Biochemical Characteristics

Extracellular Enzymes
1. Starch Hydrolysis
2. Lipid Hydrolysis
3. Casein Hydrolysis
4. Gelatin Hydrolysis

1. Starch Hydrolysis – Bacillus subtilis

Starch is a high molecular weight polymer composed of glucose molecules
linked by glycosidic bonds. Amylase is capable of breaking down starch
into smaller polysaccharides called dextrins and then into maltose
molecules. Maltase yields low molecular weight soluble glucose that can
be transported to cell and used for energy production through a process
called glycolysis.
Amylase Maltase
Starch Maltose Glucose

Grow Microbes on Starch agar

Starch with iodine gives blue black colour to the medium
This indicates absence of starch splitting enzymes
This is Negative Result
If the starch is hydrolyzed a clear zone of hydrolysis will form around the
microorganism
This is positive result
The organism is able to produce the extracellular enzyme amylase

2. Lipid Hydrolysis – Staphylococcus, Micrococcus, Pseudomonas

Lipids are high molecular weight compounds possessing lot of energy
Lipase
Triglycerides Glycerol and fatty acids

Microbes grown on tributyrin agar (opaque, emulsion forming agar)

Inoculation and incubation of agar plates
Organisms that release lipase show lipolysis
Clear area around bacterial growth. Positive reaction
Agar remains opaque – Negative reaction
3.Casein Hydrolysis
Casein is a milk protein composed of amino acid subunits linked together by
peptide bonds.
Proteins must undergo step by step degradation into peptones, polypeptides,
dipeptides and finally into amino acids.
This is called proteolysis and is mediated by proteases
The low molecular weight amino acids can be transported through the cell
membrane

Milk agar is used to show the hydrolytic activity of these enzymes

The medium is coloured, opaque and deflects white rays
Inoculated and incubated
Microbes producing protease will exhibit a zone of proteolysis – clear zone –
loss of opacity. Positive reaction
Lactococcus lactis, Bacillus cereus, Serratia marcescens
If the medium remains opaque – negative reaction

4.Gelatin Hydrolysis
Gelatin is produced by hydrolysis of collagen, a major component in the
muscle and connective tissues of animals and humans
Below 25 deg – gel
Above 25 deg – Liquid
Liquefaction is accomplished by gelatinase producing organisms which
hydrolyze protein to amino acids
After liquefaction even at 4 deg it will not gel
Bacillus anthracis, B. cereus, B. subtilis, Clostridium perfringens, C. tetani

Nutrient gelatin tubes

Inoculation and incubation for 48 hours
Kept in refrigerator for 30 min
Cultures that remain liquefied produce gelatinase and demonstrate rapid
gelatin hydrolysis

Intracellular enzymatic activities of Microorganisms

1. Carbohydrate fermentation
Some organisms ferment sugars anaerobically
Some organisms ferment sugars aerobically
In fermentation carbohydrates and alcohols undergo anaerobic fermentation
and produce lactic acid, formic acid, acetic acid along with hydrogen or
carbon di oxide

Lactose broth
Inoculate and incubate
Add phenol red indicator (neutral pH – red, yellow – acidic)
Yellow
Durham’s tube – captures gas
Positive reaction
Escherichia coli, Alcaligenes faecalis, Salmonella typhimurium, Staphylococcus
aureus

2. Litmus milk reactions

Milk substrates capable of transformation – lactose, casein, lacto-albumin
and lacto-globulin
pH indicator and an oxidation-reduction indicator litmus incorporated into
medium
Litmus Milk – Biochemical changes
1. Lactose fermentation
2. Gas production
3. Litmus reduction
4. Curd formation
5. Proteolysis
6. Alkaline reaction

1. Lactose fermentation

β-galactosidase
Lactose Glucose + Galactose Pyruvic acid
Lactic acid
Litmus turns pink at pH4 (Purple at neutral pH)

