EXPERIMENT NO.

1
Objective: Protein estimation by Folins Lowry method.
Lowrys assay for total protein is one of the most commonly performed colorimetric assays. This procedure is
sensitive because it employs two colour forming reactions. It uses the Biuret reactions in which Cu2+ in presence of a
base reacts with a peptide bond of protein under alkaline conditions resulting in reduction of cupric ions (Cu2+) to
cuprous ions (Cu+), and Lowrys reaction in which the Folin Ciocaltaeu reagent which contains phosphomolybdic
complex which is a mixture of sodium tungstate, sodium molybdate and phosphate, along with copper sulphate
solution and the protein, a blue purple colour is produced which can be assessed by measuring the absorbance at 650700nm.

The blue purple color is formed due to the reduction of phosphomolybdotungstate to hetero-polymolybdenum blue by
the copper catalysed oxidation of aromatic amino acids tryptophan and tyrosine. Thus the color intensity depends on
the amount of these aromatic amino
acids present and will thus vary for
different proteins.

The measurement of protein with

Folin's
reagent
has
certain
advantages. Firstly, it is a sensitive
assay which requires no digestion.
Secondly, It is 10 or 20 times more
sensitive than measurement of the
ultraviolet absorption at 280 nm and is much more specific and much less liable to disturbance by turbidities, thirdly,
it is several fold more sensitive than the ninhydrin reaction and is simpler, as well as much easier to adapt for small
scale
analyses.
Also
it
is
100
times
more
sensitive
than
the
biuret
reaction.
There are disadvantages also for the Folin's reaction, like the amount of color varies with different proteins, it is
lessconstant than the biuret reaction, but more constant than the absorption at 280 nm. The color is not always
proportional to concentration.Inspite of all these concerns, folin's method can be used in measurement of protein
during enzyme fractionations, mixed tissue proteins, measurement of very small absolute amounts of protein, or
highly diluted protein and analyses of large numbers of similar protein samples.

Most protein estimation techniques use Bovine Serum Albumin (BSA) as a standard protein, because of its easy
availability and low cost with improved purity. Constructing a protein standard curve is of prime importance in
studying the activity of an enzyme as this analysis relies on accurate quantitation of protein concentration.

Reagents
A. 2% Na2CO3 in 0.1 N NaOH
B. 1% NaK Tartrate in H2O
C. 0.5% CuSO4.5 H2O in H2O
D. Reagent I: 48 ml of A, 1 ml of B, 1 ml C

E. Reagent II- 1 part Folin-Phenol [2 N]: 1 part water

BSA Standard - 1 mg/ ml

Procedure:
0.2 ml of BSA working standard in 5 test tubes and make up to 1ml using distilled water.
The test tube with 1 ml distilled water serve as blank.
Add 4.5 ml of Reagent I and incubate for 10 minutes.
After incubation add 0.5 ml of reagent II and incubate for 30 minutes
Measure the absorbance at 660 nm and plot the standard graph.
Estimate the amount of protein present in the given sample from the standard graph.

Graph:
Result:

EXPERIMENT NO. 2

Objective: Reducing sugar estimation by DNSA method.

Maltose is a disaccharide made up of two subunits of glucose monomers. It is also called malt sugar. It is present in
germinating grain, in a smaller amount in corn syrup, and also is a product of the partial hydrolysis of starch. Maltose
is considered as an important constituent in making fermented barley which is used to brew beer. Adding in a third
unit of glucose produces a sugar known as maltotriose, while further units make it possible to produce maltodextrins.

 D-glucopyranosyl,(1-4) D-glucopyranose

Maltose is a reducing sugar (those carbohydrates which have a free aldehyde or keto group can reduce Fehling's and
Benedict's reagents are called reducing sugars). Maltose can be used as a standard for estimating reducing sugar in
unknown samples. Constructing a standard curve / graph for maltose helps us to estimate concentration of reducing
sugars present in an unknown sample and for determining the activity of amylase enzyme in forthcoming
experiments.The standard curve for maltose is usually constructed using 3, 5-Dinitro salicylic acid (DNS) as the
reagent. Maltose reduces the pale yellow coloured alkaline 3,5-Dinitro salicylic acid (DNS) to the orange- red
coloured, 3 amino,5 nitro salicylic acid.

