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West Virginia University at Parkersburg
Biology 110L
Microbiology Laboratory
Holly R. Martin, Professor
Written by Dr. Marshall Griffin, Professor
BACTERIOLOGICAL MEDIA
Classification of Bacteriological Media:
Bacteriological media may be classified in two ways:
1.
2.
chemical content
laboratory usage
Media based on chemical content:
1.
synthetic or defined medium-- A synthetic or defined medium is one in
which the exact chemical composition is known. In other words, one
knows exactly what and how much of each substance is present in the
medium.
For the most part, this type of medium is generally used only for highly controlled
experimental work. A synthetic or defined medium is not used routinely because
of the time and expense involved in its preparation. Also, such a specific type of
medium is not needed for the isolation of most medically important bacteria.
2.
non-synthetic medium-- A non-synthetic medium is one in which the exact
chemical composition is unknown.
A non-synthetic medium typically contains as a part of its formulation some type
of infusion. An infusion is an aqueous extract of either plant or animal tissue;
therefore, its exact chemical composition is unknown.
A commonly used infusion is called brain heart infusion (BHI). BHI is prepared
from the brains and hearts of cattle. One prepares this infusion by simply placing
fresh brains and hearts in a pot, covering with distilled water, and boiling for
about one hour. The liquid is then used as the infusion.
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Media based on laboratory usage:
1.
basal medium-- A basal medium is a type of all purpose medium on which most
organisms can grow except for those which have complex nutritional
requirements.
A basal medium is composed of an infusion which may or may not contain a
solidification agent.
An example of a basal medium is BHI (brain heart infusion) broth or BHI agar.
2.
enriched medium-- An enriched medium is a type of all purpose medium which
is formulated to the support the growth of essentially all organisms including
those which have complex organic nutritional requirements.
Organisms which will grow on a basal medium will also grow on an enriched
medium. However, organisms which require some type of complex nutrient such
as vitamins or purines will only grow on an enriched medium.
An enriched medium is generally composed of a basal medium plus added
nutritional supplements.
A common example of an enriched medium is Sheep Blood Agar (SBA). This
medium is prepared by adding sterile, defibrinated sheep red cells to a basal
medium such as blood agar base or trypticase soy agar which has been sterilized
and cooled to approximately 50C. If the blood was added to the medium at a
temperature above 50C, the blood cells would be destroyed by the excessive heat.
Chocolate agar is another example of an enriched medium. It does not contain
chocolate; rather it has a chocolate appearance. Chocolate agar is prepared by
adding sterile hemoglobin solution and yeast extract to a sterile basal medium.
3.
selective medium-- A selective medium is formulated to allow for the growth of a
desired group of organisms while inhibiting the growth of any undesired
organisms which may be present in the same specimen.
Selective media are prepared by adding substances which retard the growth of
unwanted bacteria (inhibitors) to a basal or enriched medium. Some examples of
inhibitors are dyes, salts, pH indicators, and antibiotics.
An example of a selective medium is Thayer-Martin medium. This medium is
fairly complex in that it is basically chocolate agar to which one has added
antibiotics. This medium is used for the primary isolation of the pathogenic
species of the genus Neisseria.
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4.
differential medium-- A differential medium is formulated to accentuate certain
metabolic differences between closely related bacteria.
Differential media are usually prepared by adding pH indicators, salts,
carbohydrates, etc. to a basal medium.
An example of a differential medium is eosin-methylene blue agar (EMB). This
medium is composed of a basal medium plus the dyes eosin and methylene blue
and the carbohydrates lactose and sucrose. EMB agar is used for the primary
isolation of gram negative bacteria (a group of related bacteria). It differentiates
those gram negative bacteria which can ferment the carbohydrates from those
which cannot ferment the carbohydrates (carbohydrate fermentation is the
metabolic characteristic that is accentuated in this case). Gram negative bacteria
which ferment the carbohydrates will produce acid as an end product of the
fermentation process. The acid will then react with the dyes in the medium so
that the colonies of those organisms will turn a dark color. Those bacteria which
do not ferment the carbohydrates will not produce acid, and therefore, there will
be no effect on the dyes in the medium. The colonies of the non-fermenting
bacteria will remain a clear, light pink color.
Forms of Bacteriological Media:
There are two forms of bacteriological media:
1.
Liquid which is generally referred to as a broth.
2.
Solid which is generally referred to as an agar. A solid medium is prepared by
adding a solidification agent either agar or gelatin to the other medium
ingredients.
The Use of Agar and Gelatin as Solidification Agents:
Agar is a carbohydrate substance that is extracted from the giant sea weed, a member of
the genus Gelidium. Gelatin is a protein extracted from animal tissues by boiling
collagen.
Agar is the preferred solidification agent in bacteriological media. Agar by itself contains
no usable nutrients. Since the agar is inert to the various digestive enzymes released by
growing bacteria, agar containing media remain solid during culture incubation. Gelatin,
however, can be utilized by many organisms as a nutrient. A gelatin containing medium
will often be liquefied as the bacteria grow and release the enzyme gelatinase.
Agar has a melting point of 100°C and a solidification temperature of 42-45°C. The wide
range of temperatures between the melting point and the solidification point offers a
distinct advantage in that one has a reasonable length of time for pouring the melted agar
solution before solidification occurs. Gelatin, on the other hand, has a melting point of
39-40°C and a solidification point of 32-35°C.
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Another advantage of the high melting point of agar is the fact that agar remains a solid at
37°C which is the temperature at which most bacteriological cultures are incubated. With
gelatin-containing media, the media tend to become semi-solid at incubator temperatures.