Bio f110 Manual of Biology Laboratory, Hyderabad Campus

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LABORATORY MANUAL FOR BIOLOGY (BIO F110 BIOLOGY LABORATORY) DEPRTMENT OF BIOLOGICAL SCIENCES EDUCATIONAL DEVELOPMENT DIVISION BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE PILANI 333031 (RAJASTHAN) 2011

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Transcript of Bio f110 Manual of Biology Laboratory, Hyderabad Campus

LABORATORY MANUAL FOR BIOLOGY (BIO F110 BIOLOGY LABORATORY) DEPRTMENT OF BIOLOGICAL SCIENCES EDUCATIONAL DEVELOPMENT DIVISION BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE PILANI 333031 (RAJASTHAN) 2011 Contents Preface Laboratory Instructions Index Table Experiment 1:Measurement of total protein content in the given sample.I-1 Experiment 2:Measurement of Glucose concentration in the given sample. II-1 Experiment 3:Separation of chlorophyll pigments by paper chromatography. III -1 Experiment 4:Measurement of Mitotic Index and duration of mitosis in the given plant tissue. IV -1 Experiment 5 :Determination of ABO-Rh Blood Typing.V -1 Experiment 6:To extract DNA from banana.VI -1 Experiment 7:To study the phenomenon of plasmolysis in onion peel.VII -1 Experiment 8:To quantify the level of total cholesterol present in serum.VIII -1 Experiment 9:To take measurement of blood pressure.IX -1 Experiment 10:To observe the permanent slides.X-1 Preface The spectacular progress over the past two decades in our understanding of biological processes atthemolecularlevelhasbeenmadepossiblebythecomingtogetherofphysical,chemical, biochemicalandgeneticapproachesandbytheavailabilityofawiderangeofanalytical techniques. Theneedforre-writingthislaboratorymanualwasfeltbecauseofthespecialstatusofthe course Biology Lab, which is being offered to the students across the campuses of BITS Pilaniwith the objective to give them their first Laboratory exposure in Biology. ItgivesusimmensepleasuretopresentthisLaboratorymanualforbiologyLabotatory.Some salient features of this manual are: Description of experimental set-up and working of specialized instruments. An adequate theoretical explanation of the protocol and inclusion of review questions to help the student prepare and understand the experiment. Abuilt-informatofthemanualincludingtablesandgraphsforrecordingthe experiments. Theauthorsgratefullyacknowledgethecontinuousinterestandencouragementreceivedfrom Director,ProfG.Raghurama,DeputyDirectorProfR.N.Saha,&DeanIDProf.A.P.Singh.OursincerethanksareduetoHeadofDepartmentBiologicalSciencesDr.Shibasish Chowdhuryforhiscontinuedmotivationandconstructivesuggestionsregardingthemanual. Our thanks are specially due to Dr. S.K. Verma, Dr. A.K.Das, Dr. V.N.Sharma andDr. Neeru Soodfortheircontributionindevelopingthefirsteditionofthismanual.Itwastheintense discussionandconstructivecriticismamongstthefacultymembers,speciallycourse restructuringteammembers,namely,Manoj,Pankaj,PrabhatandVishal,whohelpedby providinginputsindevelopingthecoursecontent.Alsothiseffortwouldneverhavebeen successfulwithouttheconstructivecriticismandsuggestionsfromourhighlydedicated Researchscholars&TeachingAssistantswhoascourseInstructorshavetimetotime continuouslycontributedtowardsthebettermentoftheearlierformofthiscourseandthe presentmanual. Uma S. Dubey B.Vani Ashish Runthala S.K. Verma General Laboratory I nstructions Theobjectiveoftheselaboratoryexercisesistoacquaintyouwiththetechniques emphasizingtheneedofmeasurementsinthefieldofbiology.Wewantyoutotake seriousinterestinconductingtheseexperiments,whichwillhelpyoutohaveenough exposure in answering the basic questions in modern biology. Beginyourexperiment,forgettingallpreconceivednotionsaboutwhatissupposedto happeninaparticularexperiment.Followdirectionscarefully,andseewhatactually does happen. Be meticulous in recording the true observations even though you know something else should happen. You must be on time in laboratory and come well prepared for the experiment. Every student has to have his/her individual copy of the manual. The laboratory manual has to be brought when you come to the laboratory. The laboratory work will continue for two hours. You are supposed to be fully engaged for the specified time and can not leave the laboratory before time. For accurate measurements, work carefully and co-operate with your team members. Presentation of data, tables, graphs and calculations should be neat and carefully done in the manual itself and should be verified by your instructor. Bringyourcalculator,scaleandpenciletc.andcompletethecalculationsinthe laboratory itself. Handletheapparatuswithcarewhileworking.Reportanysortofbreakagetothe instructor. ForevaluationcomponentstherelatedchaptersinthefirstyearGeneralBiologybook should be taken into account. Regularity is expected and make-ups will not be encouraged. BIOLOGY LABORATORY Semester 201_ to201_ Name:. ID No:. . Section No:. ..... Day: ..Hr: S. No.ExperimentDateInstructors Signature 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. EXPERIMENT NO. 1 OBJECTIVE Measurement of total proteins in a given sample by Lowrys Method using photometry. THEORY Lowrysmethod:InthismethodproteinsamplesaretreatedwithBiuretreagentandFolin-Ciocalteu reagent subsequently. The Biuret reaction involves the binding of Cu+2 under alkaline conditions to nitrogen in the peptide bonds of the proteins. The Folin- Ciocalteu reagent, which containsphosphomolybdotugstateacids,isreducedbyTyrosine,tryptophanandpolar aminoacidspresentinproteins.Thisprocessisfacilitatedbycopper(II)ionsfromtheBiuret reaction. In this reaction, heteropolymolybdenum blue is detected at 740nm (red filter) because at this wavelength the blue complex absorbs maximum light. The intensity of the blue colour of the solution is directly proportional to the amount of these aromatic amino acids present in the sample Photometery:Photometryinvolvesmeasurementoflighttransmittingpowerofasolutionin order to determine the concentration of light absorbing material present. Analytical instrument s basedonthedirectmeasurementofcolourintensityintermsoflightabsorbedatspecific wavelengths is called a photometer. Colorimetric procedures are limited to the visible portion of thespectrumwhereasphotometricprocedureshoweverinvolveuseofUltraviolet,visibleand Infra red portions of the spectrum. Photometers use a light filter for the wavelength at which the photometricmeasurementaremade.Whereasincaseofaspectrophotometeradiffraction gratingoraprismalongwithslitisusedforgettingmonochromaticlight.Lightfiltersdonot transmit monochromatic light but they narrow range of spectral region (8-30nm). Spectrometers havetheadvantageofgreaterflexibility,increasedsensitivity,spectralrangeandnarrowness besides flexibility and convenience over photometers but are more expensive. TheworkingofspectrometersandphotometersarebasedonBeersandLambertslaws.The simplest expression of these laws is as follows: Beerslaw:Itstatesthattheopticaldensityofasolutionisdirectlyproportionaltothe concentration of the solution. Lamberts law: It states that the optical density of a coloured solution is directly proportional to the path of light i.e., (diameter of the cuvette). Note: If the diameter of cuvette is doubled the OD will also be doubled but since cuvettes of the samediameterareusedformostanalyticalpurposessoautomaticallyLambertslawis observed.AccordingtoBeerandLambertslawtheabsorbanceofasolutioncontaininglight absorbing material depends on the follow factors:(1) The nature of the substance (2) The wavelength of light (3) The path of light (4) The amount of coloured material in the light path. If the incident light is I0 the transmitted light (light emerging from cuvette containing solution) is I then the ratio I/I0 transmittance (T) of the coloured solution. If the material does not absorb atallthenIandI0arethesamethe%T=100whereasifasubstancedoesnottransmitatall thentransmittanceiszeroandthesubstanceisopaque.ByusingnegativelogarithmofT,the