Lab Four:-
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Medical Microbiology
Prepared by: Luma J. Witwit
Bacteriological Culture Media
What are culture media?
Bacteria that are grown in the lab are referred to as “cultures”. Bacteria
are cultured using media, or materials containing the nutrients that will
support the growth and reproduction of bacteria. Media is classified
according to its form and contents.
One of the most important reasons for culturing bacteria in vitro is its
utility in diagnosing infectious diseases. Isolating a bacterium from sites in
body normally known to be sterile is an indication of its role in the disease
process. Culturing bacteria is also the initial step in studying its morphology
and its identification. Bacteria have to be cultured in order to obtain antigens
from developing serological assays or vaccines. Certain genetic studies and
manipulations of the cells also need that bacteria be cultured in vitro.
Culturing bacteria also provide a reliable way estimating their numbers
(viable count). Culturing on solid media is another convenient way of
separating bacteria in mixtures.
Classification:There are three main forms of media:A) liquid media: Liquids are usually some kind of broth contained in
tubes, and the bacteria will grow suspended in this liquid. Media that contain
only meat infusion, peptone, salt and water are in liquid state, It has some
disadvantage:
* Growth usually do not exhibit characteristic appearance.
* When there is more than one type of organisms they can not be
separated by growing in liquid media.
E.g.] nutrient broth, brain heart infusion broth
B) Semisolid media: Media that is of semisolid or solid consistency
usually contains a thickening agent called agar. Semisolid media is of a soft
jelly-like consistency, is also usually in tubes, and the bacteria will also
grow within in it. It is used to separate a mixture of a motile and non motile
organisms contain 0.2-0.5% agar. e.g.] motility media.
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Lab Four:-
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Medical Microbiology
Prepared by: Luma J. Witwit
C) Solid media: to study colonial characteristics, we must culture
organisms on solid media. Solidification may be achieved by adding agar agar (2-3%), gelatin, serum or egg albumin to other ingredients. Solid media
is the consistency of a very firm gelatin type desert, but the bacteria will tend
to grow only where it is inoculated on the media surface. Solid media that is
provided in tubes often has a slanted surface and are referred to as “slants”.
Solid media that is provided in Petri- dishes are referred to as “plates”. e.g]
Blood agar.
Media can be classified according to the function as:
1- Basic (Ordinary media).
2- Enriched media.
3- Differential and Selective media.
4- Special media.
Basic (Ordinary media):
These are the simplest media, containing only some meat extract or other
simple infusion, peptone, salt, and water. The meat extract or infusion
supplies the organisms with amino acids, vitamins, salts, and traces of
carbon, nitrogen, and other elements. Salts, usually, NaCl serve to obtain the
required isotonicity for the maintenance of constant osmotic pressures.
e.g.]nutrient agar, nutrient broth, Brain heart infusion broth
Enriched Media:
We may define enriched media as basic media that are supplemented
with body fluid, specific vitamins, amino acids, proteins, or any other
nutrients like blood, egg, serum. E.g] Blood agar: is one of the most
commonly used media in microbiology laboratory. It may by adding (510)% sterile blood to any basic agar media (use to detect hemolytic activity
of bacteria, Chocolate agar (when blood agar is heated to 80 Cº for 10
minutes, the media change to chocolate brown color. According to
hemolytic activity of bacteria, there are three types of hemolysis:
1- β hemolytic (complete hemolysis)
colony.
2- α hemolytic(partial hemolysis)
of media.
3- δ hemolytic(no hemolysis).
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a clear zone around the
a greenished coloration
Lab Four:-
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Medical Microbiology
Prepared by: Luma J. Witwit
Differential and Selective media:
Selective and differential media are usually basic or enriched agar media
to which certain reagents have been added that will prevent the growth of the
majority of bacteria.
* MacConkey agar: Lactose fermentation will be demonstrated by a
change in color of the phenol red to purplish pink. Colonies will absorb this
dye, and the bile salts, at acid PH ,will precipitate to show off this color even
more intensely. Non lactose - fermenters appear as pale, smooth, opaque
colonies. Bile salts, in combination with crystal violet, will also inhibit most
gram-positive organisms.
* Salt Manitol agar: It is highly selective for Staphylococci. Manitol
fermentation, as affected by most Staphylococcus aureus strains, is also
demonstrated by change in color, from red to yellow, of the indicator phenol
red.
* Eosin Methylene 2blue agar (EMB): It is differential media for
Enterobacteriaceae.
* Thayer - Martin medium: This medium is particularly useful for
primary isolation of Neisseria gonorrhoeae. This medium would also contain
5ml of VCN is a mixture of antibiotics (vancomycin, colistin and nystatin).
* Thiosulphate citrate bile - salts sucrose agar(TCBS): It is selective
media for isolation of vibrios.
* Salmonella - Shigella agar: It is highly selective for Salmonella
reasonably so for Shigella species.
