Yashwantrao Chavan Maharashtra
Open University
Post Graduate Degree Programme (Bio-Technology)
SBT075: Lab Course M. Sc. (Bio-Technology)
Lab
Workbook
Yashwantrao Chavan Maharashtra Open University
Vice-Chancellor-Dr. Rajan Welukar
Expert Advisory Committee
Mr. Manoj Killedar
Director, School of Science & Technology, Y.C.M. Open University, Nashik
Mrs. Sunanda More
School of Science & Technology, Y.C.M. Open University, Nashik
Mrs. Chetana Kamlaskar
School of Science & Technology, Y.C.M. Open University, Nashik
Dr. Sunil Ganatra
135, Krushnakunj, Toata Colony, Lakadganj, Nagpur
Prof. Indira Ghosh
Bio-Informatics Center, University of Pune, Pune
Prof. Urmila Kulkarni-Kale
Bio-Informatics Center, University of Pune, Pune
Prof. Dr. Piyali Kar
Maharashtra Education Foundation, Foundation Towers, Sector – 11/20, CBD Belapur,
Navi Mumbai
Course Writer
Mr. Pravinkumar Domade
G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345, Kingsway
Nagpur
Course Editor
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G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345, Kingsway
Nagpur
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School of Science & Technology, Y.C.M. Open University, Nashik
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2
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3
List of Experiments of Semester 07 Biotechnology Lab
S.
No.
1
2
3
4
5
6
7
10
11
12
13
14
15
16
17
1
1 21
22
23
24
Name of the Experiments
Page
No.
Laboratory Rules: Basic Rules of a Microbiology Laboratory
Preparation of solution of given Molarity ,Molality and Normality
Preparation of buffer of specified pH using pH meter
Determination of isoelectric PH of Protein
The detection of changes in the conformation of bovine serum albumin
(BSA) by viscosity measurement and the effect of PH on the conformation of
bovine serum albumin.
Estimation of sugar by DNSA method
Protein estimation by Lowry/Biuret method
Separation of amino acid by circular paper chromatography
Separation of amino acid by Paper Electrophoresis
The estimation of DNA by the diphenylamine reaction
a ) The estimation of DNA by the diphenylamine reaction
b ) Estimation of RNA by Orcinol reagent.
Fractionation of Protein – salt precipitation, solvent precipitation, isoelectric precipitation
a) Demonstration of starch hydrolysis by given bacterial culture
b) Demonstration of amylase production by Aspergillus niger
c) Demonstration of Protein (Gelatin) hydrolysis
d) Demonstration of Fat hydrolysis (lipase activity) by a bacterial culture
e) Demonstration of Uease Production i.e. urea Hydrolysis
a) To estimate the activity of α- amylase
To study the effect of pH on activity of α amylase.
To study the effect of temperature on activity of enzyme (α amylase)
Effect of inhibitor on enzyme activity
To determine the effect of substrate concentration on enzyme activity
Evaluation of KINETIC CONSTANT [Km and Vmax] of purified enzymes
Separation of green plant pigments by column chromatography
To calculate the mean ,median ,mode of the given problems
To find out whether the dihybrid ratio is good to fit or not in the following
two crosses from the data given using χ2 test
A bag contains 10 pink, 10 yellow, 10 orange and 20 red beads
I.
What is probability of drawing 1 yellow bead.
II.
What is probability of drawing 1 yellow and 1 red bead.
Calculate standard error (sampling error) from the observation obtained by
drawing samples randomly for 25 times of sizes 5 beads at a time from
population for getting pink beads.
4
BASIC RULES OF A MICROBIOLOGY LABORATORY
A microbiology laboratory is a place for working with a variety of microorganisms. Since
several culture media are prepared and organic materials are present, the chances exist for the
presence of high spectrum of microbial community. Secondly, while working with pure culture
one should always follow the microbiological rules so that neither the experiment should be
unsuccessful nor any hazard may occur. If a large number of students are working in a
microbiology laboratory, they should be aware what to do or what not? What are the
apparatus/instruments/equipments present in the laboratory and what is their functioning? What
are the chemical solutions and stains and how to handle? How should the students enter in the
microbiology laboratory and how should they work? Therefore, the freshers such as students,
teachers, laboratory assistants and helper must follow the following guidelines:
1) Always wear an apron (a white coat or gown) before entering the microbiology laboratory
to protect from microbial contamination and laboratory hazards. At regular intervals get the
apron washed.
2) Cut nails regularly.
3) Tie long hairs back to avoid contamination and fire hazard.
4) Keep your working laboratory bench clean of everything. Nothing should be laying on the
bench.
5) Never keep books, purses, bags, etc. on the working bench.
6) Always wash your hands with soap in running tap water before and after the work.
7) Clean your working bench with ethanol (70%) or phenol (1:100).
8) Never spit and smoke in the laboratory.
9) Don’t put anything of the laboratory(e.g. pencil, thread, labels, inoculation needle, pins,
etc) in your mouth, ears, nose and eyes.
10) Don’t put your fingers in your eyes, ears, mouth. It may facilitate the chance of infection by
pathogenic microorganisms.
11) Don’t eat or drink or talk while working with microorganisms.
12) Don’t mishandle the chemical solution, stains, spirit lamp, UV light, instruments/apparatus
or electricity.
13) Always keep the burner at distance from the organic solvents. Your sincere care will avoid
fire accident. The burner must be turned off soon after the use.
14) Always maintain aseptic condition while working with microorganisms.
15) Always use flame sterilised inoculation needle/loop.
16) Don’t open the culture tubes/plates directly and never inhale them nor observe with naked
eyes.
17) Open the culture tubes/plates near the vicinity of flame of the burner.
18) While working with broth culture don’t suck the suspension with mouth. Always use
pipette sucker.
19) After completion of work always label the cultures with names, code and date of work. It
will help recording the data.
20) Always keep plates in tiers and culture tubes in upright in basket or racks. Finally, transfer
all the culture in the incubator at desired temperature of where ever to keep.
21) Never leave your cultures on working table or seat.
22) Clean the working table/bench when the work is completed.
23) Clean lenses of objective with tissue paper.
24) Keep the stains, reagents, stock cultures to their respective places when the work is
completed.
25) After completion of work keep your slides/pipette/culture tubes/plates in container and
steam sterilize before washing.
26) Record your result at time.
27) For any difficulty, ask your laboratory assistant or concerned teachers.
5
Experiment No: 1
Aim : Preparation of solution of given Molarity, Molality and Normality.
Theory and Principle:
Normality : The number of gram equivalent of the solute present per liter of its solution .
N = Weight in gram of solute/ litre of solution
Equivalent weight in gram.
Or = equivalent weight/ 1000.
Molarity : gram molecular weight of solute per litre of solution .
M = Molecular weight of solute
1000ml of solvent .
Molality : It represent the number of moles of solute present in 1000g of solvent .
1m = Molecular weight of compound
1000g of solvent .
Procedure :
1. Weigh the required amount of solute.
2. Dissolve in requived amount of solvent .
A. Prepare 0.1N, 1M, 0.2M Solution of NaOH.
Molecular weight of NaOH = 23+16+1 = 40
Equivalent weight of NaOH =
molecular wt.
No.of replacable H/OH
= 40
a. To prepare 0.1 N solution of NaoH .
To prepare 1N solution: Dissolve 40g NaOH in 1000 ml of solvent .
0.1N NaOH 4g of NaOH in 1000 ml of solvent (Distilled Water)
Molarity = Molecular weight / 1000ml of solvent .
