Special Microbial Media Types and Examples
•
Topic –
Media
Presented by
Pallavi Mohite
Assistant Professor
Department of
Biotechnology
Deogiri College, Aurangabad
Media
Class M.Sc. Biotech
Pallavi Mohite
Asst. Prof.
DCA
Special microbial media.
•
Selective media
•
Indicator or Differential media
•
Enriched media
•
Enrichment media
•
Microbiological assay media
•
Transport media
Selective media
•
Selective media
allow certain types of organisms to grow
,
and inhibit the growth of other organisms.
•
The selectivity is accomplished in several ways. For example,
organisms that can utilize a given
sugar
are easily screened by
making that sugar the
only carbon source
in the medium.
•
On the other hand, selective inhibition of some types of
microorganisms can be achieved by adding
dyes, antibiotics,
salts or specific inhibitors
which affect the metabolism or
enzyme systems of the organisms.
MANNITOL SALT AGAR (MSA)
•
Mannitol salt agar is a selective medium used for the isolation of
pathogenic staphylococci
.
•
The medium contains mannitol, a phenol red indicator, and 7.5% sodium
chloride.
•
The high salt concentration inhibits the growth of most bacteria other
than staphylococci.
•
On MSA, pathogenic
Staphylococcus aureus
produces small colonies
surrounded by yellow zones.
•
The reason for this change in color is that
S. aureus
ferments the
mannitol, producing an acid, which, in turn, changes the indicator from
red to yellow
.
•
The growth of other types of bacteria is generally inhibited.
MacConkey Agar
•
MacConkey agar (MAC)
was the first solid differential media to be
formulated which was developed at 20th century by Alfred Theodore
MacConkey.
•
MacConkey agar
is a selective and differential media used for the
isolation and differentiation of
non-fastidious gram-negative rods
,
particularly members of the family
Enterobacteriaceae
and the
genus
Pseudomonas
.
•
Principle of MacConkey Agar
•
MacConkey agar is used for the isolation of gram-negative enteric
bacteria and the differentiation of lactose fermenting from lactose non-
fermenting gram-negative bacteria.
•
Pancreatic digest of gelatin
and
peptones (meat and casein)
provide
the essential nutrients, vitamins and nitrogenous factors required for
growth of microorganisms.
•
Lactose monohydrate
is the fermentable source of carbohydrate. The
selective action of this medium is attributed to
crystal violet
and
bile
salts,
which are inhibitory to most species of gram-positive bacteria.
•
Sodium chloride
maintains the osmotic balance in the
medium.
Neutral red
is a pH indicator that turns red at a pH below 6.8
and is colorless at any pH greater than 6.8.
Agar
is the solidifying agent.
DIFFERENTIAL MEDIA
•
Differential media are used to
differentiate closely related
organisms or groups of organisms.
•
Owing to the presence of certain dyes or chemicals in the
media, the organisms will produce
characteristic changes
or
growth patterns that are used for identification or
differentiation.
•
A variety of selective and differential media are used in
medical, diagnostic and water pollution laboratories, and in
food and dairy laboratories.
•
Most common selective and differential media are described
below and will be used in the laboratory exercise.
EOSIN METHYLENE BLUE
AGAR (EMB agar)
•
Eosin methylene blue agar is a differential medium used for the
detection and isolation of
Gram-negative intestinal pathogens
.
•
A combination of
eosin and methylene blue
is used as an indicator
and allows differentiation between organisms that ferment lactose
and those that do not.
•
Saccharose is also included in the medium because certain members
of the Enterobacteria or coliform group ferment saccharose more
readily than they ferment lactose.
•
In addition,
methylene blue acts as an inhibitor to Gram-positive
organisms. Colonies of
E. coli
normally have a dark center and a
greenish metallic sheen, whereas the pinkish colonies of
Enterobacter aerogenes
are usually mucoid and much larger than
colonies of
E. coli
.
•
Other organisms, such as Salmonella (one of the causative agents of
food poisoning), do not ferment lactose or saccharose and produce
colonies that are noncolored.
Enriched medium (added growth
factors)
•
In some cases, it is necessary to formulate an
enriched
medium.
Such a medium provides
specific nutrients
that
encourage
selected species of microorganisms to flourish in a
mixed sample.
•
Addition of extra nutrients in the form of
blood, serum, egg
yolk
, etc, to basal medium makes enriched medium.
•
Enriched media are used to grow nutritionally extracting
(fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s
serum slope, etc are a few examples of enriched media.
