V.

HYDROGEN SULFIDE TEST

Two Major fermentative pathways for hydrogen sulfide (H2S) production of some
microorganisms:
Pathway 1:

 Gaseous H2S may be produced by the reduction (hydrogenation) of organic sulfur

present in the amino acid cysteine, which is a component of peptones contained in
the medium.
 These peptones are degraded by microbial enzymes to amino acids, including the sulfur-
containing amino acid cysteine.
 This amino acid in the presence of a cysteine desulfurase loses the sulfur atom, which
is then reduced by the addition of hydrogen from water to form bubbles of hydrogen
sulfide gas (H2s c) as illustrated:

Pathway 2:

 Gaseous H2S may also be produced by the reduction of inorganic sulfur

compounds such as the thiosulfates (2S032-), sulfates (SO42-), or sulfites (SO32-).
 The medium contains sodium thiosulfate, which certain microorganisms are capable of
reducing to sulfite with the liberation of hydrogen sulfide.
 The sulfur atoms act as hydrogen acceptors during oxidation of the inorganic compound
as illustrated in the following:

SIM medium composition

 peptone and sodium thiosulfate as the sulfur substrates,

 ferrous sulfate (FeSO4), which behaves as the H2S indicator, and
 sufficient agar to make the medium semisolid and thus enhance anaerobic respiration.
Regardless of which pathway is used, the hydrogen sulfide gas is colorless and therefore not
visible.
H2S TEST
 H2S positive forming an insoluble black ferrous sulfide
precipitate that is seen along the line of
the stab inoculation
H2S negative Absence of the precipitate

 Ferrous ammonium sulfate in the medium serves as an indicator by combining with the
gas, forming an insoluble black ferrous sulfide precipitate that is seen along the line
of the stab inoculation and is indicative of H2S production.
 Absence of the precipitate is evidence of a negative reaction.

A. MOTILITY TEST

 These tests are used to determine whether an enteric organism is motile.

 An organism must have flagella to be motile.
 Motility is recognized when culture growth (turbidity) of flagellated organisms is not
restricted to the line of inoculation. (Motile)
 Growth of non motile organisms is confined to the line of inoculation. (Nonmotile)
 SIM agar may also be used to detect motile organisms.
 A gelatin-based complex agar with a colorless dye, 2,3,5-triphenyltetrazolium
chloride (TTC), may also be used to determine bacterial motility. This test is based on
the reduction of TTC by motile bacteria to form formazan, an insoluble red pigment.

STEPS
1. Aseptically inoculate each experimental organism into its appropriately labeled tube by
means of stab inoculation. The last tube will serve as a control. (Stab once to a depth of only 1/3
to 1/4 inch in the middle of the tube)
2. Incubate all cultures for 24 to 48 hours at 37°C.
3. Examine all SIM cultures for the presence or absence of black coloration along the line of the
stab inoculation.
4. Observe all cultures for the presence (+) or absence (-) of motility.
MOTILITY TEST

Media  Enzymatic digest of gelatin (10 g),

 beef extract (3 g), NaCl (5 g),
 agar (4 g) per 1000 mL,
 pH 7.3.
Positive Motile organisms will spread out into the
medium from the site of inoculation

Negative Nonmotile organisms remain at the site of

inoculation

Limitations
Some organisms will not display sufficient growth in this medium to make an accurate
determination, and additional follow-up testing is required.
Quality Control
Positive: Escherichia coli
Negative: Staphylococcus aureus
VI. UREASE TEST/ Hydrolase Test/ Christensen’s Method

Urease

 an enzyme that is especially helpful in the identification of Proteus vulgaris. Although

other organisms may produce urease, their action on the substrate urea tends to be
slower than that seen with Proteus species.
 hydrolytic enzyme that attacks the nitrogen-carbon bond in amide compounds such as
urea and forms the alkaline end product ammonia
Urease Test

 This test is used to determine an organism’s ability to produce the enzyme urease,
which hydrolyzes urea
 serves to rapidly distinguish members of Proteus from other non-lactose- fermenting
enteric microorganisms.