For 1M of NaoH : Dissolve 40g NaoH in 1000ml of solvent (D.W.)
Molality = molecular weight of compound
100g of solvent
For water; volume = mass/ density
= 1000ml = mass of water
1g/ml
=1000g = mass of water
For 1M of NaoH = 40g of NaoH dissolved in 1000ml of water
There fore, 0.2M Of NaoH= 8g of NaoH dissolved in 1000ml of water
B. To prepare 0.5N, 2M, 0.4M of HCL in 1000ml of solvent
Molecular Weight of Hcl = 1+35.5=36.5
For Hcl specific gravity = 1.18
Volume = Mass/specific gravity
=36/1.18
6
Therefore for 1N Hcl
For 0.5N Hcl
For 1M Hcl
For 2M Hcl
= 30.51ml
= Dissolve 30.51ml of Hcl in 1000ml of solvent(D.W.)
= 15.25ml Of Hcl in 1000ml of solvent (D.W.)
=Dissolve 30.51ml of Hcl in 1000ml of solvent(D.W.)
=Dissolve 61.02ml of Hcl in 1000ml of Solvent(D.W.)
Result:
7
Experiment No: 2
Aim :- Preparation of buffer of specified pH using pH meter.
Theory:- Buffers are defined as substances that resist changes in the pH of the system. Weak
acids or bases, in the presence of their salts with strong bases or strong acids respectively form
buffer system.
Ex: Phosphate/monohydrogen phosphate.
Carbonic acid/bicarbonate
Proteins/proteinate.
pH:- The pH of the solution is the value with which defines its hydrogen ion
concentration in aqueous solution, it is relative strength of hydrogen ion reach species called acid
and the hydrogen ion deficient species called base which determines the net pH of a solution.
Measurement of pH :pH indicators:- These are usually organic compounds of natural or synthetic origin whose
colour is dependent on the pH of the solution. Indicators are dependent on the pH of the solution.
Indicators are usually weak acids which dissociates in solution.
pH meter:- The most reliable and accurate method for the routine measurement of pH is the pH
meter in which a change in pH is measured as change in electrical potential. If a metal rod is
placed in solution of its salt, it acquires potential. If two dissimilar metals are dipped into the
solution of their salts, the difference in potential can be measured or calculated from the two
separate potentials. the standard electrode is thus required against which the potential of other
electrodes can be compared. this is the “standard hydrogen electrode” , consisting of platinum rod
dipped in the aqueous solution with a given H+ activity in which hydrogen gas is bubbled
continuously at 1 atmospheric pressure. But this is too cumbersome to be used, as reference
electrode for routine use, other secondary reference electrode of known potential in relation to
standard hydrogen electrode are used.
Example:- Calomel electrode , glass electrode.
Precautions :1.The glass electrode is fragile and must be handled with care.
2.Electrode must not be left to dry.
3.The temperature compensation dial must be set before it is calibrated as potential is produced
dependent on temperature.
4.The meter must be calibrated first with a standard buffer of pH. 7 and then with pH. 4 or pH. 9.
Procedure :1. Prepare the solution of given molarity as per the table.
2. Do the addition for specific buffer as per the table.
3. Make up the volume up to 1000ml.
4. Check the pH with the help of pH meter.
Observation Table:S.NO.
1.
2.
3.
4.
5.
pH
2
4
6
9
10
SOLUTION TO BE USED
0.2M KCl + 0.2M HCl
0.2M succinic acid + 0.2M NaOH
0.2M succinic acid + 0.2M NaOH
0.1M KCl + 0.1M H3BO3
1M NaHCO3 + 1M Na2CO3
VOLUME IN ml
65ml + 250ml
250ml + 100ml
250ml + 435ML
250ml+250ml+208ml
13.8ml + 12ml
Result:- The buffer solution of specific pH were prepared with the help of pH meter.
8
Experiment No: 3
Aim: Determination of isoelectric PH of Protein.
Principle: The PH at which a Protein is least soluble is it’s isoelectric pH , defined as the pH at
which the molecule has no net electric charge and fails to move in an electric field. Under these
condition there is no electrostatic repulsion between neighbouring protein molecules, and they
tend to coalesce and precipitate. However, at pH values above the isoelectric point all the protein
molecules have a net charge of the same sign. They therefore repel each other, preventing
coalescence of single molecules into insoluble aggregates. Some protein are virtually insoluble at
their isoelectric pH.
Since different proteins have different isoelectic pH values, because their content of
amino acids with ionizable R groups differs, they can often be separated from each other by
isoelectric precipitation.
Materials and reagents
1) 0.005N HCl : take 83 ul of HCl and make up the volume to 200 ml with distilled water.
2) Skimmed Milk or Casein : suspend 10 gm of Skimmed Milk in 200 ml distilled water
3) Albumin Solution : Dissolve egg white from 2 egg in 200 ml distilled water.
Procedure: Carry out the experiment as per the protocols given below in the table-1 and table-2
Table-1
Test tubes 1
2
3
4
5
6
Reagents
Skimmed 15
15
15
15
15
15
Milk
0.005N
1
2
3
4
5
6
HCl
Stand for 30 minutes and determine PH of each tube
7
8
9
10
15
15
15
15
7
8
9
10
8
9
10
15
8
15
9
15
10
Table-2
Test tubes 1
2
3
4
5
6
7
Reagents
Albumin
15
15
15
15
15
15
15
0.005N
1
2
3
4
5
6
7
HCl
Keep the tubes in water bath at 50oC and determine the PH
Result : The isoelectric PH of the given protein solution has been found to be---------
9
Experiment No: 4
Aim: The detection of changes in the conformation of bovine serum albumin (BSA) by
viscosity measurement and the effect of PH on the conformation of bovine serum albumin.
Principle: high concentration of urea causes unfolding ie denaturation of proteins by weakening
the hydrophobic bonds that maintain the tertiary structure. This change in the protein
conformation leads to a less compact molecule with a larger viscosity than the native protein.
Such changes in the tertiary structure can be readily followed using an Ostwald viscometer (see
figure),. This , essentially consist of a capillary tube down which a known volume of protein
solution is allowed to flow under gravity. The time taken for this flow is measured (t1) and also
that of the solvent (t0); the relative viscosity is then given by.
ηrel = η1/η0 = (t1/t2) x (ρ1/ρ0)
where η1 is the viscosity of the protein solution of density ρ1 and η0 the viscosity of the solvent of
density ρ0. If the densities are taken to be the same then the expression simplifies to
ηrel = t1 / t0
Einstein has shown that, for spherical molecules, the relative viscosity is related to the
concentration of the molecule ( c ) and the partial specific volume (V ), which is the volume
occupied by the molecule and its bound water:
ηrel = 1+ 2.5cV
Materials
Viscosity is very sensitive to temperature, so all solutions an the viscometer must be kept at 30oC
in the water bath.
