Bile Esculin Hydrolysis, Starch Hydrolysis,
UreaHydrolysis, Casein Hydrolysis
Hydrolysis is a chemical reaction that uses water to split complex molecules. The water molecule H2O is split in the mechanism of hydrolysis, hydrogen cations and hydroxide anions. When the enzyme catalyzes its reaction inside the cell, it is referred to as intracellular hydrolases. When the enzymes secreted from the organism to catalyze reactions outside the cell, it is referred to as exoenzymes or extracellular hydrolases. Four hydrolysis reactions are discussed in the report, bile esculin hydrolysis, starch hydrolysis, urea hydrolysis and casein hydrolysis. Bile esculin hydrolysis determines whether an organism is able to hydrolyze the glycoside esculin. Starch Hydrolysis identifies organisms that can hydrolyze starch. Urea hydrolysis tests for the ability of organisms to hydrolyze urea via the intracellular enzyme urease. Casein hydrolysis tests for organisms capable of hydrolyzing casein via the casease.
Materials and Methods
Bile Esculin Hydrolysis
The organisms Lactococcus lactis and Enterococcus faecalis were spot-inoculated on a bile esculin agar plate. The bile esculin agar plate is a both selective and differential medium contains primarily esculin. The plate was then inverted and incubated at 37 oC for 24 hours. Bile salts, the selective agent, can allow only Enterococcus and group d streptococcus to hydrolyse esculin in the presence of bile salts, and inhibit growth of other gram positive organisms. Ferric citrate is an indicator. When the hydrolytic substrate esculin molecules are split, the product esculetin reacts with ferric citrate and forms a dark brown iron ions precipitate. Positive result indicates the organism is a member of group D Streptococcus or Enterococcus, they grow in the medium and turn medium darken. Negative result indicates the organism is not a member of group D Streptococcus or Enterococcus and gives no growth and no darkening of medium.
The organisms Bacillus cereus and Escherichia coli were spot-inoculated on a starch agar plate. The starch agar plate contains primarily starch as a hydrolytic substrate having Amylose and Amylopectin. The plate was then inverted and incubated aerobically at 37 oC for 24 hours. Positive result means that the organism can produce α-amylase and 1,6-glucosidase to hydrolyze starch represents, the indicator iodine then reacted with starch and produced a dark brown color. The negative result represents no amylase is present and no clearing around growth.
The organisms Escherichia coli and Proteus vulgaris were inoculated in two Urea broths. The major component of the Urea broth is urea and an indicator phenol red. All tubes were then incubated aerobically at 37 oC for 24 hours. Positive results pink color, represents there is a rapid urea hydrolysis, strong urease production is present with acidic pH>8.4. The negative results orange or yellow color, represents there is no urea hydrolysis with neutral pH=8.4 and basic pH>8.4 respectively, organism does not produce urease or cannot live in broth.
The organisms Escherichia coli and Bacillus cereus were spot-inoculated on a milk agar plate. The major component of milk agar is casein and powdered nonfat milk. Casein is both hydrolytic substrate and indicator. The plate was then inverted and incubated aerobically at 37 oC for 24 hours. Positive result represents Casease is present and negative result represents Casease is absent.
|Test |Organism |Result(+/-) |Observation | |Bile Esculin Hydrolysis |Lactococcus Lactis |+ |Medium was darkened | | |Enterococcus faecalis |+ |Medium was darkened...