Transcript Four Quadrant straking and Initial Growth Characteristicsx

Faculty of Medicine and Health Sciences
Microbiology Lab Experiment 11
Four Quadrant Streaking and Initial Growth
Characteristics
(Second Semester 2015-2016)
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Introduction:
In nature, bacteria are usually found in mixed populations.
For examples:
Thousands of bacterial species can be found in soil
Hundreds if not thousands of bacterial species can be found among human intestinal
microbiota
Many bacterial species can be found among oral and skin microbiota
In addition:
More than one bacterial species may be involved simultaneously is causing an
infection.
A sample obtained for culture from a patient, may contain members of microbiota that
are found at the infection site.
Accordingly, when ever you culture a sample (say, a clinical sample), you may get
many bacterial species growing on your culture plate.
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Definition:
A Pure bacterial culture: it is a bacterial culture that consists only of one bacterial
species. It can only be obtained in Microbiology Labs.
It is important to have a pure bacterial culture so as to be able to conduct many bacterial
diagnostics tests or other types of experiments.
Remember that a bacterial cell is so small to be seen by our naked eyes.
However, when a bacterial cell is cultured on solid a culture medium such as Nutrient agar,
this bacterial cell will replicate during the incubation period to form a bacterial colony that
is visible by our naked eyes.
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Definition:
A Bacterial colony: it is a cluster of identical bacterial cells on the surface of (or within)
a solid medium, that is usually derived from a single parental bacterial cell (or a colonyforming unit).
In other words, a bacterial colony is an accumulation of billions of bacterial cells that
forms a mass that can be seen by our naked eyes. All of these bacterial cells are derived
in theory from a single parental cell that has replicated to generates billions of its
descendants.
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It is so important to bear in our minds all the time that a microbiology clinical sample from a
patient may contain more than one bacterial species.
However, a clinical sample from a patient that has an infection , may contains billions of
bacterial cells that may be of a single bacterial species or more than one species a patient’s .
Remember that it was mentioned before that more than one bacterial species may be involved in
causing an infection.
Another possibility is that in addition to the bacterial species that causes the infection, there may
be some bacterial species that are members of microbiota which can be found at the site of
infection.
A third possibility is that bacterial species that are members of microbiota may get included in
the sample while obtaining the sample from the patient.
Accordingly, our first priority is to culture this sample in a way that will help us to separate these
bacterial species from each other and then to subculture them in order to be able to conduct the
needed subsequent diagnostic tests, because all bacterial diagnostic tests are mainly based on
having a single bacterial species during the perforation of each test
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The main aims of this experiment are:
1- To culture a clinical microbiology sample in such a way to obtain separate individual
colonies.
2- To examine these colonies to obtain the initial growth features of these cloning in
terms of their:
Size
Shape
Color (pigment production)
Margin
Surface
Texture
In addition, some of the initial growth features can be obtained
characaestics . Some of these initial characteristics depend on the used the agar culture
medium.
Note: The colonies of deferent bacterial species may have different features
regarding their, size Shape, color (pigment production), margin, surface and
texture
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How to culture a clinical sample in a way to obtain separate individual
colonies:
It was mentioned earlier that a clinical sample from a patient may contain billions of bacterial cells
Accordingly, it is important to culture the sample onto the surface of an agar plate in such a way that the
bacterial cell on the surface are separated enough from each other so as to obtain colonies that are also
separated from each other. Is possible to do so by:
I- Preparing a serial dilution of the sample:
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2- Diluting the sample while streaking by using the FOUR QUADRANT
STREAKING METHOD:
Procedure:
By using a sterile loop, inoculate the first quadrant the agar plate.
 Flame and cool the inoculating loop.
Streak the second quadrant of the plate by touching the loop into the first quadrant and
streaking all the way across the second quadrant, making six to eight strokes.
 Flame and cool the loop.
 Streak the third quadrant by touching the loop into the second quadrant and streaking into the
third quadrant, making six to eight strokes.
Flame and cool the loop.
Streak the fourth quadrant in a manner to produce isolated colonies. Touch the loop to the third
quadrant and spread the organism into the fourth quadrant using a continuous streak..
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Initial Growth Characteristics:
Identification of a bacterial pathogen that causes an infection
can be started by examining the features of its colonies , such as
size, color, and other features (mentioned in the previous slides)
Examples:
1- Colony Size and margin:
Although both have circular margins, however, in term of the
sizes of their colonies, in general Streptococcal species have
smaller colonies that Staphylococcal species.
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2- Capsulated bacteria have mucoid colonies
Other features of bacterial
colonies that may also be
examined, is the texture of the
colony. In general, the colonies
of capsulated bacteria are
relatively large and exhibit
mucoid texture such as the
colonies of the capsulated
bacterium Klebsiella pneumonia
The colonies seen in this image are those
of E coli, which is. in most cases, not
capsulated.