1. Ostwald viscometer
2. water bath at 30oC
3. Potssium Chloride (100mmol/liter)
4. Urea solutions (0.5,1,2,34,6, and 8 mol/liter in 100 mmol/liter KCl)
5. Bovine Serum Albumin (10g/liter in 100mmol/ liter KCl and the above urea solutions)
6. stop watch accurate to at least 0.1s
Method
Always use the viscometer by one limb only and never squeeze the two arms together. Rinse the
viscometer with KCl solution and place it in position
in water bath by carefully clamping one limb. Check
that it is vertically using the plumbline and introduce
exactly 20 ml ( or the volume marked on the
viscometer) of KCl solution at 30oC in the bulb A
with a syringe or Pipette. Leave for 5 minute to
equilibrate, then either apply positive pressure to the
wide limb (I) or gentle suction to the other limb (II)
until the meniscus rises to upper graduation mark B.
release the pressure and measure the time ( to the
nearest 0.1s) for the liquid to flow between the two
graduation marks B and C. Repeat the experiment
until the flow time agree within 0.2s and calculate
the average flow time. Repeat the whole procedure
10
with the urea solution alone (t0), which are the solvents, and then with bovine serum albumin
dissolved in the urea (t1). Plot the values of t0 and t1 against the concentration of urea and join up
the points with smooth curves. Select convenient concentration of urea and calculate the relative
viscosities (t1/t0) using the values from the curves. This ensures that any slight errors involved in
the determination of t1 and t0 are not magnified on taking the ratios. Finally prepare the graphs of
the relative viscosity against the concentration of urea and comment on the results. In additions ,
calculate the partial specific volume of serum albumin in 10 mmol/literKCl and in 8 mol/ liter
urea. Assume that the molecule remains spherical so that Einstein’s equation is valid.
For the determination of PH on the conformation of bovine serum albumin
Material required as above experiment and PH meter
Method
Using the ostwald viscometer, follow the structural change in albumin dissolved in 100
mmol/KCl and distilled water as the PH is varied over the range 2-12. Comment on the results
11
Experiment No: 5
Aim: Estimation of sugar by DNSA method
Principle: This method tests for the presence of free carbonyl group (c=o), the so called
reducing sugar. This involves the oxidation of the aldehyde functional group present;for
example; in glucose and the ketone functional group in fructose. Simultaneously 3,5
dinitrosalicylic acid (DNS) is reduced to 3amino, 5 nitrosalicylic acid under alkaline conditions.
The above reaction scheme shows that one mole of sugar will react with one mole of 3,5
dinitrosalicylic acid. Different reducing sugars generally yield different colour intensities ;thus it is necessary
to caliberate for each sugar. In addition to the oxidation of the carbonyl groups in the sugar, other side
reactions such as the decomposition of sugar also competes for the availability of 3,5 dinitrosalicylic acid.
Although this is a convenient and relatively inexpensive method, due to the relatively Low
specificity, one must run blanks diligently if the colorimetric result are to be interpreted correctly and
accurately.
When the effects of extraneous compounds are not known one can effectively include a socalled internal standered by first fully developing the color for the unknown sample, then a known amount of
sugar is added to this sample. The increase in the absorbance is equivalent to the incremental amount of sugar
added.
REQUIRMENTS:
1 Test tubes, Pipettes, Spectrophotometer
2 NaoH
3 DNSA
4 Standerd giucose solution
PROCEDURE:
1 Add 3ml of DNSA reagent to 3ml of glucose sample in a lighly capped test tube.(To avoid the
loss of liquid due to evaporation, cover it with paraffin film.)
2 Heat the mixture at 90ºC for 5-15min to develop the red brown colour.
3 Add 1ml of 40% potassium tartarate(Rochelle salt)solution to stabilize the colour.
4 After cooling at room temperature in a cold water bath, record the absorbance with a
spectrophotometer at 575 nm.
RESULT:
Concentration of unknown was found to be ___________mg/ml.
12
Experiment No: 6
Aim- Protein estimation by Lowry/Biuret method.
PrincipleThe principle of this method is based on the facts that the Folin-Ciocalteu regents reacts with
aromatic residues of proteins and yields blue color which in turn is read in colorimeter. The
different proteins contain different aromatic residues. Blue color develops because the alkaline
copper reacts with proteins; tyrosin and tryptophan present in protein reduce phosphomolybdate.
(present in Folin-Ciocalteu reagent)
Requirements1.1N NaOH Solution.
2.ALKALINE SODIUM CARBONATE Solution-Dissolve 20gm of Na2 CO3 in 100 ml of 0.1N
NaoH solution, prepare fresh.
3.COPPER SULPHATE-SODIUM POTASSIUM TARTRATE Solution- Dissolve 0.5g/lt of
CuSo4.5H2o in 1% OF sod-pot. tartrate solution, prepare fresh.
4.ALKALINE COPPER REAGENT- Mix 50ml of reagent 2 and 1ml of reagent 3 only on the
day of use.
5.FOLIN-CIOCALTEAU REAGENT-It is a solution of Sodium tungstate and Sodium molybdate
in phosphoric and hydrochloric acids (available commercially). Dilute the commercial reagent
with an equal volume of distilled water only on the day of use.
Procedurea) Take a test tube, transfer 1N NaoH solution and heat up to 100o C.
b) Suspend 1 ml of protein sample into the above solution for 4-5 minutes.
c) Add 5ml of reagent 4, mix properly and leave this mixture at room temp for 10 minutes.
d) Add 0.5 ml of Folin-Ciocaltue reagent rapidly with immediate mixing.
e) Leave it for 30 minutes; thereafter measure the absorbance of solution at750 nm in the
colorimeter.
PROTOCOLSr no Stock of protein (ml)
Distilled water (ml)
Concentration of protein
Alkaline Reagent
1
2
3
4
5
6
7
0.0
0.1
0.2
0.3
0.4
0.5
0.6
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0
0.02
0.04
0.06
0.08
0.10
0.12
5ml
5ml
5ml
5ml
5ml
5ml
5ml
Fcr
(ml)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
8
9
10
11
12
13
0.7
0.8
0.9
1.0
Unknown 1
Unknown 2
0.3
0.2
0.1
0.0
-
0.14
0.16
0.18
0.20
-
5ml
5ml
5ml
5ml
5ml
5ml
0.5
0.5
0.5
0.5
0.5
0.5
O.D
Result- Concentration of protein in unknown sample was found to be –
Unknown 1 – mg/ml
Unknown 2 – mg/ml
13
Experiment No: 7
Aim: - Separation of amino acid by circular paper chromatography.
Requirement: - Whattman filter paper, Capillary tube, Cotton wicks, Spray bottle.
Formula: - Rf = Distance travelled by solute /Distance travelled by solvent.
Principle: - Chromatography is sensitive technique used for rapid and efficient analysis and
separation of components of a mixture and purification of compounds.
This experiment is based on the partition chromatography principle i.e. if to a mixture of
two immiscible liquids A and B, a substance which is soluble in both A and B is added, then it
distributes itself in such a way that the ratio of its concentration in two liquid A and B is constant
at a particular temperature. Separation of different constituents takes place because each
constituent distribute itself to different degree between the solvent which flows down the column
and stationary liquid.
In paper chromatography paper is used as an inert support with one solvent as stationary
or immobile phase.
Radial paper chromatography shows radial development. This experiment is based on
differential migration of individual components of a mixture through a stationary phase.
The movement of mobile phase is due to capillary action and its flow is outward from a
central spot.
Solvent having flow of solvent varies inversely as its viscosities.
If the components to be separated are colourless then their presence is detected by
spraying a suitable solvent called developing reagent i.e. ‘Ninhydrin’ on the chromatogram, when
various components become visible, the process is known as development.
The ratio of distance the substance moves compared with the distance reached by solvent
both measured from the point of application of sample is known as the ‘Rf’ i.e. retention factor.
Procedure:1. In this technique a circular filter is employed and then various materials to be analyzed
are placed at the centre.
2. After drying the spot paper is placed horizontally on petridish possessing the solvent so
that the wick of paper dips into solvent.
3. Solvent rises through wick and moves sufficient distance so that components get
separated.
