sbt075 lab manual

7/31/2019 SBT075 Lab Manual

1/44

Yashwantrao Chavan Maharashtra

Open University

Post Graduate Degree Programme (Bio-Technology)

SBT075: Lab Course M. Sc. (Bio-Technology)

Lab Workbook


2/44

2

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


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

Dr. Suchitra Godbole

G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345, Kingsway

Nagpur

Course Coordinator and IT Editor

Mrs. Sunanda More


E-Production

Manoj Killedar Director, School of Architecture, Science & Technology

E-Version available athttp://www.ycmou.com=>Architecture, Science and Technology=>DownloadDocuments=> SBT_LR

Yashwantrao Chavan Maharashtra Open University, Nashik

Printed & Published by: Shri. S.P. Kowale, Registrar, Y.C.M. Open University, Nashik


3/44

3

CERTIFICATE

This is to certify that Mr./Mrs./Smt..

has successfully completed experiment as per the following details:

(1) Total number of experiment completed: .

(2) Serial number of experiment completed:

Laboratory Instructor Name : ..

Signature & date : .

Programme co-ordinator Name: ..

Signature & date : .

External Examiner Name : ..

Signature & date : ...


4/44

4

List of Experiments of Semester 07 Biotechnology LabS.

No.

Name of the Experiments Page

No.

Laboratory Rules: Basic Rules of a Microbiology Laboratory

1 Preparation of solution of given Molarity ,Molality and Normality

2 Preparation of buffer of specified pH using pH meter

3 Determination of isoelectric PH

of Protein

4 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.

5 Estimation of sugar by DNSA method

6 Protein estimation by Lowry/Biuret method

7 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.10 Fractionation of Protein salt precipitation, solvent precipitation, iso-

electric precipitation

11 a) Demonstration of starch hydrolysis by given bacterial culture

b) Demonstration of amylase production byAspergillus nigerc) Demonstration of Protein (Gelatin) hydrolysis

d) Demonstration of Fat hydrolysis (lipase activity) by a bacterial culture

e) Demonstration of Uease Production i.e. urea Hydrolysis12 a) To estimate the activity of- amylase

13 To study the effect of pH on activity of amylase.14 To study the effect of temperature on activity of enzyme ( amylase)

15 Effect of inhibitor on enzyme activity

16 To determine the effect of substrate concentration on enzyme activity17 Evaluation of KINETIC CONSTANT [Km and Vmax] of purified enzymes

1 Separation of green plant pigments by column chromatography

1 -

21

To calculate the mean ,median ,mode of the given problems

22 To find out whether the dihybrid ratio is good to fit or not in the following

two crosses from the data given using 2 test

23 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.

24 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.


5/44

5

BASIC RULES OF A MICROBIOLOGY LABORATORY

A microbiology laboratory is a place for working with a variety of microorganisms. Sinceseveral culture media are prepared and organic materials are present, the chances exist for thepresence of high spectrum of microbial community. Secondly, while working with pure cultureone 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 amicrobiology laboratory, they should be aware what to do or what not? What are theapparatus/instruments/equipments present in the laboratory and what is their functioning? Whatare the chemical solutions and stains and how to handle? How should the students enter in themicrobiology 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 laboratoryto protect from microbial contamination and laboratory hazards. At regular intervals get theapron 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 thebench.

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) Dont put anything of the laboratory(e.g. pencil, thread, labels, inoculation needle, pins,

etc) in your mouth, ears, nose and eyes.10) Dont put your fingers in your eyes, ears, mouth. It may facilitate the chance of infection by

pathogenic microorganisms.11) Dont eat or drink or talk while working with microorganisms.12) Dont 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) Dont 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 dont 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, transferall 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.


6/44

6

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 solutionEquivalent weight in gram.

Or = equivalent weight/ 1000.

Molarity : gram molecular weight of solute per litre of solution .

M = Molecular weight of solute1000ml of solvent .

Molality : It represent the number of moles of solute present in 1000g of solvent .

1m = Molecular weight of compound1000g 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 = 40Equivalent weight of NaOH = molecular wt.

No.of replacable H/OH

= 40a. 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 compound100g 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 waterThere 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 solventMolecular Weight of Hcl = 1+35.5=36.5For Hcl specific gravity = 1.18

Volume = Mass/specific gravity=36/1.18


7/44

7

= 30.51mlTherefore for 1N Hcl = Dissolve 30.51ml of Hcl in 1000ml of solvent(D.W.)For 0.5N Hcl = 15.25ml Of Hcl in 1000ml of solvent (D.W.)For 1M Hcl =Dissolve 30.51ml of Hcl in 1000ml of solvent(D.W.)For 2M Hcl =Dissolve 61.02ml of Hcl in 1000ml of Solvent(D.W.)