The intensity of the colour is proportional to the concentration of maltose present in the solution [as per Beer
Lambert's law. This intensity change in colour is measured using a colorimeter as the absorbance at 540nm
wavelength. Wave length is set to 540 nm because it is the region where orange-red colour absorbs. A series of
solutions containing varying concentrations of maltose are prepared in test tubes and a known quantity of DNS is
added to each. These test tubes are then heated on a water bath for few minutes and their optical densities are
measured using a colorimeter. A graph is then plotted with amount of maltose on X axis and the observed optical
density at Y axis. The plot thus obtained is called a standard maltose curve.

Preparation of Reagents:

1.

3,5-dinitrosalicylic acid [DNS]: About 1g of DNS is dissolved in 50ml of distilled water. To this
solution add about 30g of sodium potassium tartarate tetrahydrate in small lots, the solution turns milky
yellow in colour. Then add 20ml of 2N NaOH, which turns the solution to transparent orange yellow colour.

The final volume is made to 100 ml with the distilled water. This solution is stored in an amber coloured
bottle.
2.
Maltose working solution : 180mg of maltose is weighed and made up to 100ml with distilled
water.
Procedure:

1.

Pipette out standard maltose solution in the range of 0.2, 0.4, 0.6, 0.8 and 1 ml, into 5 separate test
tubes.

2.
3.
4.
5.
6.
7.
8.
9.

A test tube containing a blank solution is also prepared.

Using distilled water, bring the volume up to 2ml in each test tube, including the test tube containing
the blank solution.
Add 1 mL of DNS reagent to each tube and cover the test tubes with aluminum foil.
Heat the contents in the test tubes in a boiling water bath for 5 minutes.
Cool the test tubes to room temperature, after taking them out of the water bath.
Then add 9ml distilled water to each test tube and mix well.
Take 1ml from each test tube into different cuvettes and place each cuvette in a colorimeter and
record the intensity of dark orange red colour at 540 nm as the 'absorbance' or OD.
Plot a graph with the amount of maltose on X axis Vs OD at 540nm (A540nm ) on Y axis.
EXPERIMENT NO. 3

Objective: Extraction of amylase enzyme from potato and qualitative detection by starch iodide
method
Amylase is an extracellular enzyme. When plant material is crushed in buffer and then centrifuged, enzyme
is obtained in supernatant.
Iodine reacts with starch (substrate for amylase enzyme) to give blue color. As enzyme acts on starch, its
quantity reduces, thus reducing the blue color. This is indicative of enzyme activity.
Carbohydrates account for the major storage form of energy in plants and in animals. Starch is a homopolysaccharide
which is the most important storage polysaccharides in plant cells. It is composed of two types of glucose polymer,
amylose and amylopectin. The amylose consists of long, unbranched chains of D-glucose residues joined by (-1,4)
linkages. The successive glucose residues in amylopectin are joined by -1,4 glycosidic linkages and -1,6 linkages
for branch points.

Amylases are enzymes that hydrolyze starch. The enzyme -amylase catalyses the hydrolysis of -1,4 glycosidic
linkages from the non-reducing end of the polysaccharides ( starch amylose, amylopectin ), to yield maltose units.
The glucose residues at the nonreducing ends of the outer branches are removed enzymatically to facilitate the
mobilization of starch for energy production. Thus it is also known as 1-4--D-Glucan maltohydrolase. -Amylase is
specific for amylose chains of six glucose units.

Procedure:
1. Prepare acetate buffer
Sodium acetate 36.2ml
Glacial Acetic acid 14.8ml
Vol make up 100ml
2. Take pieces of potato in buffer and crush in mortar and pestle.
3. Centrifuge and take supernatant.

4.
5.
6.
7.
8.

Add 5ml,10ml,15ml of supernatant in different test tubes.

Add 2ml starch in each.
Add 1 drop of iodine.
Incubate at 35 degrees centigrade for 15min.
Observe for reduction in blue color.

Result:

EXPERIMENT NO.4
Objective: Estimation of amylase activity by DNS method.
Amylases are important hydrolase enzymes which have been widely used since many decades. These enzymes
randomly cleave internal glycosidic linkages in starch molecules to hydrolyze them and yield dextrins and
oligosaccharides. Among amylases -Amylase is in maximum demand due to its wide range of applications in the
industrial front. With consumers growing increasingly aware of environmental issues, industries find enzymes as a
good alternative over other chemical catalysts. -Amylase can be produced by plant or microbial sources. Due to the
advantages that microbial production offers, -Amylase from microorganisms has been focused upon and preferred to
other sources for production. The ubiquitous nature, ease of production and broad spectrum of applications make Amylase an industrially important enzyme.
The DNS method for estimating the concentration of reducing sugars in a sample is used. Reducing sugars contain
free carbonyl group, have the property to reduce many f the reagents. All monosaccharides and some disaccharides are
reducing sugars When alkaline solution of 3,5-dinitrosalicylic acid reacts with reducing sugars it is converted into 3amino-5 nitrosalicylic acid with orange color.

