Faculty of Medicine and Health Sciences

General Microbiology Lab (7105404)

Lab-3
Four Quadrant Streaking and Initial Growth
Characteristics

Second Semester 2021-2022

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Introduction: Deduction G. ' Ii
wise
In nature, bacteria are usually found in mixed-populations. For examples:

➢Thousands of bacterial species can be found in soil and among intestinal-microbiota in both human and
animals.
➢Many bacterial species can be found among oral and skin microbiota.

In addition, during infections, more than one bacterial species may share in causing one infection.
Moreover, in certain infections, a clinical-sample obtained from an infected-patient, may contain the
bacterial pathogen that causes the infection and members of microbiota that are found at that site such as
pharyngitis, bacterial gastroenteritis, urinary tract infection.

Accordingly, when ever a clinical-sample is cultured,

one or more bacterial species is/are expected to grow.

Definition: Mixed Bacterial Culture

A Pure Bacterial Culture: it is a bacterial culture

that consists only of one bacterial species. It can
only be obtained in Microbiology Labs, but it is
unlikely to be found under natural circumstances.

Pure Bacterial Culture

Experiment- Part-A:
Preparation of a pure culture (s) from a microbiological sample:
It is important to have a pure bacterial culture so as to be able to conduct many bacterial tests,
such as:
Diagnostic tests, Antibiotic's susceptibility or any other type of bacteriological experiments.

A clinical sample may contain billions of bacterial cells of one or more different bacterial
species. So as to be able to conduct the needed tests for the purpose of identification or other
issues, it is important initially to obtain a pure culture(s) of the infecting agent (s).

Because of the fact that a clinical-sample may contain billions of bacterial cell of one or more
species, it is important initially to dilute the sample so that upon culturing the bacterial cells
on the surface of an agar medium, they will be separately-seeded and consequently,
individuals separated-colonies will be obtained by the end of the incubation period.
Methods used to dilute microbiological samples:
se
I- By preparing a serial dilution: (as shown in the figure given below)

Clinical sample
(for example Urine)
II- By Four Quadrant Streaking:

It is a simple and a practical-method that is commonly used to obtain separate colonies that does
not involve preparation of a serial dilution from the original sample to obtain separate colonies.
Instead, the bacterial cells are diluted while culturing the sample on the surface of the agar plate.
This can be a achieved by streaking a loopful of the sample by a method that is known as the Four
Quadrant Streaking.
●By using a sterile loop, inoculate the first quadrant of 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.
●Flame and cool the loop.
● Streak the third quadrant by touching the
loop into the second quadrant and streaking
into the third quadrant.
●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.
What is the importance of obtaining separated individual colonies:
1- It may be possible to know how many types of bacterial species are there in the
original sample based on the variation in the characteristic of the obtained colonies.
2- To subculture individual colonies to obtain pure culture(s) to further conduct other
experiments as mentioned earlier.
Experiment- Part-B:

Initial characteristics of a bacterial culture: colonial and growth characteristics

Diagnosis of bacteria by the classical methods is a laborious work that may require many types of
different tests such as Gram-staining, staining for certain cellular structural components such as the
capsule and the endospore, examining the biochemical and the enzymatic properties of the isolated
bacterium…….

However, before of all of these steps, bacterial diagnosis indeed may initially start by collecting
information about:

1- The characteristics of bacterial colony morphology of the isolated bacterium.

2- The initial growth characteristics of the isolated bacterium based on the used culture medium.

3- Other growth characteristics such as the odor of the culture of the isolated bacterium.

These information can be very useful and in some cases in is possible to identify the isolated bacterium
based on them.

Note: in many other cases, additional tests must be conducted to identify the isolated bacterium (to be
discussed during next labs).
1- The morphological characteristics of bacterial colonies
EXAMPLE

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Pigmentations:
some bacterial species produce characteristic pigments, which can be helpful in bacterial diagnosis. .
To see these pigments, it is preferred that the pigment-producing bacterium is cultured on transparent
agar medium such as Nutrient agar.

Examples:
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)

Green nail syndrome Ulcer

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Other bacterial species that are commonly-known to produce characteristic pigments:

Serratia merscence is known to produce a characteristic Red pigment.

Porphyromonas gingivalsis is known to produce a characteristic Black pigment.

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2- The initial growth characteristics of an isolated bacterial
species depends on the used culture medium
The initial growth characteristics of a grown bacterial species can be obtained from its initial culture,
which depend on the used culture medium such as:

I- Blood-Agar: which is an enrich medium that can support the growth of many types of Gram-positive
and Gram-negative bacteria. However, certain bacterial species may exhibit certain features on Blood-
agar that can be helpful in its identification and diagnosis, such as:
A-Hemolytic activity: which can classified as follows:

●Beta-Hemolysis
●Alpha hemolysis
●Gamma hemolysis
Hemolytic activity
●Beta-Hemolysis: it is characterized by the formation
of a transparent zone around the grown colonies, due to
complete lysis of RBCs. The complete hemolysis of
the RBCs in the medium is mediated by a hemolytic
enzyme produced by the bacterial cells of the grown
bacterium such as Staphylococcus aureus,
Streptococcus pyogenes and Streptococcus agalactiae

●Alpha hemolysis: it is characterized by the formation

of a greenish around the grown colonies, due to
chemical modification of hemoglobin of the RBCs in
the culture medium, by some bacterial species such as
Streptococcus pneumoniae and Streptococcus mutans.