Result:


8/44

8

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. Weakacids or bases, in the presence of their salts with strong bases or strong acids respectively form

buffer system.Ex: Phosphate/monohydrogen phosphate.

Carbonic acid/bicarbonateProteins/proteinate.

pH:- The pH of the solution is the value with which defines its hydrogen ionconcentration in aqueous solution, it is relative strength of hydrogen ion reach species called acidand 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 whosecolour 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 pHmeter in which a change in pH is measured as change in electrical potential. If a metal rod isplaced in solution of its salt, it acquires potential. If two dissimilar metals are dipped into thesolution of their salts, the difference in potential can be measured or calculated from the twoseparate potentials. the standard electrode is thus required against which the potential of otherelectrodes can be compared. this is the standard hydrogen electrode , consisting of platinum roddipped in the aqueous solution with a given H+ activity in which hydrogen gas is bubbledcontinuously at 1 atmospheric pressure. But this is too cumbersome to be used, as referenceelectrode for routine use, other secondary reference electrode of known potential in relation tostandard 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 produceddependent 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. pH SOLUTION TO BE USED VOLUME IN ml

1. 2 0.2M KCl + 0.2M HCl 65ml + 250ml2. 4 0.2M succinic acid + 0.2M NaOH 250ml + 100ml3. 6 0.2M succinic acid + 0.2M NaOH 250ml + 435ML4. 9 0.1M KCl + 0.1M H3BO3 250ml+250ml+208ml5. 10 1M NaHCO3 + 1M Na2CO3 13.8ml + 12ml

Result:- The buffer solution of specific pH were prepared with the help of pH meter.


9/44

9

Experiment No: 3

Aim: Determination of isoelectric PH

of Protein.

Principle: The PH at which a Protein is least soluble is its isoelectric pH, defined as the pH atwhich the molecule has no net electric charge and fails to move in an electric field. Under thesecondition there is no electrostatic repulsion between neighbouring protein molecules, and theytend to coalesce and precipitate. However, at pH values above the isoelectric point all the proteinmolecules have a net charge of the same sign. They therefore repel each other, preventingcoalescence of single molecules into insoluble aggregates. Some protein are virtually insoluble attheir isoelectric pH.

Since different proteins have different isoelectic pH values, because their content ofamino acids with ionizable R groups differs, they can often be separated from each other byisoelectric 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 water3) 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 tubesReagents

1 2 3 4 5 6 7 8 9 10

SkimmedMilk

15 15 15 15 15 15 15 15 15 15

0.005N

HCl

1 2 3 4 5 6 7 8 9 10

Stand for 30 minutes and determine PH of each tube

Table-2

Test tubesReagents

1 2 3 4 5 6 7 8 9 10

Albumin 15 15 15 15 15 15 15 15 15 150.005NHCl

1 2 3 4 5 6 7 8 9 10

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---------


10/44

10

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 weakeningthe hydrophobic bonds that maintain the tertiary structure. This change in the proteinconformation 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 (seefigure),. This , essentially consist of a capillary tube down which a known volume of proteinsolution is allowed to flow under gravity. The time taken for this flow is measured (t 1) and alsothat of the solvent (t0); the relative viscosity is then given by.

rel = 1/0 = (t1/t2) x (1/0)

where 1 is the viscosity of the protein solution of density 1 and 0 the viscosity of the solvent ofdensity 0. If the densities are taken to be the same then the expression simplifies to

rel = t1 / t0

Einstein has shown that, for spherical molecules, the relative viscosity is related to theconcentration of the molecule ( c ) and the partial specific volume (V ), which is the volumeoccupied by the molecule and its bound water:

rel = 1+ 2.5cVMaterialsViscosity is very sensitive to temperature, so all solutions an the viscometer must be kept at 30oCin the water bath.

1. Ostwald viscometer2. water bath at 30oC3. 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

MethodAlways use the viscometer by one limb only and never squeeze the two arms together. Rinse the

viscometer with KCl solution and place it in positionin water bath by carefully clamping one limb. Checkthat it is vertically using the plumbline and introduce

exactly 20 ml ( or the volume marked on theviscometer) of KCl solution at 30oC in the bulb A

with a syringe or Pipette. Leave for 5 minute toequilibrate, then either apply positive pressure to thewide 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 thenearest 0.1s) for the liquid to flow between the twograduation marks B and C. Repeat the experimentuntil the flow time agree within 0.2s and calculatethe average flow time. Repeat the whole procedure


11/44

11

with the urea solution alone (t0), which are the solvents, and then with bovine serum albumindissolved in the urea (t1). Plot the values of t0 and t1 against the concentration of urea and join upthe points with smooth curves. Select convenient concentration of urea and calculate the relativeviscosities (t1/t0) using the values from the curves. This ensures that any slight errors involved inthe determination of t1 and t0 are not magnified on taking the ratios. Finally prepare the graphs ofthe 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 Einsteins 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 P

H

is varied over the range 2-12. Comment on the results


12/44

12

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 calledreducing sugar. This involves the oxidation of the aldehyde functional group present;forexample; in glucose and the ketone functional group in fructose. Simultaneously 3,5dinitrosalicylic 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 3,5dinitrosalicylic acid. Different reducing sugars generally yield different colour intensities ;thus it is necessaryto 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 Lowspecificity, one must run blanks diligently if the colorimetric result are to be interpreted correctly andaccurately.