DNS Reagents

DNS reagent contained DNS (1%), potassium sodium tartarate (Rochelle salt, (1 M) and NaOH (0.4 M) and ddH2O.

Procedure for 100 mL Reagent

Dissolve by stirring (Remi, India) at room temperature (RT) 1g DNS in 50 mL ddH2O, then added 20 mL 2 M NaOH
and 28.2 g Rochelle salt, finally made up to 100 mL by ddH2O. The reagent was stored at RT.

Other solutions

Buffer: 0.02 M sodium phosphate buffer (pH, 6.9) with 0.006 M sodium chloride.
Starch solution: 1.0% starch solution was prepared fresh by dissolving 1.0 g soluble starch in 100 mL 0.02 M sodium
phosphate buffer (pH, 6.9).
Maltose stock solution: Dissolved 50 g maltose in 50 mL ddH2O in a standard flask and stored at 4 oC.
Protocol:

Pipette out 0.5 mL enzyme solution (prepared as explained in 3.5.1.) and incubate tubes at 25 oC for 3 min.
Add 0.5 mL starch solution and incubate for 5 min (RT).
Stop the reaction by adding 1 mL DNS reagent.
Heat the solution in a boiling water bath for 5 min.
Cool it in running tap water.
Make up the volume to 10.0 mL by the addition of ddH2O.
Read the absorbance at 540 nm using UV-Vis Spectrophotometer
Blank is prepared without enzyme.
Prepare a standard graph with 0 -100 g maltose.

Calculation of enzyme activity activity (U/ml/min)

= (Quantity of product released x total assay volume x DF)/ (Vol of enzyme used x vol in cuvette x incubation time)

EXPERIMENT NO 5 & 6
Objective: To study the effect of pH and temperature on enzyme activity.
This study is to be performed with amylase enzyme.
For pH studies: acetate and phosphate buffers with different pH is to be made. And quantification of enzyme activity
is done by DNS method as previously.
For temperature studies: reaction at different temperature was performed and then activity calculated using DNS
method.

1. 0.5ml of the substrate solution and 0.5ml of the enzyme solution was pipette into two separate test tubes. Then
both the substrate and the enzyme were preincubated in separate test tubes at 10C for 5 minutes.
2. After preincubation at that temperature, the substrate solution from the test tube mixed and incubated for
5minutes at that temperature for the reaction to take place.
3. At the end of the incubation time, 0.5ml of the 2N NaOH was added to arrest the reaction followed by 1.0ml
of DNS reagent.
4. The above three steps were repeated to study the effect of temperature at 25C, 37C, 55C and 80C by
preincubation followed by the incubation at that respective temperature.
5. The control at respective temperature was set up by adding the 0.5ml of the substrate, 0.5ml of 2N NaOH.
6. After the incubation, all the tubes were placed in a boiling water bath for 5min and cooled to room
temperature.
7. The absorbance of all the tubes against respective control was read at 540nm.
8. The amount of product liberated was determined from maltose calibration curve and hence activity at each
temperature was obtained.
9. The graph was plotted with activity versus time and optimum temperature was determined.
10. The graph was plotted with the activity versus 1/Temperature in K , the slope of which gives the value of
activation E/R.
11. For pH study, similar protocol is followed but the enzyme solution and substrate are incubated in acetate
buffer of different pH
EXPERIMENT NO. 7
Objective: To analyze the effect of substrate concentration on the activity of enzymes.

Principle:
The enzyme Amylase can catalyze the hydrolysis of internal -1,4-glycosidic bond present in starch with the
production of reducing sugars. In the study of substrate concentration on enzyme kinetics, the enzyme is kept
constant where as the concentration of Starch is taken in increasing order. As the substrate concentration
increases, the amount of products produced in every successive tube also increases. This was explained by
Michealis and others that an enzyme catalyzed reaction at varying substrate concentrations is diphasic i.e. at low
substrate concentration the active sites on molecules (enzyme) are not occupied by substrate and the enzyme rate
varies with substrate molecules concentration (phase1). As the number of substrate molecules increases, the
enzyme attains the saturation level, since there is no more reaction sites remaining for binding. So the enzyme
can work with full capacity and its reaction rate is independent of substrate concentration. (Phase II).

