AS Biology Practical Assessment Booklet
AS Biology PracticalAssessment BookletRyan Mercer-Southern Regional College
INDEX1. FOOD TESTSCarry out practical work to detect the presence of carbohydrate and proteins using biochemical tests: iodine test; Benedict’s test; glucose specific tests; Biuret test2. CHROMATOGRAPHYCarry out practical work to identify amino acids using paper chromatography: prepare, run and develop the chromatogram; calculate Rf values.3. ENZYME ACTIVITYCarry out practical work: investigating factors, such as temperature, pH, substrate concentration and enzymeconcentration that affect enzyme activity; illustrating enzyme immobilisation; using a colorimeter to follow the course of a starch–amylase catalysed reaction (or otherappropriate reaction).4.Homogenisation and CentrifugationCarry out practical work to demonstrate knowledge and understanding of the use of homogenisationand centrifugation;5. MICROSCOPESCarry out practical work: examining photomicrographs and electron micrographs; recognising cell structures from photomicrographs and electron micrographs; drawing individual cells or cell sections; staining tissues to aid observation when using a microscope (for example using iodine or methyleneblue); calculating true size (in μm) and magnification, including using scale bars; and using a graticule and stage micrometer to measure cell length.Ryan Mercer-Southern Regional College
6. Measuring Water and Solute PotentialCarry out practical work to include: measuring the average water potential of cells in a plant tissue: using a weighing method for apotato or other suitable tissue; calculating the percentage change in mass; and determining theaverage water potential from a graph of percentage change in mass against solute potential ofimmersing solution; measuring the average solute potential of cells at incipient plasmolysis by: using onion epidermisor other suitable tissue; calculating percentage plasmolysis; and determining the average solutepotential from a graph of percentage plasmolysis against solute potential of the immersing solution(at 50% plasmolysis the average pressure potential is zero);7. OBSERVING MITOSIScarry out practical work: preparing and staining root tip squashes; recognising chromosomes at different stages of cell division; identifying the stages of mitosis; and examining prepared slides or photographs of the processes of mitosis and meiosis and identifyingthe structures visible and the different stages;8. Ileum and Mesophyll LeafCarry out practical work to include: examining stained sections of the ileum using the light microscope and electron micrographs orphotomicrographs to identify the villi (and associated blood capillaries and lacteals), crypts ofLieberkühn (and Paneth cells), mucosa, columnar epithelium, goblet cells, muscularis mucosa,submucosa, muscularis externa and serosa; examining sections of a mesophytic leaf using the light microscope or photographs to identify theepidermal layers, waxy cuticles, palisade mesophyll, chloroplasts, spongy mesophyll, vascular tissue(xylem and phloem), and guard cells and stomata; and making accurate drawings of sections of the ileum and the leaf to show the tissue layers anddrawing block diagrams of tissues within the ileum and the leaf.9. RESPIROMETERCarry out practical work to include understanding how to use a simple respirometer to: measure O2 consumption (with KOH present); and measure the net difference between CO2 production and O2 consumption (with no KOH present)and so determine CO2 production.Ryan Mercer-Southern Regional College
10. BLOOD VESSEL SLIDES AND HEARTCarry out practical work: examining prepared slides and/or photographs of blood vessels (in section) to distinguish betweenarteries, veins and capillaries; and dissecting the mammalian heart to identify heart chambers, AV valves, semilunar valves, chordaetendinae, papillary muscles, interventricular septum and major blood vessels: vena cavae,pulmonary artery and aorta;11. BLOOD CELL IDENTIFICATIONCarry out practical work examining stained blood films using light microscopes and/orphotomicrographs to identify red blood cells, polymorphs, monocytes, lymphocytes and platelets;.12. ECOLOGICAL SAMPLINGCarry out practical work to include the qualitative and quantitative techniques used to investigatethe distribution and relative abundance of plants and animals in a habitat: sampling procedures to include: random sampling; line transect; and belt transect; sampling devices, including quadrats, pin frames, pitfall traps, sweep nets and pooters; estimating species abundance, density, frequency and percentage cover; and appreciating and, where possible, measuring the biotic and abiotic factors that may be influencingthe distribution of organisms.Ryan Mercer-Southern Regional College