When the effects of extraneous compounds are not known one can effectively include a so-called internal standered by first fully developing the color for the unknown sample, then a known amount ofsugar is added to this sample. The increase in the absorbance is equivalent to the incremental amount of sugaradded.

REQUIRMENTS:

1 Test tubes, Pipettes, Spectrophotometer2 NaoH3 DNSA4 Standerd giucose solution

PROCEDURE:

1 Add 3ml of DNSA reagent to 3ml of glucose sample in a lighly capped test tube.(To avoid theloss of liquid due to evaporation, cover it with paraffin film.)

2 Heat the mixture at 90C 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.


13/44

13

Experiment No: 6

Aim- Protein estimation by Lowry/Biuret method.

Principle-

The principle of this method is based on the facts that the Folin-Ciocalteu regents reacts witharomatic residues of proteins and yields blue color which in turn is read in colorimeter. Thedifferent proteins contain different aromatic residues. Blue color develops because the alkalinecopper reacts with proteins; tyrosin and tryptophan present in protein reduce phosphomolybdate.(present in Folin-Ciocalteu reagent)

Requirements-

1.1N NaOH Solution.2.ALKALINE SODIUM CARBONATE Solution-Dissolve 20gm of Na2 CO3 in 100 ml of 0.1NNaoH solution, prepare fresh.3.COPPER SULPHATE-SODIUM POTASSIUM TARTRATE Solution- Dissolve 0.5g/lt ofCuSo4.5H2o 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 theday of use.5.FOLIN-CIOCALTEAU REAGENT-It is a solution of Sodium tungstate and Sodium molybdatein phosphoric and hydrochloric acids (available commercially). Dilute the commercial reagentwith an equal volume of distilled water only on the day of use.

Procedure-a) 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.

PROTOCOL-Sr no Stock of protein (ml) Distilled water (ml) Concentration of protein Alkaline Reagent Fcr

(ml)O.D

1 0.0 1.0 0 5ml 0.5

2 0.1 0.9 0.02 5ml 0.5

3 0.2 0.8 0.04 5ml 0.5

4 0.3 0.7 0.06 5ml 0.5

5 0.4 0.6 0.08 5ml 0.5

6 0.5 0.5 0.10 5ml 0.5

7 0.6 0.4 0.12 5ml 0.5

8 0.7 0.3 0.14 5ml 0.5

9 0.8 0.2 0.16 5ml 0.5

10 0.9 0.1 0.18 5ml 0.5

11 1.0 0.0 0.20 5ml 0.5

12 Unknown 1 - - 5ml 0.5

13 Unknown 2 - - 5ml 0.5

Result- Concentration of protein in unknown sample was found to be Unknown 1 mg/mlUnknown 2 mg/ml


14/44

14

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 andseparation of components of a mixture and purification of compounds.

This experiment is based on the partition chromatography principle i.e. if to a mixture oftwo immiscible liquids A and B, a substance which is soluble in both A and B is added, then itdistributes itself in such a way that the ratio of its concentration in two liquid A and B is constantat a particular temperature. Separation of different constituents takes place because eachconstituent distribute itself to different degree between the solvent which flows down the columnand stationary liquid.

In paper chromatography paper is used as an inert support with one solvent as stationaryor immobile phase.

Radial paper chromatography shows radial development. This experiment is based ondifferential 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 acentral 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, whenvarious components become visible, the process is known as development.

The ratio of distance the substance moves compared with the distance reached by solventboth 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 be-

Glutamic acid -Tryptophan -

Tyrosine -


15/44

15

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 isillustrated in the case of three amino acids: Aspartic acid, Histidine and lysine. Electrophoresis atlow voltage is not usually used to separate low molecular weight compounds because ofdiffusion, but it is easier to illustrate the relationship between charge and PH with amino acidsthan 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, anuncharged molecule, is included in the mixture to check for any movement of the origin due toelectro-osmosis.

Materials

1 horizontal electrophoresis apparatus\2 Power packs3 amino acids (aspartic acid, histidine, lysine and a mixture of all three in tris-aetate buffercontaining 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 reagent8 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 byarranging 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 thepaper. Streak three other paper strips with three other paper strips with only one amino acid and


16/44

16

run all five electrophoresis strips together. Wet the paper from each electrode compartment towithin a few centimeters of the point of application, leave the rest to be wetted by capillaryattraction,, and immediately switch on the current . this way there is minimum spreading of thesample. Carry out electrophoresis for 3h at 8 V/cm, remove the strips , and dry in an oven at110oC.Develop one of the strips containing mixture for glucose and dip the remaining four strips rapidlyin 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 anyelectro-osmosis.Repeat the experiment with 0.07 mol/liter citrate buffer, PH 3.0 .