This Enzyme substrate reaction can be determined by measuring the increase in reducing sugars using the 3, 5
Dinitro salycilic acid reagent. In an alkaline condition, the pale yellow colored the 3, 5- dinitro salicylic acid
undergo reduction to yield orange colored 3- amino -5-nitrosalicylic acid. The absorbance of resultant solutions is
read at 540nm. The intensity of color depends on the concentration of reducing sugars produced.

Amylase
Starch

Maltose + glucose

Materials Required:
1.
2.
3.
4.
5.

10ml

of

0.5%,

10ml
10ml
15ml
Distilled Water 150ml

1%,

2%,

3%,

of
of
of

4%,

5%

2N
DiNitro

Sodium
Salicylic

Soluble
Starch
Amylase
hydroxide
acid

solution.
solution
(NaOH)
(DNS)

Reagent preparation:

1)

Amylase solution:

0.5% starch solution- 0.5g starch soluble in 100ml deionised water.

1% starch solution- 1g of starch soluble in 100ml deionised water
2% starch solution- 2g of starch soluble in 100ml deionised water
3% starch solution- 3g of starch soluble in 100ml deionised water
4% starch solution- 4g of starch soluble in 100ml deionised water
5% starch solution- 5g of starch soluble in 100ml deionised water

2)

2N NaOH Solution:

8g NaOH in 100ml distilled water.

3)

DNS Solution:

1g of DNS is dissolved in 50ml of distilled water. 30g of sodium potassium tartarate tetrahydrate is added
in small lots. The solution turns milky yellow in colour. Then 20ml of 2N NaOH is added, which turns the solution to
transparent orange yellow colour. The final volume is raised to 100 ml with the distilled water. This solution is
stored in an amber coloured bottle.

Procedure

1.
2.

Make different concentration of starch soluble (0.5%, 1%, 2%, 3%, 4% and 5%,).
Take 12 clean and dry boiling tubes. Label tube as control C and Test T for each concentration. Add
0.5ml (500l) of starch solution to all the tubes.
3.
Preincubate the starch solutions of all concentrations and amylase solutions for 10 minutes at 37C.
4.
Add 0.5ml (500l) of Amylase enzyme to the tubes labeled T of respective concentration.
5.
Incubate all the tubes for at 37C for 10 minutes
6.
After incubation, Immediately add 2N NaOH to test tubes containing test solution and then to the test
tubes containing control solution. Mix the solutions in each test tubes.
7.
Pipette out 0.5ml (500l) of Amylase to the test tube containing control solutions. Mix the solutions
well.
8.
Add 1 ml of DNS reagent to all tubes. Mix the solutions in the test tubes well.
9.
Keep in boiling water bath for 5 minutes at 100C and cool it.
10.
Dilute all the tubes by adding 9.5 ml of distilled water.
11.
Mix the solutions in each test tubes by using vortex mixer.
12.
The absorbance of test solutions was read at 540nm against the Control.
Graph
Result

EXPERIMENT NO. 8
Objective: Enzyme encapsulation/immobilization in alginate-sodium chloride system.

Alginate, a naturally occurring biopolymer, finds increasing applications in various fields. It has been
successfully used for many years in the food and beverage industry as a thickening agent, gelling agent, and
a colloidal stabilizer. It also has several unique properties that have opened the field of its use as a matrix for
the entrapment and/or delivery of a variety of proteins, drugs, and cells.
Alginate is a water-soluble linear, polyanionic; polysaccharide extracted from brown seaweed and is composed of
alternating blocks of 14 linked -L-guluronic and -D-mannuronic acid residues. The gel beads are prepared through
the sol-gel transformation of alginate which is brought about by cross-linking the alginate with divalent cations like
Ca2+.Guluronic acid is responsible for the formation of gel by the alginate with the cations in the solution. The
alginate matrix consisting of an open lattice structure forms porous beads. The beads have low retention capacity for
encapsulating low molecular weight and water soluble drugs.
It is a very popular pH-responsive polymer due to its shrinkage in lower pH, enabling encapsulated drug retention in
the stomach while protecting it against enzymatic deactivation. Properties of biodegradability, biocompatibility, low
toxicity, low immunogenicity and good muco-adhesion facilitate its application in oral drug delivery.11Micro or nano
particles of ALG can be formed either by physical or chemical cross linking for the sustained drug release. As some
cross-linkers are toxic in nature, physical cross linking is usually preferred over chemical cross linking. Calcium, a
divalent cation used for crosslinking of ALG, is reported to maintain the biological efficiency of the drug molecules.

