EXERCISE 15 NITROGEN AND
SULFURMETABOLISM
Post Lab Discussion
A.)UTILIZATION OF ORGANIC AND
INORGANIC NITROGEN
Media used: NMA and NMB
Positive Results:
 Growth in NMA: bacteria can utilize
INORGANIC Nitrogen like Nitrate.
 Growth in NMB: bacteria can utilize
ORGANIC Nitrogen.
 Growth in NMA and NMB: bacteria
can utilize both.
Negative result: No growth on plates:
bacteria cannot utilize any form of Nitrogen.
A.)UREA HYDROLYSIS
Media used: Christensen’s medium or
Urea broth
Urease: a hydrolytic enzyme that attacks the
nitrogen and carbon bond in amide
compounds like urea which forms ammonia.
Significance: This can be used in identifying
the genus Proteus. This genus can produce
enough ammonia to give the reaction a more
alkaline pH of 8.1.
Positive result: Pink to red or violet color on
broth
Negative result: Light orange to yellow color
on broth
Reaction involved:
* The alkaline reaction causes the pH indicator to
turn hot pink.
B.)INDOLE PRODUCTION FROM
TRYPTOPHAN
Media to be used: Tryptone Broth (TB)
Reagent to be used: Kovac’s Reagent
Principle: Microorganisms with
tryptophanase cleave tryptophan which
produces indole, ammonia and pyruvic acid.
Amyl alcohol which serves as a solvent for
indole reacts with para-
dimethylaminobenzaldehyde producing a red
resindole dye.
Positive result: Appearance of red ring on
the interface or top of the media.
Negative result: Brown or yellow ring on top
or no color change when Kovac’s reagent is
added
Reaction involved:
RESULT:
C.)PHENYLALANINE DEAMINATION TEST
Media to be used: Phenylalanine Agar Slant
(PA slant)
Reagent to be used: 10 % FeCl3 Solution–
used to determine
Leuconostoc mesenteroides
which is can produce
phenylpyruvic acid from
phenylalanine
Principle: Phenylpyruvic acid is detectable
thru a reaction with FeCl3 producing avocado
green colored product.
Positive result: Dark green color on slant
Negative result: No color change
Reaction involved:
D.)LYSINE DECARBOXYLATION TEST
Media to be used: Lysine Decarboxylase
Broth (LDB)
– used in differentiating
Enterobacteriaceae
*Bromcresol purple is the pH indicator.
Principle: The acid-end product of the
fermentation activates lysine decarboxylase.
This enzyme decarboxylates lysine forming an
alkaline product which is detected by
bromcresol purple. As a result, the sample
should change from neutral (purple) to acidic
(yellow) then back to purple (basic).
Positive result: Light to purple color on
broth
Negative result: The sample and control
tubes are yellow
Reaction involved:
 control (+): purple (-):
yellow
color color

E.)NITRATE REDUCTION
Media to be used: Nutrient Broth with 0.1%
KNO3 with inverted Durham tube.
Reagents to be used:
• Nitrate Reagent A -(5N Acetic
acid: Sulfanilic acid)
• Nitrate Reagent B- (5N Acetic
acid: α-naphtol)
• Powdered zinc

Principle: Once NO3- was reduced to NO2-,

the presence of NO2- will be detected by the
Nitrate reagents A and B. NO2- will react with
sulfanilic acid forming a colorless complex
(NO2-sulfanilic acid). When Nitrate reagent B
is added, a red precipitate (prontosil) will
appear resulting to a red colored solution.
Positive result: Gas production/Red colored
solution which can turn to brown
Negative result:
 No gas production/Absence of
red color: Nitrate was not
reduced to nitrite orniitrate was
reduced to nitrite but have
been further reduced to NO,
N2O or N2.
 Confirmation for negative
result: Add zinc to catalyze the
reduction process which can
produce red colored solution
that indicates NO3- was reduced
to NO2-.
Principle: If no red color appeared after
adding zinc, the bacteria have not only
reduced nitrate to nitrite, but have also
reduced nitrite to nitrogenous gases NO or N2.
These gases can be trapped on the inverted
Durham tube.
A.)HYDROGEN SULFIDE (H2S) PRODUCTION
Media to be used: Lead Acetate Agar (LAA
slant)
- solid LAA enhance aerobic respiration
-Lead serves as the H2S indicator
- Sodium Thiosulfate provides sulphur
Principle: Sulfate when activated by ATP,
accepts electrons from acetate and is reduced
to Adenosine Phosphosulfate (APS). APS is
then reduced to sulfite and will be reduced
further to H2S. H2S will then react to lead
producing lead sulfite (PbS).
Enzyme: Cysteine desulfhydrase
Positive result: Brown to black color of
inoculated portion: bacteria can reduce sulfur
to H2S.
Negative result: Absence of brown to black
color: bacteria cannot reduce sulfur.
Reaction involved:

Black color

References:
http://www.microvet.arizona.edu/courses/MIC205/Labs/Man
ual/12Microbial%20Metabolism2.htm

http://www.microvet.arizona.edu/.../lab
%2011%20%20Microbial%20metabolism%20part%20II
%20.ppt
http://www.microvet.arizona.edu/Courses/MIC285/LabManu
al285.pdf

http://www.cdc.gov/std/gonorrhea/lab/tests/nitrate.htm

By: SARAH JANE A.

IBASCO
MCB 101 F-3L