Result: - The Rf value of following amino acid was found to beGlutamic acid Tryptophan
Tyrosine
-
14
Experiment No:
Aim : The separation of amino acid by Paper Electrophoresis
Principle: the charge carried by the molecule depends on the PH of the medium and this is
illustrated in the case of three amino acids: Aspartic acid, Histidine and lysine. Electrophoresis at
low voltage is not usually used to separate low molecular weight compounds because of
diffusion, but it is easier to illustrate the relationship between charge and PH with amino acids
than with proteins or other macromolecules.
At PH 7.6, Histidine will carry zero net charge, Aspartic acid will be negatively charged and
lysine will be positively charged
Structures
These three amino acids can therefore be readily separated by electrophoresis. Glucose, an
uncharged molecule, is included in the mixture to check for any movement of the origin due to
electro-osmosis.
Materials
1 horizontal electrophoresis apparatus\
2 Power packs
3 amino acids (aspartic acid, histidine, lysine and a mixture of all three in tris-aetate buffer
containing 10 g/ liter glucose)
4 tris-acetate buffer(0.007 mol/liter, PH 7.6)
5 Ninhydrin location reagent (Dissolve 0.2 g I 100ml of acetone just before use )
6 Paper strips (10 cm x 2.5 cm)
7 Aniline-diphenylamine reagent
8 Citrate buffer (0.07 mol/liter, PH 3.0)
9 Oven at 110 oC
Method
Fill both parts of each electrode compatment with buffer solution to the same level: check this by
arranging the siphon between them. Remove the siphon, place five filter paper strips as shown,
and carefully apply a streak of the amino acid mixture to two of these , avoiding the edge of the
paper. Streak three other paper strips with three other paper strips with only one amino acid and
15
run all five electrophoresis strips together. Wet the paper from each electrode compartment to
within a few centimeters of the point of application, leave the rest to be wetted by capillary
attraction,, and immediately switch on the current . this way there is minimum spreading of the
sample. Carry out electrophoresis for 3h at 8 V/cm, remove the strips , and dry in an oven at
110oC.
Develop one of the strips containing mixture for glucose and dip the remaining four strips rapidly
in freshly prepared Ninhydrin Solution: allow the acetone to evaporate in the air and develop the
colours by heating in the oven for a few minutes. Identify the amino acids and check for any
electro-osmosis.
Repeat the experiment with 0.07 mol/liter citrate buffer, PH 3.0 .
16
Experiment No: 10 a
Aim : The estimation of DNA by the diphenylamine reaction
Principle : when DNA is treated with diphenylamine reaction under acid conditions, a blue
compound is formed with a sharp absorption maximum at 595 nm. This reaction is given by 2deoxypentoses in general and is not specific for DNA. In acid solution, the straight chain form of
a deoxypentose is converted to the highly reactive β-hydroxylevulinaldehyde that reacts with
diphenylamine to give blue complex. In DNA, only the deoxyribose of the purine nucleotides
reacts, so that the value obtained represents half of the total deoxyribose present.
Materials
1) DNA (commercial sample)
10 mg
2) RNA (commercial sample)
10 mg
3) pig spleen DNA solution
10 mg
4) yeast RNA solution
10 mg
5) buffered saline
500 ml
( 0.15 mol/liter NaCl: 0.015 mol/liteer sodium citrate, PH 7) 1 liter
6) diphenylamine reagent. ( dissolve 10g of pure diphenylamine in 1 liter of glacial acetic acid
and add 25 ml of concentrated sulphuric acid. This solution must be prepared fresh)
7) boiling water bath
Method
Dissolve 10 mg of nucleic acid in 50ml of buffered saline, remove 2 ml and add 4 ml of
diphenylamine reagent. Heat on a boiling water bath for 10 minutes, cool and read the extinction
at 595 nm. Read the test and standards against water blank. Assay the isolated nucleic acids and
the commercial samples for DNA.
17
Experiment No: 10 b
Aim: Estimation of RNA by Orcinol reagent.
Principle: It is a general reaction for pentoses and depends on the formation of furfural. When
the pentose is heated with concentrated HCl, the Orcinol reacts with furfural in the presence of
ferric chloride as a catalyst, giving a green colour. Only the purine nucleotide gives significant
reactions.
Requirements:
A. REAGENTS:
1. Orcinol reagent: Dissolve 1gm of Ferric chloride in 1litre of Concentrated HCl and add 35ml
of 6 %(w/v) Orcinol in absolute alcohol.{total volume 1litre/35ml}.
2. Standard RNA: Dissolve 10mg of RNA in 100ml of 10%TCA (10% TCA in distilled water)
B.OTHERS
Test tubes, Beaker, pipettes, burner, boiling waterbath etc.
Procedure:
1. Pipette out differently 0.2-1ml of Std RNA solution and make up the volume upto 2ml by
adding 10% TCA.
2. Use 2ml of TCA for the blank.
3. Add 3ml of Orcinol reagent in all the tubes, Mix well.
4. Boil the tubes in boiling water bath for 10 mins and cool it.
5. Take the OD at 665nm.
Observation:
Students are expected to write the protocol and observation of this experiment.
Result:
The concentration of the given unknown was found to be-------- mg/ml
.
18
Experiment No: 11
Aim : Fractionation of Protein – salt precipitation, solvent precipitation, iso-electric
precipitation
Salt Precipitation
Principle: the charges on a protein in solution can also be neutralized by the addition of salts and
this also has been used for purification of proteins. Theoretically any salt can be used but
generally ammonium sulphate is preferred for two reasons . one, it has a high solubility , 840
g/liter, and secondly , its dissolution in water is exothermic or the solution gets cooled.
Method : carry out the experiment given in the flow chart
Estimate the amount of protein in each fraction and calculate back to per gram tissue weight. Care
must be exercised to carry out the protein estimation, as ammonium sulphate will interfere with
the assay. Therefore, it is preferable to adopt the modified biuret method after TCA precipitation.
Alternatively, each fraction can be dialyzed to remove the salt and then estimation carried out.
Solvent precipitation
Another method of neutralizing the protein in solution and precipitating them out is by adding
solvents like acetone or alcohol.
Method : fractionate a tissue homogenate into different proteins by adding different amounts of
ice cold acetone ( 30%, 40% and 50%). Tabulate the results.
Isoelectric precipitation
Take about 10 ml of milk in a beaker. Slowly add 1N acetic acid in drops. At a particular stage a
sudden flocculent precipitation takes place. Measure the PH. It will be found to be about 4.5. the
major protein of milk is casein and its isoionic point is 4.5. At this PH, the net charge on the
molecule is zero.
Remember that proteins remain in the solution mainly because of the charges present on them
which makes them hydrophilic, once this charge is neutralized, the protein precipitate out. This is
how cured is prepared. The inoculum added contains lactobacilli which utilize the lactose of milk
to produce lactic acid. When the PH reaches 4.5, casein is precipitated out.
19
Experiment No: 12
Aim : Demonstration of starch hydrolysis by given bacterial culture
Principle : starch ( C6 H6 O5)n is an insoluble polymer of glucose which acts as a source of
carbon for microorganisms which have an ability to degrade them. Starch degrading
microorganism transport the degraded form across the cytoplasmic membrane of the cell. Some
bacteria posses the ability to produce amylase that breaks starch into maltose. The amylase is an
extracellular enzyme which is released from the cell of the microorganisms.