measurementsaretransformedtothelightabsorbed1/T.TheequationnowbecomesA=2 log T and since 2 is the log of 100, the % T formula becomes A = 2 log T.According to Beer- Lamberts law T= 10-kcl Where. T = Transmittance k = Molar extinction coefficient constant (depends on the characteristic of the solution) c = Concentration of the coloured solution l = Path of light through the coloured solution By converting this expression through logarithmic form Log T = - kcl - log T = kcl O.D. = kcl O.D.isconstantforaparticularsolutionandpathlengthisalsoconstantasthediameterof cuvetteisfixedthereforetheopticaldensityisdirectlyproportionaltotheconcentration. Absorbance is also called optical density.A = kIc Molarextinctioncoefficientistheextinction(O.D.orabsorbance)givenbyasubstance concentration1mol/litreinapathlengthof1centimetre.Graphsofabsorbancevs. concentration,orlog%Tvs.concentrationareknownasBeersLawplots.Theyaremadeby measuringthelightabsorbedbysolutionofvaryingconcentration.Thecellwidthandthe wavelengthofthewavelengthofthelightaremaintainedconstant.Ifalinearplotisobtained (showingthattheBeer-Lambertrelationshipholdsforthesolutionatthatwavelength),itthan may be used to determine the concentration of unknown solutions. Applications:Photometryhasaveryvastapplicationrangesuchasstudiesonchemical compositions of substances, enzyme analysis, soil sample analysis, microflora studies etc. Most interestingly it can be use in diagnosis of many diseases. Diagnosis of diseases: Body fluids such as blood (serum and plama ),cerebrospinal fluid (CSF) andurinecontainseveralorganicandinorganicsubstances.Forexamplebloodcontains glucose,urea,uricacid,creatinine,proteins,etc.Italsocontainsinorganicanionscationslike sodium,potassium,calcium,etc.andanionssuchasphosphorus,chlorides,bicarbonates,etc. Most chemicals are in equilibrium is there rate of production and rate of loss (by degradation or excretion) are equal. The normal concentration of most of these substances varies within narrow limits. Diseases alter this equilibrium in many ways. Thus the alteration and ists extent is can be useful in the diagnosis of a verybroad range ofdisease likediabetes, anemia liverand kidney dysfuctions.(See Table 1 for commonly used colorimetric assays). Table 1: Common colorimetric assays: Substance ReagentWavelength(nm) Inorganic Phosphate Ammoniun molybdate; H2SO4; 1,2,4-aminonaphthanol; NaHSO3; Na2SO3 600 Amino acidsa.Ninhydrin b.Cupric salts 570(proloine 420) 620 Peptide bonds Phenols, tyrosine Biuret (alkaline tartarate buffer, cupric salt) Folin(phosphomolybdate,phosphotungstate, cupric salt) 540 660 or 750 (750more sensitive) Proteina.Folin b.Biuret c.BCA reagent(Bicinchoninic acid) d.Coomassie Brilliant Blue 660 540 562 595 Carbohydratesa.Phenol, H2SO4 b.Anthrone, H2SO4 varies,e.g.glucose 490, xylose 480 620 or 625 Reducing sugarsDinitrosalicilate, alkaline tartarate buffer540 Pentosesa.Bial ( orcinol, ethanol, FeCl3, HCl)665 b.Cysteine,H2SO4380-415 Hexosesa.Carbazol, ethanol, H2SO4 b.Cysteine, H2SO4 c.Arsenomolybdate 540 or 440 380 415 Usually 500-570 GlucoseGlucose oxidase, peroxidase, O-dianisidine, Phosphate buffer 420 Ketohexosea.Resorcinol, thiourea, ethanoic acid, HCl b.Carbazole, ethanol, cysteine, H2SO4 c.Diphenylamine,ethanol,ethanoicacid, HCl 520 560 635 HexosaminesEhrlich(dimethylaminobenzylaldehyde,ethanol, HCl) 530 DNA RNA Diphenylamine Bial ( orcinol, ethanol, FeCl3, HCl) 665 - Oxo acidsDinitrophenylhydrazine, Na2CO3, ethyl acetate435 SterolsLiebermann-Burchardt reagent ( acetic anhydride, H2SO4, chloroform) 625 DescriptionofMINISPECSL171:MINI SPEC, SL 171 is a table top, ruggedly engineered instrument with its components inside and the controls. Readouts etc., on the panels outside laid outforeasymaintainabilityandoperation.MINISPEC,SL171mainlycomprisesoflight source, a monochromator, a photo diode, processing electronics and readout. Lightfromthetungstenfilamenthalogenlampisfocusedontotheentranceslitofthe monochromator(whichalsoadiffractiongratingandanexitslit)bycondensingoptics.The light from the slit is collimated and directed on to Czerny Turner type ruled diffraction grating with 600 lines/mm. This disperses the beam and the spectrum obtained as a result is focused on to the exit slit. Variouswavelengthsare scanned byrotating thegratingon its axis by rotating thewavelengthdiscwhichiscoupledtothegratingmounting.Themonochromaticlight, isolatedbyexitslit,passesthroughBlank,StandardorSample(heldinacuvette)andthe transmittedlightfallsonaphoto-diode.Theoutputsignalfromthephoto-diodeisamplified, processed,readtoPercentageTransmittance(%T),Absorbance(Abs.)andConcentration (Conc.) as selected.WhitelightemanatingfromthetungstenhalogenlampoftheMINISPECSL171passes through the monochromator containing entrance slit ,diffraction gratings ruled 600lines/mm and an exit slit. Of the dispersed beam a narrow band of similar wavelength light passes through a secondslitintoasamplesolutionbeingmeasured.Anyofthislightwhichisnotabsorbedby thesamplesolution,butwhichpassesthroughthesolution,fallsuponthephototubeofthe instrument, where the intensity of the transmitted light is measured electronically. DescriptionoftheSpectronic20:Whitelightemanatingfromthetungstenlampofthe Spectronic20passesthroughanentranceslitandisdispersedbyadiffractiongrating.Ofthe dispersedbeamanarrowbandofsimilarwavelengthlightpassesthroughasecondslitintoa sample solution being measured. Any of this light which is not absorbed by the sample solution, butwhichpassesthroughthesolution,fallsuponthephototubeoftheinstrument,wherethe intensity of the transmitted light is measured electronically. Thediffractiongratingisaprecisionreplicagratinghaving600groovestothemillimetreand accurately spaced. The white light falling upon the grating is dispersed into a horizontal fan of beams(violetandultra-violet)atoneendandthe longwavelengths(redand infrared) at the other. Thespectrumoflightfalls onadarkscreenwithaslit cut in it. Only that portion of thespectrumwhichhappens tofallontheslitgoes throughontothesample, andwecanprojectanypart ofthespectrumontotheslit thatwewishsimplyby turningthegrating.The grating is turned by the knob onthetopoftheinstrument (thewavelengthcontrol knob). Attached to this knob isadialcalibratedin wavelengths.Thisdialmay besettothewavelength wanted.Thewavelengthsaregiveninmillimicrons(1m=10A=10-7cm).Theslitofthe instrumentpassesabandofwavelengthsof20m.Becauseofthelineardiffractionofthe grating, this bandwidth of 20m is constant over the entire wavelength region. The colorimeter is turned on by rotating the amplifier control (left hand knob) clockwise. This shouldbedoneatleast20minutesbeforemeasurementsaremade.Aftertheinstrumenthas warmedup,theamplifiercontrolknobmaybeadjustedsothatthemeterneedlewillread0 on the percentage transmission scale when no light is striking the phototube. Theright-handknobregulatestheamountoflightpassingthroughthesecondslittothe phototube. The need for this control knob arises from the fact that the light source does not emit light of different wavelengths at equal intensities and the phototube is not equally responsive to lightofvaryingwavelength.Besidesthis,theblanksolution(themediuminwhichthe substance being measured is located) may itself absorb light of certain wavelengths. In order to measuretheabsorbanceduetoonlyaparticularspeciesinsolution,suchsideeffectswhich affectthe%Treadingmustbecompensatedfor.Therefore,afterthecolorimeterhasbeen zeroed (by means of the amplifier control knob), a blank solution is placed in the light path andthelightcontrolknobisrotateduntilthedialreads100%T,toachievethisdesired compensation.Ifasamplesolutionisnowplacedinthelightpath,anychangeinthe%T readingisduetotheparticularlightabsorbingspeciesinthesampleandthe%Treadingisa measure of the quantity of that species present. Whenever a wavelength is changed, the 0 %T and 100 %T must then be reset, since the amount ofcompensationneededvarieswithwavelength.Themeterneedleshouldgoto0%T whenever the cuvette is removed from the