1. FOOD TESTSCarry out practical work to detect the presence of carbohydrate and proteins using biochemicaltests: iodine test; Benedict’s test; glucose specific tests; Biuret test1. Starch (iodine test). Add a few drops of iodine/potassium iodide solution to the sample. Ablue-black colour indicates the presence of starch as a starch-polyiodide complex is formed.2.Reducing Sugars (Benedict's test). All monosaccharides and most disaccharides (exceptsucrose) are called reducing sugars because they will reduce ions like Cu2 . Add a few mL ofBenedict’s reagent (which is a copper (II) sulphate solution) to the sample. Shake, and heatfor a few minutes at 95 C in a water bath. A coloured precipitate of copper (I) oxideindicates reducing sugar. The colour and density of the precipitate gives an indication of theamount of reducing sugar present, so this test is semi-quantitative. The original pale bluecolour means no reducing sugar, a green precipitate means relatively little sugar; a brown orred precipitate means progressively more sugar is present.Non-reducing Sugars (Benedict's test). Sucrose is called a non-reducing sugar because itdoes not reduce copper sulphate, so there is no direct test for sucrose. However, if it is firsthydrolysed to its constituent monosaccharides (glucose and fructose), it will then give apositive Benedict's test. So sucrose is the only sugar that will give a negative Benedict's testbefore hydrolysis and a positive test afterwards. First test a sample for reducing sugars, tosee if there are any present before hydrolysis. Then, using a separate sample, boil the testsolution with dilutehydrochloric acid for a few minutes to hydrolyse the glycosidic bond. Neutralise the solutionby gently adding small amounts of solid sodium hydrogen carbonate until it stops fizzing,then test as before for reducing sugars.3.Clinistix - used to detect glucoseClinistix strips are impregnated with chemicals that change colour in the presence ofglucose. Clinistix is specific (unlike Benedict's test). Strips turn purple/blue if glucose ispresentCan be a way for doctors to detect the presence of glucose in the urine (symptom ofdiabetes)4. Protein (biuret test).Add a few mL of biuret solution to the sample. Shake, and the solutionturns lilac-purple,indicating protein. The colour is due to a complex between nitrogen atoms in the peptidechain and Cu2 ions, so this is really a test for peptide bonds.Ryan Mercer-Southern Regional College
2. CHROMATOGRAPHYCarry out practical work to identify amino acids using paper chromatography: prepare, run and develop the chromatogram; calculate Rf values.Amino acids have no colour. Therefore all of these procedures need to be carried out"blind", and the results will be seen when a revealing agent (ninhydrin) is sprayed on theresulting chromatogram.You are provided with a number of solutions of amino acids, and solution X (a mixture of 2amino acids). Do not use the same pipette for more than one liquid.Chromatography paper must not be touched with the hands (at the bottom, at least). Useplastic/rubber gloves, and work on a clean surface (e.g. inside page of pad of paper).Cut a suitable length of chromatography paper (slightly longer than the glass chamber) andmark it with a pencil line about 1.5 cm from the bottom. Again using a pencil, put 3 markson the line forming crosses, the outer ones labelled with (3 letter codes for) the amino acidsyou are going to use, and X in the middle. Put your name at the top.Using 3 different pipettes, place a drop of each amino acid, and the mixture X, at theappropriate positions on the line. If you have time, gently warm the paper and repeat thespotting process (on the same positions) to raise the concentration of the amino acids, butensure that the paper is completely dry before proceeding.Partially fold the paper in half lengthways, to remove its tendency to curl up.In a fume cupboard or a secluded (well ventilated?) area of the lab :Using a funnel, pour a small amount of chromatography solvent (butanol/ethanoic acid) intothe glass chamber (to about 1 cm depth). Place on the lid to allow the atmosphere tobecome saturated with vapour. Leave the chamber on the bench in its final position, so thatit does not splash up; bring the paper to meet it.Line up the paper with the (outside of the) chamber, and either fold over the top or cut it sothat it will fit. The solvent should touch the lower part of the paper but not cover the dropson the line.Attach the paper to the lid, and then place the lid on. The paper should not touch the sidesof the chamber.The solvent should gradually rise up the paper, passing the line and heading upwards.After about 3 hours, the liquid should have risen about three-quarters of the height of thepaper. If the solvent nears the top of the paper, proceed immediately to the next stage.Remove the paper from the apparatus, and use a pencil to mark the position of the solventfront. Up to 5 pieces of chromatography paper can be placed across a clean A4 sheetof paper, stapled at the top of the chromatograms.Place the chromatograms into an oven at about 45 C to dry.Either in a fume cupboard or outside on a still day, spray ninhydrin evenly over the paper.Care: ninhydrin is dissolved in an inflammable solvent.Ryan Mercer-Southern Regional College
