Biochemical

tests
(Biooxidations)

Dr. Hebat Allah Youssef

1-Sugar fermentation test:
The purpose is to see if the microbe can ferment the
Sugars as a carbon source to produce acid end
products .
- the pH of the medium will drop. A pH indicator in the
medium changes color to indicate acid production.
- Phenol red sugar broth is used with Durham tube
inverted to collect gas.
- inoculated tube is incubated at 37 °C for 24 hours and
the results are determined. A positive test consists of a
color change from red to yellow, indicating a pH change
to acidic.
 2- Mixed acid fermentation
(Methyl red)
 Principle : to test the ability of the
organism to produce acid end product
from glucose fermentation, this is a
qualitative test for acid production.
 3-Butanediol fermentation
(Voges-Proskauer)
 To determine the ability of the organisms
to produce neutral end product
(acetoin) from glucose fermentation.
Procedure:
1.Inoculate the tested organism into 2 tubes of MR-
VP broth
2. Incubate the tubes at 37°C for 24 hours
3. AFTER INCUBATION: Run the MR test in the tube
1, and the VP test in tube 2.
– For methyl red: Add 6-8 drops of methyl red
reagent.
– For Voges-Proskauer: Add 12 drops of Barritt's A
(α-naphthol), mix, 4 drops of Barritt's B (40%
KOH), mix
– Let sit, for at least 1hour
 Methyl Red test

Glucose ferm. Mixed acids Add MR Red color

Glucose Alcoholic ferm Acetoin Add VP (Barrit) reagent

Scarlet Red color

 4-Catalase test

 Enzymes that decompose hydrogen

peroxide into water and oxygen.
 Hydrogen peroxide forms as one of the
oxidative end products of aerobic
carbohydrate metabolism. If this is allowed
to accumulate in the bacterial cells it
becomes lethal to the bacteria.
 Reagent:
 3% hydrogen peroxide stored in dark brown
bottle under refrigeration,
 18 to 24 hrs. culture of the organism to be
tested.
 The catalase enzyme neutralizes the
bactericidal effects of hydrogen peroxide
and protects them.
 2H2O2 Catalase H2O+ O2 (effervescence).
5- Oxidase test
 This laboratory test is based on detecting
the production of the enzyme cytochrome
oxidase

 This enzyme play a role in electron

transport chain during aerobic respiration

 Cytochrome oxidase, part of an intracellular

system that perform the final step in
electron transport, oxidation of the reduced
cytochrome by molecular oxygen.
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent)
colourless

Cytochrome oxidase enzyme

Indophenol
(Purple colour)
- Put a filter paper in a petridish containing tested
culture, place several drops of freshly prepared
solution of “ oxidase reagent” and observe
color change.
- Positive results will apear with in 10 -30 sec as
color will change to deep purple.
6- Nitrate Reduction Test

 Used to detect the ability of an organism to

reduce nitrate (NO3) to nitrite (NO2) using
the enzyme nitrate reductase

Nitrate Reductase Forms red color once

- -
NO3 NO2 solutions A and B are added.
 Materials

 Broth media containing : peptone , beef

extract and KNO3)
 Reagents:

Solution A
(Sulfanilic acid – Conc. H2SO4 – Distilled water)
Solution B
(α-naphthylamine - Conc. H2SO4 – Distilled water)
 Procedure:
 Inoculate Nitrate broth tubes with loop of
the tested organisms then incubate at
37oC for 24 hrs.
 Results:
 After inoculation and incubation, the ability of an
organism to reduce nitrate to nitrite is detected by
adding two reagents:

Solution A , Solution B

3 ml of culture to clean tube + 1 ml of solution

A + 0.5 ml of solution B

 If a red color appears after addition of solution A

and B, this is considered a positive result
 If there is no color change occurs after
additions of solutions A & B, two possibilities
must be considered:

1) nitrates were not reduced by the organism

2) the organism possessed such potent nitrate

reductase enzymes that nitrates were reduced
to ammonia or molecular nitrogen
NO3 - NO3 -

Nitrate Reductase N2 (Nitrogen Gas)

NO3- NO2-
NH3+ (Ammonia)
 Next, (zinc dust) is added to the tubes that showed no
color change after addition of reagents A & B

 Zinc, a reducing agent, is capable of converting nitrate to

nitrite; the development of a red color after addition of zinc
dust means that nitrates were present and reduced by the
zinc not the organism; this is a negative result

 If the addition of zinc does not produce a color change, the

nitrates were reduced to ammonia or nitrogen gas; this is a
positive results