lab_8 photosynthesis

8/10/2019 lab_8 photosynthesis

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Lab #8: PhotosynthesisOBJECTIVES:

After completion of this exercise, you should be able to:

1. Identify the structures of a monocot and dicot leaf involved in photosynthesis.2. Demonstrate how leaf pigments are extracted from leaves.

. Identify the pigments involved in photosynthesis by thin layerchromatography and!or paper chromatography.

". Determine the wavelengths most useful for photosynthesis bydetermining the absorption spectrum of a pigment extract.

#. Describe the roles played by light and pigment in photosynthesis.$. Demonstrate how light intensity affects the rate of photosynthesis using

spinach leaf dis%s.

INTRODUCTION:hotosynthesis is a fundamental biochemical process that sustains all life on &arth.'hotosynthetic plants, protists and bacteria are the producers in a food chain

manufacturing foods for themselves and the consumers (animals and fungi). &vencarnivorous animals are ultimately dependent on photosynthesis since carnivores eatherbivores who have fed upon plants. In addition to producing food materials (sugar andstarch), photosynthetic organisms are the source of 2* + of the air that is oxygen whichma%es respiration possible.

'

hat is common to all photosynthetic organisms is the green pigmentchlorophyll , a compound that can be activated by light. 'hotosynthesis is the conversion

of carbon dioxide and water into organic compounds. -he energy that drives this processcomes from light activated chlorophyll. hat sets photosynthetic organisms apart fromthe rest of the living world is their ability to synthesi/e from inorganic compounds andfrom the energy of the sun all the organic compounds they re0uire for building andmaintaining their protoplasm. A summary e0uation for photosynthesis is presented

below:

$ 2 3 12 4 2 3 5ight 3 hlorophyll $4 12 $ 3 $ 2 3 $ 4 2

r balanced as:

$ 2 3 $ 4 2 3 5ight 3 hlorophyll $4 12 $ 3 $ 2

rganisms having this independent nutritional status are %nown as autotrophs (self feeders). All other living organisms must depend upon the organic compoundssynthesi/ed by autotrophs6 these compounds provide them with energy (throughrespiration and fermentation) and protoplasmic building bloc%s. rganisms having thisdependent nutritional status are called heterotrophs . hy autotrophic organisms must bethe first lin% in any food chain and assume a central and crucial role in the community oflife should now be clear to you.

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In this lab exercise, you will first examine leaf structure where photosynthesis begins. ;ou will next extract the pigments from a leaf and learn how to separate these pigments by a techni0ue called chromatography . ;ou and your team will also determinethe wavelengths of light important in photosynthesis and measure the rate of

photosynthesis using spinach leaves. -his lab exercise will conclude with two activities

involving starch production in covered tructure of a 5eaf

-he leaf is the principal organ of photosynthesis. -he flattened shape is anadaptation to expose large numbers of chloroplasts to sunlight. -he leaf must also providea moist cell surface for exchange of gases with the environment yet prevent excessivewater loss. 5astly, the leaf must contain extensions of the vascular system of the stem tomove the products of photosynthesis from the leaf to the stem and roots and to movewater and minerals from the roots into the leaves. As you examine examples of monocotand dicot leaves in cross section, as% yourself which of its structural features are

particularly important in ma%ing it an efficient site for photosynthesis.

Mater als Needed : ompound microscope ?odel of leaf structure >amples of live monocot and dicot leaves 'repared slide with leaf cross section of Ligustrum , Lilium, or generali/ed

dicot 'repared slide with leaf cross section of Zea mays or a generali/ed monocot

"rocedure:

1. btain a prepared slide of a leaf cross section of Ligustrum (privet hedge), Lilium or generali/ed dicot. @iew under low and high power, using igure .1 (A) as aguide to identify the structures mentioned below.