Compare the colonies of E coli with the
mucoid colonies of Klebsiella
pneumoniae (capsulated bacterium) in
the image shown above.
Notice that the colonies of E coli are
not mucoid
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Some of the initial growth characteristics (features)
may be obtained based on the used culture media
I- Blood agar:
Blood agar is an enriched medium that allows the growth of many types of both Grampositive and Gram-negative bacteria.
Bacterial species that produce hemolysins, which are a pore forming exotoxin, which
have a transparent zone around their colonies (this is known as Beta-hemolysis).
As you know, a single bacterial colony contains millions or billions of bacterial cells.
When all bacterial cells in a single colony produce hemolysin, this exotoxin will diffuse
into the Blood agar medium around the colony and causes complete hemolysis of RBC
(Beta hemolysis) found in the Blood agar. This explains that why you see a transparent
zone around the colonies that produce hemolysin).
Some bacterial species produce substances that oxidize hemoglobin. So, on Blood agar,
the colonies of these will have a greenish zone around them.
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This greenish zone is appears because of the oxidation of hemoglobin around the colonies mediated by
the substance that is produced by billions of bacterial cells in the colony, which diffuses into the blood
agar medium around the colony (This is known as alpha hemolysis)
Other bacterial species do not produce any hemolysin exotoxin or any substance that oxidizes
hemoglobin. In this case, you will not see any change in the Blood agar around the colonies of these
bacterial species. (This known as Gamma hemolysis, which means NO HEMOLYSIS
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Swarming Motility of Proteus on Blood agar:
Certain species of Proteus show a characteristic swarming motility on some
types of culture media such as blood agar
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MacConkey agar:
It is a selective agar medium that inhibit the growth of Gram-positive bacteria.
The inhibition of Gram-positive bacteria is because this medium has bile salts and crystal
violet.
In addition, this medium has Lactose and a pH indicator.
The appearance of fuchsia colored colonies indicates that the growing bacterium is a
lactose ferementer. Lactose fermentation generates acidic products that change the pH of
the agar medium and thus, the pH indicator changes into its acidic color.
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EMB agar:
It is a selective agar medium that allows the allows of only Gram-negative bacteria.
However, E coli exhibits a characteristic colonies on this agar medium that have a
characteristic metallic sheen appearance
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Manitol Salt Agar:
It is both a selective and a differential agar medium that inhibit the all bacterial pathogens
except those of the genus Staphylococci.
This inhibition of all other bacterial pathogens is mainly because of the high salt concentration
that this medium has.
In addition, this agar medium contains the sugar Manitol and a pH indicator.
Although both Staphylococcus aureus and Staphylococcus epidermidis, it is only that the species
Staphylococcus aureus ferments Manitol.
Fermentation of Manitol changes the pH of the agar medium and thus the color of the pH
indicator (the color of the agar medium).
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The Growth Culture of Certain Bacterial Species Has a Characteristic
Odor (Smell).
These characteristic odors, can help in the identification (Diagnosis) of certain
bacterial species with specific odor
 A Pure culture of E coli has a characteristic rotten apple-like smell
A Pure culture of Proteus species have a characteristic smell (Fishy)
A Pure culture of Pseudomonas spp have a characteristic smell (Aromatic )
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Production of Certain Characteristics Pigments
Some bacterial species produce characteristic pigments.
To see these pigments, it is preferred that the pigment-producing bacterium is cultured on
an agar medium that has no pH indicator in order to be confused with the color of
colonies that may be cause of the change in the color of the pH indicator.
Nutrient agar is recommended to detect the production of pigments by certain types of
bacteria.
These characteristic pigments can help in identifying (diagnosis) of the grown bacterial
species.
Example:
Pseudomonas aeruginosa produces several types of fluorescent pigments:
1- Blue-green pigments (Known as Pyocyanin)
2- Yellow-green pigment (known as Pyoverdine)
3- Red-brown pigment (Known as Pyorubin)
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Once you get separate colonies , the following information can be obtained:
1- If the grown colonies are of different sizes or colors…. you may tell how many
pathogens are causing the infection ( specially if the infection is at a sterile body
location)
2- Some bacterial pathogens have colonies with particular and specific characteristics
that would reveal the identity of the infecting pathogen
3- Single colonies are pick up using a sterile loop or needle so that each colony can be
sub-cultured on a new plate in order to obtain a pure culture. This pure culture is
needed to carry out consequent experiments ( tests) needed to identify the infecting
bacterial pathogen or to carry out antibiotic sensitivity tests. .
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SUBCULTING INDIVIDUAL BACTERIALCOLONIES ONTO A
SEPARATE PLATE TO OBYAINE A PURE CULTURE
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