Biochemical

tests
(Hydrolysis)

Dr. Hebat Allah Ibrahim

 1-STARCH hydrolysis
 Differentiates bacteria based on their ability
to hydrolyze starch with the extra cellular
enzyme amylase

 Starch (a polysaccharide) is to large to pass

through the actinomycetes cell wall; to be
of metabolic value to them, starch must be
split into smaller fragments or individual
glucose molecules
Materials
 Starch agar medium
 Iodine solution
 Procedure:
 Pour 2 petri dishes of starch agar medium
, cool and allow to solidify
 With a sterile loop streak the surface of a
plate with the tested organism and leave
the second plate as control (uninoculated)
 Incubate at 37oC for 24 hrs.
 After incubation flood the plates with
iodine solution and allow reacting for few
minutes, drain excess and examine
 Results:

 Iodine reacts with starch and

produces a blue color.

 After addition of iodine to the plate

media the blue area represents the
unhydrolyzed starch , while the clear
zone indicate areas in which the
starch has been hydrolyzed.
2- Lipiolytic activity
 Used to identify bacteria capable of
producing the exoenzyme lipase

 Fat Lipase enzyme glycerol + fatty acids

(Olive oil)
Materials
 Nutrient agar medium
 Olive oil
 20% CuSo4 solution
 Procedure

 The medium is melted and cooled to 45oC

 2 ml of sterilized olive oil is added to the melted
medium
 Pour the medium in plates and allow to solidify
 Inoculate the plates and incubate at 37oC for 24
hrs
 after incubation flood the plates with 20%
CuSo4 solution and left for 10 mins. Then
remove the excess reagent
 Results:

 The occurance of blue

crystals along the line of
inoculation means
positive results.
 3- Casein Hydrolysis Test

 Identifies bacteria capable of hydrolyzing casein

(protein) with the enzyme caseinase

 Casein is the protein that gives milk it’s color.

 To be utilized by certain bacteria, casein must

be broken down in to its smaller subunits, amino
acids

 Casein caseinase smaller peptides + amino acids

 Procedure:
 Inoculate skim-milk agar plates with the test
organism

 incubate at 37oC for 24 hrs

 Examine plates after incubation , colonies of

actinomycetes which digest casein will be
surrounded by clear zone, while areas in which
casein hasn’t been digested will remain opaque
Results:
 4- Gelatin Hydrolysis
 Test for the ability of an organism to
produce the exoenzyme gelatinase which
digests and liquefies gelatin
 Neutrient gelatine medium solidify when
cooled below 25oC and liquifies when
heated to 25oC or more.
 When gelatin is hydrolized it liquifies and
doesnot solidify even cooles below 20oC.
 Procedure:

 Nutrient gelatin tubes are stab inoculated

with the tested organisms then incubated
at 37 o C for 24 hrs
 leave one tube uninoculated as control
 To examine gelatin hydrolysis, cool the
tubes in ice water, the control tube and
negative tubes will solidify while positive
tubes will remain liquid
 Results:
 Since nutrient gelatin melts at 37°C, care must be
taken to distinguish between organisms capable of
producing gelatinase and gelatin tubes that are
affected by incubator temperature.

 After incubation, tubes should be refrigerated;

tubes inoculated with gelatinase positive
organisms remain liquid after refrigeration
 After refrigeration
5- Indole test:
to detect the ability of the M.os to degrade the amino
acid tryptophane (tryptone).
Tryptophane + water Indole +Pyruvate+Ammmonia
Inoculate tryptone broth medium and incubate for 24
hrs at 37°C.
After incubation add drops of “ Kovac’s reagent”
(p-dimethylaminobenzaldehyde)
Positive result appear as red ring on medium surface.
6-Urease test:
The urease test identifies those organisms that are
capable of hydrolyzing urea to produce ammonia
and carbon dioxide.
(NH2)2CO + H2O → CO2 + 2NH3
Urease test media contain 2% urea and phenol red as a pH
indicator inoculated and incubated at 37°C for 24 - 48
hrs. An increase in pH due to the production of ammonia
results in a color change from yellow to bright pink.