2. Gas production
End product of fermentation of lactose is carbon di oxide and hydrogen. Seen
as cracks or fissures

3. Litmus reduction
Fermentation is anaerobic reaction, takes place in the absence of oxygen
Removal of hydrogen from substrate
Hydrogen is accepted by acceptor litmus, which turns white
Oxidised state – litmus is purple
Reduced state – white

4. Curd formation
Organisms grown in litmus milk show two types of curds (clots)- acid or
rennet depending on biochemical mechanism
Acid Curd – lactic acid causes precipitation of milk protein casein as calcium
caseinate to form insoluble clot. The clot is hard and immobile .
Rennet Curd – some organisms produce rennin that acts on casein to form
paracasein which is converted to calcium paracaseinate and forms an
insoluble clot. This is soft, semi solid clot that is mobile when the test tube is
tilted.

5. Proteolysis
Certain organisms use proteolytic enzymes to hydrolyze milk proteins casein
into amino acids. In the process there is evolution of ammonia making the pH
alkaline.
Litmus turns purple – upper portion of tube
Translucent brown whey like appearance – below

6. Alkaline reaction
Medium unchanged or changes to blue colour – indicative of partial
degradation of casein

3.Hydrogen Sulphide Production

Ability of microbes to produce hydrogen sulphide
Pathway 1
Cysteine desulphurase
Cysteine Pyruvic acid + Hydrogen Sulphide gas + ammonia

Pathway 2
Thiosulphate reductase
Thiosulphate + 4H +4e-
+
Sulphate + Hydrogen Sulphide gas

SIM medium has peptone and sodium thiosulphate

Ferrous ammonium sulphate in the medium serves as an indicator. Combines
with hydrogen sulphide and forms an insoluble black ferrous sulphide
precipitate. Salmonella typhi

4.Nitrate Reduction
Ability of microbes to reduce nitrates NO3- to nitrites NO2-
Reduction of nitrates by aerobes, facultative anaerobes occurs in the absence
of oxygen, an anerobic process

Nitrate Reductase
Nitrate + Hydrogen + electrons Nitrite+ Water

• Nitrate broth supplemented with 0.1% potassium nitrate + 0.1% agar

• Inoculate and incubate
• Add sulfanilic acid and -naphthylamine
• Production of cherry red colour – positive test
No colour – negative test
E.coli, Alcaligenes faecalis, Pseudomonas aeruginosa

5. Catalase Reaction
Catalase Test - Determine the ability of the microbes to degrade hydrogen
peroxide by producing catalase. If hydrogen peroxide is not degraded it will
produce toxic superoxide resulting in the death of organisms. These are
produced by aerobes, facultative anaerobes and microaerophiles.
catalase
Hydrogen Peroxide water and oxygen
Aerobic organisms that lack catalase can degrade toxic superoxides using the
enzyme superoxide dismutase. The end product is hydrogen peroxide which
is less toxic than superoxide.
Anaerobes cannot produce catalase, peroxidase or superoxide dismutase. So
oxygen is poisonous to them.
Add hydrogen peroxide to substrate in trypticase soy agar
Inoculate and incubate
Bubbles of free oxygen – catalase present – positive test
Absence of bubbles – negative catalase test
Staphylococcus aureus, Micrococcus luteus, Lactococcus lactis

6. Urease Test
Ability to degrade urea by urease
Urease produced by Proteus vulgaris is helpful for identification
urease
Urea +2H2O Carbon di oxide + water + ammonia
Urea broth containing phenol red indicator
Deep pink – presence of urea
No pink – negative test

