Medium selective for Neisseria gonorrhoeae
• EMB agar: selective for gram-negative bacteria. The dye methylene blue in
the medium inhibits the growth of gram-positive bacteria; small amounts
of this dye effectively inhibit the growth of most gram-positive bacteria
• nutrient agar supplemented with the antibiotic penicillin can be used to
select gran negative bacteria
• Campylobacter Agar (CAMPY): selective isolation of Campylobacter jejuni
from fecal or rectal swabs
• LJ medium – M.tuberculosis
• Wilson and Blair medium – S.typhi
• Potassium tellurite medium– Diphtheria bacilli
• Mac Conkey’s medium for gram -ve bacteria
Differential media
• Differential media are widely used for differentiating closely
related organisms or groups of organisms
• Media are designed in such a way that different bacteria can be
recognized on the basis of their colony colour
• Because of the presence of certain dyes, metabolic substrates,
or chemicals in the media, those bacteria that utilize them
appear as differently coloured colonies
• The organisms will produce certain characteristic changes or
growth patterns that are used for identification or differentiation
of microorganism.
• Ex: Mac Conkey agar, Eosin Methylene Blue (EMB) agar
• Differential media
• A media which has substances incorporated in it
enabling it to distinguish between bacteria
• Eg: Mac Conkey’s medium Distinguish between lactose
fermenters & non lactose fermenters
• Lactose fermenters – Pink colonies
• Non lactose fermenters – colourless colonies
• Anaerobic media
• These media are used to grow anaerobic organisms
• Eg: Robertson’s cooked meat medium, Thioglycolate medium
• Transport media
• Media used for transporting the samples
• Delicate organisms may not survive the time taken for
transporting the specimen without a transport media
• Eg: Stuart’s medium – non nutrient soft agar gel containing a
reducing agent
• Buffered glycerol saline- enteric bacilli
• CHOCOLATE AGAR: Non-selective, enriched growth
medium. containing red blood cells that have been lysed by
slowly heating to 80 °C
• Used for growing fastidious bacteria, such as Haemophilus
influenzae
• BLOOD AGAR: Contains mammalian blood (usually sheep or
horse), typically at a concentration of 5– 10%
• Blood agar plates are enriched, differential media used to isolate
fastidious organisms and detect hemolytic activity
• Enrichment media liquid media that also
serves to inhibit commensal in the clinical
specimen. Selenite F broth and alkaline
peptone water are used to recover pathogens
from fecal specimens
Mannitol Salt Agar (MSA)
• Mannitol salt agar is both a selective and differential media used for the
isolation of pathogenic Staphylococci from mixed cultures.
•
• Components:
• 7.5% NaCl – selects for species of Staphylococcus. This concentration of
salt is too high for most other bacteria to withstand and , therefore,
inhibits their growth.
•
• Mannitol – alcohol of the carbohydrate mannose. Mannitol fermentation
produces acid end products which turn the medium yellow. Yellow
indicates mannitol positive and no color change indicates mannitol
negative.
•
• Phenol red pH indicator – yellow in acid pH (The same indicator that is
used in phenol red carbohydrate fermentation broths).
Culture Media for different Microorganism
Culture Media for different Microorganism

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Culture Media for different Microorganism

  • 2.
  • 3. Microbiological Culture Method of cultivating microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions
  • 4. Culture Media • Study of microorganisms depends on its ability to grow in the laboratory • Growth of microorganisms is possible only if suitable culture media is available • A culture medium is defined as a solid or liquid preparation used for the growth, transport and storage of microorganisms • The effective culture medium must contain all the nutrients required for the growth of the microorganism.
  • 5. How many types of growth media? • There are two major types of growth media: • Cell culture: which use specific cell types derived from plants or animals • Microbiological culture: which are used for growing microorganisms, such as bacteria or yeast.