Beads were prepared by syringe/ dropper extrusion method. Extrusion is a process of creating an object of a
fixed dimension by pushing or drawing the material through a die of the desired cross-section. The solution
of sodium alginate is mixed with the protein to be immobilized and added dropwise in calcium chloride
solution being gently stirred at 300rpm in magnetic stirrer by means of syringe/dropper.

EXPERIMENT NO. 9
Objective: To separate and identify amino acids by thin layer chromatography.
Chromatography:

Chromatography is by far the most useful general group of techniques available for the separation of closely related
compounds in a mixture. Here the separation is effected by differences in the equilibrium distribution of the
components between two immiscible phases, viz., the stationary and the mobile phases. These differences in the
equilibrium distribution are a result of nature and degree of interaction of the components with these two phases. The
stationary phase is a porous medium like silica or alumina, through which the sample mixture percolates under the
influence of a moving solvent (the mobile phase). There are a number of interactions between the sample and the
stationary phase and these have been well exploited to effect the separation of compounds.

Thin layer chromatography [TLC]:

Thin layer chromatographic (TLC) technique readily provides qualitative information and with careful attention to
details, it is possible to obtain quantitative data. Thin layer chromatography is a technique used to separate and
identify compounds of interest. A TLC plate is made up of a thin layer of silica adhered to glass or aluminum for
support. The silica gel acts as the stationary phase and the solvent mixture acts as the mobile phase. In the ideal
solvent system the compounds of interest are soluble to different degrees. Separation results from the partition
equilibrium of the components in the mixture.
In the simplest form of the technique, a narrow zone or spot of the sample mixture to be separated is applied near one
end of the TLC plate and allowed to dry. The strip or plate is then placed with this end dipping in to the solvent
mixture, taking care that the sample spot/zone is not immersed in the solvent. As the solvent moves towards the other
end of the strip, the test mixture separates into various components. This is called as the development of TLC plates.
The separation depends on several factors; (a) solubility: the more soluble a compound is in a solvent, the faster it will
move up the plate. (b) attractions between the compound and the silica, the more the compound interacts with silica,
the lesser it moves, (c) size of the compound, the larger the compound the slower it moves up the plate.

The plate is removed after an optimal development time and dried and the spots/zones are detected using a suitable
location reagent. An important characteristic used in thin layer chromatography is Rf value.

Since amino acids are

colourless compounds, ninhydrin
is used for detecting them. To identify this, after development, the TLC plate is sprayed with ninhydrin reagent and
dried in an oven, at 105C for about 5 minutes. Ninhydrin reacts with - amino acids that results in purple coloured
spots [ due to the formation of the complex - Rheuman's purple. Rf values can be calculated and compared with the
reference values to identify the amino acids. [The Rf value for each known compound should remain the same
provided the development of plate is done with the same solvent, type of TLC plates, method of spotting and in
exactly the same conditions].
Reagents:

1.
2.
3.

2% solution of individual amino acids.

Solvent mixture of normal butanol, acetic acid and water in the ratio 12:3:5 by volume.
Ninhydrin reagent.

Requirements:

1.
2.
3.
4.
5.
6.

TLC plate.
TLC chamber.
Capillary tubes.
Reagent spray bottle.
Conical flasks.
Beakers.

Procedure:

1.
2.
3.
4.
5.
6.
7.
8.

9.
10.
11.
12.
13.

Pour the solvent mixture in to the TLC chamber and close the chamber.
The chamber should not be disturbed for about 30 minutes so that the atmosphere in the jar becomes saturated
with the solvent.
Cut the plate to the correct size and using a pencil (never ever use a pen) gently draw a straight line across the
plate approximately 2 cm from the bottom.
Using a capillary tube, a minute drop of amino acid is spotted on the line.
Allow the spot to dry.
Spot the second amino acid on the plate [enough space should be provided between the spots].
Repeat the above step for spotting the unknown acid.
Place the plate in the TLC chamber as evenly as possible and lean it against the side(immerse the plate such
that the line is above the solvent). Allow capillary action to draw the solvent up the plate until it is approximately 1 cm
from the end.
Remove the plate and immediately draw a pencil line across the solvent top.
Under a hood dry the plate with the aid of a blow dryer.
Spray the dry plate with ninhydrin reagent.
Dry the plates in hot air oven at 105C for 5 min. [Ninhydrin will react with the faded spots of amino acids
and make them visible as purple coloured spots.]
After some time, mark the center of the spots, then measure the distance of the center of the spots from the
origin and calculate the Rf values.

