FMB 2111 Aquatic microbiology

Topic 3.2
Biochemical Tests
Dr. Leong Sui Sien
LEARNING OUTCOME
• Provide an understanding on biochemical tests used in
microbiological study.
• Explain the principle of each biochemical tests used in
characterization of bacteria.
• Learn to perform basic biochemical tests on bacterial samples.
• Discuss on the interpretation of the result of each biochemical
tests.
Biochemical tests
• Definition: the tests used for the identification of
bacteria species based on the differences in the
biochemical activities of different bacteria.
Carbohydrate/ Sugar Fermentation Test
• Aim: To determine the ability of microbes to ferment
carbohydrates with the production of an acid and/or gas.

• Principle:

Sugars are metabolized through different metabolic pathways

depending on types of microbial species/ aerobic /anaerobic
environment .
• If fermenting bacteria are grown in a liquid culture medium
containing the carbohydrate, they may produce organic acids
as by-products of the fermentation.
• These acids are released into the medium and so lower pH.
• pH indicator (phenol red or bromocresol blue) will change the
medium from its original colour to yellow.
• Gases produced in Durham tube in the form of a bubble.
Interpretation:
• Positive : Yellow
• Negative : Red
• Gas production positive : gas bubble is found in Durham’s
tube
Indole Production Test
• Aim: To determine the ability of microbe to degrade the
amino acid tryptophan.

• Principle:
Interpretation
• Positive : cherry red colour ring formed within seconds after
adding the Kovacs’ reagent
• Negative : No ring formation
Methyl Red Test
• Aim: To differentiate E.coli and E. aerogen and to determine the
ability of microbes to oxidize glucose with production and
stabilization of high content of acid end product.

• Principle:
Interpretation
• Positive: Red (E. coli)
• Negative : Yellow (E. aerogen)
Voges-Proskauer (VP) Test
• Aim: To differentiate the E.coli and Enterobacter aerogen by the
production of 2,3–butanediol and acetoin via glucose
fermentation.
• Principle:
• This test determines the capability of some organisms to
produce non-acidic or neutral end products, such as acetyl
methyl corbinol (acetoin), from the organic acid that results
from glucose metabolism.
• This test is characterizes E. aerogen. Test identifies bacteria that
ferment glucose and leading to 2,3-butanediol accumulation in
the medium.
Interpretation
• Positive: crimson red colour (E. aerogen)
• Negative : No change
Citrate Utilization Test
• Aim: To determine the ability of the microbes to ferment
citrate as sole carbon source.
• Principle:
• Citrate as a sole carbon source for their energy needs.
• Presence of a citrate permease that facilitates transport of
citrate into the bacterium.
• Sodium citrate as the carbon source, NH4+ as a nitrogen
source.
• pH indicator - bromothymol blue.
• This test is done on slants since O2 is necessary for citrate
utilization.
• When bacteria oxidize citrate, they remove it from the
medium and liberate CO2.
• CO2 combines with sodium (supplied by sodium citrate) and
water to form sodium carbonate - an alkaline product.
• This raises the pH, turns the pH indicator to a blue color, and
represents a positive citrate test
• Citrate-negative cultures will also show no growth in the medium and
the medium remains green.
Interpretation
• Positive : blue (E. aerogens)
• Negative : green
Nitrate Reduction Test
• Aim: To determine the ability of some microbes to reduce
nitrate (NO3-) to nitrites (NO2-) or beyond the nitrite stage.

• Principle:
• Certain organisms like Chemolithoautotrophic bacteria and
many chemoorganoheterotrophs can use nitrate (NO3-) as a
terminal electron acceptor during anaerobic respiration.
• In this process, nitrate is reduced to nitrite (NO2-) by nitrate
reductase.
• Further reduce the nitrite to either the ammonium ion or
molecular nitrogen.
• Nitrate broth medium containing 0.5% potassium nitrate
(KNO3).
• Examined for the presence of gas and nitrite ions in the
medium.
Interpretation
• Positive : Red (E. coli)
• Negative : No change
Urease Test
• Aim: To determine the ability of microbes to degrade urea by
urease.

• Principle:
• Urea is diamide carbonic acid often referred as carbamide.
• The hydrolysis of urea is catalysed by specific enzyme urease
to yield 2 moles of ammonia.
• Urease attacks the nitrogen and carbon bond in urea and
forms ammonia.
• The presence of urease is detected, when the organisms are
grown in urea broth.
• Medium containing the pH indicator phenol red.
• Splitting of urea creates the alkaline condition which turns
phenol red to deep pink in colour.
• Mainly used for identification of Proteus spp. from other genus of
lactose non-fermenting enteric organisms.
Interpretation:
• Positive : Deep pink (Proteus sp.)
• Negative : Yellowish
Triple Sugar Iron (TSI) Test
• Aim: To differentiate among and between the members of
Enterobacteraceae family.