sample holder, because removing the cuvette releases anoccluder,whichdropsintothelightbeamandpreventsthebeamfromreachingthe phototube. Adjusting the zero point o the scale is therefore always done with the sample holder empty.Thephototubeisacesium-antimonysurfacetypephotoemissivecell(typeS-4).The relative response of the phototube to a beam of monochromatic light of constant intensity is: Wavelength Response (Relative) 350 90% 375 98 400 100 450 91 475 81 500 68 512 61 525 53 550 37 575 21 600 10 6127 625 5 Thus it is seen that the phototube is much more sensitive to light of wavelength 400 m than to light of wavelength 600m.This means that the phototube will require a greater flux of 600m than of 400m monochromatic light, in order for the same %T reading to be registered upon the colorimeter dial. Handling of Cuvettes: The handling of cuvettes is extremely important. Often two cuvettes are used interchangeably; one for the blank solution and one for the samples to be measured. Yet anyvariationinthecuvette(suchasachangeincuvettewidthorthecurvatureoftheglass, stains,smudgesorscratches)willcausevaryingresults.Thus,itisessential,indealingwith cuvettes, to follow several invariant rules: Do not handle the lower portion of a cuvette (through which the light beam will pass). Always rinse the cuvette with several portions of the solution before taking a measurement cuvette, with a clean Scott Wiper before placing the cuvette in the instrument. NEVER wipe cuvettes with towels or handkerchiefs. Wipeoffanyliquiddrops,orsmudgesonthelowerhalfoftheStandardSolution (200g/ml) when inserting a cuvette into the sample holder: Toavoidanypossiblescratchingofthecuvetteintheopticalpath,insertthecuvettewith the index line facing towards the front of the instrument. After the cuvette is seated, line up the index lines exactly. The cuvette should be removed in the reverse manner. Whenusingtwocuvettesinterchangeably,useoneofthecuvettesalwaysfortheblank solutionandtheothercuvettealwaysforthevarioussamplesbeingmeasured.Under apparatus, these are called matched tubes. Theserulesmustbeobservedinallexperimentalworkwiththecolorimeter.Theequipment usedintheseexperimentsisexpensiveandrequiresareasonableamountofcare.Sloppy techniques will not be tolerated. REQUIREMENTS 1.Reagent A: Dissolve 20 g Na2CO3 and 4 gm NaOH in 1 litre of distilled water. 2.Reagent B: Dissolve 0.5 g CuSO4.5H2O and 1 g sodium potassium tartarate in 80ml of distilled water. Make up the volume to 100 ml. (PRECAUTI ON: This reagent is not to be shaked before use only its supernatant should be used).3.Reagent C: To 50 ml reagent A, add 1 ml reagent B (freshly prepared). 4.Reagent D: Diluted (1N) Folins and Ciocalteu reagents. 5. Examining samples: 6.Protein Standard Solution: 1.StockSolution:Dissolve50mgofbovineserumalbumin(BSA)in50mlofdistilled water and store in refrigerator. 2.Working Standard: Dilute the stock solution 5 times so that final protein concentration is 200g/ml. PROCEDURE 1.Take6cleantesttubesandmarkthemasB(blank),S1,S2,S3,S4(different concentrationsofstandard)andT(testsolutions).Fillinthesetubesinthefollowing way: BS1S2S3S4T Standard Solution --0.25 ml0.50 ml0.75 ml1.0 ml-- Water1.0 ml0.75 ml0.50 ml0.25 ml---- Test Solution ----------1.0 ml Final conc.(g/ml) 0.0050.0100.0150.0200.0? 2.Add 5 ml of alkaline copper solution (reagent C) in each tube.Allow them to stand for 10 min at room temperature. 3.Add0.5mlofreagentD(dilutedFolinsreagent)ineachtube.Miximmediatelyand incubate at room temperature for 20 min, until blue colour develops. 4.Read OD at 740 nm using blank solution to set zero. OBSERVATION 1.Read OD in the following table: Tube NumberConcentration of proteinOD at 740 nm B S1 S2 S3 S4 T 2.PlotthevaluesofOD740nmagainstdifferentconcentrationsofstandardprotein solution in the graph paper for preparation of standard curve. 3.MatchtheODofT(testsample)onthestandardcurve.Thecorresponding concentrationonX-axiswillbetheactualproteinconcentration(ing/ml)inthegiven sample. RESULTS The protein concentration in the given sample is ------------------- g/ml. MARKS OBTAINED SIGNATURE OF INSTRUCTOR REVIEW QUESTIONS 1.Name two invasive and one non-invasive methods of protein estimation. 2.Name two aromatic amino acids which help in protein estimation by Lowrys method. 3.What protein have you used in Lowrys method to obtain your standard curve? 4.What is the function of serum albumin in blood? 5.Why does a too densely coloured sample fail to give proper results using the instrument? 6.How can photometry be used in disease diagnosis? 7.Which disease is caused due the deficiency of proteins in humans? 8.What are 5 common sources of protein in our diet? EXPERIMENT No. 2 OBJECTIVE To determine the concentration of glucose present in the given sample of blood, using Folin-Wu method. THEORY Glucose is an aldohexose, belonging to the carbohydrate family, and is found in large quantities throughout, the living world. It is the primary fuel for living cells. Dietary sources include plant starch, lactose, maltose and sucrose. In nature, glucose exists as D-glucose (dextrose). Glucose is a simple sugar (monosaccharide). In solution, it exists as open chain form (less than 1%), and cyclized ring forms - D-glucose (36%) and -D-glucose (63%). Glucoseisareducingsugar.Itisabletofunctionasareducingagent,becausefreeor potentiallyfree,aldehydegroupispresentinthemolecule.Thisaldehydegroupisreadily oxidized to glucoronic acid at neutral pH by mild oxidizing agents and enzymes. This property is utilized in detecting and quantitating glucose in biological fluids such as urine or blood. Thecuprousoxideformedisbrownishincolor.Foropticalquantification,thiscompoundis reactedwithphosphomolybdicacid,togivebluecolor(whoseintensityisproportionaltothe concentration of Cu2O and thereby glucose). Theoxidizingagentusedhereistartratecomplexofalkalinecopper(II)sulphate,knownas Fehlings solution. A precaution needs to be taken when used with blood, since it reacts with blood proteins and may give false results; blood is deproteinized (using tungstic acid) before use. Glucoseisaprimarysourceofenergyinmostorganisms.Itisbrokendownstepwise(during the processes of glycolysis and Krebs cycle) to give CO2 and water, and 7300 kCal of energy permole.Excessglucoseisstoredasglycogeninanimalsandasstarchinplants.Inman,a constantlevelofglucoseinmaintainedinbloodbytheinterplayofhormonessuchasinsulin, glucagon and epinephrine. Glucose determination is mainly useful in diagnosis of diabetes mellitus, the condition in which bloodglucoselevelsareelevated(hyperglycemia).Otherdiseaseslikehyperthyroidismand hyperpituitarismalsoleadstohyperglycemia.Hyperglycemiaoccursfrequentlyasaresultof over dosage of insulin antidiabetes treatment. If untreated, it may lead to coma. Yetanothermethodofglucoseestimation,whichismorerapidtheFolin-Wusmethod,isthe GOD/PODmethod.Here,glucoseoxidase(GOD)istheoxidizingagentperoxidase(POD) catalyzes the subsequent reaction to produce red coloration. REQUIREMENTS 1.Alkaline copper sulphate: Reagent A: 2% Na2CO3 in 0.1 N NaOH. Reagent B: 0.5% CuSO4 in 1 % sodium potassium tartrate. ReagentC:alkalinecoppersulphatesolution:Mix50mlof reagent A with 1.0 of reagent B prepare fresh. 2.Phophomolydic acid: Take 35gm molybic acid and add 5gm Na-Tungstate and dissolve itin100mlwater.Tothisadd200mlof1NNaOH(madeby dissolving 8g NaOH in 200ml water). Boil this solution for 30-45 minutes. When you observe a brown colour than cool the solution andmakeupto350mlbyaddingdistilledwater.Finallyadd 125ml of 85% phosphoric acid and make up the volume to 500ml. 3.Glucose standard 100mg/100ml= 1mg/ml (working). PROCEDURE 1.Making protein free filtrate of blood: takea volume of whole blood andass 9 volumes ofdeproteinizingsolution(8partsN/2sulfuricacidonepart10%sodiumtungstate). Shakeandfilter.Takethesupernatantwhichshouldbeaclearsolution.Bloodfiltrate preparedrepresents1:10dilutionofsample,or1mloffiltraterepresents0.1mlof sample. 