Rf value can be calculated using the formula:

The Rf values with butanol-acetic acid- water solvent are as follows: alanine 0.24, glutamic acid 0.25, glycine 0.2,
leucine 0.58, valine 0.4, lysine 0.58, tyrosine 0.42.

EXPERIMENT NO. 10
Objective: Isoelectric precipitation of protein: Casein from milk.
Milk is a mixture of many types of proteins, most of them present in very small amounts. Milk proteins are classified
into three main groups of proteins on the basis of their widely different behaviors and forms of existence. They are
caseins (80%), whey proteins and minor proteins. Casein is a heterogeneous mixture of phosphorous containing
proteins in milk. Casein is present in milk as calcium salt and calcium caseinate. It is a mixture of alpha, beta and
kappa caseins to form a cluster called micelle. These micelles are responsible for the white opaque appearance of
milk.
Casein, like proteins, are made up of many hundreds of individual amino acids. Each may have a positive or a
negative charge, depending on the pH of the [milk] system. At some pH value, all the positive charges and all the
negative charges on the [casein] protein will be in balance, so that the net charge on the protein will be zero.

That pH value is known as the isoelectric point (IEP) of the protein and is generally the pH at which the protein is
least soluble. For casein, the IEP is approximately 4.6 and it is the pH value at which acid casein is precipitated. In
milk, which has a pH of about 6.6, the casein micelles have a net negative charge and are quite stable. During the
addition of acid to milk, the negative charges on the outer surface of the micelle are neutralized (the phosphate groups
are protonated), and the neutral protein precipitates.

The same principle applies when milk is fermented to curd. The lactic acid bacillus produces lactic acid as the major
metabolic end-product of carbohydrate [lactose in milk] fermentation. The lactic acid production lowers the the pH
of milk to the IEP of casein. At this pH, casein precipitates.

Materials required:

1) Raw milk - 100ml

2) 0.2N HCl - 50ml
3) Diethyl ether - 50ml
4) 50% Ethanol - 50ml
5) Whatman No 1 filter paper strip (Size 2550mm) - 2 no.

Procedure:

1.

Measure 100ml of milk in a measuring cylinder and transfer 25ml of milk to four Oakridge centrifuge tubes
each.

2.
3.
4.
5.
6.
7.
8.
9.

Centrifuge the milk in a centrifuge at 4000rpm at room temperature (25- 30o C) for 20 minutes. This is done
to remove the fats and lipids from the mixture.
After centrifugation, carefully remove the fats and lipids from the surface of the milk with a spatula.
Then transfer the milk from all the tubes into a beaker and add equal volume of distilled water and stir well.
Now check the pH.
Start adding 0.2N HCl drop by drop into the milk mixture and stir well.
Note the PH at which precipitation (white curdy substances) appears. The pH should be 4.6.
Take the curdy precipitate and allow it to sediment.
Now decant the supernatant using a filter paper and funnel and wash the precipitate with distilled water to
remove the salts, then wash with diethyl ether and ethanol.
Dry the precipitate and take the weight of the casein and record it.

EXPERIMENT NO.11
Objective: Fermentation of simple carbohydrate.
A metabolic process performed by almost all types of bacteria is known as fermentation. This will result in the
production of ATP, the ultimate energy source of the organism. This will happen either in the presence or absence
of atmospheric oxygen. Bacteria utilize the nutrients in their environment to produce ATP for their biological
processes such as growth and reproduction. The enzyme systems in bacteria allow them to oxidize environmental
nutrient sources. Bacteria will use different energy sources in the medium depends on the specific enzymes of
each bacteria. Many bacteria possess the enzymes system required for the oxidation and utilization of the simple
sugar, glucose. Some bacteria have the ability to degrade complex carbohydrates like lactose, sucrose or even
polysaccharides. Such bacterium should possess the enzymes that should cleave the glycosidic bonds between the
sugar units and the resulting simple carbohydrate can be transported into the cell. Lactose is a disaccharide

consisting of the glucose and galactose connected by glycosidic bond. The bacteria which produce the enzyme
lactase will break this bond and thus release free glucose that can be easily utilized by the organism. The
characteristics feature of the enzyme production in the bacteria enables them to use diverse carbohydrates and
this will aid in the identification of unknown bacteria. Fermentation is best described by the degradation of glucose
by Embden- Meyerhof pathway or Glycolytic pathway.

The sugar fermentation pattern may be unique to a particular species or strain (Figure 2).