Requirements
1) Bacterial culture
2) Inoculation loop
3) Starch agar medium*
4) Petri dish
5) Iodine
*Starch Agar medium
starch
20 g
Beef extract
3 g
Peptone
5g
Agar
15 g
Distilled water
1 liter
Iodine solution ( as used in gram staing)
Method
a) Prepare starch agar plate and streak with suitable culture.
b) Allow the microbe to grow at 37oC for 48 hours.
c) Pour iodine solution in the plate.
d) The blue-black colour appear due to formation of starch-iodine comples. If the area
around streaked culture remain clear it indicates the degradation of starch has occurred
due to production of amylase.
Results
The starch and iodine make a complex of blue colour. Iodine does not react with maltose or with
any other product of starch degradation. Hence, no color is formed in such cases and clear area is
visible.
The given bacterial culture has been found to amylase positive
Or
The given bacterial culture has been found to be amylase negative.
20
Aim-
Demonstration of amylase production by Aspergillus niger
PrincipleStarch is broken into monomer units by the enzyme amylase produced by the organism.
However starch reacts with iodine and develops blue coloured complex. Moreover, intensity of
the colour developed is directly proportional to the concentration of starch present in the sample.
RequirementsGrowth Medium
Yeast extract
Malt extract
Peptone
- 0.5g
Soluble starch
Distilled water
pH = 6.0
- 0.3g
- 0.3g
- 1.0g
- 1000ml
Reagents
Iodine solution - 0.01N
Starch solution - 0.1%
Procedure1. Prepare growth medium, dispense 200 ml medium in flask and autoclave it.
2. Prepare pure culture of Aspergillus niger and prepare fresh culture in tubes.
3. Transfer 10ml of inoculum in sterilized growth medium and incubate at 30ºC for 24
to 48 hours.
4. Filter the filterate through sterile Whatman filter paper no.42. Collect supernatant and
measure amylase activity of filterate by starch iodine method.
5. Take different aliquots of starch solution ranging from 0-2ml and make initial
volume to 16ml by adding distilled water. Blank will lack starch.
6. Add 4ml of 0.01N iodine solution. Measure O.D after 10min of incubation at 578nm.
7. Draw graph between O.D and starch concentration.
ObservationStudents are expected to write the observation of this experiment, which will be based upon the
findings of the experiment.
Result-
21
Aim : Demonstration of Protein (Gelatin) hydrolysis
Principle: Gelatin is a polymer of amino acids and the protein is used as nitrogen and carbon
source for microorganisms. The gelatin or protein is generally broken down into peptides of short
amino acid polymers and amino acids can be transported into the cell. The enzyme that acts on or
degrade the protein is called protease. The property gelatin is to remain solid below 22oC, while
the degraded form of gelatin i.e. amino acids and peptides is to remain liquid below 22oC
Requirements
a) bacterial cultures
b) trichloroacetic acid
c) Nutrient gelatin broth/ agar
d) Ice flakes
e) Incubaors
Nutrient gelatin broth/ agar medium:
Gelatin
120 g
Beef extract
3g
Peptone
5g
Distilled water
1 liter
Agar ( if required)
15 g
Method
1) prepare gelatin agar medium and streak the plates with given bacterial cultures.
2) Incubate the streaked plates at 20 oC for about a month and check for gelatin liquefaction.
If gelatin is used as liquid. Inoculate and incubate the tubes at 35oC
3) After a month keep in ice to check liquefaction.
4) The third possibility is to use solidify medium and incubate the plates at 35oC
5) Flooded the plates with trichloroacetic acid
Result
Trichloracetic acid will precipitate the gelatin and plate will become opaque.
In the same way casein hydrolysis can be performed. Growth of protease producing bacteria
results in hydrolysis of casein by forming clear zone around the colonies.
22
Aim : Demonstration of Fat hydrolysis (lipase activity ) by a bacterial culture
Oil is used as substrate. Some microorganisms have the ability to hydrolyse fats and results into
rancidity in some food products. The ability of organisms to hydrolyse fat is accomplished due to
the enzyme lipase. Fat molecules are degrade resulting in glycerol and fatty acids. Lipases are one
of the most important classes of industrial enzymes. Many important biotechnological
applications have been explored in paper industry units, organic chemical processing and agrochemical industry. Lipases have been used in food, dairy , beverages, and detergent formulation.
Requriments
a) Bacillus species
b) Lipase producing broth*
c) Nutrient agar medium
d) Tween 80 (1%)
* lipase producing broth medium for Bacillus Species
NaCl
5g
CaCl2
0.05 g
Yeast extract
5g
Tween 80 (1%)
5ml
Distilled water
1 liter
PH
8.0
Method
1)
2)
3)
4)
5)
grow 6 hour old culture if Bacillus sp. On nutrient agar slants containing 1% Tween 80.
Add inoculum in flask containing lipase producing broth medium.
Incubate the flask for 15 hours at 50 oC under continuous shaking condition i.e. 200 rpm.
Centrifuge the contents at 1000 g for 20 minutes at 4 oC
Meaure enzyme activity in cell free supernatent using p-nitrophenol palmitate as
substrate by colorimetric assay.
Result
Express the result in terms of units ( U). one unit of activity is defined as the amount of enzyme
releasing 1mg of p-nitrophenol/minute.
23
Aim : Demonstration of Uease Production i.e. urea Hydrolysis
Principle: urea is the waste nitrogenous material and excreted out by mammals. Some bacteria
degrade the urea into ammonia and CO2. due to production of ammonia , the urease production
can be easily demonstrated by the following reaction:
NH2 – CO2 - NH2
2 NH3 + CO2
Requirements
a)
b)
c)
d)
Proteus vulgaris, E. coli and Serratia marcecsens
Urea broth medium*
Inoculation tube
Incubator
* Urea broth medium
urea
yeast extract
K2HPO4
K2HPO4
Phenol red
Distilled water
PH
20 g
0.1 g
9g
9.5 g
0.01 g
1 liter
6.8
Method
1) Incubate the urea broth medium with bacterial culture
2) Incubate the culture at 37oC for 48 hours.
3) The phenol red indicator will turn to pink due to alkaline nature of the medium of
ammonia production as seen in case of P. vulgaris. Otherwise, indicator will remain
yellow at acidic range of PH. then it shows no urease production by the given
microorganisms as in case of E. coli and serratia marcescens.
Results
P. vulgaris can be distinguished by E. Coli and S. marcescens by its ability to produce large
amount of the enzyme urease.
24
Experiment No: 12
Aim: - To estimate the activity of α- amylase.
Principle: - α -amylase E.C. No. 3.2.1.1 i.e α endo α 1,4 glucan hydrolase. It occurs widely in
bacteria and fungi. All α amylase are endo active enzymes which specially cleaves α 1,4
glycosidic linkages in amylose , amylopectin and glycogen yielding sugar in α configuration.
They are unable to hydrolyse α 1-6 branch points in amylopectin but are able to bypass this
branch point. Two types of microbial α amylase are existing i.e. saccharifying α amylase and
liquefying α amylase. They are distinguished by the fact that saccharifying α amylase produce a
increased quantity of reducing sugar about twice of liquefying α amylase.
Properties
Molecular wt.
Optimum pH
Optimum temperature
Metal ion req.
Km(milimoles)
Specificity
Substrate
Amylase,glycogen
amylopectin
Beta amylase
55,000 – 1,60,000
5.5 -7.5
37° -55°C
0.2
Alpha 1,4 limkages
cannot bypass alpha
1,6 linkages
Maltose and beta unit
dextrin
Amylase, glycogen,
amylopectin
Nature
Endosplitting
Exosplitting
Product
Αlpha amylase
50,000
3.5 -7
35° -90° C
Ca++
1
Alpha 1,4 linkages
can bypass alpha 1,6
linkages
Maltodextrin
Amyloglucosidase
50,000 – 1,12,000
4.0 -6.0
40° – 70°C
-18.5
Alpha 1,4 and alpha
1,6 linkages
Beta glucose, amylase
and amylopectin
glycogen,
amylopectin,dextrin
maltose.
