BACTERIAL METABOLISM

Citrate utilization test ;

Principle
Citrate utilisation test is used to detect the ability of an organism to utilize sodium citratre as a
sole source of carbon and ammonium salt as a sole source of nitrogen. Bacteria that grow in the
medium turn the medium alkaline. This is indicated by the change of color of bromothymol blue
indicator from green to blue.
Citrate utlilization test is used to determine the ability of bacteria to utilize sodium citrate as its
only carbon source and inorganic (NH4H2PO4) is the sole fixed nitrogen source.

Citrate utilization test (Image source: Microbelibrary)

A: Negative
B: Positive

Procedure of citrate utilization test:

1. Inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a
colony that is 18 to 24 hours old.
2. Incubate at 35oC to 37oC for 18 to 24 hours. Some organisms may require up to 7 days of
incubation due to their limited rate of growth on citrate medium.
3. Observe the development of blue color; denoting alkalinization.

Expected results in citrate utilization test:

Citrate positive: growth will be visible on the slant surface and the medium will be an intense
Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate
catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change
from the original green color to blue .

Citrate negative: trace or no growth will be visible. No color change will occur; the medium will
remain the deep forest green color of the uninoculated agar. Only bacteria that can utilize citrate
as the sole carbon and energy source will be able to grow on the Simmons citrate medium, thus
a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant
Triple Sugar Iron Agar test
Principle:
Triple sugar iron agar test is used to determine whether gram negative bacilli utilize glucose and
lactose or sucrose fermentatively and produce hydrogen sulfide (H2S). It contains 10 parts of
lactose: 10 parts of sucrose: 1 part of glucose and peptone. Phenol red and ferrous sulphate
serves as an indicator for acidification of medium and H2S production respectively.
Glucose is utilized first by a fermentative organism and the entire medium becomes acidic
(yellow) in 8 to 12 hours. Butt remains acidic even after 18 to 24 hours incubation peroid because
of the presence of organic acids resulting from the fermentation of glucose
under anaerobic conditions in the butt of the tube.The slant reverts to alkaline state that is
indicated by red color as the fermentation products gets oxidised to carbon dioxide (CO2) and
water (H2O) and peptone in aerobic condition the slant undergoes oxidation releasing
alkaline amines(Phenol red in alkaline pH turns red while in acidid pH turns yellow).
 If the organism ferments glucose but does not ferment lactose and/or sucrose, then the
slant becomes red and butt remains yellow (K/A) within 18 to 24 hrs.
 If the organism in addition to glucose ferments lactose and/or sucrose, the fermentation
product formed on the slant will more than neutralize the alkaline amines rendering the slant
acidic (yellow) (A/A) provided the reaction is read within 18 to 24 hours.
 If the organism is non fermenter, instead of sugars, peptone is utilised as an alternate
source of energy under aerobic condition on the slant which makes it alkaline indicated by
the red color while there is no change in the color of the butt. K/NC
Reactions in TSI should not be read after 24 hours of incubation, because aerobic oxidation of
fermentation products form lactose and/or sucrose will occur and the slant will eventually revert
to alkaline state.
The formation of CO2 and H2 is indicated by the presence of bubbles or cracks in the medium or
by the separation of the agar from sides or bottom of the tube. The production of H2S rqires an
acidic condition and is indicated by blackening of the butt of the medium in the tube.

Procedure:
1. Touch a well isolated colony with a sterile straight wire.
2. Inoculate TSI by first stabbing through the centre of the medium to the bottom of the tube
and then streak the surface of the slant.
3. Leave the cap loose and incubate the tube at 35 in ambient air for 18 to 24 hours.
4. Observe the reaction
Expected results:

