Culture Media

Use of differential, selective and enriched media:
EMB, blood and starch agar.

OBJECTIVES:

distinguish between different bacterial species based on colony morphology on agar plates
To distinguish the growth characteristics of microorganisms in various differential, and selective media.
Differentiate bacteria based on their ability to hydrolyze starch.

Materials:

Plates of EMB, Starch and blood agar.
Stool sample.
Inoculating loop.
Bunsen burner.
Soil sample.
Cotton soap.
Skin sample.
Gram iodine.

Results:

Starch agar:

Special types of media:

Type of medium
Medium
Appearance of medium and growth
Selective and differential
Eosin-methylene blue

Klebsilla
E.coli
Enriched
Blood agar

Discussion:

Starch Agar is a differential medium used for detection of starch hydrolyzing microorganisms.
The starch agar plate is inoculated by a microorganism. Then, it is incubated for 24 hours at an appropriate temperature. iodine is added to the surface of the agar, Iodine turns blue-black in the presence of starch( Negative results – No hydrolysis) . Absence of the blue-black color indicates that starch is no longer present in the medium. Bacteria which show a clear zone around the growth produce the exoenzyme amylase which cleaves the starch into di- and monosaccharide’s. These simpler sugars can then be transported into the cell to be catabolized. Bacillus species are known to produce the exoenzyme, amylase.
In our experiment, we concluded that Bacillus cereus from soil can hydrolyze starch in order to use these starches as a carbon source and use it. But Staphylococcus cannot.

Blood agar is an Enriched Media which have been supplemented with 5% sheep red blood cells. BAP tests the ability of an organism to produce hemolysing enzymes that damage/lyses red blood cells (erythrocytes). The degree of hemolysis is helpful in differentiating members of the genera Streptococcus. Which Hemolytic activity, classified as:
Beta-hemolysis is complete hemolysis. It is characterized by a clear (transparent) zone surrounding the colonies. In our experiment Streptococcus pyogenes is b-hemolytic.
Partial hemolysis is termed alpha-hemolysis. Colonies typically are surrounded by a green, opaque zone. Streptococcus pneumonia is a-hemolytic
If no hemolysis occurs, this is termed gamma-hemolysis. There are no notable zones around the colonies. Staphylococcus epidermidis is gamma-hemolytic. The majority of EMB plates we isolated contain gamma-hemolytic bacteria.

Eosin-methylene blue agar (Levine) is selective and Differential agar medium. In that it promotes the growth of coliforms (selective for gram negative) and differentiates between E. coli and other coliforms (differential) ,lactose and the dyes eosin & methylene blue permit differentiation between enteric lactose fermenters and non-fermenters.

Eosin changes color, to a dark purple, when the medium around the colony becomes acidic. EMB contains lactose.
Lactose-fermenting bacteria (E. coli and other coliforms) produce acid from lactose fermentation, and the combination of the dyes (which serve as pH indicators) produces color variations in the colonies because of the acidity.
Strong acidity produces a deep purple colony with a green metallic sheen.
Less acidity may produce a brown-pink coloration of colony.
Non- lactose fermenters appear in white color.

E. coli can be can be distinguished from other coliforms by its growth and color reaction on eosin methylene blue (EMB) agar.
E. coli generates a large amount of acid while all other coliforms produce a small amount of acid. On EMB, the large amount of acid released by E. coli will cause the precipitation of both the eosin and methylene blue dyes, causing the E. coli colony to become metallic green in color. All other coliform colonies will be pink (or pink with a dark center) since the small amount of acid they release will only precipitate the eosin (red) dye.