2.Take 5 test tubes and mark them as B, S1, S2, S3, S4 and T (B=blank, S=standard and T=test solution). Fill in these test tubes in the following way: BS1S2S3S4T Standard solution-0.50 ml1.00 ml1.50 ml2.00 ml- Water2.0 ml1.50 ml1.00 ml0.50 ml-- Test solution-----2.0 ml Concentration(mg/ml)0.00.51.01.52.00? a.Add 2ml of alkaline copper solution in each tube and keep in a boiling water bath for 6 minutes. b.Cool the tubes under tap water and add 2 ml of phosphomolybdic acid solution to all the tubes. c.Mix the contents by gentle shaking and read the optical density at wavelength of 420 or 680 nm. Use blank solution to adjust zero setting on the instrument. OBSERVATION 1.Record the optical density in the following table: TubeBS1S2S3S4T OD420 2.Preparestandardcurveandmatchtheopticaldensityofthetestsample.The corresponding concentration on X-axis will give the concentration of glucose present in the blood. RESULTS The concentration of glucose was found to be ___________________. MARKS OBTAINED SIGNATURE OF THE INSTRUCTOR REVIEW QUESTION 1.Whatistheroleheattreatmentat80 Cinglucoseestimationfrombloodserumby Folin Wus method? 2.What is the normal concentration of blood sugar in human? 3.How the level of sugar is kept constant in vertebrate blood? 4.Why is glucose alone used for intravenous infusion, not fructose or sucrose? 5.What is the difference between reducing and non-reducing sugar? EXPERIMENT No. 3 OBJECTIVESeparation of chlorophyll pigments by paper chromatography. THEORY Themeanstocarryoutphotosynthesisinplantsisthegreenpigmentchlorophyll.Awhole series of chlorophylls called Chl a, b, c, d and e is known. Chlorophyll a is the primary photosynthetic pigment and is found widely distributed in the plant kingdom.Higherplantscontainpredominantlytwotypesofchlorophylls;chlorophyllaand chlorophyllb.However,chlorophyllc,dandeareencounteredonlyinalgaeandin combination with chlorophyll a. Chl b is absent in blue green algae and red algae. The basic molecule of chlorophyll consists of a tadpole like structure with a porphyrin head and a phytol tail. The porphyrin head is formed of four pyrrole rings linked together by methane (-CH=)groupsformingaringsystem.Thisproducesasequenceofconjugateddoublebonds(-C=C-C=C- .e. alternating single and double bonds) in the porphyrin ring. The skeleton of eachPyrrole ring comprises of five atoms four carbons and one nitrogen, which lie towards centre. In the centreof the porphyrin head isa bivalentMg++ (non-ionic)atom. It iscomplexed with thenitrogenatomsofthefourpyrroleringsbytwocovalentandtwocoordinatebonds.In additiontofourpyrrolerings,afifthisocyclicring(cyclopentanonering)isalsopresent.It comprisesofonlythecarbonatomsandischemicallythereactivesiteofmolecule.The porphyrinheadbearsanumberofcharacteristicsidegroupsatvariouspoints.Theidentityof the side groups provides the identity of various chlorophylls. Phytol tail is a 20-C alcohol attached to carbon 7 of the pyrrole ring IV through a propionic acid esterbond.Itisconsideredasthemostimportantsidegroup.Thelonglipophilictailis extremelyusefulintheorientationandanchoringofthechlorophyllmoleculesinthe chloroplast lamellae. Chlorophyll a differs from chlorophyll b in the nature of groups attached at carbon 3 of pyrrole ring II. Chlorophyll a has a methyl group (-CH3) while chlorophyll b has an aldehyde group (-CHO), chlorophyll a has the molecular formula C55H72O5N4Mg (mol. Wt. 893) and chlorophyll bC55H70O6N4Mg(mol.Wt.907).Chlorophyllaisbluegreenwhilechlorophyllbisyellow green.Chlorophyllaissolubleinpetroleumwhilechlorophyllbisbestsolubleinmethyl alcohol. Chlorophyll a is called primary photosynthetic pigment since it is responsible forthe emission ofelectronsduringcyclicandnon-cyclicphotophosphorylation.Chlorophyllbontheother handisanaccessorypigmentbecauseittransferstheenergyabsorbedbyittochlorophylla. Mostplantscontain2or3morechlorophyllathanchlorophyllb.Chlorophyllabsorbslight nearbothendsofvisiblespectrum.Theblueandredlight,andtransmitorreflectgreenlight and that is why the chlorophyll appears green. Thesechlorophyllsareveryeffectivephotoreceptorsbecausetheycontainnetworksof alternating single and double bonds. Such compounds are called POLYENES. These have very strongabsorptionbandsinthevisibleregionofspectrum,wherethesolaroutputreachingthe earthismaximal.ThepeakmolarabsorptioncoefficientofChlaandChlbare105cm-1M-1, among the highest absorbed for organic compounds. PAPER CHROMATOGRAPHY Thebasisofallformsofchromatographyis thepartitionanddistributioncoefficient(Kd) which describes the way in which a compound distributesitselfbetweentwoimmiscible phases.Foracompounddistributingitself betweenequalvolumesoftwoimmiscible solvents A and B, the value for this coefficient isaconstantatagiventemperatureandis given by the expression: Concentration in solvent A = Kd Concentration in solvent B The distribution of a compound can, however, bedescribednotonlyintermsofits distribution between two solvents, but also by itsdistributionbetweenanytwophases,such assolid/liquidorgas/liquidphases.Thusa distribution coefficient of a substance between silicicacidandbenzenemightbe0.5,which meansthattheconcentrationofthesubstance in benzene is twice than that in the silicic acid. Thecellulosefibresofchromatographypaper act as the supporting matrix for the stationary phase. The stationary phase may be water, a non polarmaterialsuchasliquidparaffinorimpregnatedparticlesofsolidadsorbent.Paper chromatographyisthemethodinwhichtheanalysisofanunknownsubstanceismainlydone bytheflowofsolventsonspeciallydesignedfilterpaper.Paperchromatographyseparates compounds on paper as solvent carries the mixture up the paper by capillary action. Compounds which are highly soluble in the solvent move along with the advancing solvent front, while less solublecompoundstravelslowlythroughthepaper,wellbehindthesolventfront.Asaresult the different compounds are separated on the basis of their solubility in the chosen solvent Rfisdefinedastheratioofthedistancetravelledbycompoundatitspointofmaximum concentrationtothedistancetravelledbysolvent.Boththedistancesaremeasuredfromthe point of application of the sample. Rf value has no unit. Rf =Distance travelled by substance Distance travelled by solvent REQUIREMENTS 1.Coin 2.Petroleum Ether 3.Acetone 4.Distilled Water 5.Solvent preparation: Mix Petroleum Ether, Acetone and water in the ratio of 3: 1: 1. PROCEDURE Sample Loading:1.Take the rectangular sheet of Whatman No. 1 filter paper. 2.Mark a pencil line to mark the locus of putting the leafextract.Make sure to keep this line almost 5 cm. from any of the longer side corner.3.Placetheleafonthepencilline,andstartrollingthecoinwithlesserforce,totransfer the leaf constituents on the marked line.Sample chromatographic run:1.Take 25 ml of solvent in a jar and cover with lid. 2.Put down the paper into the solvent, making sure that marked line portion does not come in direct contact with the solvent. 3. Run chromatogram for 1 hour. 4.Take paper out and mark solvent front with a pencil. The different pigments will appear like lines on the paper at different distance from the starting point, measure Rf value and record. OBSERVATION ParameterDistance travelled(cm) Solvent Chl a Chl b Others (carotene & xanthophylls) Rf. of various pigments is calculated by using formula: Rf = Distance travelled by pigment Distance travelled by solvent Rf of Chl a = Rf of chl b= Rf of carotene= RESULTS Retention Factor ofChl a = Chl b= Carotene= MARKS OBTAINED SIGNATURE OF INSTRUCTOR REVIEW QUESTIONS 1.What will happen if plants are exposed to green light? 2.Name the central element present in chlorophyll. 3.What is the partition coefficient? 