*Loewenstein - jensen agar: This medium is an enriched selective
medium for isolation of Mycobacterium species.
Special media:
Media that cannot be easily grouped under one of the foregoing heading
will be discussed here. Most of these are used to ascertain one or more
biochemical characteristics.
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Lab Four:-
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Medical Microbiology
Prepared by: Luma J. Witwit
A) Triple sugar iron agar (TSI):
This medium let to solidify in slant tubes, will detect an organism’s
ability to ferment glucose, lactose, sucrose, or combination of these, and to
produce H2S and gas.
B) Indole motility medium:
this may be readily produced from tryptophan in peptone and can be
detected by adding few drops of Kovac’s reagent.
C) MR-VP medium:
1) Methyl red test: Some organisms produce acid end products when
fermenting glucose; these end products are readily demonstrable by adding a
few drops of a 1% solution of MR to a portion of a 48 hours, positive - red;
MR negative - yellow.
2) Voges-Proskauer test: Other organisms produce acetyl methyl carbinol
from glucose.VP positive-red; VP negative- yellow.
D) Urea base agar:
This medium let to solidify in slant tubes, members of proteus group,
Klebsiella, will rapidly produce urease, is detected by a change in color of
the indicator phenol red from pale to a bright purplish pink in less than six
hours.
E) Simmon’s Citrate agar:
This medium let to solidify in slant tubes, to differentiate different
species of Enterobacteriaceae. It changes from green to blue with drop of
bromothymol blue.
* Transport media: are devised to maintain the viability of pathogen
and to avoid overgrowth of the contaminants during transit from the patient
to the Lab.
*Storage media : to available condition for the maintenance of bacterial
culture for long time .
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Lab Four:-
________________
Medical Microbiology
Prepared by: Luma J. Witwit
Preparation of Agar Plate:
Most of agar are present in powder form . they dissolved in distilled
water as per their instructions as follow:1- In a conical flask media dissolved in distilled water .
2- It usually necessary to gently boil the mixture to facilitate dissolving
by oven or over gently flame burner.
3- Sealed the top mouth of flask with a cotton , and finally covering the
cotton with loose layer of aluminum foil.
4- To sterile the media autoclaved for (15) minutes , (121) in autoclave
5- The sterile media is then allowed to cool to (45 Co) pouring at this
temperature prevent condensation forming on the lid .
Before plates are poured , every care is taken not to contaminate:
1- The bench wiped with ethanol.
2- A Bunsen burner is set up with gentle blue flame.
3- The number of plates are placed on bench with their lids.
4- The aluminum foil , cotton are removed with little finger.
5- The mouth of flask is flamed to kill bacteria .
6- The lids of plates are lifted just enough to be poured , and is quickly
half filled with media.
Cultivation with anaerobic bacteria:
Anaerobic bacteria will not grow in the presence of O 2 for growth and
metabolism but obtain their energy for fermentation reactions because the
bacteria lack mechanism of oxidation through respiratory enzymes(like
cytochrome oxidase, catalase, peroxidase) resulting H2O2 accumulation.
The H2O2 is toxic for bacterial growth.
The most commonly used system for creating specialized anaerobic
atmosphere is the anaerobic jar.
The introduction of a gas mixture containing H 2 into a jar is followed by
catalytic conversion of the O2 in the jar with hydrogen to water.
Anaerobic jars can be set up by using a commercially available H 2 and
CO2 generator envelop, which is activated by simple adding 10 ml of water.
The open envelop is placed in the jar with inculcated plates, water is added
and the jar is sealed. Production of heat within a few minutes (detecting by
touching the top of the jar) and subsequent development of moisture on the
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Lab Four:-
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Medical Microbiology
Prepared by: Luma J. Witwit
walls of the jar are indications that the catalyses and generator envelope are
functioning properly.
Figure(1) Anaerobic jar
Morphologies of Bacterial Cells
Viewed under the light microscope, most bacteria appear in variations of
four different shapes:1- Cocci: they are usually round but they may also be oval shape .
each coccus being elongated and pointed at on end but rounded at the
other.
A) Chains.
B) Clusters : There are three types:
* Grape Like Clusters called (Staphylococcus).
* Cube like packet : Clusters of eight cocci.
* Tetrad (4 coccus ) : called (Micro coccus).
* Diplococci or lancet shape
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Lab Four:-
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Medical Microbiology
Prepared by: Luma J. Witwit
2- Rod shape (Bacilli) : They are sub divided as follow:* single bacilli.
* Cocco bacilli: the length of individual cell approximating its width.
* Chinese letter shape or ( V ) shaped arranged at angles to each.
* Comma Shaped bacilli . curved appearance cells.
3- Spiral Shape: Have corkscrew shape with hair like flagellum that
assistant movement.
4- Filamentous shape: are branching bacteria like fungi called
(Actinomyces).
Figure 6: Bacterial shape
sshashapes
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