Phenol Red Carbohydrate Fermentation Broth:

Phenol red broth is a general purpose fermentation media comprising of trypticase, sodium chloride, phenol red
and a carbohydrate. The trypticase provides amino acids, vitamins, minerals and other nitrogenous substances
making it a nutritious medium for a variety of organisms. Sodium chloride helps in maintaining the osmotic
balance and provides the essential electrolytes for the transport into the cell while the carbohydrate acts as the
energy source. The phenol red is the pH indicator and is initially neutral (pH 7). It supports the growth of most
organisms whether they are able to ferment sugar or not. When the bacterium is inoculated into the tube, the
bacterium which ferments the sugar will result in the production of acid that will change the color of phenol red.
Fermentation reactions often begin with glycolysis. Glucose acts as an electron donor in the fermentation reaction,
pyruvate, and metabolic product of glucose act as an electron acceptor. The other disaccharides and
polysaccharides are hydrolyzed into glucose or converted into glucose and then the fermentation reaction will
occur. Finally the reaction will result in the end products such as acid, ethanol, Hydrogen and Carbon dioxide and
other compounds. This depends on the species of bacteria. Phenol red broth is a test is differential for gram
negative
bacteria.
When the organism ferments carbohydrates, acidic organic by products (Lactic acid, formic acid or acetic acid) is
accumulated which turns the medium into yellow color with reduction in the pH (acidic). The inverted Durham
tubes will detect the presence of gas. The degradation of peptones in the broth may result in the production of
alkaline end products, which will change the broth color to pink often at the top of the tube.

Most bacteria produce energy (ATP) by one or more of three mechanisms: 1. Aerobic
respiration. 2. Anaerobic respiration. 3. Fermentation. Aerobic respiration is an oxidative
process which uses oxygen as a final electron acceptor. Anaerobic respiration is similar to
aerobic respiration, but it uses an inorganic molecule other than oxygen as the final electron
acceptor. Fermentation uses an organic molecule as a final electron acceptor. Aerobic
respiration produces 36-38 ATP per glucose molecule, and is the most efficient form of energy
production. Fermentation is the least efficient means of energy production; it produces only
two ATP per glucose molecule. Anaerobic respiration is more efficient than fermentation, but
less efficient than aerobic respiration. The ATP yield per glucose molecule varies, depending
on the final electron acceptor used.

Materials Required:

1.
2.
3.
4.

Phenol Red Carbohydrate Fermentation Broth.

Bacterial culture.
Inoculation loop.
Incubator(370 C).
Procedure:

I. Preparation of Carbohydrate Fermentation Broth

1.
2.
3.
4.
5.

6.

Weigh and dissolve trypticase, Sodium chloride, and Phenol red in 100 ml distilled water and transfer into
conical flasks.
Add 0.5% to 1% of desired carbohydrate into all flasks.
Insert inverted Durham tubes into all tubes, the Durham tubes should be fully filled with broth.
Sterilize at 1150 C for 15 minutes.
Important: Do not overheat the Phenol red Carbohydrate fermentation broth. The overheating will result in
breaking down of the molecules and form compounds with a characteristic color and flavour. The process is known as
caramelization of sugar (the browning of sugar).
Transfer the sugar into screw capped tubes or fermentation tubes and label properly.

Ingredients of the Fermentation Broth:

1.

Trypticase: 1g

2.
3.
4.

Carbohydrate: 0.5g
Sodium Chloride: 0.5g
Phenol red : 0.0189mg

*Autoclave at 115o C for 15 minutes

II. Inoculation of Bacterial Culture into the Phenol Red Carbohydrate Broth

1.

Aseptically inoculate each labeled carbohydrate broth with bacterial culture.(keep uninoculated tubes as
control tubes).
2.
Incubate the tubes at 18-24 hours at 37oC.
3.
Observe the reaction.
Precautions:

1.

After inoculation into a particular sugar, sterilize the loop in order to avoid cross contamination of the tube
with other sugars.
2.
Keep uninoculated sugar tubes as control tubes.
3.
Do not use the tubes with Durham tubes that are partially filled or with bubbles.
4.
Over incubation will help the bacteria to degrade proteins and will result give false positive results.

Expected Results:

Results of carbohydrate fermentation test

1.

Acid production: Changes the medium into yellow color- organism ferments the given carbohydrate and
produce organic acids there by reducing the ph of the medium into acidic.
2.
Acid and Gas production: Changes the medium into yellow color-organism ferments the given Carbohydrate
and produce organic acids and gas. Gas production can be etected by the presence of small bubbles in the inverted
durham tubes.
3.
Absence of fermentation: The broth retains the red color. The organism cannot utilize the carbohydrate but
the organism continues to grow in the medium using other energy sources in the medium.