  • 6. Nutrient Requirements • Nutritional requirements vary greatly among the microorganisms • In common all microorganisms need sources of energy, nitrogen, carbon, water, phosphorus, sulfur, and various minerals • The exact composition of a satisfactory medium will rely on the species to be identified and cultivated
  • 7. Reasons for Culturing • Bacteria have to be grown (cultured) for them to be identified and subsequent clinical diagnosis • Culturing on solid media is another convenient way of separating bacteria in mixture • Bacteria have to be cultured in order to obtain antigens from developing serological assay for vaccines • Certain genetic studies and manipulations of the cells also need that bacteria be cultured invitro
  • 8. History of culture media • Louis Pasteur used simple broths made up of urine or meat extracts • Robert Koch realized the importance of solid media and used potato pieces to grow bacteria • It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media
  • 9. History of culture media • Before the use of agar, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems…. • It existed as liquid at normal incubating temperatures (35-37incubating temperatures (35-37oC) • Digested by certain bacteria
  • 10. Agar • Used for preparing solid medium • Polysaccharide extract obtained from seaweed • 2% agar is employed in solid medium • Agar is an ideal solidifying agent as it ia: • No nutritive value • Bacteriologically inert i.e, no affected on the growth of bacteria • Remains solid at 370C • Melts at 980C • It is transparent
  • 11. Composition of culture media • Provide similar environmental and nutritional conditions that exist in its natural habitat • An artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat • A culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors • The pH of the medium must be set accordingly
  • 12. Classification of Media • Bacterial culture media can be classified based on 1.Consistancy 2.Nutritional component 3.Functional use
  • 13. Classification of Culture Media I. Based on the consistency a) solid medium b) liquid medium c) semi solid medium II. Based on the ingredients a) simple medium b) complex medium c) synthetic or defined medium d) Special media
  • 14. III. Based on functional use or application 1) Basal media 2) Special media • Enriched media • Selective media • Enrichment media • Differential media • Transport media • Indicator media • Anaerobic media
  • 15. Classification based on consistency A) Solid media: • An agar plate is a Petri dish that contains a growth medium (typically agar agar plus nutrients)used to culture microorganisms. • Agar is the most commonly used solidifying agent • Used for separating mixed bacteria • Bacteria can be identified basing on colony character • Used for the isolation of bacteria as pure culture • Colony morphology, pigmentation, hemolysis can be appreciated. • Eg: Nutrient agar, Blood agar
  • 16. B) Liquid media: • Contains no agar • Used for profuse growth, inoculum preparation • Eg: Nutrient broth • Available for use in test-tubes, bottles or flasks • Referred to as broths (e.g nutrient broth) • Bacteria grow uniformly producing general turbidity • Mixed organisms cannot be separated C) Semi-solid agar: • Contains 0.5% agar • fairly soft and useful in demonstrating bacterial motility and separating motile from non-motile strains • Eg: SIM
  • 17. Different Types of Media • Basal/Simple media: Basal media are basically simple media that supports most non- fastidious bacteria • Ex: Peptone water, nutrient broth and nutrient agar • NB consists of peptone, yeast extract, NaCl NB + 2% agar = Nutrient agar
  • 18. • Complex media • Media other than basal media • They have added ingredients • Provide special nutrients • specially prepared for research purposes • composition of every component is well known. • Synthetic or defined media • Media prepared from pure chemical substances and its exact composition is known • Eg: peptone water – 1% peptone + 0.5% NaCl in water
  • 19. • Enriched media • Enriched media are media that have been supplemented with highly nutritious materials such as blood, serum, egg yolk or yeast extract for the purpose of cultivating fastidious organisms. • Addition of extra nutrients to basal medium makes them enriched media • Used to grow bacteria that are exacting in their nutritional needs • Ex: Chocolate agar, Blood agar • Enrichment media • Liquid media used to isolate pathogens from a mixed culture • Media is incorporated with inhibitory substances to suppress the unwanted organism • Eg: Selenite F Broth –for the isolation of Salmonella, Shigella • Alkaline Peptone Water – for Vibriocholerae
  • 20. • Enriched media contains the nutrients required to support the growth of a wide variety of organisms including some fastidious ones • Used to grow as many different types of organisms as are present in the specimen • Enrichment media promotes the growth of a particular organism by providing it with essential nutrients and rarely contains certain inhibitory substances to prevent the growth of normal competitors • Ex: Selenite F Broth –favors the growth of Salmonella and also prevent the growth of normal competitors like E.coli. • E.coli does not die in the medium but they donot flourish like salmonella does
  • 21. Blood agar and Chocolate agar
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  • 23. • Selective media enrichment media • Selective media allows the growth of certain type of organisms, while inhibiting the growth of other organisms. • Designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria • This selectivity is achieved in several ways. For example, organisms that have the ability to utilize a given sugar are screened easily by making that particular sugar the only carbon source in the medium for the growth of the microorganism. • Like wise Inhibitory substance is added to solid media • Any agar media can be made selective by addition of certain inhibitory agents (antibiotics, dyes, chemicals, alteration of pH or a combination of these) that don’t affect the pathogen.