•
Blood agar is prepared by adding 5-10% (by volume) blood to
a blood agar base.
Chocolate agar
is also known as heated
blood agar or lysed
blood agar
.
Blood agar
•
Blood agar, like most other nutritional
media, has one or more protein sources,
salt, and beef extract for vitamins and minerals. Besides these
components, 5% defibrinated mammalian blood is also added to the
medium.
•
Blood agar is an enriched nutritious medium that supports the
growth of
fastidious organisms by supplementing it with blood
or
as a general medium without the blood.
•
The blood added to the base provides more nutrition to the medium
by providing
additional growth factors
required for these fastidious
organisms.
•
The blood also aids in visualizing
hemolytic reactions
of different
bacteria. The hemolytic reactions, however, depend on the type of
animal blood used.
•
Sheep blood is mostly used for Group A Streptococci as it provides
the best results, but it fails to support the growth of
Haemophilus
haemolyticus
. It is because sheep blood is deficient in pyridine
nucleotides.
•
The growth and hemolysis of
H. haemolyticus
are best with horse
blood, and the pattern of hemolysis might even mimic
Streptococcus
pyogenes
on sheep blood.
Blood Agar and
Hemolysis
•
Hemolysis is the lysis of red blood cells in the blood due to the
extracellular enzymes produced by certain bacterial species. The
extracellular enzymes produced by these bacteria are called
hemolysins which radially diffuse outwards from the colonies,
causing complete or partial lysis of the red blood cells.
1. Alpha hemolysis
•
Alpha hemolysis is defined by a
greenish-grey or brownish
discoloration around the colony
as a result of the partial lysis of the
red blood cells.
•
During α-hemolysis,
H
2
O
2
produced by the bacteria causes
hemoglobin
present in the RBC of the medium is converted into
methemoglobin
.
•
Some of the α-hemolytic species are a part of the human normal
flora, but some species like
Streptococcus pneumonia
causes
pneumonia and other such severe infections.
•
2. Beta hemolysis
•
Beta hemolysis is defined by a
clear zone of hemolysis
under
and around the colonies when grown on blood agar.
•
The clear zone appears as a result of the
complete lysis of the
red blood
cells present in the medium, causing denaturation of
hemoglobin to form colorless products.
•
β-hemolytic bacteria include the Group A streptococci like
S.
pyogenes
and
S. agalactiae
, both of which are associated with
severe infections in humans.
•
3. Gamma hemolysis
•
Gamma hemolysis is also called
non-hemolysis
as no lysis of
red blood cells occurs.
•
As a result, no change of coloration or no zone of hemolysis os
observed under or around the colonies.
•
Species like
Neisseria meningiditis
are non-hemolytic or
gamma-hemolytic.
Special microbial media play a crucial role in microbiology for selective growth, inhibition, and differentiation of microorganisms. Explore various types like selective media, enrichment media, transport media, and more. Discover examples such as Mannitol Salt Agar (MSA) and MacConkey Agar, along with their applications and principles. Enhance your understanding of microbial culture techniques with these specialized media formulations.
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Presentation Transcript
Topic Media Presented by Pallavi Mohite Assistant Professor Department of Biotechnology Deogiri College, Aurangabad
Media Class M.Sc. Biotech Pallavi Mohite Asst. Prof. DCA
Special microbial media. Selective media Indicator or Differential media Enriched media Enrichment media Microbiological assay media Transport media
Selective media Selective media allow certain types of organisms to grow, and inhibit the growth of other organisms. The selectivity is accomplished in several ways. For example, organisms that can utilize a given sugar are easily screened by making that sugar the only carbon source in the medium. On the other hand, selective inhibition of some types of microorganisms can be achieved by adding dyes, antibiotics, salts or specific inhibitors which affect the metabolism or enzyme systems of the organisms.
MANNITOL SALT AGAR (MSA) Mannitol salt agar is a selective medium used for the isolation of pathogenic staphylococci. The medium contains mannitol, a phenol red indicator, and 7.5% sodium chloride. The high salt concentration inhibits the growth of most bacteria other than staphylococci. On MSA, pathogenic Staphylococcus aureus produces small colonies surrounded by yellow zones. The reason for this change in color is that S. aureus ferments the mannitol, producing an acid, which, in turn, changes the indicator from red to yellow. The growth of other types of bacteria is generally inhibited.