2. Bote that the leaf is composed of a complicated arrangement of cells. ;ou willfirst notice sections of vascular bundles that are recogni/ed as veins in the wholeleaf. A vascular bundle is composed of two types of vascular tissue: #ylem andphloem . -hese are continuous with the same tissues in the stem and root. bservefrom the top of the leaf down.

a) -he cut cle , a waxy layer on the upper and lower leaf surface. In Ligustrum it is thic%er on the upper surface. hat is its functionC hy is

it thic%er on the upper surfaceC

b) -he upper ep derm s forms a protective layer and secretes a waxysubstance called cut n . -he continuous layer of cutin forms the cut cle .4ow many cells thic% is the upper epidermisC Does this layer containchloroplastsC

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igure .1: 5eaf cross sections. (A) Diagram of a dicot such as Ligustrum or Lillium .( ) Diagram of a monocot such as corn ( Zea mays ).

c) -he mesophyll is the region between the upper and lower epidermallayers excluding the vascular tissue. -he cells that ma%eup the mesophyllor parenchyma are characteri/ed by thin walls, a well developed centralvacuole, and a maEority of the leafFs chloroplasts. Chloroplasts containseveral different green or blue green chlorophyll pigments plus yellow or orange pigments called caroteno ds . -he electron micrograph of achloroplast ( igure .2)

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igure .2: >tructure of the chloroplast.

reveals groups of membranes which loo% li%e a stac% of coins called agranum . Bote that chlorophyll is found between the layers %nown asthyla$o ds . hat photosynthetic reactions occur in the chloroplastsC

ould you consider an isolated chloroplast a living unitC ecause themesophyll contains so many chloroplasts, it is the primary photosynthetictissue of the plant. It has two distinct regions: pal sade mesophyll ,cylindrical cells located nearest the upper epidermis6 and spongymesophyll , a region of irregularly shaped cells beneath the palisade layer,containing many ntercellular a r spaces .

d) -he lo%er ep derm s . Do these cells contain chloroplastsC 5ocate verysmall cells called guard cells that surround stomata (pores) leading tointercellular spaces in the mesophyll. Are there chloroplasts in the guardcellsC 4ow do you thin% guard cells functionC

. 'lants that grow in temperate climates have leaves that are structurally similarto Ligustrum or the dicot that you examined above. -hese types of plants arecalled C& plants because of the nature of their biochemical pathway. In these

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plants, carbon fixation ta%es place in the mesophyll cells and produces a threecarbon compound, < '.

". ompare the structure of your leaf with that of the demonstration model.Identify each of the modelGs components and %now at least one function ofeach.

#. 'lants that survive in drier climates and carry on photosynthesis under intenseheat and low 2 levels have special adaptations. -hese plants, such as corn,sugar cane, and some grasses, are called C' plants because their first productafter the addition of 2 is a four carbon compound (oxaloacetate). arbonfixation occurs in the (undle)sheath cells that surround the vascular bundlesrather than in the mesophyll cells, as in plants.

$ btain and examine a prepared slide of a corn ( Zea mays ) leaf H igure .1( ) . >can the slide under low and high power, and observe the vascular

bundles. Bote that all the bundles are visible only in cross section because ofthe parallel arrangement of veins in the leaf. 4ow does the mesophyll layerdiffer from that of the Ligustrum leafC In corn, the mesophyll layer contains

en/ymes needed to pump in 2 under low carbon dioxide tension. Bote thatthe (undle)sheath cells in corn contain chloroplasts while those in Ligustrum do not. ompare the si/e of the bundle sheath cells in privet and corn. hywould the si/e of these cells be importantC hat other structural adaptationscan you find in the corn cross section that would ma%e it suitable for survivalin drier climatesC

Answer the following 0uestions at home:

1. (a) hat is the function of the cuticleC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

(b) hy is it thic%er on the upper surfaceC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

2. 4ow many cells thic% is the upper epidermisC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

(a) Does this layer contain chloroplastsC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

. Is an isolated chloroplast a living unitC JJJJJJ

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". (a) Do cells of the lower epidermis contain chloroplastsC JJJJJJJJJJJJJJJJJJJJJJJJ

(b) Are there chloroplasts in the guard cellsC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

(c) 4ow do you thin% guard cells functionC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

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#. 4ow does the mesophyll layer of corn differ from that of the privet ( Ligustrum )C

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$. hy would the si/e of the bundle sheath cell be important in cornC


K. hat other structural adaptations can you find in corn that would ma%e it suitable forsurvival in drier climatesC

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ . Lsing igure .1 as a guide and label the diagram below:

E#erc se *+, - E#tract on o. "lant " gments

A variety of pigments are found in plants. Anyone who has lived up Borth duringautumnGs outburst of colors can recall. A p gment is a substance that absorbs light. If a

pigment absorbs all wavelengths of visible light it appears blac%. ther pigments absorbsome wavelengths and reflect others. ;ellow pigments reflect light wavelengths in theyellow portion of the visible light spectrum, green reflects in the green portion, etc. >omecolors are produced by only one pigment, but a great diversity of colors can be produced

by the cumulative effects of different pigments in cells.