Alkaline slant/no change in butt (K/NC) or Alkaline slant/Alkaline butt (K/K) = glucose, lactose
and sucrose nonfermenter.(red/red)
Alkaline slant/acidic butt (K/A)- glucose fermentation only. (red/yellow)
Acidic slant/acidic butt (A/A)- glucose, lactose and/or sucrose fermenter. (yellow/yellow)
A black precipitate in the butt indicates production of H2S . H2S produced reacts with ferric salt to
produce black precipitate of ferrous sulfide.
Bubbles or cracks in the tube indicate the production of CO2 or H2. Drawing of circle around butt
indicates that gas is produced by glucose and sucrose or glucose and lactose and glucose,
sucrose and lactose fermenter.
Hydrogen Sulfide Test
Some microorganisms have an ability to reduce sulfur (Sulphur) containing
compounds to hydrogen sulfide during metabolism which is commonly
employed as a test measure for their identification in laboratories. Numerous
methods are used to detect H2S production by micro-organisms which vary
with the source of sulfur and the metal salts used to indicate H2S formation.
SIM is more sensitive in the detection of H 2S than either TSI or KIA, because of
its semisolid nature, its lack of interfering carbohydrates, and the use of
peptonized iron as an indicator. However, Lead acetate paper is 10 times more
sensitive than other media.

Objectives
To determine whether the microbe reduces sulfur-containing
compounds to sulfides to produce hydrogen sulfide gas.

Principle
An iron compound and a sulfur compound are included in the test medium to
test for the production of hydrogen sulfide gas. Hydrogen sulfide is produced
if the sulfur compound is reduced by the bacterial strain. This test thus
determines whether the microbe reduces sulfur-containing
compounds to sulfides during the process of metabolism. H2S is produced by
certain bacteria through reduction of sulphur containing amino acids like
cystine, methionine or through the reduction of inorganic sulphur compounds
such as thiosulfates, sulfates or sulfites during protein degradation or when
anaerobic respiration shuttles the electrons to sulfur instead of to oxygen. In
either case H2S is produced (hydrogen sulfide gas) which reacts with the iron
compound to form the black precipitate of ferric sulfide. The black color acts
as an indicator for the presence of hydrogen sulfide. The detection of
hydrogen sulphide (H2S) gas produced by an organism. is used mainly to
assist in the identification of that particular organism.

Media:
This test can be performed with the use of several media including Triple
Sugar Iron (TSI), Kligler’s Iron Agar (KIA), SIM medium and Lead Acetate Paper.
  Sulphite indole motility (SIM) medium to detect H2S
This medium contains ferrous ammonium sulfate and sodium
thiosulfate, which then together serve as indicators for the production
of hydrogen sulfide. Production of hydrogen sulfide can be detected
when ferrous sulfide, a black precipitate, is produced as a result of
ferrous ammonium sulfate reacting with H2S gas.

Composition:
Beef extract 3.0 g Peptone 30.0 g Ferrous ammonium sulphat 0.2 g Sodium
thiosulphate 0.025 g Agar 3.0g Final pH ( at 25°C) 7.3±0.2 Distilled water
1000ml

 Iron agars to detect H2S

This medium is suitable for detecting H2S production by enterobacteria.
H2S is detected by the ferric citrate contained in the medium
 Lead acetate paper test to detect H2S
When a sensitive technique for detecting H2S production is required,
the lead acetate paper test is recommended.

Procedure
I. In Sulphite indole motility (SIM) medium

1. Inoculate the organism into labeled tube by means of stab inoculation.

2. Incubate the inoculated tubes at 37°C for 24-48 hours.
3. Observe for the formation of black precipitate on the medium.
II. In Kligler iron agar (KIA) and Triple Sugar Iron Agar (TSIA)

1. Inoculate the test organism into KIA and incubate it at appropriate

temperature over night.
2. Observe for blackening of the medium.
III. Lead acetate paper test

1. Inoculate a tube or bottle of sterile peptone water or nutrient broth with

the test organism.
2. Insert a lead acetate paper strip in the neck of the bottle or tube above
the medium, and stopper well.
3. Incubate the inoculated medium at 35-37oC, and examine daily for a
blackening of the lower part of the strip.
Results
 Positive result:Blackening on the medium
 Negative result:No blackening on the medium