17/44

17

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 bluecompound is formed with a sharp absorption maximum at 595 nm. This reaction is given by 2-deoxypentoses in general and is not specific for DNA. In acid solution, the straight chain form ofa deoxypentose is converted to the highly reactive -hydroxylevulinaldehyde that reacts withdiphenylamine to give blue complex. In DNA, only the deoxyribose of the purine nucleotidesreacts, so that the value obtained represents half of the total deoxyribose present.

Materials

1) DNA (commercial sample) 10 mg2) RNA (commercial sample) 10 mg3) pig spleen DNA solution 10 mg4) yeast RNA solution 10 mg5) buffered saline 500 ml( 0.15 mol/liter NaCl: 0.015 mol/liteer sodium citrate, PH 7) 1 liter6) diphenylamine reagent. ( dissolve 10g of pure diphenylamine in 1 liter of glacial acetic acidand 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 ofdiphenylamine reagent. Heat on a boiling water bath for 10 minutes, cool and read the extinctionat 595 nm. Read the test and standards against water blank. Assay the isolated nucleic acids andthe commercial samples for DNA.


18/44

18

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 offerric chloride as a catalyst, giving a green colour. Only the purine nucleotide gives significantreactions.

Requirements:

A. REAGENTS:

1. Orcinol reagent: Dissolve 1gm of Ferric chloride in 1litre of Concentrated HCl and add 35mlof 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.OTHERSTest 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 byadding 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 .


19/44

19

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 andthis also has been used for purification of proteins. Theoretically any salt can be used butgenerally ammonium sulphate is preferred for two reasons . one, it has a high solubility , 840g/liter, and secondly , its dissolution in water is exothermic or the solution gets cooled.

Method : carry out the experiment given in the flow chartEstimate the amount of protein in each fraction and calculate back to per gram tissue weight. Caremust be exercised to carry out the protein estimation, as ammonium sulphate will interfere withthe 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 addingsolvents like acetone or alcohol.

Method : fractionate a tissue homogenate into different proteins by adding different amounts ofice 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 asudden flocculent precipitation takes place. Measure the PH. It will be found to be about 4.5. themajor protein of milk is casein and its isoionic point is 4.5. At this PH, the net charge on themolecule is zero.Remember that proteins remain in the solution mainly because of the charges present on themwhich makes them hydrophilic, once this charge is neutralized, the protein precipitate out. This ishow cured is prepared. The inoculum added contains lactobacilli which utilize the lactose of milkto produce lactic acid. When the PH reaches 4.5, casein is precipitated out.


20/44

20

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 degradingmicroorganism transport the degraded form across the cytoplasmic membrane of the cell. Somebacteria posses the ability to produce amylase that breaks starch into maltose. The amylase is anextracellular enzyme which is released from the cell of the microorganisms.

Requirements

1) Bacterial culture2) Inoculation loop3) Starch agar medium*4) Petri dish5) Iodine

*Starch Agar mediumstarch 20 gBeef extract 3 gPeptone 5 gAgar 15 gDistilled water 1 literIodine 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 occurreddue to production of amylase.

Results

The starch and iodine make a complex of blue colour. Iodine does not react with maltose or withany other product of starch degradation. Hence, no color is formed in such cases and clear area isvisible.The given bacterial culture has been found to amylase positiveOrThe given bacterial culture has been found to be amylase negative.


21/44

21

Aim- Demonstration of amylase production byAspergillus niger

Principle-

Starch 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.

Requirements-

Growth Medium

Yeast extract - 0.3gMalt extract - 0.3gPeptone - 0.5gSoluble starch - 1.0gDistilled water - 1000mlpH = 6.0

ReagentsIodine solution - 0.01NStarch solution - 0.1%

Procedure-

1. Prepare growth medium, dispense 200 ml medium in flask and autoclave it.2. Prepare pure culture ofAspergillus niger and prepare fresh culture in tubes.3. Transfer 10ml of inoculum in sterilized growth medium and incubate at 30C 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 initialvolume 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.

Observation-

Students are expected to write the observation of this experiment, which will be based upon thefindings of the experiment.