Exosplitting
25
Assay: - α amylase are generally quantified by beta amylation of reducing group formed due to
hydrolysis of soluble starch. The simplest method is dinitrosalicyclic acid method. Starch iodine
colorimetric method is also used..
β amylase: - β amylase i.e. 1-4 β 1-4 glucanmalto hydrolase E.C. No. 3.2.1.2. It is a saccharifying
amylase i.e. widely distributed in plants and also in microorganisms. β amylase are exosplitting
enzymes which attacks amylase , amylopectin, glycogen at non reducing terminal resulting in
formation of maltose in β configuration, β amylases is specific for , α 1-4 linkages and is unable
to bypass α 1,6 branch point.
Assay: - Since the main product for β amylase hydrolysis are the disaccharides β maltose units , β
unit dextran, the reducing sugar assay described for alpha amylase may be employed.
Amyloglucosidase E.C. No. 3.2.1.3 also called glucoamylase. Amyloglucosidase are
exosplitting amylopectinamylopecic enzyme which attack amylase, amylopectin and glycogen are
specifically hydrolysed α 1, 4 and α 1, 6 linkages from non reducing end.
However α 1,6 glucosidic linkages are cleaved by less readily than α 1,4 glucosidic linkages.
The product of reacton is glucose.
Assay: - The major product of amylase action is glucose. The spectrometric assay for glucose
may be employed.
Procedure: - Take 0.5 ml of α amylase enzyme in a test tube and 1 ml of buffer and 0.5 ml of
starch. The glucose production in this tube is to be determined. Incubate these tubes for 10 min at
37 C then add 1 ml of DNSA reagent in it. Boil test tube for 10-20 min in water bath. A control
tube is also performed in which DNSA is added before addition of the starch so, as to deactivate
the enzyme. Arrange the test tubes as per protocol. After addition boil these test tubes for 10 – 15
min. Optical density of these tubes are to be read at 520 - 540 nm in colorimeter.
Protocol: Reagents
Starch
Buffer
Enzyme
NaOH
Enzyme
DNSA
C1 (ml) E1 (ml) C2 (ml) E2 (ml) C3 (ml)
2.5
2.5
3.5
3.5
4.5
3.5
3.5
2.5
2.5
1.5
1
1
INCUBATE FOR 30 MIN ONLY FOR EVP.
1
1
1
1
1
1
1
1
1
1
1
1
1
E3 (ml)
4.5
1.5
1
C4 (ml)
5.5
0.5
-
E4 (ml)
5.5
0.5
1.0
1
1
1
1
1
1
1
OBSERVATION:
1
2
3
4
5
6
7
8
9
10
OD(450nm)
Result: - The activity of α amylase was found to be
µg/ml from the graph.
26
Experiment No: 13
Aim: - To study the effect of pH on activity of α amylase.
Theory: - Enzyme is a protein, every protein exists in active state at an optimum pH If pH is less
than or greater than this optimum pH the enzyme will Undergo structural change which will
decrease its activity. Thus if graph is plotted, a bell shaped curve is obtained; showing pH
optimum at the top with gradual decrease of enzyme activity on either side.
Procedure :- 1) Take 7 clean and dry test tube and label them for control, 1-6.
To these add 1ml of buffered starch.
2) Now from 1-6 add different pH solution starting from 5.5-8
Respectively. 5ml of pH solution is added.
3) Now add 0.5ml enzyme solution in test tube from 1-6.
4) In the control add 5ml of any buffer and then add 0.5 ml enzyme
Add 0.5ml NaOH so that reaction is terminated.
5) Now keep for incubation at 300C for 20min.
6) Now add 0.5ml NaOH in the tubes labeled 1-6, then add 1ml of
DNSA solution in all the tubes and keep for boiling in water
Bath for 15min.
7) Take the O.D after cooling the tubes at 546nm.
Reagents: -
1) Phosphate buffer (0.1M) - Add 40ml 0.2N NaOH to 50ml of 0.2M
NaH2PO4 and dilute to 100ml.
2) Starch solution – 1% in phosphate buffer
3) Enzyme solution -1% in bufferd saline
4) DNSAreagent.
5) pH variation- prepare pH from 5.5-8 by the help of a ph meter.
The solutions used are 0.2M NaH2PO4 and 0.2M
Na2HPO4 is mole toward acidic and it has a pH
Around 4.5 .so with proper addition of discreat
pH of 5.5, 6, 6.5,7,7.5,8 is obtained.
Result: - As the pH increase, activity of enzyme also increase, it reaches the maximum pH 6
after that the activity gradually decrease.
27
Experiment No: 14
Aim: To study the effect of temperature on activity of enzyme (α amylase).
Principle: α-Amylase catalyses the hydrolysis of α-1, 4 linkage of starch with production of
reducing sugars .The reaction is followed by measuring the increase or decrease in the reducing
sugars using DNSA reagent. As the temperature increases the activity of the enzyme initially
increases, attains a maximum and then gradually decreases thus produces a bell shaped curve.
REAGENTS:
1. Buffered starch-1% in phosphate buffer
2. Enzyme solution -0.5% in buffered saline
3. DNSA reagent -5g DNSA reagent in 100ml of 2M NaOH. Add 250ml of 150g Na-K
tartarate . Make up the volume to 500ml with distilled water.
4. Phosphate buffer (0.1M): add 40ml 0.2 N NaOH to 50ml of 0.2 M NaH2 PO4. Mix well
make up the volume to 500ml with distilled water.
5. 0.9% NaCl
Procedure:
1. Take 7 clean and dry test tubes and label them as control and rest as 1 to 6.
2. To these tubes add 2.5ml starch solution followed by 1.5ml buffered saline.
3. Add 0.5 ml of 0.5 % enzyme solution to all the experimental tubes.
4. Prepare 6 water baths equilibrated at 5ºC, 15 ºC, 25 ºC, 35 ºC, 45 ºC, 55 ºC.
5. Incubate 1-6 tubes in respective water bath
6. Control can be incubated at room temperature or a separate control for each water bath
is prepared.
7. Incubate for 20mins and then add 0.5ml NaOH in each of the experimental tubes
8. In control enzyme is added only after the addition of NaOH.
9. Add DNSA to each tube including control
10. Boil for 15 mins and read at 540nm.
Observation:
TEMPERATURE
OPTICAL
DENSITY(540nm)
30 ºC
45 ºC
60 ºC
75 ºC
90 ºC
105 ºC
Result: As the temperature of the solution increases the activity of the enzyme also increases at
75 ºC it shows optimum activity then as the temperature further increases the activity declines.
28
Experiment No: 15
Aim:- Effect of inhibitor on enzyme activity.
Principle- Inhibitors are classified into reversible and irreversible .Reversible inhibition are
competitive or uncompetitive that can be effectively studied using bitterguard (karela).
TUBES C1
E1
C2
E2
C3
E3
C4
E4
O.D
1.COMPETITIVE INHIBITION –It is characterized by decrease in enzyme activity and
increase in Km value, Vmax remains unaltered.
Vo =Vmax[S]/Km(1+I/Ki) +[S]
2.NONCOMPETITIVE INHIBITION –It is characterized by decrease in Vmax , Km being the
same.