MacConkeyAgar MacConkey agar (MAC) was the first solid differential media to be formulated which was developed at 20th century by Alfred Theodore MacConkey. MacConkey agar is a selective and differential media used for the isolation and differentiation of non-fastidious gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas. Principle of MacConkey Agar MacConkey agar is used for the isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non- fermenting gram-negative bacteria. Pancreatic digest of gelatin and peptones (meat and casein) provide the essential nutrients, vitamins and nitrogenous factors required for growth of microorganisms. Lactose monohydrate is the fermentable source of carbohydrate. The selective action of this medium is attributed to crystal violet and bile salts, which are inhibitory to most species of gram-positive bacteria. Sodium chloride maintains medium. Neutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8. Agar is the solidifying agent. the osmotic balance in the
DIFFERENTIAL MEDIA Differential media are used to differentiate closely related organisms or groups of organisms. Owing to the presence of certain dyes or chemicals in the media, the organisms will produce characteristic changes or growth patterns that are used for identification or differentiation. A variety of selective and differential media are used in medical, diagnostic and water pollution laboratories, and in food and dairy laboratories. Most common selective and differential media are described below and will be used in the laboratory exercise.
EOSIN METHYLENE BLUE AGAR (EMB agar) Eosin methylene blue agar is a differential medium used for the detection and isolation of Gram-negative intestinal pathogens. A combination of eosin and methylene blue is used as an indicator and allows differentiation between organisms that ferment lactose and those that do not. Saccharose is also included in the medium because certain members of the Enterobacteria or coliform group ferment saccharose more readily than they ferment lactose. In addition, methylene blue acts as an inhibitor to Gram-positive organisms. Colonies of E. coli normally have a dark center and a greenish metallic sheen, whereas the pinkish colonies of Enterobacter aerogenes are usually mucoid and much larger than colonies of E. coli. Other organisms, such as Salmonella (one of the causative agents of food poisoning), do not ferment lactose or saccharose and produce colonies that are noncolored.
Enriched medium (added growth factors) In some cases, it is necessary to formulate an enriched medium. Such a medium provides specific nutrients that encourage selected species of microorganisms to flourish in a mixed sample. Addition of extra nutrients in the form of blood, serum, egg yolk, etc, to basal medium makes enriched medium. Enriched media are used to grow nutritionally extracting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler s serum slope, etc are a few examples of enriched media. Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. Chocolate agar is also known as heated blood agar or lysed blood agar.
Blood agar Blood agar, like most other nutritional media, has one or more protein sources, salt, and beef extract for vitamins and minerals. Besides these components, 5% defibrinated mammalian blood is also added to the medium. Blood agar is an enriched nutritious medium that supports the growth of fastidious organisms by supplementing it with blood or as a general medium without the blood. The blood added to the base provides more nutrition to the medium by providing additional growth factors required for these fastidious organisms. The blood also aids in visualizing hemolytic reactions of different bacteria. The hemolytic reactions, however, depend on the type of animal blood used. Sheep blood is mostly used for Group A Streptococci as it provides the best results, but it fails to support the growth of Haemophilus haemolyticus. It is because sheep blood is deficient in pyridine nucleotides. The growth and hemolysis of H. haemolyticus are best with horse blood, and the pattern of hemolysis might even mimic Streptococcus pyogenes on sheep blood.
Blood Agar andHemolysis Hemolysis is the lysis of red blood cells in the blood due to the extracellular enzymes produced by certain bacterial species. The extracellular enzymes produced by these bacteria are called hemolysins which radially diffuse outwards from the colonies, causing complete or partial lysis of the red blood cells. 1. Alpha hemolysis Alpha hemolysis is defined by a greenish-grey or brownish discoloration around the colony as a result of the partial lysis of the red blood cells. During -hemolysis, H2O2 produced by the bacteria causes hemoglobin present in the RBC of the medium is converted into methemoglobin. Some of the -hemolytic species are a part of the human normal flora, but some species like Streptococcus pneumonia causes pneumonia and other such severe infections.
2. Beta hemolysis Beta hemolysis is defined by a clear zone of hemolysis under and around the colonies when grown on blood agar. The clear zone appears as a result of the complete lysis of the red blood cells present in the medium, causing denaturation of hemoglobin to form colorless products. -hemolytic bacteria include the Group A streptococci like S. pyogenes and S. agalactiae, both of which are associated with severe infections in humans. 3. Gamma hemolysis Gamma hemolysis is also called non-hemolysis as no lysis of red blood cells occurs. As a result, no change of coloration or no zone of hemolysis os observed under or around the colonies. Species like Neisseria meningiditis are non-hemolytic or gamma-hemolytic.