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mentioned above, absorb wavelengths of light different from those absorbed by thechlorophylls6 this energy is then transmitted to the chlorophylls. In order to separate thevariety of colored pigments in leaves, we must first extract the pigments by brea%ingdown the cell walls.

Mater als: Acetone, # m5 Distilled water, # m5 ine sand et the stoppered tube aside in the test tube rac% for minutes.1*. Add " m5 of petroleum ether to the leaf suspension using the graduate

cylinder.

11. >topper the tube and again sha%e vigorously for * seconds.12. >et it in the test tube rac% and watch as the solvents and pigments separate.1 . 5eaf pigments are more soluble in non)polar (petroleum ether) solvents than

in polar (water li%e) solvents such as acetone. -he leaf pigments dissolved in petroleum either floats on top of the water acetone as a clear dar% green layer.

1". -he leaf pigments extracted here will next be separated from each other because of their d ..erent al solu( l ty by a techni0ue %nown aschromatography.

E#erc se *+& - Separat on o. " gments (y Th n)0ayerChromatography

In this exercise, you will use commercially prepared strips, consisting of silica gelon acetate sheet bac%ing, to ensure uniformity of adsorbent thic%ness. -here are severaladvantages of thin layer chromatography: first is the speed at which separation occurs6secondly, it is especially useful when the compounds to be separated are very closelyrelated chemically and, therefore, difficult to separate.

In thin layer chromatographic (-5 ) separation, the compounds to be separatedare spotted along a line near one end of a plastic slide coated with an inert material such

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as silica. After the spot is allowed to dry, the same end of the slide is placed in a closedchamber containing the chosen solvent. hromatography ta%es advantage of subtledifferences in the relative solubility of various substances between a stationary phase anda mobile phase. -he greater the solubility of a given pigment in the mobile organic phaserelative to its solubility in the stationary phase (silica), the greater its tendency to remain

in the organic phase and the farther its ascent as the solvent rises by capillary action.-hus, the different pigments in the original mixture will travel at different rates, and theoriginal spot will separate into a number of different bands. &ach band will move acertain fraction of the total distance traversed by the solvent front. -his fraction is acharacteristic of the particular pigment ma%ing up the band and will always be the samewhenever this pigment is chromatographed in the same solvent system. If a different

pigment or solvent is used, the distance traveled by each compound would differ fromthat in the original system. y careful choice of mobile solvent and supportive medium, itis possible to separate the components of almost any mixture of compounds.

Mater als Needed:

-5 strip, 1 x inch hlorophyll extract apillary pipette oplin Ear (chromatography chamber)

"rocedure:1. btain, from the supply area or your tray, a 1F x F thin layer chromatography

(-5 ) strip. 4andle it by the edges only and note its two sides: one shiny and theother chal%y white.

2. ?a%e a small pencil dot (not in%) on the white chal%y side 2 cm from the end andhalfway between the side edges.

. Lsing the capillary pipette draw up a small amount of the clear green li0uid fromthe top layer in the grinding tube. C1UTION : you will only get one -5 strip sodonGt ruin it by dripping or scratchingM

". 4olding the pipette vertically, touch the tip to the pigment spot and lightly pressthe pipette (not the bulb). low on the pigment spot as it forms to evaporate thesolvent.

#. 7epeat this operation as often as necessary (at least # times) to produce a dar%green spot, allowing the pigment to dry between applications. Bote the greencenter of the spot and the orange yellow ring around it. low the spot completelydry.

