BIOCHEMISTRIES

What are biochemical tests?

Biochemical tests consist of different biochemical reactions between

microorganisms, enzymes and nutrients in the culture medium, which allows us to
identify different microorganisms, as well as know their physiological behavior in
different culture conditions.

Its operating system generally consists of determining the activity of a metabolic

pathway from a substrate that is incorporated into a culture medium and that the
bacteria incorporates or does not incorporate when growing.
(Mac Faddin, 1984)
oxidase test

Technique

It allows detecting the presence in the

microorganism of enzymes of the cytochrome
oxidase system, capable of catalyzing the
transport of electrons between a donor present
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Slide Oxidase
Catalase test

Catalase is an enzyme that protects cells Techni

against hydrogen peroxide produced in que
oxygen metabolism. Catalyzes the
formation of water and oxygen from
hydrogen peroxide.

• Transfer a sample of the culture

• Add one or two drops of hydrogen
peroxide
Oxidation-Fermentation (OF) Test

Fermentation is an anaerobic process Techni

and bacterial fermenters of a Two
que
tubes with OF medium enriched
carbohydrate are generally facultative with 1% glucose are inoculated by deep
anaerobes. Through the fermentation pitting, to which 1 mL is added to one of
process a carbohydrate is metabolized them. of mineral oil and incubated at
or fermented; the key intermediate is 35ºC ± 2ºC for 24 to 48 hours.
pyruvic acid.
Methyl red test

Technique
The methyl red test is based on a pH
Inoculate the RM-VP broth by roasting with a
indicator, methyl red, to determine
pure culture of no more than 24 hours of the
the concentration of hydrogen ions
microorganism under study. Incubate at 35°C for
present when an organism ferments
3-5 days.
glucose.
Methyl red test
Interpretation
Add two to three drops of methyl red solution to the culture medium for 3-5 days of
incubation; The test is positive if there is a development of red color, this indicates
that the acid production is sufficient to produce the change of the indicator and the
microorganism fermented glucose through the mixed acid route, and negative if there
is development of a yellow color.

No organic acids are

The production of
produced, the pH is
organic acids lowers
greater than 4.3 and the
the pH below 4.3 and
methyl red remains
the methyl red turns
yellow.
red.
Voges-Proskauer test

It is based on the production of acetylmethylcarbinol (acetoin), a neutral final product

derived from the glucose product. This is metabolized into pyruvic acid, a primary
intermediate of glycolysis.

Technique
Inoculate the RM-VP broth with a pure culture of no more than 24 hours of the
microorganism under study. Incubate at 35 ± 2°C for 24 to 48 hours. Add 0.6 mL of 5%
alpha-naphthol and 0.2 mL of 40% KOH and wait 20 min.
Voges-Proskauer test
Interpretation
After having incubated for 2 hours at 35 ± 2°C or 4 hours at room temperature, the
test is considered positive if there is a brick red color development in the tube and as
negative if there is no color change in the tube.

The reagents If there is no acetoin a, the medium does not change color when KOH and alpha-naphthol are
convert I to added.
acettina into
diacetyl and a red
color is formed.
citrate test

The use of citrate as the sole carbon source is detected in a culture medium with
citrate as the sole carbon source, alkalization of the
medium. through growth and

Technique
Simmons citrate agar is inoculated with pit and streak
inclination. Incubate at 35°C for 24 to 48 hours.
citrate test
Interpretation
The test is positive when growth is observed along the stria, accompanied by a change
of the indicator from green to blue, and negative when the tube remains green.

Positive result. Citrate is Negative reaction . Citrate is

used as a carbon not used. V the pH indicator
source. The pH of the (bromothymol blue) remains
medium I increases green.
and the indicator turns
from green to blue.
Indole Production

Indole is one of the metabolic degradation products of the amino acid tryptophan.
Bacteria that possess tryptophanase are capable of hydrolyzing and deaminating
tryptophan with the production of indole, pyruvic acid and ammonia.

Technique
Sow by pitting in SIM medium and incubate at
35 ± 2°C for 24 to 48 hours.
Indole test
Interpretation
Add 5 drops of ether to extract the indole and 5 drops of Kovac reagent. It is
considered positive when there is development of a red ring on the surface of the
medium in the alcoholic layer and a negative test does not produce color in the layer.

alcoholic.
Tests for Decarboxylases
(Lysine-Ornithine-Arginine)

The decarboxylation of amino acids is carried out by decarboxylases, forming amines

and CO 2 . The amino acids commonly tested for amino acid identification are lysine,
ornithine and arginine.