Result-


22/44

22

Aim : Demonstration of Protein (Gelatin) hydrolysis

Principle: Gelatin is a polymer of amino acids and the protein is used as nitrogen and carbonsource for microorganisms. The gelatin or protein is generally broken down into peptides of shortamino 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 22o

C, whilethe degraded form of gelatin i.e. amino acids and peptides is to remain liquid below 22oC

Requirements

a) bacterial culturesb) trichloroacetic acidc) Nutrient gelatin broth/ agard) Ice flakese) Incubaors

Nutrient gelatin broth/ agar medium:Gelatin 120 g

Beef extract 3 gPeptone 5 gDistilled water 1 literAgar ( 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 35oC3) 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 bacteriaresults in hydrolysis of casein by forming clear zone around the colonies.


23/44

23

Aim : Demonstration of Fat hydrolysis (lipase activity ) by a bacterial cultureOil is used as substrate. Some microorganisms have the ability to hydrolyse fats and results intorancidity in some food products. The ability of organisms to hydrolyse fat is accomplished due tothe enzyme lipase. Fat molecules are degrade resulting in glycerol and fatty acids. Lipases are oneof the most important classes of industrial enzymes. Many important biotechnological

applications have been explored in paper industry units, organic chemical processing and agro-chemical industry. Lipases have been used in food, dairy , beverages, and detergent formulation.

Requriments

a) Bacillus speciesb) Lipase producing broth*c) Nutrient agar mediumd) Tween 80 (1%)

* lipase producing broth medium for Bacillus Species

NaCl 5 g

CaCl2 0.05 g

Yeast extract 5 g

Tween 80 (1%) 5ml

Distilled water 1 liter

PH 8.0

Method

1) grow 6 hour old culture if Bacillus sp. On nutrient agar slants containing 1% Tween 80.2) Add inoculum in flask containing lipase producing broth medium.3) Incubate the flask for 15 hours at 50 oC under continuous shaking condition i.e. 200 rpm.4) Centrifuge the contents at 1000 g for 20 minutes at 4 oC5) 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 enzymereleasing 1mg of p-nitrophenol/minute.


24/44

24

Aim : Demonstration of Uease Production i.e. urea Hydrolysis

Principle: urea is the waste nitrogenous material and excreted out by mammals. Some bacteriadegrade the urea into ammonia and CO2. due to production of ammonia , the urease productioncan be easily demonstrated by the following reaction:

NH2 CO2 - NH2 2 NH3 + CO2

Requirements

a) Proteus vulgaris, E. coli and Serratia marcecsensb) Urea broth medium*c) Inoculation tubed) Incubator

* Urea broth medium

urea 20 gyeast extract 0.1 gK2HPO4 9 gK2HPO4 9.5 gPhenol red 0.01 gDistilled water 1 literPH 6.8

Method

1) Incubate the urea broth medium with bacterial culture2) 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 ofP. vulgaris. Otherwise, indicator will remainyellow at acidic range of PH. then it shows no urease production by the givenmicroorganisms as in case ofE. coli andserratia marcescens.

Results

P. vulgaris can be distinguished by E. Coli and S. marcescens by its ability to produce largeamount of the enzyme urease.


25/44

25

Experiment No: 12

Aim: - To estimate the activity of- amylase.

Principle: - -amylase E.C. No. 3.2.1.1 i.e endo 1,4 glucan hydrolase. It occurs widely inbacteria and fungi. All amylase are endo active enzymes which specially cleaves 1,4glycosidic linkages in amylose , amylopectin and glycogen yielding sugar in configuration.

They are unable to hydrolyse 1-6 branch points in amylopectin but are able to bypass thisbranch point. Two types of microbial amylase are existing i.e. saccharifying amylase andliquefying amylase. They are distinguished by the fact that saccharifying amylase produce aincreased quantity of reducing sugar about twice of liquefying amylase.

Properties lpha amylase Beta amylase Amyloglucosidase

Molecular wt. 50,000 55,000 1,60,000 50,000 1,12,000Optimum pH 3.5 -7 5.5 -7.5 4.0 -6.0Optimum temperature 35 -90 C 37 -55C 40 70CMetal ion req. Ca++ - --Km(milimoles) 1 0.2 18.5

Specificity Alpha 1,4 linkagescan bypass alpha 1,6linkages

Alpha 1,4 limkagescannot bypass alpha1,6 linkages

Alpha 1,4 and alpha1,6 linkages

Product Maltodextrin Maltose and beta unitdextrin

Beta glucose, amylaseand amylopectin

Substrate Amylase,glycogenamylopectin

Amylase, glycogen,amylopectin

glycogen,amylopectin,dextrinmaltose.

Nature Endosplitting Exosplitting Exosplitting


26/44

26

Assay: - amylase are generally quantified by beta amylation of reducing group formed due tohydrolysis of soluble starch. The simplest method is dinitrosalicyclic acid method. Starch iodinecolorimetric method is also used..

amylase: - amylase i.e. 1-4 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. amylase are exosplittingenzymes which attacks amylase , amylopectin, glycogen at non reducing terminal resulting information of maltose in configuration, amylases is specific for , 1-4 linkages and is unableto bypass 1,6 branch point.