Urea broth containing phenol red indicator

Deep pink – presence of urea
No pink – negative test

7. Oxidase Test
Distinguish bacteria on the basis of cytochrome oxidase activity
Cytochrome oxidase catalyses the oxidation of a reduced cytochrome by
molecular oxygen, resulting in the formation of water or hydrogen peroxide.
Aerobic bacteria, facultative anaerobes, microaerophiles show oxidase
activity
Oxidase positive – Neisseria, Pseudomonas
Oxidase negative – Enterobacteriaceae
Trypticase soy agar Medium + p-aminodimethylaniline oxalate
Inoculate and incubate
Pink reagent turns maroon, then oxidized to black colour on surface of
colonies – indicates cytochrome oxidase production – positive test
No colour change – absence of oxidase production – negative test
IMViC Test
Identify enteric bacilli
Gram negative, non-spore forming bacilli
Pathogens – Salmonella, Shigella, Proteus, Klebsiella
Normal intestinal flora – Escherichia, Enterobacter

I – Indole
M – Methyl Red
VP - Voges Proskauer
C – Citrate Utilization

Enteric, lactose fermenters – Escherichia coli, Enterobacter aerogenes.

Klebsiella pneumoniae
Enteric, Lactose non-fermenters – Salmonella typhi, Shigella dysenteriae,
Proteus vulgaris, Pseudomonas aeruginosa, Alcaligenes faecalis
Non-enteric – Corynebacterium xerosis, Micrococcus luteus, Lactococcus
lactis, Staphylococcus aureus, Bacillus cereus

Indole Production Test

• Ability of microbes to degrade tryptophan amino acid

Tryptophanase

• Tryptophan Indole + Pyruvic acid + ammonia

• Ability to hydrolyze tryptophan with the production of indole serves as
a biochemical marker
• SIM agar containing tryptophan is used
• Indole detected by adding Kovac’s reagent
• Produces a cherry red layer
• Colour is produced by the reagent which is composed of p-dimethyl
aminobenzaldehyde, butanol and hydrochloric acid.
• Escherichia coli
Methyl Red Test
 • Determines the ability of the microorganisms to oxidise glucose to acid
end products
• Indicator methyl red detects the acid products
• pH range 4 – red – positive test
• pH range 6 – yellow – negative test
• E.coli

Voges Proskauer Test

• Determines the capacity of organisms to produce acetyl methyl
carbinol
• MR – VP Medium
• Pink Colour is formed – positive result
• Absence of pink colour – negative result
• Enterobacter aerogenes

Citrate Utilization Test

• Ability to ferment citrate as a solid source of carbon
• In the absence of glucose or lactose, some organisms use citrate as
carbon source for energy
• Simmon’s citrate medium
Citrase
• Citrate Oxaloacetic acid + Acetic acid Pyruvic acid+
Carbon di oxide

During this reaction the medium becomes alkaline, the carbon di oxide that
is generated combines with sodium and water to form sodium carbonate, an
alkaline product. Sodium carbonate changes bromothymol blue indicator in
the medium from green to deep Prussian blue.
Enterobacter aerogenes
Citrate negative organisms – green

Triple Sugar – Iron Agar Test

• Differentiates the Enterobacteriaceae members
• Gram negative bacilli that ferment glucose producing acid
• TSI slant (has sucrose, glucose and lactose) with phenol red indicator

1. Alkaline Slant (Red) and acid butt (yellow) – only glucose fermentation
has taken place
2. Acid slant (yellow) and acid butt (yellow) – Lactose fermentation has
taken place
3. Alkaline slant (red) and alkaline butt (red) or no change in butt – no
carbohydrate fermentation has taken place
The TSI agar medium also has sodium thiosulphate, a substrate for
hydrogen sulphide production
Only organisms that can produce hydrogen sulphide will show
blackening in the butt

TSI reactions for differentiation of enteric microbes

1. Acid slant, acid butt, no H2S – Escherichia, Klebsiella, Enterobacter
2. Acid slant, acid butt, H2S produced – Citrobacter, Proteus
3. Alkaline Slant, acid butt, no H2S (Red) – Shigella, Proteus
4. Alkaline slant, acid butt, H2S produced – Salmonella, Citrobacter
5. Alkaline slant, alkaline or no change in butt – Alcaligenes, Pseudomonas