Vo=Vmax/(1+I/Ki)[S]/Km+[S]
3.UNCOMPETITIVE INHIBITION- It is characterized by modification of both Km and
Vmax.
Vo= Vmax/(1+Io/Ki)[S]/[So]+Km(1+Io/Ki)
REAGENTS- 1. Source of inhibitor:- a. Bitterguard pulp
b. Seed pulp
2.Phospate buffer:- Add 40ml 0.2N NaOH to 50ml 0.2 NaH2PO4 and dilute it to
100ml.
3. Starch solution .
4. Enzyme solution
5. DNSA
Procedure:- As per the protocol
REAGENT
EN YME
BUFFER
INHIBITOR
NaOH
C1(ml)
0.5
1.0
1
C2(ml)
0.5
0.8
0.2
1
C3(ml)
0.5
0.6
0.4
1
C4(ml)
0.5
0.4
0.6
1
E1(ml)
0.5
1.0
-
E2(ml)
0.5
0.8
0.2
-
E3(ml)
0.5
0.6
0.4
-
E4(ml)
0.5
0.4
0.4
-
Boil for 10 min. in waterbath and read at 540nm
STARCH
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Incubate for 15min. at 37°C .
NaOH
DNSA
1
1
Boil for 15min.in waterbath at 540nm.
Observation:Result:-
29
Experiment No: 16
Aim: To determine the effect of substrate concentration on enzyme activity.
Principle:- Amylase acts on starch to produce glucose. In this particular experiment, the amount
of starch i.e the substrate is kept constant where as the concentration .of amylase is taken in
increasing order. It is observed that the amount of glucose produced in every successive tubes
also increases. This is due to reason that as the enzyme concentration increases the number of
active site also increases. Thus substrate readily binds to enzyme thereby increasing the amount
of glucose produced.
Thus increase in concentration of enzyme increases activity of amylase. Care should be taken
that:1) Amount of substrate should be in excess, so that becomes rate-determining factor.
2) All other parameters like time, PH and temperature should be kept constant.
3) Tubes should be clean and dry to avoid contamination due to inhibition and dilution by
reagents.
Procedure:1)
2)
3)
4)
5)
6)
7)
8)
Take 7 clean and dry test tubes and label them, add 2.5 ml of starch solution.
Add gradually increasing concentration of enzyme solution in tube 1-6 as per protocol.
Incubate tubes for 30 mins.
After incubation add 0.2N NaoH to each tube.
Add 1ml of DNSA to each tubes.
Keep in boiling water bath for 15mins and cool it.
If required dilute it to 10ml with distilled water.
Read OD at 590nm.
Observation Table:C1/E1
C2/E2
C3/E3
C4/E4
Optical Density
Result:-
30
Experiment No: 17
Aim: Evaluation of KINETIC CONSTANT [Km and Vmax] of purified enzymes.
Principle: When all the other parameters including enzyme concentration, time and temperature
are kept constant and the initial substrate concentration is varied between wide limits, the changes
in the rate of enzyme-catalyzed reaction may be described in the following fig:
When more than one substrate are involved then, the concentration of second substrate
should also be kept constant. As the concentration of substrate increases the rate of reaction is
also increased upto a certain limit. This is apparent from the initial linear curve with further
increase in substrate concentration. Thus linearity does not persist as the increase in rate of
reaction gradually goes on decreasing, finally when the rate of reaction is not affected by increase
in substrate concentration the curve shows plateau.
The velocity indicating this splitting is called as Vmax. Km is substrate concentration at
half the Vmax.
REAGENTS:
1. Phosphate buffer
2. Enzyme
3. Starch solution
4. DNSA
5. Sodium hydroxide (NaOH)
31
Procedure: Arrange the test tubes and perform according to the protocol.
Sr
no.
Reagents
control 1
2
3
4
5
6
7
8
9
10
1
Enzyme
-
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
2
Phosphate
buffer
4
4.8
4.6
4.4
4.2
4.0
3.8
3.5
3.0
2.5
2.0
3
Starch
(1%)
1
0.2
0.4
0.6
0.8
1.0
1.2
1.5
2.0
2.5
3.0
INCUBATE FOR 20 MINS
4
NaOH
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5
Enzyme
0.5
-
-
-
-
-
-
-
-
-
-
6
DNSA
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Boil in water bath for 15 mins and cool .Take the OD at 450nm.
Observation:
1
2
3
4
5
6
7
8
9
10
OD(450nm)
Result: The value of Km was found to be
µg/ml.
32
Experiment No: 1
Aim:- Separation of green plant pigments by column chromatography.
Principle:- Column chromatography using alumina gel works on the basis of physical factor of
adsorption only and hence it is called as column chromatography. Adsorption is the phenomenon
whereby a substrate gets bound to the surface of unique particle. If the compound is in the
solution, particles insoluble in solvent then part of substrate is adsorbed and part remain in the
solution. The ratio between amount adsorbed and amount remained in the solution is constant
called adsorption coefficient. The extent of binding between solute and adsorbent depends on
charge, van der Waals' forces, dipole interaction, hydrogen bonding and steric forces and depends
upon the structure of compounds.
The mass of solute adsorbed per unit weight of adsorbent [m] depends on the
concentration of solute.
According to Langmuir equationm = K 1K 2c
1+K2c
Where, m = Mass of solute adsorbed per unit weight of adsorbent.
K1= Number of active adsorption sites per unit and depend upon nature of
Adsorbent.
K2= Affinity of solute for adsorbent and depends upon component of system.
Langmuir assumed only one binding site on the adsorbent. Hinshelwood equation for
number of different binding sites is as followsm = ∑ K 1K 2c
1+K2C
The molecule with least adsorbtivity goes along with the solvent and these are eluted
first followed by other component.
Materials :1) Chromatographic Column
2) Fresh Spinach Leaves
3) Alumina
4) Calcium Carbonate
5) Sodium Sulphate
6) Petroleum Ether
7) Methanol, Benzene, Waring Blender.
Procedure:Preparation of extract:
Homogenize 5 to 10gm of leaves in Waring blender adding 20 to 40gm mixture of
petroleum ether, methanol and benzene in 45:15:5 proportions. Filter the extract through
separating funnel. Add 10 to 20 ml of water, shake well and allow layer to separate. Remove the
lower layer containing methanol. Repeat the addition of water, shaking and removing the aqueous
layer 3 to 4 times till the lower layer becomes colorless and only faintly colored. Avoid the
vigorous shaking to avoid emulsion formation. Remove the last traces of water by adding
anhydrous sodium sulphate. Filter to remove the solid and concentrate the extract to few ml by
careful evaporation up to dryness. If presence of H2O is suspected, repeat the above steps.
II] Preparation of column:
33
Alumina was used as adsorbent. Column was filled with 5 cm alumina, 7cm
calcium carbonate, 7cm sucrose in petroleum ether. A small amount of glass wool is pushed
down to the bottom as a pad. About 2 to 5 g of adsorbent [previously dried at 120oC overnight for
activation] is taken in a beaker. A small amount of C6H6 added to slurry. The slurry is carefully
poured into a burette; use more benzene if necessary to pour the extra slurry. The adsorbent
should never be allowed to dry. After all the adsorbent has settled add 20ml of more benzene and
allow it to pass through the column. When the solvent level is just about 1cm above the solid
layer close the stop cork.
III] Separation of solution of pigment:
Take extract as prepared from (I) part in 1ml of benzene add quantitatively, transfer
it with a pipette to the top of the column taking care not to disturb the surface of the layer. Open
the stop cork and allow the solution to separate. When the level of the solvent has just reached the
surface of adsorbent, a small amount of benzene is added to develop the column.