Urease Test
Medium Urea Broth Medium

pH indicator Phenol red.

Positive: Change in color of slant from light orange to magenta/ deep pink
Negative: No color change (agar slant and butt remain light orange)

As the substrate urea is split into its products, the presence of ammonia creates an alkaline
environment that causes the phenol red to turn to a deep pink. This is a positive reaction for
the presence of urease.
Failure of a deep pink color to develop is evidence of a negative reaction.
To eliminate false-positive reactions, perform a control test with the base medium without
urea.
Quality Control
Positive: Proteus vulgaris
Weak positive: Klebsiella pneumoniae
Negative: Escherichia coli

STEPS
1. using aseptic technique, inoculate each experimental organism into its appropriately labeled
tube by means of loop inoculation. The last tube will serve as a control.
2. Incubate cultures 24 to 48 hours at 37°C.
3. Examine all urea broth cultures for color.
VII. NITRATE REDUCTION TEST

 The reduction of nitrates occurs in the absence of molecular oxygen / anaerobic

process
 Nitrates (NO3) or Sulfates (SO4) are used to supply oxygen that is subsequently utilized
as a final hydrogen acceptor during energy formation.
 The biochemical transformation may be visualized as follows:

 Some organisms possess the enzymatic capacity to act further on nitrites to reduce
them to ammonia (NO3) or molecular nitrogen (N2).
 These reactions may be described as follows:

Limitations
Alkaline reactions may appear after prolonged incubation and may be the result of peptone or other
protein utilization raising the pH. To eliminate false-positive reactions, perform a control test with the
base medium without urea.

NITRATE REDUCTION TEST

Nitrate Reduction Test is an anaerobic process that determines an organism's ability to
reduce nitrate to nitrites.

Medium: Nitrate Broth Medium composed of:

.  0.1% potassium nitrate (KNO3) - acts as a nitrate substrate.

 0.1% agar - makes medium into a semisolid form

 Solution A- Sulfanilic Acid

Reagents:  Solution B- Alpha-naphthylamine
 Positive Cherry Red Color/ Red Color after the addition of reagents
Reaction:

Negative No color development

Reaction:

Note:
 Zinc powder is used to make sure the result of NRT is true negative
 This should not be confused with Barrt's reagent.

 To determine whether the result is true negative (NRT negative), a small amount of zinc
powder is added to the basically colorless cultures already containing Solutions A and B

 Zinc reduces nitrates to nitrites, checks presence unreduced nitrites.

 The development of red color therefore verifies that nitrates were not reduced to nitrites
by the organism. If nitrates were not reduced, a negative nitrate reduction reaction has
occurred.
 If the addition of zinc does not produce a color change, the nitrates in the medium were
reduced beyond nitrites to ammonia or nitrogen gas. This is a positive reaction,

Negative Result/ No Color Developments suggest either:

1. Nitrates were not reduced by the organism, or

2. organism possessed sucli potent nitrate reductase enzymes that nitrates were rapidly
reduced beyond nitrites to ammonia or even molecular nitrogen.

STEPS:
1. Using aseptic technique, inoculate each experimental organism into its appropriately labeled
tube by means of a loop inoculation. The last tube will serve as a control.
2. Incubate all cultures for 24 to 48 hours at 37°C.
3. Add five drops of Solution A (sulfanilic acid) and then five drops of Solution B (a-
naphthylamine) to all nitrate broth cultures.
4. Observe whether a red coloration develops in each of the cultures.
5. Add a minute quantity of zinc to the cultures in which no red color developed.

Expected Results
The nitrate reduction test is read for the presence or absence of three metabolic products: gas,
nitrate (NO3 ), and nitrite (NO2 ). The expected results can be summarized as follows:
 Quality Control Positive:
NO3+, no gas: Escherichia coli
Positive: NO3+, gas: Pseudomonas aeruginosa
Negative: Acinetobacter baumannii

Limitations

Nitrate reduction is a supportive test for identification of Enterobacteriaceae to the genus level; however,
additional follow-up confirmatory testing is required for final identification.