4.Mention one structural difference between Chl a and Chl b. 5.What are Carotenoids? EXPERIMENT No. 4 OBJECTIVE(i)TodeterminetheMitoticIndexanddurationofmitosisinthegivenplanttissue(ii) To observe various stages of mitosis through readymade slides THEORY Mitosisissomaticcelldivision.Thecelldivisiontakesplaceintwostages;karyokinesis (division of cell nucleus and cytokinesis (division of the cytoplasm contents). The karyokinesis is divided into four phases- prophase, metaphase, anaphase and telophase. Inanypopulationofmitoticallyactivecells,onlysomecellsareindividingphases (karyokinesis)atanyonetime,whileothercellsareininterphase(non-dividingphase).The fraction or percentage of dividing cells is defined as the mitotic index (MI). MI is important to determine the duration of mitosis in the cell cycle of growing tissue/cells and also for scaling up of cell culture for various biotechnological purposes. ToobtainMI,oneshouldexamineamitoticallyactivecellpopulation,forexample,growing root tips of Alium cepa (onion). The mitotic stages are generally studied by squash preparation. MICROSCOPE DESIGN Microscopesareindispensableinbiologicalstudies.Thehumaneyehassomeintrinsic limitations as a magnifying instrument, the eye cannot focus on objects brought closer to it than approximately 25 cm. This is the distance of maximal effective magnification. Secondly, to be visible an object must subtend an angle of 1o or greater at the eye. As can be seen from the figure, the compound microscope consists of three lenses: the objective lens, the condenser and the eyepiece (ocular) lens. The specimen is mounted on a stage that can bemovedatrightanglestothemicroscopeaxis,whichisalsoreferredtoasz-axis.Thelight sourceisfocusedbyadjustingthecondensersothatlightpassesthroughtheobjectiveocular lens, which is rigidly connected to the microscope column. Focusing of the specimen is done by adjusting the distance between the specimen and the objective. WORKING PRINCIPLE OF MICROSCOPE Theprinciplefunctionofamicroscopeisthusmagnification.Inotherwords,themicroscope increases the apparent angle subtended at the eye by objects within the microscopic field. This property of a microscope is quantified as the Magnification (M). Resolving power specifies the smallestdetailthatamicroscopecanresolveinimaginganidealspecimen.Thedistance betweentwopointsinthemicroscopicfieldthatcanjustbedistinguishedfromoneanotheris called the minimum resolvable distance, Dmin. This is defined by the equation Dmin = 0.5 . N sin where, is Wavelength of light source. is the Aperture angle of the objective lens N is the Refractive index of the medium between the specimen and the objectivelens. Inordertobevisiblethroughamicroscopeanobjectmustpossesacertaindegreeofcontrast withitssurroundingmedium.Thiscontrastisaresultofthefactthatlesslightistransmitted through the object than through the medium. This decreased light transmission is caused by two factors- light absorbed by the object and light refracted out of the optical path of the microscope by a difference in the refractive index between the object and the surrounding medium. Contrast can be greatly increased by staining procedures: treatment with dyes that bind selectively either tothewholecellortocertaincellcomponents,thusproducingamuchgreaterabsorptionof light. Thusweseethatmagnification,resolutionandcontrastarethreeimportantfactorsin microscopy. OBJECTIVE I To determine the Mitotic Index and duration of mitosis in the given plant tissue. REQUIREMENTS Reagents: 1.10% Hydrochloric Acid- Take 9 ml water and add 1 ml HCl to it slowly. 2.AcetocarmineStainMix90mlAceticAcidwith110mldistilledwater(45%acetic acid). Add 2gm carmine to 100 ml 45%aceticacid and boilfor 30 minutes. Cool and make up the volume of filtered stain to 100 ml by adding 45% acetic acid and store. 3.Farmers Fixative Mix absolute alcohol and glacial acetic acid in 3:1 ratio. 4.Glycerin/ Liquid paraffin. Materials: 1.Microscope with 40X and 100X objectives. 2.Microslides. 3.Coverslips no. 1 (Square/ round) 4.Tooth picks/ matchsticks. 5.Tissue paper/ filter paper. 6.Root tip of Alium cepa (Onion 7.Slides of various stages of cell division PROCEDURE 1.Cuttheroottipfromonionbulbs,about3-4mmfromthetip(atthebaseofthe meristem) and rinse briefly in distilled water. 2.Placethetipin10%HCl(v/v)for5minutesatroomtemperatureandrinseagainin distilled water. 3.Put the tip on acetocarmine solution for 5 minutes and rinse again in distilled water. 4.Place the stained tip in a drop of water on a microslide and cover with a coverslip. 5.Gentlytaptheroottissuewiththeflatendofaglassrod/toothpick/matchstickto produceasquashhavinghomogeneouscellsuspension.Donottaphardonthe coverslip, it may break the coverslip. 6.Remove the excess liquid from under the coverslip by placing tissue paper/ filter paper overthecoverslipandpressgentlywithindexfinger.Sealedgeswithglycerin/liquid paraffin. 7.Examine under the microscope. OBSERVATION Countthenumberofmitoticandinterphasecellsunderthemicroscopeat10X40 magnification from at least three different places. No. of cells Cell TypeSite ISite IISite IIITotal Mitotic Cells Interphase Cells CALCULATION 1.Mitotic Index = No. of mitotic cells = frequency of mitotic cell Total no. of cells counted 2.Duration of Mitosis = Mitotic Index X Duration of Cell Cycle (Given that cell cycle duration = 19 hours) RESULTS Mitotic Index = Mitotic Duration = OBJECTIVE II To observe various stages of mitosis through readymade slides. MARKS OBTAINED SIGNATURE OF THE INSTRUCTOR REVIEW QUESTIONS 1.What does S-Phase of cell cycle represent? 2.What is the difference between cell division in plant cell and an animal cell? 3.Describe different stages of mitosis. 4.What is a kinetochore? EXPERIMENT No. 5 OBJECTIVE To determine the blood group and Rh status of the given sample of blood THEORY Thebiologicaluniquenessthateachindividualattainsisfrequentlynotedinthereactionsthat occur when it receives biological material from other organisms. In animals the tissues that are removedfromoneindividualandgraftedtoanotherarefrequentlysloughedofforrejected because of incompatibility between the introduced material and that of host. ABO blood typing is an excellent example of the serological principle of agglutination. Around the turn of century itwasdeterminedbyKarlLandsteinerthattherewerefourdifferentimmunologicalhuman blood types. This theory was based on the fact that two distinct antigens (agglutinogens), A and B, could be present on the surface of RBCs. Depending on the presence or absence of either or boththeantigens,bloodtypeswereestablished:A,B,ABorO.theyconstitutetheABO classification system as illustrated in the table- RBC antigen (agglutinogen) Plasma antibodies (agglutinins) Blood group A Anti BA B Anti -A B A and B None AB None Anti A and Anti-B O Of medical importance is the fact that the fluid portion of the blood, the plasma may contain antibodies(agglutinins).Ifpresenttheseantibodiesarenotreactiveagainsttheindividuals own RBCs. When mixed with the red blood cells antigens of a different blood type, however as during the course of a blood transfusion, a violent, incompatible agglutination reaction may result. Thus ABO blood typing is a routine prerequisite to blood transfusions. RH FACTOR IN HUMANS K. Landsteiner and S. Weiener discovered the Rh factor in 1940 from rabbits immunized with thebloodofthemonkeyMacacarhesus.Theresultingantibodieswerefoundtoagglutinate notonlytheRBCsofmonkeybutthoseofthehighpercentageofhumanpopulationsalso. Individuals whose blood cells react with Rh antibody are termed as Rh positive; those who do not react are termed Rh negative. The symbol Rh came from the first two letters of the species name of the monkey. A test for Rh incompatibility is accomplished by placing a drop of blood fromthesubjectonaslideandintroducinganti-Rhserum.Agglutinationoferythrocytes indicates incompatibility, whereas an even distribution of erythrocytes indicates no reaction. Theoriginalantigen,nowsymbolizedRhishighlyantigenictohumans.Thus,cross matchingofRhfactor,aswellasABOtypesofdonorandrecipientbloodisnowusedto avoidincompatibilityagglutinationreactionsfollowingtransfusions.Bloodisfrequently exchanged between the mother and the fetus during childbirth. Thus, Rh negative mothers are oftenimmunizedbybloodfromRhpositivefetuses(whichmayresultwhenfathersareRh positive)towhichtheygavebirth.Usuallynoilleffectsareassociatedwiththeexposureof the mother to the Rh- positive antigen during the first childbirth (unless the mother has been exposed to Rh antigen by transfusion). Subsequently Rh- positive children carried by the same motheragainsttheRhantigen,whicharecarriedacrosstheplacentainbloodserum.Such children may develop symptoms of hemolytic jaundice and anemia, a condition referred to as erythroblastosis fetalis . The symptoms may be mild or severe, even resulting in the death of the fetus or new born infants if appropriate steps are not taken by the physician. Intheexperimenttofollow,studentswillperformanABOtypingprocedurebyseparately mixingadropoftheirbloodwithanti-Aandanti-Bandanti-D-seraonaglassslide.The determinationofthebloodtypeismadebyobservingforagglutinationontheslide preparation as illustrated in the figure Anti A Anti B Anti A Anti B Group AGroup B Anti AAnti BAnti AAnti B Group ABGroup O REQUIREMENTS 1. Anti-A, Anti-B and Anti-D blood typing sera and 70% alcohol. 