Assay: - Since the main product for amylase hydrolysis are the disaccharides maltose units , 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 areexosplitting amylopectinamylopecic enzyme which attack amylase, amylopectin and glycogen arespecifically hydrolysed 1, 4 and 1, 6 linkages from non reducing end.

However 1,6 glucosidic linkages are cleaved by less readily than 1,4 glucosidic linkages.

The product of reacton is glucose.

Assay: - The major product of amylase action is glucose. The spectrometric assay for glucosemay be employed.

Procedure: - Take 0.5 ml of amylase enzyme in a test tube and 1 ml of buffer and 0.5 ml ofstarch. The glucose production in this tube is to be determined. Incubate these tubes for 10 min at37 C then add 1 ml of DNSA reagent in it. Boil test tube for 10-20 min in water bath. A controltube is also performed in which DNSA is added before addition of the starch so, as to deactivatethe enzyme. Arrange the test tubes as per protocol. After addition boil these test tubes for 10 15min. Optical density of these tubes are to be read at 520 - 540 nm in colorimeter.

Protocol: -

Reagents C1 (ml) E1 (ml) C2 (ml) E2 (ml) C3 (ml) E3 (ml) C4 (ml) E4 (ml)Starch 2.5 2.5 3.5 3.5 4.5 4.5 5.5 5.5Buffer 3.5 3.5 2.5 2.5 1.5 1.5 0.5 0.5Enzyme - 1 - 1 - 1 - 1.0

INCUBATE FOR 30 MIN ONLY FOR EVP.NaOH 1 1 1 1 1 1 1 1Enzyme 1 - 1 - 1 - 1 -DNSA 1 1 1 1 1 1 1 1

OBSERVATION:

Result: - The activity of amylase was found to be g/ml from the graph.

1 2 3 4 5 6 7 8 9 10

OD(450nm)


27/44

27

Experiment No: 13

Aim: - To study the effect of pH on activity of amylase.

Theory: - Enzyme is a protein, every protein exists in active state at an optimum pH If pH is lessthan or greater than this optimum pH the enzyme will Undergo structural change which willdecrease its activity. Thus if graph is plotted, a bell shaped curve is obtained; showing pHoptimum 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 5.5-8Respectively. 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 waterBath 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 0.2MNaH2PO4 and dilute to 100ml.

2) Starch solution 1% in phosphate buffer3) Enzyme solution -1% in bufferd saline4) 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 0.2MNa2HPO4 is mole toward acidic and it has a pH

Around 4.5 .so with proper addition of discreatpH 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 6after that the activity gradually decrease.


28/44

28

Experiment No: 14

Aim: To study the effect of temperature on activity of enzyme ( amylase).

Principle: -Amylase catalyses the hydrolysis of -1, 4 linkage of starch with production ofreducing sugars .The reaction is followed by measuring the increase or decrease in the reducingsugars using DNSA reagent. As the temperature increases the activity of the enzyme initiallyincreases, attains a maximum and then gradually decreases thus produces a bell shaped curve.

REAGENTS:

1. Buffered starch-1% in phosphate buffer2. Enzyme solution -0.5% in buffered saline3. 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 5C, 15 C, 25 C, 35 C, 45 C, 55 C.5. Incubate 1-6 tubes in respective water bath6. 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 tubes8. In control enzyme is added only after the addition of NaOH.9. Add DNSA to each tube including control10. Boil for 15 mins and read at 540nm.

Observation:

TEMPERATURE 30 C 45 C 60 C 75 C 90 C 105 COPTICALDENSITY(540nm)

Result: As the temperature of the solution increases the activity of the enzyme also increases at75 C it shows optimum activity then as the temperature further increases the activity declines.


29/44

29

Experiment No: 15

Aim:- Effect of inhibitor on enzyme activity.

Principle- Inhibitors are classified into reversible and irreversible .Reversible inhibition arecompetitive or uncompetitive that can be effectively studied using bitterguard (karela).

1.COMPETITIVE INHIBITION It is characterized by decrease in enzyme activity andincrease in Km value, Vmax remains unaltered.

Vo =Vmax[S]/Km(1+I/Ki) +[S]

2.NONCOMPETITIVEINHIBITION It is characterized by decrease in Vmax , Km being thesame.

Vo=Vmax/(1+I/Ki)[S]/Km+[S]

3.UNCOMPETITIVE INHIBITION- It is characterized by modification of both Km andVmax.

Vo= Vmax/(1+Io/Ki)[S]/[So]+Km(1+Io/Ki)

REAGENTS- 1. Source of inhibitor:- a. Bitterguard pulpb. Seed pulp

2.Phospate buffer:- Add 40ml 0.2N NaOH to 50ml 0.2 NaH2PO4 and dilute it to100ml.