EXPERIMENT NO.12
Objective: To quantify the amount of amino acids by using ninhydrin reaction.
Theory:
Amino acids are known as the building blocks of all proteins. There are 20 different amino acids commonly found in
proteins. Amino acids are comprised of a carboxyl group and an amino group attached to the same carbon atom (the
carbon).
They vary in size, structure, electric charge and solubility in water because of the variation in their side chains ( R
groups). Detection, quantification and identification of amino acids in any sample constitute important steps in the
study of proteins.
Alpha amino acids react with Ninhydrin involved in the development of color which is explained by the following
five steps.
1. alpha-amino acid + Ninhydrin ---> Reduced ninhydrin +Alpha amino acid +H2O
This is an oxidative deamination reaction that elicit two hydrogen from the alpha amino acid to produce an alpha
imino acid. Also the ninhydrin reduced and loses an oxygen atom with the formation of water molecule.
2.

alpha-amino acid + H2O ---> alpha-keto acid +NH3

The rapid hydrolysis of NH group in the alpha imino acid will cause the formation of an alpha- keto acid with an
ammonia molecule. This alpha-keto acid further involved in the decarboxylation reaction of step.
3.

alpha-keto acid + NH3 ---> aldehyde + CO2

Under a heated condition to form an aldehyde that has one less carbon atom than the original amino acid.
A carbon dioxide molecule is produced along with aldehyde. These first three steps produce the reduced
ninhydrin and ammonia that are required for the production of color .The overall reaction for the above reactions
is simply explained in Reaction (4) as follows:
4.

alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BLUE) + 3H2O

In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. It is this purple color
that is detected in this method. Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group
is present in all amino acids, proteins or peptides. Whereas, the decarboxylation reaction will proceed for a free
amino acid, it will not happen for peptides and proteins. Theoretically only amino acids produce color with
ninhydrin reagent. However, one should always check out the possible interference from peptides and proteins
by performing blank tests especially when such solutions are readily available. For example, one can simply

add the ninhydrin reagent to a solution of only proteins and see if there is any color development. There is
no excuse for failing to perform such a vital test when the sample mixture contains both proteins and amino
acids. There are also reports that chemical compounds other than amino acids also respond positively to this
reaction.
The ninhydrin reaction, one of the most important method of detecting amino acids, both technically and historically,
has been conventionally used to detect their microgram amounts. When amino acids with a free alpha amino groups
are treated with an excess of ninhydrin, they yield a purple colored product. Under appropriate conditions, the color
intensity produced is proportional to the amino acid concentration.

Several other convenient reagents are available which can react with the alpha amino group to form colored or
fluorescent derivatives. These include fluorescamine, dansyl chloride, dabsyl chloride, etc., used in the detection of
trace amounts of amino acids at the nanogram level.
In the quantitative estimation of amino acid using Ninhydrin reagent, the absorbance of the Ruhemann's purple formed
by the reaction at 570nm is measured.
Reagents Required:
1.
2.
3.
4.
5.

Standard amino acid stock solution (150 micrograms of Standard amino acid stock solution (150g/ml)).
0.2M Acetate buffer (pH=5.5).
8% w/v of Ninhydrin reagent [ Preparation: Weigh 8g of ninhydrin and dissolve in 100ml of acetone].
50% v/v ethanol.
Distilled water.

Apparatus and Glasswares Required:

1.
2.
3.

Test / Boiling tubes.

Pipettes [glass / micropipette].
Waterbath.
Colorimeter.

4.

Procedure:
1.

Pipette out different volumes(0.1ml-1ml) of standard amino acid solution to the respective labelled test
tubes.

2.
3.
4.

Add distilled water in all the test tubes to make up the volume to 4ml.
Add 4ml of distilled water to the test tube labelled Blank.
Now add 1ml of ninhydrin reagent to all the test tubes including the test tubes labelled 'blank' and
'unknown'.
5.
Mix the contents of the tubes by vortexing /shaking the tubes.
6.
Put a few marble chips in each tube.
7.
Cover the mouth of the tubes with aluminium foil.
8.
Place all the test tubes in boiling water bath for 15 minutes.
9.
Cool the test tubes in cold water and add 1ml of ethanol to each test tube and mix well.
10.
Now record the absorbance at 570 nm.