$. ith a paper towel, thoroughly dry the inside of the oplin (-5 ) Ear. low it dry

also.K. ?easure the runn ng sol2ent by dispensing 1 m5 of acetone into the 1* m5

graduate cylinder and then adding m5 of petroleum ether to it up to the " m5line. 3 thout %ett ng the inside walls of the Ear, empty the running solvent ontothe bottom of the -5 Ear.

. arefully lower the -5 strip (pigment spot down) between the glass ribs insidethe -5 Ear until it is standing in the solvent on the bottom. 7eplace the lid, notethe time, and do not move the Ear during the run (see igure . ).

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9. bserve through the rib less sidewal1 of the Ear the rapid separation of the leaf pigments, occurring in a matter of minutes.

1*. Bote that the first color band moving up is orange yellow. It consists of thecarotenes (pro vitamin A), further down the blue green of chlorophyll a , afterwhich follows the yellow green of chlorophyll b, and finally the several bright

yellow bands of the #anthophylls . ( -he grey layer between carotene and chl. a is pheophytin, achlorophyll molecule lac%ing the central ?g 23 ion. )11. hen the uppermost orange yellow band is within a centimeter of the top (about

K to 1* minutes), lift the strip out, replace the lid, and blow the strip dry. bservehow rapidly the fresh colors fade.

1ns%er the .ollo% ng 4uest ons:A. 4ow many different pigments were there in the extractCJJJJJJJJJJJJJJJJJJJJ

. n the basis of color, try to identify them: JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

. 7emove the chromatogram, allow it to dry, and then attach to this page.

E#erc se *+' - "aper Chromatography o. "hotosynthet c" gments

5ight must be absorbed before its energy can be utili/ed. A substance that absorbslight is a p gment . -he primary photosynthetic pigments that absorb light for

photosynthesis are chlorophylls a and b. 4owever, chlorophyll is not the only photosynthetic pigment6 accessory p gments , such as carotenes and #anthophylls ,absorb light and transfer it to chlorophyll a . -he light energy absorbed by accessory

pigments is transmitted to chlorophyll."aper chromatography is a techni0ue used for separating dissolved compounds

such as plant pigments. hen a solution of these pigments is applied to strips of paper,the pigments adsorb onto the cellulose fiber matrix of the paper. hen the tip of the paper is immersed in a solvent, the solvent is absorbed and moves through the paper. As the

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igure . : -hin layer chromatography.(A) Application of pigment. ( ) hromatography chamber.


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solvent moves through the spot where the mixture of pigments was applied, the pigmentsdissolve in the moving solvent some pigments move almost as fast as the solvent, whileothers move more slowly. -his differential movement of pigments is the result of each

pigment having a characteristic tendency to stic% to the cellulose fibers of the paper. A pigmentGsmolecular s 5e6 polar ty6 and solu( l ty determine the strength of this

tendency. 'igments adsorbed strongest move slowly, while those wea%ly adsorbed movefastest. -hus, each pigment has a characteristic rate of movement, and the pigments can be separated from each other. our bands of color will appear on the strip a yellow bandof carotenes, a yellow band of xanthophylls, a blue green band of chlorophyll a , and ayellow green band of chlorophyll b.

-he relationship of the distance moved by the pigment to the distance moved bythe solvent front is specific for a given set of conditions. e call this relationship the 7 f ,and define it as follows:

7 f N Distance moved by pigmentDistance from pigment origin to solvent front

-hus paper chromatography can be used to identify each pigment by itscharacteristic 7 f . -his 7 f value is constant for a given substance in a particular solventmatrix mixture.

Mater als Needed: >pinach leaf ilter paper strip and scissors -est tube with stopper and

pin ?illimeter ruler

>olvent (9 parts petroleumether: 1 part acetone)

-est tube rac% oin

"rocedure:1. btain a strip of chromatography paper from the supply area. 4andle the paper by

its edges so that oil on your fingers does not contaminate the paper. ?a%e a faint pencil line across the paper approximately 2 cm from the tip of the paper ( igure

.").

2. Bow place a spinach leaf on top of the chromatography paper. 7oll a coin severaltimes on top of the leaf across the pencil line in your chromatography paper. ;ouneed a concentrated green line.