• Principle:
• Study and identify the organisms belonging to
Enterobacteraceae family.
• It is also used to distinguish the Enterobacteriaceae from
other gram-negative intestinal bacilli (by their ability to
catabolize glucose, lactose or sucrose, and to liberate
sulfides from ferrous ammonium sulfate or sodium
thiosulfate)
• TSI agar slants contain a 1% concentration of lactose and
sucrose, and 0.1% glucose.
• The pH indicator phenol red, is also incorporated into the
medium to detect acid production from carbohydrate
fermentation.
• The uninoculated medium is red in colour due to presence of
phenol red dye.
• Yeast extract, beef extract and peptone provides nitrogen,
sulphur, trace elements and vitamin B complex etc.
• NaCl maintains osmotic equilibrium.
• Lactose, Sucrose and Dextrose are the fermentable
carbohydrates.
• Sodium thiosulfate and ferrous sulfate make H2S indicator
system.
• Thiosulfate is reduced to H2S by several species of bacteria
and H2S combines with and form insoluble black precipitates.
FeSO4 present in the medium
• Blackening usually occurs in butt of tube.
• Incubation is for 18 to 24 hours in order to detect the
presence of sugar fermentation, gas production, and H2S
production.
• The indicator is pink at alkaline pH and yellow at acidic pH, at
neutral pH it remains red.

• The following reactions may occur in the TSI tube:

• Yellow butt (A) and red slant (K) : due to the fermentation of
glucose (phenol red indicator turns yellow due to the
persisting acid formation in the butt). The slant remains red
(alkaline) (K) because of the limited glucose in the medium
and, therefore, limited acid formation, which does not
persist.
• Yellow butt (A) and slant (A) : due to the fermentation of
lactose and/or sucrose (yellow slant and butt due to the high
concentration of these sugars) leading to excessive acid
formation in the entire medium.

• Gas formation noted by splitting of the agar.

• Gas formation (H2S) seen by blackening of the agar.

• Red butt (K) and slant (K) : indicates that none of the sugars
were fermented and neither gas nor H2S were produced.
Interpretation
Oxidase Test
• Aim: To determine the ability of microbes to produce Oxidase
enzyme

• Principle:
• Oxidase enzyme plays a key role in Electron Transport Chain
during aerobic respiration.
• Cytochrome Oxidase catalyzes the oxidation of reduced
Cytochrome by molecular oxygen (O2), resulting in the formation
of H2O and H2O2.
• Aerobic as well as some facultative anaerobes and
microaerophilic bacteria shows oxidase activity.
Interpretation
• Positive : Purple
• Negative: No change
Catalase Test
• Aim: To determine the ability of an organism to produce
catalase.

• Principle:
• Certain organisms produce hydrogen peroxide during aerobic
respiration and sometimes extremely toxic superoxide
radicals.
• These are extremely toxic because they are powerful
oxidizing agents and destroy cellular constituents very
rapidly.
• A bacterium must be able to protect itself against such O2
products or it will be killed.
• Many bacteria possess enzymes that afford protection against
toxic O2 products.
• Obligate aerobes and facultative anaerobes usually contain the
enzymes superoxide dismutase, which catalyzes the destruction
of superoxide.
• And either catalase or peroxidase, catalyze the destruction of
hydrogen peroxide.
• Catalase production and activity can be detected by adding
the substrate H2O2 to an appropriately incubated (18- to 24-
hour) tryptic soy agar slant culture.
• If catalase was produced by the bacteria, they will liberate
free O2 gas on reaction.
• Bubbles of O2 represent a positive catalase test; the absence
of bubble formation is a negative catalase test.
Interpretation
• Positive : Burbles formed (Staphylococcus aureus)
• Negative : No change (Enterococcus faecalis)
Starch Hydrolysis Test
• Principles:
• Many bacteria produce enzymes called hydrolases.
• Hydrolases catalyze the splitting of organic molecules into
smaller molecules in the presence of water.
• The starch molecule consists of two constituents:
-Amylose, an unbranched glucose polymer (200 to 300
units)
- Amylopectin, a large branched polymer.
• Both amylopectin and amylose are rapidly hydrolyzed by
certain bacteria.
• Using their α-amylases, to yield dextrins, maltose, and
glucose.
• Gram’s iodine can be used to indicate the presence of starch.
• When it contacts starch, it forms a blue to brown complex.
• Hydrolyzed starch does not produce a colour change.
• If a clear area appears after adding Gram’s iodine to a medium
containing starch and bacterial growth:
• Amylase has been produced by the bacteria.
• If there is no clearing, starch has not been hydrolyzed.
Interpretation
• Positive : Clear zone formed
• Negative : No change
Lipid hydrolysis Test
• Principles:
• Lipids are high molecular weight compounds possessing
large amounts of stored energy.
• The two common lipids catabolized by bacteria are the
triglycerides (triacylglycerols) and phospholipids.
• Triglycerides are hydrolyzed by the enzymes called lipases
into glycerol and free fatty acid molecules.
• Glycerol and free fatty acid molecules can then be taken up by
the bacterial cell and further metabolized through reactions of :
glycolysis, β-oxidation pathway, and the citric acid cycle.

• These lipids can also enter other metabolic pathways where they
are used for the synthesis of cell membrane phospholipids.
• Since phospholipids are functional components of all cells,
the ability of bacteria to hydrolyze host-cell phospholipids is
an important factor in the spread of pathogenic bacteria.

• The culture medium contains tributyrin as a reactant

• Degradation of this compound gives rise to clear zones
surrounding the lipolytic colonies in the otherwise turbid
culture medium.
Interpretation
• Positive : Clear zone formed
• Negative : No change
Thank you