3. Starch solution .4. Enzyme solution5. DNSA

Procedure:- As per the protocol

REAGENT C1(ml) C2(ml) C3(ml) C4(ml) E1(ml) E2(ml) E3(ml) E4(ml)EN YME 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5BUFFER 1.0 0.8 0.6 0.4 1.0 0.8 0.6 0.4INHIBITOR - 0.2 0.4 0.6 - 0.2 0.4 0.4NaOH 1 1 1 1 - - - -

Boil for 10 min. in waterbath and read at 540nm

STARCH 1 1 1 1 1 1 1 1

Incubate for 15min. at 37C .NaOH - - - - 1 1 1 1DNSA 1 1 1 1 1 1 1 1

Boil for 15min.in waterbath at 540nm.

Observation:-

Result:-

TUBES C1 E1 C2 E2 C3 E3 C4 E4

O.D


30/44

30

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 amountof starch i.e the substrate is kept constant where as the concentration .of amylase is taken inincreasing order. It is observed that the amount of glucose produced in every successive tubesalso increases. This is due to reason that as the enzyme concentration increases the number ofactive site also increases. Thus substrate readily binds to enzyme thereby increasing the amountof glucose produced.

Thus increase in concentration of enzyme increases activity of amylase. Care should be takenthat:-

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) Take 7 clean and dry test tubes and label them, add 2.5 ml of starch solution.2) Add gradually increasing concentration of enzyme solution in tube 1-6 as per protocol.3) Incubate tubes for 30 mins.4) After incubation add 0.2N NaoH to each tube.5) Add 1ml of DNSA to each tubes.6) Keep in boiling water bath for 15mins and cool it.7) If required dilute it to 10ml with distilled water.8) Read OD at 590nm.

Observation Table:-

C1/E1 C2/E2 C3/E3 C4/E4Optical Density

Result:-


31/44

31

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 temperatureare 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 substrateshould also be kept constant. As the concentration of substrate increases the rate of reaction isalso increased upto a certain limit. This is apparent from the initial linear curve with furtherincrease in substrate concentration. Thus linearity does not persist as the increase in rate ofreaction gradually goes on decreasing, finally when the rate of reaction is not affected by increasein substrate concentration the curve shows plateau.

The velocity indicating this splitting is called as Vmax. Km is substrate concentration athalf the Vmax.

REAGENTS:1. Phosphate buffer2. Enzyme3. Starch solution4. DNSA5. Sodium hydroxide (NaOH)


32/44

32

Procedure: Arrange the test tubes and perform according to the protocol.

Boil in water bath for 15 mins and cool .Take the OD at 450nm.

Observation:

Result: The value of Km was found to be g/ml.

Srno.

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 Phosphatebuffer

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

1 2 3 4 5 6 7 8 9 10

OD(450nm)


33/44

33

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 ofadsorption only and hence it is called as column chromatography. Adsorption is the phenomenonwhereby a substrate gets bound to the surface of unique particle. If the compound is in thesolution, particles insoluble in solvent then part of substrate is adsorbed and part remain in thesolution. The ratio between amount adsorbed and amount remained in the solution is constantcalled adsorption coefficient. The extent of binding between solute and adsorbent depends oncharge, van der Waals' forces, dipole interaction, hydrogen bonding and steric forces and dependsupon the structure of compounds.

The mass of solute adsorbed per unit weight of adsorbent [m] depends on theconcentration of solute.

According to Langmuir equation-

m = K1K2c1+K2cWhere, m = Mass of solute adsorbed per unit weight of adsorbent.

K1= Number of active adsorption sites per unit and depend upon nature ofAdsorbent.

K2= Affinity of solute for adsorbent and depends upon component of system.Langmuir assumed only one binding site on the adsorbent. Hinshelwood equation fornumber of different binding sites is as follows-

m = K1K2c1+K2C

The molecule with least adsorbtivity goes along with the solvent and these are elutedfirst followed by other component.

Materials :-1) Chromatographic Column2) Fresh Spinach Leaves3) Alumina4) Calcium Carbonate5) Sodium Sulphate6) Petroleum Ether7) 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 throughseparating funnel. Add 10 to 20 ml of water, shake well and allow layer to separate. Remove thelower layer containing methanol. Repeat the addition of water, shaking and removing the aqueouslayer 3 to 4 times till the lower layer becomes colorless and only faintly colored. Avoid thevigorous shaking to avoid emulsion formation. Remove the last traces of water by addinganhydrous sodium sulphate. Filter to remove the solid and concentrate the extract to few ml bycareful evaporation up to dryness. If presence of H2O is suspected, repeat the above steps.