. 'lace the chromatography strip in a test tube containing # m5 of chromatographysolvent (9 parts petroleum ether: 1 part acetone). 'osition the chromatographystrip so that the tip of the strip (but not the stripe of plant extract) is submerged inthe solvent. ;ou can do this by hoo%ing the strip of paper with a pin inserted inthe tubeGs stopper ( igure ."). C1UTION : -his solvent is highly flammable, soextinguish all flames, hotplates, etc. before wor%ing with this solventM

". 'lace the tube in a test tube rac% and watch as the solvent moves up the paper.

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#. 7emove your chromatography strip before the solvent reaches the top of the strip(about # 1* minutes). ?ar% the position of the solvent front with a pencil, and setthe strip aside to dry. bserve the bands of color and draw your results.7777Repeat steps 8 - 9 se2eral ' to 9; t mes n preparat on .or the ne#te#erc se+ The more o. these str ps that are a2a la(le the more ntense the

colors % ll (e .or e#erc se *+9+ 777777

$. or one of these strips, measure the distance from the origin to the solvent frontand from the origin to each pigment band. alculate the 7 f value for each

pigment6 enter results in the table provided below.

ig. .": >etup for 'aper hromatography1. ?a%e a diagram of your chromatogram in the diagram provided on the right. Identify

and label the color of each pigment band.

2. omplete -able .1 below:

-able .1: 7 f @alues for 'igments found in hloroplasts" gment Band D stance .rom

Or g nR . Value

arotene

Oant hophyllhlorophyll ahlorophyll b

. hat does a small 7 f value tell you about the characteristics of themoving moleculesC

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hromatogram

ut paper to

approximatelythis length

hoo%

cor%

solvent


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". hich are more soluble in the chromatography solvent: xanthophyllsor chlorophyll aC

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#. ould you expect the 7 f value of a pigment to change if we altered the composition ofthe solventC hy or why notC

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$. If yellow xanthophylls were present in the extract, why did the extract appear greenC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

K. Is it possible to have an 7f value greater than 1C hy or why notC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

E#erc se *+9 - Determ n ng the 1(sorpt on Spectrum .or 0ea." gments

In &xercises . and .", you used chromatography to isolate and separate pigments that were involved in photosynthesis.

hich of these pigments do you thin% support photosynthesisC JJJJJJJJ

In this exercise, you will wor% in teams of " students to carry the previousinvestigation a step further and plot the a(sorpt on spectrum of leaf pigments that you

separated by paper chromatography. -he absorption spectrum is the absorption pattern for a particular pigment, showing relative absorbance at different wavelengths of light. orexample, we %now that chlorophyll a is a green pigment, and thus reflects or transmitsgreen wavelengths of light. 4owever, we donFt %now the relative proportions ofwavelengths of light absorbed by chlorophyll a . -his information is of interest because itsuggests that those wavelengths showing greatest absorbance are important in

photosynthesis.

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-he absorption spectrum can be determined with an instrument called aspectrophotometer+ A spectrophotometer measures the proportions of light of differentwavelengths absorbed and transmitted by a pigment solution. It does this by passing a

beam of light of a particular wavelength through the pigment solution being tested. -hespectrophotometer then measures the proportion of light transmitted or absorbed by that

particular pigment and shows the reading on the calibrated scale.efore measuring the absorption spectrum of the four pigments separated by paper chromatography, consult the diagram of the electromagnetic spectrum in your textand predict the wavelengths of light at which absorption will be greatest for each

pigment. 7ecord your predictions below in -able .2.

-able .2: 'redicted avelengths of pectrophotometer 2 small test tubes or cuvettes Pimwipes 1#* m5 bea%er to hold test tubes Acetone 2* m5 bea%er to elute pigments # spec 2* tubes labeled , O, A, , - (for carotene, xanthophylls,

chlorophyll a , and chlorophyll b, total pigment) filled with acetone."rocedure:

1. ut out the pigments you separated by paper chromatography in &xercise ."and placed them in the tubes at the instructorFs table as follows:

-ube labeled carotene-ube labeled O xanthophylls-ube labeled A chlorophyll a

-ube labeled chlorophyll b -ube labeled - total pigment solution

2. -he instructor will decide when the tubes have enough pigments dissolved ineach tube. ollowing this, measure the absorption spectrum and record yourmeasurements in the space provided.