Return the chromatograms to the oven to develop the colour. Spots should be visible aspurplish smears on the paper.The image of the spots may be enhanced if the sheet is photocopied on a darker thannormal setting.Calculation of Rf valuesMeasure the distance from the start line to the solvent front and to the front of each spot.For each spot, calculate the Rf value (Rf means relative to front):distance moved by spotdistance moved by solvent frontCompare the values you obtain with reference Rf values. Different solvents and differenttypes or makes of chromatogaphy papers will give slightly different results.One or both of the spots from solution X may be at the same level as another (known)amino acid alongside it. This should assist in identification.Ryan Mercer-Southern Regional College
3. ENZYME ACTIVITYCarry out practical work: investigating factors, such as temperature, pH, substrate concentration and enzymeconcentration that affect enzyme activity; illustrating enzyme immobilisation; using a colorimeter to follow the course of a starch–amylase catalysed reaction (or otherappropriate reaction).USING A COLORIMTER: Investigate how enzyme concentration affects the initial rate of anenzyme-controlled reactionPROCEDUREMilk protein (casein) is broken down by protease enzymes such as trypsin. The opaquewhite colour of the milk is replaced by a clear solution. Light passes more easily through thefinal solution and so the reaction can be monitored using a colorimeter (see diagram) orlight sensor.1. Plan how you will dilute the 1% trypsin stock solution with distilled water to produceadditional test solutions of 0.2%, 0.4%, 0.6% and 0.8%. Aim to produce 10 cm3 of eachconcentration. Once checked, make up the solutions as planned.2. Place 2 cm3 of trypsin solution and 2 cm3 of distilled water into a cuvette. Use this as areference cuvette to set the colorimeter absorbance to zero.3. Measure 2 cm3 of milk suspension into a second cuvette.4. Add 2 cm3 of trypsin solution to the milk in the cuvette. Working quickly, mix and placethe solution into the colorimeter and start the stop clock.5. Measure absorbance immediately and then at 15 second intervals (or more frequently ifrecording electronically) for 5 minutes, or until there is little change in absorbance.6. Rinse the cuvette with distilled water and repeat for each concentration.Ryan Mercer-Southern Regional College
ANALYSIS OF RESULTS1. Record your results in a suitable table.2. Plot a graph of absorbance against time. It should be possible to plot each concentration as adifferent line on the same axes.3. Use the graph to determine the initial rate of reaction for each concentration. Do this by drawinga tangent to the initial part of each curve and calculating the gradient of each line.4. Draw a second graph to show the initial rate of reaction against the concentration of the enzyme.5. Write a short conclusion to describe and explain the result of this investigation.Ryan Mercer-Southern Regional College
Ryan Mercer-Southern Regional College
Better milk for cats: immobilised lactaseused to make lactose-reduced milkMethod1. Mix the enzyme with the sodium alginate solution, then draw it up into a 10 ml syringe.2. Add the alginate-enzyme mixture a drop at a time from the syringe to the calcium chloridesolution and observe the formation of small beads. Do not allow the tip of the syringe tocome into contact with the calcium chloride solution, as this will cause the alginate toharden, blocking the outlet. The beads, which contain the enzyme immobilised in a matrixof calcium alginate, should be allowed to harden for a few minutes.3. Attach a short length of tubing to the tip of a syringe barrel. Place a small disc of nylongauze inside the barrel, to prevent the beads from blocking the syringe outlet.4. Separate the beads of immobilised enzyme from the liquid with the tea strainer.5. Carefully tip the beads into the syringe barrel.6. Close the tubing on the syringe barrel using a tubing clip.7. Test the milk before treatment using the glucose test strips, to ensure that it does notcontain any glucose.8. Pour a small volume of milk over the enzyme beads, then undo the clip and allow thetreated milk to run into a small beaker.9. Test the milk leaving the column using the glucose test strips. If necessary, return thetreated milk to the column until the desired concentration of glucose is achieved.Ryan Mercer-Southern Regional College
4.Homogenisation and CentrifugationCarry out practical work to demonstrate knowledge and understanding of the use ofhomogenisation and centrifugation;Homogenization:The suspended cells are then disrupted by the process of homogenization.It is usually done by:(i) Grinding(ii) High Pressure (French Press or Nitrogen Bomb),(iii) Osmotic shock,(iv) Sonication (ultrasonic vibrations). Grinding is done by pestle and mortar or potter homogenizer(a high-speed blender). The later consists of two cylinders separated by a narrow gap.The shearing force produced by the movement of cylinders causes the rupture of ceils. Ultrasonicwaves are produced by piezoelectric crystal. They are transmitted to a steel rod placed in thesuspension containing cells. Ultrasonic waves produce vibrations which rupture the cells. The liquidcontaining suspension of cell organelles and ether constituents is called homogenate. Sugar orsucrose solution preserves the cell organelles and prevents their clumping.Ryan Mercer-Southern Regional College