●Gamma hemolysis: which indicates no alteration in

the Blood-Agar medium by the grown bacterium such
as Enterococci and Streptococcus bovis
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crystal salt
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B- Swarming motility:

It is a characteristic motility behavior of certain bacterial species.

Most motile bacterial species have a flagellum or more that enable them to swim in liquid
media (swimming-motility), but not on the surface of a solid agar-medium.

However, certain bacterial species exhibit a highly-active motility on the surface of certain agar
medium that enables them to swam on the surface of the medium.

Example: Proteus merabilis on the surface of Blood Agar.

 II- MacConkey Agar:

It is a selective-medium that allows the growth only of Gram-negative bacteria (not all). It does so
because it contains bile salts and Crystal violet that inhibit the growth of Gram-positive bacteria.

➢MacConkey Agar contains Lactose and a pH indicator. A grown Gram-negative bacterium that
ferments Lactose will have a characteristic pink colonies on this medium. The pink color that is
exhibited by the colonies is due to the change in the color of the pH indicator by the acid generated
during the fermentation process.

➢If the grown Gram-negative bacterium does not ferment Lactose, it will have a colorless
colonies.
Examples:

➢Gram-negative/ Lactose fermenters:

E. coli, Klebsiella and Enterobacter

➢Gram-negative/Non Lactose-fermenters:
Proteus, Pseudomonas and Salmonella

Note: the pink color is not a color of a

pigment produced by the grown bacterium
III- Eosin Methylene Blue (EMB) Agar:

It can be considered as both a selective- and a differential-medium.

As in the case of MacConkey Agar, EMB is a selective-medium that allows the growth of many types of
Gram-negative bacteria but it inhibits the growth of Gram-positive bacteria. This inhibitory effect on
Gram-positive bacteria is mediated by the two dyes that EMB contains, which are Eosin and Methylene-
Blue (hence the name EMB agar).
EMB contains both Lactose and Sucrose as well as pH indicators Lactose or Sucrose fermenter
(Eosin and Methylene Blue also function as pH indicators).
E coli

The original color of EMB is purple. However, a grown Gram--

negative bacterium that ferments Lactose or Sucrose (or both) will
have colonies with a dark-purple color.

This color reflects the change of the pH indicator, which becomes

dark-purple in acidic pH, which results due to the fermentation of
the sugars.

On the other hand, a grown Gram-negative bacterium that does

not ferment any of the sugars will have colorless colonies.
Interestingly, E coli exhibits a characteristic greenish-colonies
with a metallic-sheen appearance. This is attributed to its strong Lactose or Sucrose non-fermenter
fermenting-activity that generates a large amount of acids. Ii
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IV-Manitol Salt Agar (MSA):

It a selective- and a deferential-medium that inhibits

growth of most bacterial species but allows the Growth
of Staphylococci.

The inhibitory effect of this medium is mediated by its

high level of salt content (7.5%).

In Addition, MSA contains the sugar Manitol and a pH

indicator (Phenol Red).

Staphylococcus epidermidis and , Staphylococcus

aureus, both can grow on this medium. However,
Staphylococcus aureus ferments Manitol, but
Staphylococcus epidermidis does not do so.

Manitol fermentation generates acid, which turn the

color of the pH indictor into yellow.

Accordingly, Staphylococcus aureus exhibits yellow

colonies on this medium, while Staphylococcus
epidermidis exhibits white colonies

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V- Salmonella Shigella (SS) agar: selective
This medium is widely used to isolate mainly Salmonella and some species of Shigella
from clinical samples, specially, during stool culture. It allows the growth of Salmonella
or Shigella bacterial species, while inhibits the growth of almost all other types of
bacteria.
Differential medium
Salmonella typhi Produce Hydrogen sulfide (H2S) gas Black centered colonies
Shigella flexneri Doesn’t produce H2S gas Colorless colonies
3-The Culture of Certain Bacterial Species Has a
Characteristic Odor (Smell).
These characteristic odors, can help in the identification (Diagnosis) of certain bacterial
species

Example:

➢ E coli has a characteristic rotten apple-like smell

➢ Proteus species have a characteristic foully smell (Fishy)

➢ Pseudomonas spp have a characteristic grape-like aromatic smell

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Experimental Procedure:

Different bacterial species will be cultures on different culture media by

Four Quadrant Streaking:

●To obtain separate colonies and to examine their characteristics

●To examine initial growth characteristics of the cultured bacterial species

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