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. -he instructions that follow are for the spectrophotometer present in lab. -hemachine should have been turned on prior to class by your instructor.7earrange the tubes to A, , O, , Q - to agree with the column order on-able . (next page).

a. >elect the beginning wavelength using the up and down arrows. egin

your measurements at "** nm. Insert the tube labeled Rblan%S from thesupply counter into sample holder and close the lid. b. Tero the instrument by pressing R* Abs!1**+ -S. ;ou are now ready

to ma%e your first reading.c. -o begin your readings, remove the blan% tube and insert tube of

solution A. lose the cover and record the reading of absorbance atthis wavelength. 7emove tube of solution A, and read tubes , O, ,Q - accordingly.

d. Insert the blan% tube and set the wavelength to "2* nm (use the up anddown arrows). Again, /ero the instrument by pressing R* Abs!1**+-S.

e. 7emove the blan% and insert solution A and ta%e your second readingat "2* nm. 7epeat for , O, , Q - as in step c above.f. ontinue to ma%e readings by increasing the wavelength by 2* nm

increments, inserting the blan% and /eroing every time the wavelengthis changed, until you reach K2* nm.

". 7ecord the class data in the chart provided below.-able . : Absorbance of 'hotosynthetic 'igments

3a2elengthnm; Chlorophyll

a Chlorophyll b


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$$*

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K**

K2*

#. Lsing the readings recorded in -able . , plot the absorption spectrum foreach pigment in the graph below. hoose appropriate scales for the axes,determine dependent and independent variables and plot data points. Dra%smooth cur2es to . t the 2alues plotted+ "repare a legend and use coloredpenc ls to dent .y each o. the .our p gments+ (>ample data from previous labscan be seen in Appendix .A .D)

$. hich pigment is most important in the process of photosynthesisC JJJJJJJJJJJJJJ >upport your answer with evidence from your results:

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JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ K. hlorophyll b and the carotenoids are called accessory pigments. Lsing data from

your results, speculate about the role of these pigments in photosynthesis: JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ E#erc se *+=: The Necess ty o. Car(on D o# de n "hotosynthes s

arbon dioxide ( 2) is essential for photosynthesis. It is re0uired in the dar%reactions or alvin enson cycle that occurs in the stroma of the chloroplasts. In thisexperiment you will test the necessity of 2 in photosynthesis using Elodea and anindicator for the presence of 2.

Mater als: test tubes -est tube rac% romothymol blue Elodea sprig

>oda straw 1#* watt flood lamp with

ring stand 'reviously heated water

"rocedure:1. 'our the previously heated (to remove any air present) water into a

small bea%er. 'lace 1* drops of bromothymol blue (p4 range of $.2 =K.$) into the bea%er. Decant about a third of the colored water into a test

tube labeled U . low air with the straw through the remaining water inthe bea%er until it turns yellow. 'our an e0ual volume of this solutioninto two test tubes. 5abel these test tubes as 1, and 2. (Bromothymol blueis an indicator here for the presence of C ! " Bromothymol blue turns yello in anacid such as carbonic acid ($ ! C % & formed hen C ! combines ith ater" 'hen noC ! is present, the indicator is blue"&

2. 'lace a sprig of Elodea in tube U1.. Attach a 1#* watt flood lamp to the support stand so that the lamp is

approximately 2# cm from the base of the stand. 'lace all three tubesunder bright light for * minutes.

". After * minutes, record your data and answer the 0uestions below.Complete Ta(le *+'

-able .": olor changes before and after exposure to lightTu(e Color (e.ore e#posure to l ght Color a.ter e#posure to l ght

-ube U1-ube U2-ube U

1. 4ave any color changes ta%en placeC hichC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

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2. 4as the control tube U changed colorC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

. 4as tube U2 changed colorC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

a. hat does this indicateC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

". 4as tube U1 with the plant changed colorC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

#. Is there any 2 in the li0uid of tube U1 after exposure to lightC JJJJJJJJJJJJJJ

$. hat can you conclude happened to the 2C

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E#erc se *+>: Measur ng "hotosynthet c Rate n Sp nach 0ea.+

In the light dependent reactions of photosynthesis, light energy is used to splitwater molecules. >ome of this energy is captured by adding phosphate (' i) to AD' toma%e A-' and by adding hydrogen to BAD' 3. 2 is released as a by product. -herate of these reactions can be estimated by measuring 2 production in dis%s cut fromspinach leaves.