2. Microscopic slides, sterile blood lancets, absorbent cotton, wooden applicator sticks andwax pencils. PROCEDURE 1.Using a wax pencil, divide a microscopic slide in half. Label one anti-A and the other anti-B. 2.Take another slide and mark it as anti-D. 3.Place one drop of each antiserum on the appropriately labeled section of the slides. 4.Using a piece of absorbent cotton moistened with 70% ethyl alcohol, wipe the tip of the middle finger. 5.Using a sterile bold lancet, prick the disinfected area of the finger. 6.Allow one drop of blood to flow into each of antiserum on the slides. 7.With separate applicator sticks, mix each drop of blood with its respective antiserum. 8.Rock the slide between your fingers in a to and fro motion and observe both mixtures for one minute for clumping (agglutinations). 9.Observe the slides under the microscope. OBSERVATIONIn the following diagram, draw the observed antibody response against the three antigens A, B and D. Anti-A Anti-B Anti-D RESULTS 1. Determine and indicate the ABO blood type 2. Indicate the agglutinogen present 3. Indicate the agglutinin present 4. Determine and indicate the Rh type MARKS OBTAINED SIGNATURE OF INSTRUCTOR REVIEW QUESTIONS 1.Explain why type B blood cannot be transfused into a person with type blood group A? 2.Why there are no agglutinins present in the plasma of an individual with type AB blood? 3.Can blood typing be performed using only the serum portion of blood? 4.What happens to the red blood cells when they are kept in 75mM NaCl, 150mM NaCl and in distilled water? 5.What is the significance of hematinic acid in hemoglobin estimation? 6.Why hemoglobin content is more in males than in females? EXPERIMENT No. 6 OBJECTIVETo extract DNA from ripened banana fruit THEORYDNA is in the nucleus of almost every cell. The length of DNA per cell is about 100,000 times as long as the cell itself. However, DNA only takes up about 10% of the cells volume. This is because DNA is specially packaged through a series of events to fit easily in the cells nucleus. ThestructureofDNA,thedoublehelix,iswrappedaroundproteins,foldedbackontoitself, and coiled into a compact chromosome.Individualchromosomescanbestudiedusingmicroscopes,butthedoublehelixofa chromosomeissothinthatitcanonlybedetectedthroughprocedureslikeX-Ray Crystallography,InfraRedandNuclearMagneticResonancespectroscopy.Chromosomal DNA froma single cellis not visible to the naked eye. However,whenchromosomal DNA is extracted from multiple cells, the amassed quantity can easily be seen and looks like strands of mucous-like, translucent cotton. DNA is a macromolecule that is one of four that are necessary forlife(sugars,proteins,fats,andnucleicacids).DNAispolymernucleotidesincluding4 nitrogenous bases, deoxyribose sugar and phosphate (Fig 1-DNA Diagram). These nucleotides arelinkedby(i)Covalentbondbetweenphosphatesandsugarsand(ii)Hydrogenbond betweencomplementarybasepairs.DNAprovidestheblueprintfortransmissionofgenetic information.ThestrandsofDNAinsidethenucleusaredirectionsforcreatingallother components of the cell necessary for living. Detergents solubilize and break down the lipids and proteins that form the primary cell membrane and disrupt the bonds that hold the membrane together. The cell contents, including thenucleus,arethusreleasedandbecomeavailableforfurthertreatmentorisolation.Sodium laurylsulphate(SDS)isanactiveingredientindetergents.Thefinalstepinthepresent procedurerequiresalcohol.ThesolubilizedDNAcomesincontactwiththealcoholwherethe two liquid layers meet (Interface). The alcohol dehydrates and precipitates the DNA, as DNA is insoluble in the alcohol. If the procedure is done properly, fine, long strands of DNA will form at the interface and can be easily spooled onto a stirring rod. Fig. 1 Structure of a DNA molecule REQUIREMENTS 1.Table salt 2.Liquid detergent containing EDTA (Cedopol) 3.100g piece of peeled banana 4.Pestle and mortar and pestle 5.60C water bath, ice-water bath, beakers 6.Autoclaved distilled water 7.Rubber band8.Measuring cylinder (100ml)9.Pippete or graduated cylinders capable of measuring volumes of 115 ml 10. Muslin cloth11. Test tubes 12. Isopropyl alcohol (7095%) kept on ice during the DNA purification 13. Rubber policeman or other stirring implement, glass rod or wooden stir stick. PROCEDURE Preparation of Detergent Solution: 1.Combine 3 g of table salt and 80 ml of distilled water. Mix to dissolve the salt. 2.Add 10 ml of liquid detergent. Add water to bring the final volume to 100 ml. Mix gently toavoid foaming. Preparing the Lysate: 3.Mash 100 g of banana in a mortar and pestle until you obtain a smooth pulp. Place the pulp in a 250 ml beaker and add 100 ml of detergent solution. Mix gently but thoroughly. 4.Place the beaker containing the homogenized banana in a 60 C water bath for 15 minutes, mixing frequently. 5.Remove the beaker to an ice-water bath for 5 minutes to cool the pulp. 6.Insert a coffee filter into a clean beaker, fold the edge of the filter over the beaker rim and secure the coffeefilter with a rubber band. The bottom of the coffee filtershould be about an inch from the bottom of the beaker. 7.Carefully pour approximately 25 ml of homogenized banana into the coffee filter. After 510minutes,youshouldhaveatleast5mloffilteredsolution.Ifthevolumeislessthan5 ml, add more homogenized banana into the coffee filter, being careful not to tear the coffee filter. Precipitating The DNA: 8.Tilt the test tube containing 15 ml of ice-cold isopropyl alcohol and gently pipet the filtered solution down the side of the tube so that the mixture forms a layer on top of the isopropyl alcohol. Do not mix. 9.Incubatethetesttubeatroomtemperaturefor45minutesoruntiltheDNAbeginsto precipitate.10. Insert the glass rod or stir stick into the tube, and slowly rotate it to spool the DNA onto the rod. Carefully remove the glass rod, and observe the purified DNA. Record the appearance of the DNA in your laboratory notebook. OBSERVATIONS RESULT MARKS OBTAINED SIGNATURE OF INSTRUCTOR REVIEW QUESTIONS 1.What are the 5 elements that make up DNA? 2.What is the function of DNA from day to day? 3.Describe how long strands of double-helical DNA fit into the nucleus of a single cell.4.What was the purpose of using the cell lysis solution? 5.Why does the DNA become visible once the alcohol is added?6.If DNA is so thin, how is it that we are able to see it during this simple lab exercise? 