II]Preparation of column:


34/44

34

Alumina was used as adsorbent. Column was filled with 5 cm alumina, 7cmcalcium carbonate, 7cm sucrose in petroleum ether. A small amount of glass wool is pusheddown to the bottom as a pad. About 2 to 5 g of adsorbent [previously dried at 120oC overnight foractivation] is taken in a beaker. A small amount of C6H6 added to slurry. The slurry is carefullypoured into a burette; use more benzene if necessary to pour the extra slurry. The adsorbentshould never be allowed to dry. After all the adsorbent has settled add 20ml of more benzene andallow 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. Openthe stop cork and allow the solution to separate. When the level of the solvent has just reached thesurface 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, butmost of the material will not remove from the top. This is because benzene is not polar enough torelease all adsorbed compound when 20ml of benzene has just been run then, add 5ml of5%acetone in benzene from the top. Note the changes by continuously increasingly the acetoneconcentration 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. Pigments Color of bands

1. Chlorophyll Green2. Xanthophylls Yellow3. Carotenoids Orange

RESULT: - The three colored layers of chlorophyll (green), xanthophylls (yellow) andCarotenoids (orange) were separated by column chromatography.


35/44

35

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 frequency

10-20 220-30 730-40 1740-50 2950-60 2960-70 1070-80 380-90 290-100 1

Arithmatic mean :It is sum of all data divided by number of observation .

Arithmatic mean forUngrouped data = X = X1+X2+X3+Xn

n

x =sum of all the value of observationn = total number of observation .

Arithmatic mean

For grouped data = X = fxn

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 repeatedMaximum times in the given data.

MEDIAN : When the data is arranged in ascending or descending order theMedian 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 - 48.40Mode is found to be -15Median 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.


36/44

36

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 2 test.

Principle:

When stastical test is used to compare on observed ratio or value with anExpected or theoretical ratio or value and determine how clearly the former fits intolatter. It is known as Testing the goodness of fit One measure commonly usedfor it 2 test.[Chi-squareTest]

{ [Observed] - [Expected] }22

Expected value

To accept or reject Null Hypothesis, this calculated 2 is compared with standardValue . If the value of 2 is less than that of table 0.05 probability , than NullHypothesis is accepted.

Di-hybrid Cross:When the cross is made between two pair of contrasting character, it is said tobe dihybrid ratio RRYY and rryy.

When plants with the above character were crossed pollinated in F1 generationthey produced hybrid plan [Rr Yy] with round and yellow seed.

When these (F1) plants were allowed to self pollinate, progeny were formedin (F2) generation in the ratio 9:3:3:1


37/44

37

Round Tellow - 9

Round Green - 3

Wrinkle Yellow - 3

Wrinkle Green - 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 AssotrmentSometimes if more than 2 genes are located on same chromosome independentassortment 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 dihybridCross [Accept Alternate Hypothesis]




38/44

38

Experiment No: 23

Aim: A bag contains 10 pink,10 yellow,10 orange and 20 red beads .

III. What is probability of drawing 1 yellow bead .

IV. What is probability of drawing 1 yellow and 1 red bead.

Theory: If an experiment has an m mutually exclusive equally , likely and exhaustivecases , out of which m are favourable to the happening of the event A , then theprobability of the happening of A is denoted by P(A) and is defined as:-

P(A) = mn

= 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 animpossible event is zero .

The probability of occurrence of any event lies between zero & one [0-1] , bothinclusive.

Procedure:

1. Withdraw a single bead at randomly from a mixture of 50 beads [yellow+pink+red]

2. Record the observation of withdrawal of yellow bead.3. Withdraw one yellow and one red bead at a time from a mixture of 50 beads and the

observation of it.4. In the first case beads one withdrawn 50 times and in second case beads are withdrawn

25 times.5. Calculate the probability.

Observation Table:-

Sr no. Beads Tally Frequency Probablity

1. Yellow 15 3/10

2. 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


39/44

39

1 yellow bead

Practically numbers of favourable case (m) =15

probability of drawing of = m =15 = 3

n 30 101 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 = 250 50 25

Practically number of favourable cases m = 5

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 andpractically 5/25.




40/44

40

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 deviationNo 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 Sample drawn Number of pink beads x- x

1 1 1 322 2 2 303 3 3 314 4 4 325 5 5 326 6 6 317 7 7 328 8 8 29

9 9 9 2910 10 10 3011 11 11 3212 12 12 3013 13 13 3014 14 14 3115 15 15 3116 16 16 3017 17 17 3118 18 18 3219 19 19 3020 20 20 3221 21 21 29

22 22 22 3023 23 23 3124 24 24 2825 25 25 32

Where, x=32 x- x = 768

Result: Standard error was found to be = 4718.5.




41/44

41

Equipments generally required in the Biotechnology laboratory

Micropipettes Micropipettes kept in micropipette stand

Petriplates with different media

Binocular Microscope


42/44


43/44

43

Distilled Water Assembly Mono-pan balance

Single-pan manual balance Centrifuge


44/44

Autoclave Distilled Water Assembly

Tipbox

Incubator Shaker