Technique
Inoculate a tube of Moeller base LIA medium with the
amino acid to be studied (lysine-ornithine-arginine) and a
control tube without amino acid. Incubate at 35°C for 24
hours to 4 days with daily examinations.
Tests for Decarboxylases
(Lysine-Ornithine-Arginine)
Interpretation
In both cases, the test is considered positive if there is a color change in the medium,
from violet to purple in the case of ornithine, as well as a purple background in lysine,
and as negative, the presence of yellow color in the medium for ornithine, and
presence of yellow color at the bottom of the tube for lysine.

Positive tubes turn violet If the pH is not raised , bromocreso purple I remains yellow .
or purple, indicating an
increase in pH.
TSI (Triple Sugar) Test
Iron)
Triple sugar iron agar (TSI) is a nutrient and differential medium that allows studying
the capacity for acid and gas production from glucose, sucrose and
lactose in a single medium. It also allows the detection of H 2 S production.

Technique

Reseed the Kligler medium by pitting and streaking

Incubate at 35°C for 24 hours.
TSI (Triple Sugar) Test
Iron)

Acid/Acid/Gas
Negative for Negative for Positive for Positive for
fermentation of fermentation H 2 S production fermentation of
glucose, lactose lactose and glucose, lactose
and sucrose; and
saccharose positive for sucrose and
glucose gas production
Sugar Fermentation

Anaerobic or facultative anaerobic bacteria often ferment carbohydrates to organic

acids and gas (H 2 or CO 2 ).

Technique
The bacteria are inoculated in a medium containing a small amount of peptone, a pH
indicator (bromothymol blue) and a 1-2% fermentable carbon source and a Durham
bell is placed inside the tubes.
Sugar fermentation
Interpretation

When he
Microorganism ferments the
studied sugar, the pH of the
medium drops; then the
bromothymol blue turns from
blue to yellow.
Starch hydrolysis

Starch hydrolysis is analyzed in media containing plate starch. After incubation, the
plates are flooded with lugol which, when combined with the intact starch, forms a
purple complex.

Technique
Plates with culture medium containing 0.1% starch are used and incubated for 48 h.
They are developed with lugol or better with 95° ethyl alcohol for 30 to 60 min.
Starch hydrolysis
Interpretation
The reaction is positive when a halo appears lighter than the rest of the plate around
the growth zone.
Gelatin hydrolysis

Protease production is evaluated by incorporation of a protein (gelatin or casein) in a

solid plate medium. The plate is flooded with acid which precipitates the
unhydrolyzed protein.

Technique

The nutrient gelatin medium is seeded by pitting in a

vertical tube, incubated at 24 ± 2°C for 48 ± 2 hours and
refrigerated for 20 minutes.
gelatin test

Interpretation
If there is liquefaction of the gelatin, the test is positive, otherwise if the gelatin
remains solid, the test is considered negative.
urease test

Urease is an important microbial enzyme linked to the breakdown of organic

compounds. This test determines the ability of an organism to split urea forming two
molecules of ammonia by the action of the enzyme urease.

Technique
For this test, the media used are urea broth pH 6.8 and phenol red as a pH indicator. It
is seeded using two roasts and incubated at 35 to 37 °C for 24 to 48 hours.
urease test

Interpretation
The test is positive when a pinkish red color appears throughout the broth, only in
Proteus species, and the reaction is negative when no color change occurs in the tube
(orange-yellow).

If the bacteria does not hydrolyze the urea, an increase in pH

The hydrolysis of does not occur and the medium remains yellow neces
urea causes alkaline
pH and the
indicator turns red.
Hydrogen Sulfide Test
Proteolysis of protein gives individual amino acids; Some
heterotrophic bacterial species are capable of releasing
sulfur enzymatically from the different amino acids that
contain them, producing hydrogen sulfide gas (H 2 S).

Technique
It is seeded by pitting in SIM medium and by pitting and
streaking in Kliger or TSI medium, and incubated at 35°C
for 24 hours.
Hydrogen Sulfide Test

It is considered positive when blackening of the medium is observed following the

inoculation line throughout the surface layer and we will consider it negative if no
blackening is observed.