7.Why is DNA referred to as your genetic fingerprint? EXPERIMENT No. 7 OBJECTIVETo study the phenomenon of plasmolysis in onion peel THEORY Plasmolysisistheprocessinplantcellswheretheplasmamembranepullsawayfromthe cellwallduetotheloss ofwaterthroughosmosis.Thereverseprocess,cytolysis,canoccur if the cell is in a hypotonic solution resulting in a higher external osmotic pressureand a net flowofwaterintothecell.Throughobservationofplasmolysisitispossibletodetermine thetonicityofthecell'senvironmentaswellastheratesolutemoleculescrossthecellular membrane. Ifaplantcellisplacedinahypertonicsolution,theplantcellloseswaterandhenceturgor pressure,makingtheplantcellflaccid.Plantswithcellsinthisconditionwilt.Furtherwater losscausesplasmolysis:pressuredecreasestothepointwheretheprotoplasmofthecell peelsawayfromthecellwall,leavinggapsbetweenthecellwallandthemembrane. Eventuallycytorrhysisthecompletecollapseofthecellwallcanoccur.Therearesome mechanismsinplantstopreventexcesswaterlossinthesamewayasexcesswatergain, but plasmolysis can be reversed if the cell is placed in a weaker solution (hypotonic solution). Stomata help keep water in the plant so it does not dry out. Wax also keeps water in the plant. The equivalent process in animal cells is called crenation. Plasmolysis only occurs in extreme conditions and rarely happens in nature. REQUIREMENTS 1.Onion, cut into slices approximately 1 cm wide, 1or 2 2.Microscope 3.Microscope slides,1per specimen 4.Cover slips, 1 per specimen 5.Distilled water 6.Salt solution (sodium chloride) 5% w/v 7.Rubber bulb 8.Pipettes 9.Forceps 10. Filter paper PRECAUTIONS 1.Onion may irritate some students eyes to the point of discomfort. It may be dipped in water to avoid it.2.Take care with microscope slides and (especially) cover slips which are fragile and break easily. Ensure students know how to deal with broken glass. 3.Sodium chloride is described as low hazard on Hazard 47B. PROCEDURE 1.Cuta1cmsquareofonion.Thenpeeloffasinglelayeroftheredcellsfromaninner fleshy leaf of the onion. 2.Place the strip on a slide. Cover it with a drop or two of distilled water. Add a cover slip. 3.Look at the cells through a microscope, starting with the low power lens. 4.Takeanotherstripofcellsfromyourplantmaterial.Thistimemountthecellswitha couple of drops of 5% sodium chloride solution. 5.Examinethroughthemicroscopeandcomparethecellstothosemountedwithdistilled water. 6.Afterafewminutesdrawoutthesodiumchloridesolutionwithapieceoffilterpaper placed at the edge of the coverslip. Replace it with distilled water added at the other side of the coverslip. 7.See what happens to the cells. OBSERVATION A. Cells mounted in presence of water General features Diagram B. Cells mounted in presence of sodium chloride General features Diagram RESULTS

MARKS OBTAINED SIGNATURE OF INSTRUCTOR REVIEW QUESTIONS 1.Explain what happened to the cells in sodium chloride solution using biological terms. Try to include these words.i.Cytoplasmii.Diffusion iii.Wateriv.Solventv.Dissolved saltsvi.Solute vii.Cell membraneviii.Vacuole ix.Cell wallx.Osmosisxi.Plasmolysisxii.Turgidxiii.Flaccidxiv.Turgor 2.What prevents the plant cells from bursting when they take in lots of water? 3.Youveseenwhathappenstocellsinepidermaltissuewhentheylosewater.How does a whole plant look when it is short of water? How does it change when you give it water? Try to explain these observations using the ideas above. 4.Animalcellsdonothavethesamestructureasplantcells.Whatdoyouthinkcould happen to an animal cell in water? 5.What would you do to investigate this process further? EXPERIMENT No. 8 OBJECTIVETo determine the level of total cholesterol present in serum. THEORY Cholesterol is a major sterol in animal tissues, is amphipathic, with a polar head (the hydroxyl group at C-3) and nonpolar hydrocarbon body (the steroid nucleus and hydrocarbon side chain at C-17) about as long as 16-carbon fatty acid. Virtually all cells and body fluids contain some steroid molecule although almost 90% of synthesis takes place in the liver and gut. A portion of thebodyscholesterolisderivedfromdietaryintake,mosttissueandplasmacholesterolis synthesizedendogenouslybytheliverandothertissuesfromsimplermoleculeslikeacetate.. Oncesynthesizedcholesterolisreleasedintothecirculationfortransportincombinationwith specificationlipoproteins.Thyroidhormonesplayaveryimportantroleinthesynthesisand degradation ofcholesterol and triglycerides.Bile acids are polar derivatives of cholesterol that act as detergents in the intestine, emulsifying dietary fats to make them more readily accessible todigestivelipases.Itisanessentialprecursorofseveralhormonesandcellmembranesand assistsinestablishingpropermembranepermeabilityandflexibility.Steroidhormonesare potent biological signals that regulate gene expression. Normal range of cholesterol in a person is 150-250 mg/dlThoughcholesterolisanessentialmoleculein mammals, its higher levels in the blood can be lethal.Elevationofthetotalcholesterolvalues is considered to be be a primary risk factor for coronaryheartdisease.Highercholesterol concentrationinbloodcandamagearteries, whichcanultimatelycauseheartdiseases linkedtocardiovascularsystem.Elevated levelsofserumcholesterolareassociatedwith atherosclerosis,necrosis,diabetesmellitus, obstructivejaundiceandmyxedema.However,thedecreasedlevelsareobservedin hyperthyroidism, malabsorption and anemia.Cholesterol reacts with acetic anhydride in the presence of glacial acetic acid and concentrated sulfuricacidtoformgreencoloredcomplex.Intensityofthecolorisproportionaltothe cholesterol concentration and can be measured at 575nm (Green-yellow filter: 520-580 nm). REQUIREMENTS 1.Test tubes 2.0.2 ml pipettes 3.Dispensers 4.Push-button pipette 5.Photometer REAGENTS 1.CholesterolReagent1:Mix5.6gof2,5dimethylbenzensulfonicacidin200mlof glacialaceticacidand300mlofaceticanhydride.Storeitinambercoloredbottleat room temperature.2.Cholesterol Reagent 2: Concentrated sulphuric acid.3.Cholesterol standard: Prepare 200 mg/dl of cholesterol in glacial acetic acid. PRECAUTIONS 1.Cholesterol reagents 1 and 2 are corrosive. Therefore should not be pipette by mouth.2.Contact with skin or clothing should be avoided.3.Use only dry glassware. The reaction is sensitive to temperature.4.Use dispensers or burettes. PROCEDURE 1.Dispense the solutions in the properly labelled tubes as follows TestStandardBlank Cholesterol reagent 1 (ml)2.52.52.5 Serum (ml)0.1-- Cholesterol standard (ml)-0.1- Distilled water (ml)--0.1 Mixwellandcoolatroomtemperaturebyplacinginawaterbath(atroom temperature). Add following reagent. Cholesterol reagent 2 (ml)0.50.50.5 2.Mix thoroughly and keep in waterbath for 10 minutes at room temperature.3.Read the absorbance of test and standard against blank at 575nm (Green yellow filter). OBSERVATION Serum total cholesterol (mg/dl) = ODtest x 200 ODstandard RESULT The concentration of serum cholesterol was found to be ___________________. MARKS OBTAINED SIGNATURE OF THE INSTRUCTOR REVIEW QUESTION 1.What is the function of cholesterol in the following (i) Bile (ii) blood (iii) Membrane 2.What will happen if the experimental temperature is increased or decreased? 3.How are LDL, VLDL and chylomicrons related to cholesterol. 4.Which molecule is the source of all carbon atoms in cholesterol. 5.Canplantsterolsbeofanytherapeuticpotentialtoremediatelevelsofcholesterol..Justify your answer.EXPERIMENT No. 9 OBJECTIVEMeasurement of Blood Pressure by using digital blood pressure measuring instrument. THEORYBloodpressure(BP)isthepressureexertedbycirculatingblooduponthewallsofblood vessels. For each heartbeat, BP varies between systolic and diastolic pressures. Systolic pressure ispeakpressureinthearteries,whichoccursneartheendofthecardiaccyclewhenthe ventricles are contracting. Diastolic pressure is minimum pressure in the arteries, which occurs near the beginning of the cardiac cycle when the ventricles are filled with blood. The mean BP, due to pumping by the heart and resistance to flow in blood vessels, decreases as the circulating bloodmovesawayfromtheheartthrougharteries.Alongwithbodytemperature,respiratory rate,andpulserate,BPisoneofthefourmainvitalsignsroutinelymonitoredbymedical professionals and healthcare providers. Pressuredropsgraduallyasbloodflowsfromthemajorarteries,throughthearterioles,the capillariesuntilbloodispushedupbackintotheheartviathevenules,theveinsthroughthe vena cava with the help of the muscles. At any given pressure drop, the flow rate is determined bytheresistancetothebloodflow.Inthearteries,withtheabsenceofdiseases,thereisvery little or no resistance to blood. The vessel diameter is the most principal determinant to control resistance. Compared to other smaller vessels in the body, the artery has a much bigger diameter (4mm), therefore the resistance is low. Thephysiologicalparametersaffectingbloodpressurearegivenbelow.Theseareinturnbe influencedbyphysiologicalfactors,suchasdiet,exercise,disease,drugsoralcohol,stress, obesity, etc.