It is likely that one or two yellow bands will appear and move down the column, but
most of the material will not remove from the top. This is because benzene is not polar enough to
release all adsorbed compound when 20ml of benzene has just been run then, add 5ml of
5%acetone in benzene from the top. Note the changes by continuously increasingly the acetone
concentration till only pure acetone is added. Collect the fraction by taking it in test tube below
the burette and plot the adsorption spectrum of each colored peak.
Note : The observation and result should be written as given below.
OBSERVATION:Three colored layers were observed.
Sr. no.
1.
2.
3.
Pigments
Chlorophyll
Xanthophylls
Carotenoids
Color of bands
Green
Yellow
Orange
RESULT: - The three colored layers of chlorophyll (green), xanthophylls (yellow) and
Carotenoids (orange) were separated by column chromatography.
34
Experiment No: 1 -21
Aim: To calculate the mean ,median ,mode of the given problems.
I.Calculate the arithmetic mean for following data.
Class Interval
10-20
20-30
30-40
40-50
50-60
60-70
70-80
80-90
90-100
frequency
2
7
17
29
29
10
3
2
1
Arithmatic mean :It is sum of all data divided by number of observation .
Arithmatic mean for
Ungrouped data
= X = X1+X2+X3+………Xn
n
∑ x =sum of all the value of observation
n = total number of observation .
Arithmatic mean
For grouped data
= X = ∑ fx
n
f =frequency i.e number of repeatition
II. Calculation mode of following data and median.
Age of students: 10, 12 15 ,20,20,22,22,15,25,15,14,19,20,15,15
MODE: It is the maximum frequency observation i.e the value which is repeated
Maximum times in the given data.
MEDIAN : When the data is arranged in ascending or descending order the
Median value is called median.
1. If observation are odd Median = [ n+1 / 2 ]th
2.If the observation are even Median = [ n /2 ]th +[n+2/2]th/2
Result:
From Calculation:
Mean is found to be - 48.40
Mode is found to be -15
Median is found to be -15
Note: The values given here are for better understanding of student but lecturer should give
to the student different values for practice.
35
Experiment No: 22
Aim: To find out whether the dihybrid ratio is good to fit or not in the following two crosses from
the data given using χ2 test.
Principle:
When stastical test is used to compare on observed ratio or value with an
Expected or theoretical ratio or value and determine how clearly the former fits into
latter. It is known as “Testing the goodness of fit’’ One measure commonly used
for it χ2 test.[Chi-squareTest]
χ
2
{ [Observed]
- [Expected] }2
Expected value
To accept or reject Null Hypothesis, this calculated χ 2 is compared with standard
Value . If the value of χ2 is less than that of table 0.05 probability , than Null
Hypothesis is accepted.
Di-hybrid Cross:
When the cross is made between two pair of contrasting character, it is said to
be dihybrid ratio RRYY and rryy.
When plants with the above character were crossed pollinated in F1 generation
they produced hybrid plan [Rr Yy] with round and yellow seed.
When these (F1) plants were allowed to self pollinate, progeny were formed
in (F2) generation in the ratio 9:3:3:1
36
Round Tellow
-
9
Round Green
-
3
Wrinkle Yellow Wrinkle Green
-
3
1
The Mendelian cross ratio is expected for every dihybrid cross [Null Hypothesis]
The ratio (F2) as cited above is actually based on Law of Independent Assotrment
Sometimes if more than 2 genes are located on same chromosome independent
assortment does not occur, instead they may be inherited together in a group.
this process is called linkage. The Mendelian ratio varies in such cases –
Alternate Hypothesis.
Result:
From the above observation, data obtained from cross I is supported statistically,
To be dihybrid cross [Accept Null Hypothesis]
While data obtained from cross II is supported statistically not be dihybrid
Cross [Accept Alternate Hypothesis]
Note: The values given here are for better understanding of student but lecturer should give
to the student different values for practice.
37
Experiment No: 23
Aim: A bag contains 10 pink,10 yellow,10 orange and 20 red beads .
III.
IV.
What is probability of drawing 1 yellow bead .
What is probability of drawing 1 yellow and 1 red bead.
Theory: If an experiment has an ‘m ’ mutually exclusive equally , likely and exhaustive
cases , out of which ‘m’ are favourable to the happening of the event ‘A’ , then the
probability of the happening of ‘A’ is denoted by P(A) and is defined as:-
P(A) = m
n
= no. of cases favorable to A
Total[exhaustive] no. of cases
Probability of an event which is certain to occur is one and the probability of an
impossible event is zero .
The probability of occurrence of any event lies between zero & one [0-1] , both
inclusive.
Procedure:
1. Withdraw a single bead at randomly from a mixture of 50 beads [yellow
+pink+red]
2.
3.
4.
5.
Record the observation of withdrawal of yellow bead.
Withdraw one yellow and one red bead at a time from a mixture of 50 beads and the
observation of it.
In the first case beads one withdrawn 50 times and in second case beads are withdrawn
25 times.
Calculate the probability.
Observation Table:Sr no.
Beads
1.
2.
Tally
Frequency
Probablity
Yellow
15
3/10
Yellow+ red
5
1/5
Calculations:1. The total number of equally , likely and exhaustive case = n = 10+10+10+20= 50
No. of favourable cases = m = 10
۟  probability of drawing = m = 10 = 1
n 50
5
38
1 yellow bead
Practically numbers of favourable case (m) =15
۟  probability of drawing of = m =15 = 3
n
30
10
1 yellow bead
2. The total no. of equally , likely an exhaustive case = n = 10+10+10+ 20 = 50
Number of favourable case (m1)
Probability of drawing one yellow and one red bead together = 10 * 20
50
50
Practically number of favourable cases m = 5
= 2
25
Result :
i)
The probability of drawing a yellow bead is theoretically 1/5 and practically it is 15/50
ii)
Probablity of drawing of yellow & red bead together is theoretically 2/25
practically 5/25.
and
Note: The values given here are for better understanding of student but lecturer should give
to the student different values for practice.
39
Experiment No: 24
Aim: Calculate standard error (sampling error) from the observation obtained by drawing
samples randomly for 25 times of sizes 5 beads at a time from population for getting pink
beads.
Theory:
Standard error = Standard deviation
√No of observations
Requirements: Beads of different colors essentially having pink colored beads
Procedure:
1. From the population of 50 beads, withdraw 5 beads randomly at a time.
2. Calculate the number of pink beads in each withdraw.
3. Withdraw the beads 25 times.
4. Calculate the mean for the observation of pink beads.
5. Calculate its mean deviation and then calculate standard error.
Observation Table:
Sr.no
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Sample drawn
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Number of pink beads
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
x- x¯
32
30
31
32
32
31
32
29
29
30
32
30
30
31
31
30
31
32
30
32
29
30
31
28
32
Where, x=32
∑ x- x¯ = 768
Result: Standard error was found to be σ = 4718.5.
Note: The values given here are for better understanding of student but lecturer should give
to the student different values for practice.
40
Equipments generally required in the Biotechnology laboratory
Micropipettes
Micropipettes kept in micropipette stand
Petriplates with different media
Binocular Microscope
41
Colorimeter
Laminar Air Flow
Waterbath
Spectrophotometer
Incubator
Spectrophotometer
42
Distilled Water Assembly
Single-pan manual balance
Mono-pan balance
Centrifuge
43
Autoclave
Distilled Water Assembly
Tipbox
Incubator Shaker
44