Uses
 It is used mainly to assist in the identification of members of family
Enterobacteriaceae and occasionally to differentiate other bacteria such
as Bacteroidessps and Brucella sps.
 The test aids in the identification and differentiation of members
of Enterobacteriaceae (enterics) from other Gram- bacilli.
 It is especially helpful in identifying Salmonella, Francisella,
and Proteus species.
Limitations
 H2S production may be inhibited on TSI for organisms that utilize
sucrose and suppress the enzyme mechanism that results in production
of H2S.
 Lead acetate is toxic to bacteria and may inhibit the growth of some
bacteria. Do not allow the media to touch the strip.
 It is recommended that biochemical, immunological, molecular, or mass
spectrometry testing be performed on colonies from pure culture for
complete identification.
Nitrate Reduction TesT
Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on the basis
of their ability to produce nitrate reductase enzyme that hydrolyze nitrate (NO3–) to nitrite (NO2–)
which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide
and ammonia (NH3) depending on the enzyme system of the organism and the atmosphere in
which it is growing.

Fig: Nitrate Reduction Pathway

Other uses of Nitrate Reduction Tests are:

1. Differentiating Mycobacterium species.

2. Identifying species of Neisseria and separating them
from Moraxella and Kingella species. The nitrate reduction test is a critical test for
differentiating between N. gonorrhoeae and K. denitrificans, particularly when strains of K.
denitrificans appear to be gram-negative diplococci in stained smears.
3. Facilitating species identification of Corynebacterium

Principle:Heavy inoculum of test organism is incubated in nitrate broth. After 4 hrs incubation, the
broth is tested for reduction of nitrate (NO3–) to nitrite (NO2–) by adding sulfanilic acid reagent
and α- naphthylamine.

1. If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous
acid. When sulfanilic acid is added, it will react with the nitrous acid to produce diazotized
sulfanilic acid. This reacts with the α-naphthylamine to form a red-colored compound.
 Therefore, if the medium turns red after the addition of the nitrate reagents, it is considered

a positive result for nitrate reduction.

2. If the medium does not turn red after the addition of the reagents, it can mean that the
organism was unable to reduce the nitrate, or the organism was able to denitrify the nitrate
or nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in
the test. Add a small amount of powdered zinc. If the tube turns red after the addition of the
zinc, it means that unreduced nitrate was present*.Therefore, a red color on the second step
is a negative result.

*Note: The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed
nitrous acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby
produced reacted with the α-naphthylamine to create the red complex.

If the medium does not turn red after the addition of the zinc powder, then the result is called a
positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite
present in the medium, either, that means that denitrification took place and ammonia or
molecular nitrogen were formed.

Requirements:

1. Media: Nitrate Broth with inverted Durhams tube

 2. Reagents: Sulphalinic acid reagent, Alpha napthylamine reagent, zinc dust
3. Others: Inoculating loop, burner, dropper

Procedure:

1. Inoculate nitrate broth with a heavy growth of test organism using aseptic technique.
2. Incubate at an appropriate temperature for 24 to 48 hours
3. Add one dropperfull of sulfanilic acid and one dropperfull of a α-naphthylamine to each
broth.
1. At this point, a color change to RED indicates a POSITIVE nitrate reduction test. If
you get a red color, then you can stop at this point.
2. No color change indicates the absence of nitrite. This can happen either because
nitrate was not reduced or because nitrate was reduced to nitrite, then nitrite was
further reduced to some other molecule. If you DO NOT get a red color, then you
must proceed to the next step.
4. Add a small amount of zinc (a toothpick full) to each broth. Zinc catalyzes the reduction of
nitrate to nitrite.
1. At this point, a color change to RED indicates a NEGATIVE nitrate reduction test
because this means that nitrate must have been present and must have been
reduced to form nitrite.
2. No color change means that no nitrate was present. Thus no color change at this
point is a POSITIVE result.

Fig. Diagrammatic representation of the detection of

nitrite in medium

Result and Interpretation

1. Nitrate Reduction Positive: (Red after sulfanilic acid + alpha-naphthylamine; no color after
zinc)
2. Nitrate Reduction Negative: (No color after sulfanilic acid + alpha-naphthylamine followed
by Red after zinc)