(i)Heartrate:Therateatwhichbloodispumpedbytheheart.Thevolumeofbloodflow fromtheheartiscalledthecardiacoutputwhichistheheartrate(therateof contraction) multiplied by the stroke volume (the amount of blood pumped out from the heart with each contraction).(ii)Volume of fluid or blood volume, the amount of blood that is present in the body. The more blood present in the body, the higher the rate of bloodreturn to theheartand theresultingcardiacoutput.Thereissomerelationshipbetweendietarysaltintake and increased blood volume, potentially resulting in higher arterial pressure, though this varies with the individual.(iii) Resistanceofthebloodvessels.Thehighertheresistance,thehigherthearterial pressureupstreamfromtheresistancetobloodflow.Resistanceisrelatedtovessel radius(thelargertheradius,thelowertheresistance),vessellength(thelongerthe vessel,thehighertheresistance),bloodviscosity,aswellasthesmoothnessofthe bloodvesselwalls.Smoothnessisreducedbythebuildupoffattydepositsonthe arterialwalls.Substancescalledvasoconstrictorscanreducethesizeofblood vessels, thereby increasing BP. Vasodilators (such as nitroglycerin) increase the size of blood vessels, thereby decreasing arterial pressure.(iv) Viscosityofthefluid.Ifthebloodgetsthicker,theresultisanincreaseinarterial pressure.Certainmedicalconditionscanchangetheviscosityoftheblood.For instance,anemia(lowredbloodcellconcentration),reducesviscosity,whereas increased red blood cell concentration increases viscosity. MEASUREMENT Bloodpressuresareusuallyexpressedasaratioofsystolictodiastolicpressure.Thenormal bloodpressureofanaduyltmaleisabout120mmHgsystolicand80mmhgdiastolic, abbreviatedto120/80.Thedifferencebetweensystolicanddiastolicpressureiscalledpulse pressure.Pulsepressuremaybeusedclinicallytoindicateseveralphysiologicaland pathologicalparameters,andusuallyaverages40mmHginahealthyindividual.Theratioof systolicpressuretodiastolicpressuretopulsepressuremayalsobeutilizedclinicallyasa diagnostic tool, and is usually 3:2:1. Arterialpressureismostcommonlymeasuredviaasphygmomanometer,whichhistorically usedtheheightofacolumnofmercurytoreflectthecirculatingpressure.BPvaluesare generally reported in millimetres of mercury (mmHg), though aneroid and electronic devices do notusemercury.Averagebloodpressureofadultsis110/65140/90mmHg.A sphygmomanometerconsistsofaninflatablerubbercuffattachedbyarubbertubetoa compressiblehandpumporbulb.Anothertubeattachestoacuffandtoamercurycolumn markedoffinmillimetersorananaeroidgaugethatmeasuresthepressureinmmHg.The soundsofbloodflowareheardwithinastethoscope.Bloodflowinanarteryisimpededby increasingpressurewithinasphygmomanometer.Whenthecuffofthesphygmomanometer appliessufficientpressuretocompletelyoccludebloodflow,nosoundscanbehearddistalto thecuffbecausenobloodcanflowthroughtheartery.Whencuffpressuredropsbelowthe lowest (diastolic) pressure in the vessel, the sound becomes muffled and usually disappears. The sounds heard through the stethoscope via this procedure are termed korotkoff sounds. T he present exercise employs the oscillometric for measurement of blood pressure. This method involvestheobservationofoscillationsinthesphygmomanometercuffpressurewhichare caused by the oscillations of blood flow, i.e., the pulse. It uses a sphygmomanometer cuff, like theauscultatorymethod,butwithanelectronicpressuresensor(transducer)toobservecuff pressureoscillations,electronicstoautomaticallyinterpretthem,andautomaticinflationand deflation of the cuff. The pressure sensor should be calibrated periodically to maintain accuracy. Oscillometricmeasurementrequireslessskillthantheauscultatorytechniqueandmaybe suitable for use by untrained staff and for automated patient home monitoring. In this method the cuff is inflated to a pressure initially in excess of the systolic arterial pressure andthenreducedtobelowdiastolicpressureoveraperiodofabout30 seconds.Whenblood flowisnil(cuffpressureexceedingsystolicpressure)orunimpeded(cuffpressurebelow diastolic pressure), cuff pressure will be essentially constant. WhenmeasuringBP,anaccuratereadingrequiresthatonenotdrinkcoffee,smokecigarettes, orengageinstrenuousexercisefor30 minutesbeforetakingthereading.Afullbladdermay haveasmalleffectonBPreadings;iftheurgetourinateexists,oneshoulddosobeforethe reading.For5 minutesbeforethereading,oneshouldsituprightinachairwithone'sfeetflat onthefloorandwithlimbsuncrossed.TheBPcuffshouldalwaysbeagainstbareskin,as readingstakenoverashirtsleevearelessaccurate.Duringthereading,thearmthatisused should be relaxed and kept at heart level, for example by resting it on a table. Anexampleofnormalmeasuredvaluesforaresting,healthyadulthumanis120 mmHg systolicand80 mmHgdiastolic(writtenas120/80mmHg).SystolicanddiastolicarterialBPs arenotstaticbutundergonaturalvariationsfromoneheartbeattoanotherandthroughoutthe day(inacircadianrhythm).Theyalsochangeinresponsetostress,nutritionalfactors,drugs, disease,exercise,andmomentarilyfromstandingup.Sometimesthevariationsarelarge. Hypertensionreferstoarterialpressurebeingabnormallyhigh,asopposedtohypotension, when it is abnormally low. REQUIREMENTS 1.Blood pressure Instrument 2.Volunteer PROCEDURE 1.Thevolunteershouldbecomfortablyseatedwitharmbared,slightlyflexed,abducted, andperfectlyrelaxed.Youmay,forconvenience,resttheforearmonatableinthe supinated position.2.WrapthedeflatedcuffoftheInstrumentaroundthearmwiththeloweredgeabout1 inch above the antecubital space. Close the valve on the neck of the rubber bulb. 3.Clean the earpieces of the stethoscope with alcohol before using it. Using the diaphragm ofthestethoscope,findthepulseinthebranchialarteryjustabovethebendofthe elbow, on the inner margin of the biceps branchii muscle.4.Inflatethecuffbysqueezingthebulbuntiltheairpressurewithinitjustexceeds170 mm Hg. At this point the wall of the branchial artery is compressed tightly, and no blood should be able to flow through. 5.Gradually release the pressure from the cuff using the knob. 6.Record all results in the observation table.7.Now,assumingthatthevolunteerdoesnothaveanyapparentcardiacorotherhealth problems,andiscapableofsuchanactivity,askhimtodosomeexercise,suchas jogginginthesameplacefor40to50timesandmeasurethebloodpressureagain immediatelyafterthecompletionoftheexercise.Alsorecordtheseresultsinthe observation table provided at the end of the exercise. OBSERVATIONS RESULTS MARKS OBTAINED SIGNATURE OF INSTRUCTOR REVIEW QUESTIONS: These are in turn be influenced by physiological factors, such as diet, exercise, disease, drugs or alcohol, stress, obesity, etc.1.How will the following factors effect the blood pressure. Explain why? (i)Diet (ii)Exercise(iii)Alcohol, (iv)Stress (i)Obesity 2.What dietary restrictions are imposed on patients suffering from hypertension. Why? 3. WhatistheAusculatorymethodfordeterminingbloodpressure?Whatadditional Instrument is required.? 4.DoyouthinkitissafeforoatientswithHypotensiontodonateblood.Justifyyour answer. EXPERIMENT No. 10 OBJECTIVETo observe the given slides, draw a diagram and write the general characteristic features it. SLIDE 1. Diagram Characteristic features SLIDE 2.Diagram Characteristic features SLIDE 3. Diagram Characteristic features SLIDE 4.Diagram Characteristic features SLIDE5. Diagram Characteristic features SLIDE 6. Diagram Characteristic features SLIDE 7.Diagram Characteristic features SLIDE 8. Diagram Characteristic features SLIDE 9.Diagram Characteristic features SLIDE 10. Diagram Characteristic features MARKS OBTAINED SIGNATURE OF INSTRUCTOR