  • 24. • Examples of Selective media • Thayer Martin Medium selective for Neisseria gonorrhoeae • EMB agar: selective for gram-negative bacteria. The dye methylene blue in the medium inhibits the growth of gram-positive bacteria; small amounts of this dye effectively inhibit the growth of most gram-positive bacteria • nutrient agar supplemented with the antibiotic penicillin can be used to select gran negative bacteria • Campylobacter Agar (CAMPY): selective isolation of Campylobacter jejuni from fecal or rectal swabs • LJ medium – M.tuberculosis • Wilson and Blair medium – S.typhi • Potassium tellurite medium– Diphtheria bacilli • Mac Conkey’s medium for gram -ve bacteria
  • 25. Differential media • Differential media are widely used for differentiating closely related organisms or groups of organisms • Media are designed in such a way that different bacteria can be recognized on the basis of their colony colour • Because of the presence of certain dyes, metabolic substrates, or chemicals in the media, those bacteria that utilize them appear as differently coloured colonies • The organisms will produce certain characteristic changes or growth patterns that are used for identification or differentiation of microorganism. • Ex: Mac Conkey agar, Eosin Methylene Blue (EMB) agar
  • 26. • Differential media • A media which has substances incorporated in it enabling it to distinguish between bacteria • Eg: Mac Conkey’s medium Distinguish between lactose fermenters & non lactose fermenters • Lactose fermenters – Pink colonies • Non lactose fermenters – colourless colonies
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  • 30. • Anaerobic media • These media are used to grow anaerobic organisms • Eg: Robertson’s cooked meat medium, Thioglycolate medium • Transport media • Media used for transporting the samples • Delicate organisms may not survive the time taken for transporting the specimen without a transport media • Eg: Stuart’s medium – non nutrient soft agar gel containing a reducing agent • Buffered glycerol saline- enteric bacilli
  • 31. • CHOCOLATE AGAR: Non-selective, enriched growth medium. containing red blood cells that have been lysed by slowly heating to 80 °C • Used for growing fastidious bacteria, such as Haemophilus influenzae • BLOOD AGAR: Contains mammalian blood (usually sheep or horse), typically at a concentration of 5– 10% • Blood agar plates are enriched, differential media used to isolate fastidious organisms and detect hemolytic activity
  • 32. • Enrichment media liquid media that also serves to inhibit commensal in the clinical specimen. Selenite F broth and alkaline peptone water are used to recover pathogens from fecal specimens
  • 33. Mannitol Salt Agar (MSA) • Mannitol salt agar is both a selective and differential media used for the isolation of pathogenic Staphylococci from mixed cultures. • • Components: • 7.5% NaCl – selects for species of Staphylococcus. This concentration of salt is too high for most other bacteria to withstand and , therefore, inhibits their growth. • • Mannitol – alcohol of the carbohydrate mannose. Mannitol fermentation produces acid end products which turn the medium yellow. Yellow indicates mannitol positive and no color change indicates mannitol negative. • • Phenol red pH indicator – yellow in acid pH (The same indicator that is used in phenol red carbohydrate fermentation broths).