Centrifugation:The separation (fractionation) of various components of the homogenate is carried out by a series ofcentrifugations in an instrument called ultracentrifuge. The ultracentrifuge has a metal rotorcontaining cylindrical holes to accommodate centrifuge tubes and a motor that spin the rotor at highspeed to generate centrifugal forces. Theodor Svedberg (1926) first developed die ultracentrifugewhich he used to estimate the molecular weight of hemoglobin.Present day ultracentrifuge rotate at speeds up to 80,000 rpm (rpm rotations per minute) andgenerates a gravitational pull of about 500,000 g, so that even small molecules like t-RNA, enzymescan sediment and separate from other components. The chamber of ultracentrifuge is kept in a highvacuum to reduce friction, prevent heating and maintain the sample at 0-4 C.During centrifugation, the rate at which each component settle down depends on its size and shapeRyan Mercer-Southern Regional College
5. MICROSCOPESCarry out practical work: examining photomicrographs and electron micrographs; recognising cell structures from photomicrographs and electron micrographs; drawing individual cells or cell sections; staining tissues to aid observation when using a microscope (for example using iodine ormethylene blue); calculating true size (in μm) and magnification, including using scale bars; and using a graticule and stage micrometer to measure cell length.Observing Onion CellsMethod:1) Collect all your apparatus (carrying the microscope carefully).2) Using tweezers carefully peel off a thin layer of epidermis from the onion.3) Lay the membrane on the microscope slide in a single flat layer.4) Place a very small drop of iodine on to the membrane.5) Carefully lower a cover slip on top of the membrane (make sure there are no air bubbles).6) Place the slide on the stage of the microscope.7) Make sure the lowest objective lens is over the specimen.8) Carefully use the course focusing knob to lower the objective lens to just above the slide.9) Look through the eye piece and carefully use the fine focusing knob to focus the image.Ryan Mercer-Southern Regional College
Observing Cheek CellsMethod:1. Gently scrape the inner side of the cheek using a toothpick, which will collect some cheekcells.2. Place the cells on a glass slide that has water on it.3. Mix the water and the cheek cells using a needle and spread them.4. Take a few drops of Methylene blue solution using a dropper and add this to the mixtureon the slide.5. After 2-3 minutes remove any excess water and stain from the slide using a blotting paper.6. Take a few drops of glycerine using a dropper and add this to the test mixture.7. Take a clean cover slip and lower it carefully on the mixture with the aid of a needle.8. Using a brush and needle, press the cover slip gently to spread the epithelial cells.9. Remove any extra liquid around the cover slip using a blotting paper.10. Place this glass side on the stage of the compound microscope and view it.Ryan Mercer-Southern Regional College
Ryan Mercer-Southern Regional College
Ryan Mercer-Southern Regional College
Eyepiece graticuleThe eyepiece graticule is a glass disc fitted into the eyepiece of the microscope. These canbe fitted to existing eyepieces or eyepieces can be purchased with graticules already fitted.The disc is marked with a fine scale from 0 to 100. The absolute size of the scale is notimportant as this is what will be calibrated.Stage micrometerThe stage micrometer is used to calibrate the eyepiece graticule. A stage micrometerconsists of a microscope slide on which is engraved a fine and accurate scale. Because thescale has to be accurately produced to give reference dimensions, stage micrometers aremuch more expensive than eyepiece graticules.Method 1When carrying out calibration, each objective lens has to be separately calibrated. This willresult in separate calibration factors for each objective.Start with the lowest power objective on the microscope. The scale on the stage micrometeris aligned with the scale of the eyepiece graticule and then a reading is taken from thescales. These readings are then used to calculate the calibration factor for the objective lensin use. The following example shows how to calibrate the graticule for the
iodine test; enedict’s test; glucose specific tests; iuret test 1. Starch (iodine test). Add a few drops of iodine/potassium iodide solution to the sample. A blue-black colour indicates the presence of starch as a starch-polyiodide complex
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