5eaf tissue is riddled with gas filled intercellular spaces ( ig. .#), so they floatwhen they are placed in solution. ut when leaf dis%s in a solution are subEect to avacuum, the gases in the leaves are pulled out and the spaces are filed by the li0uid.>ince fluids are heavier than gas, replacing the gas causes the leaf dis%s to sin% to the

bottom of the flas%. -hen as 2 is produced by the light reactions of photosynthesis, itdiffuses into the intercellular spaces and replaces the li0uid with gas. hen enough

2 has accumulated, each leaf dis% will regain its buoyancy and float to the surface .;our team will use this techni0ue as a method of measuring photosynthesis. >odium

bicarbonate (Ba4 ), which supplies 2 for photosynthesis, will be the solutionused to infiltrate the leaf dis%s.

ig. .# 5eaf dis% method for measuring the rate of photosynthesis.

Mater als: >pinach leaves or% borer (U") 2#* m5 &rlenmeyer flas% 'etri plates per team with one pair of forceps ottle of *.2+ (or higher) Ba4 solution @acuum flas% setup

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1#* att flood lamp with ring stand."rocedure:1. or% in teams of " students. Attach a 1#* watt flood lamp to the support stand so

that the lamp is approximately 2# cm from the base of the stand.2. ill a large bea%er (1 2 5) with cold water to act as a heat filter for the dish you will

place under the lamp. -he lamp can produce a lot of heat, which may affect the rate of photosynthesis. >et the bea%er aside for the moment.. 'our *.2+ Ba4 solution into 'etri plates so that they are about 2! full. 'our

approximately 1** m5 of *.2+ Ba4 solution into a 2#* m5 flas%.". tyrofoam board to cut on. As the dis%s are cut out, put them in the flas%with the bicarbonate solution.

#. Lse a water aspirator or the fume hood aspirator, as directed by your instructor, tosin% the discs.

a) Attach the vacuum tubing to the sidearm of the flas%. 'ut a rubber stopper

firmly in the mouth of the flas% and press a piece of tape securely over thehole in the stopper if it has one. 4old the flas% steady while you apply thevacuum.

b) -urn on the water faucet (or fume hood vacuum control) all the way. After several seconds, you should see bubbles coming out of the edges of thedis%s. 5eave the dis%s under the vacuum for approximately 1# = 2*seconds, and then release the vacuum by peeling bac% the tape or byturning off the vacuum control.

c) >wirl the flas% and wait to see whether the dis%s sin%. 7emember, theleaves will continue to float as long as they are under a vacuum.

d) ;ou may have to apply the vacuum two or three times. It is not necessary

to sin% all the dis%s. ;ou will damage the spinach tissue if youoveraspirate. ?eep the d s$s a%ay .rom (r ght l ght (y mmed atelyplac ng the .las$ n the des$ dra%er .or a .e% m nutes@@@@ After a fewminutes chec% to see if the maEority of the dis%s have sun%.

$. 'our the contents of the flas% into a large dish. Discard any dis%s that arefloating. Lse forceps to carefully transfer 1* 1# dis%s to each 'etri 'late. 'utlids on the 'etri plates. 'ut one plate in a dar% place such as a drawer6 putanother one under the flood lamp. >et the larger bea%er of water on top of the'etri plate6 then turn on the lamp. 'lace the third 'etri plate on the lab benchwhere it will receive only room light.

K. ait 2* minutes for photosynthesis to occur. hile the experiment is running,

go on to the next exercise.. After 2* minutes, count the number of dis%s that are either floating or turned onedge in each 'etri plate. 7ecord your data in table .# and answer the 0uestions.

-able .#: 7esults of >ubEecting >pinach 5eaf Dis%s to Different 5ight onditions0 ght A d s$s .loat ng d s$s .loat ng

Dar% 7oom 5ight

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Lnder 5amp1. hat hypothesis is being tested with this experimentC JJJJJJJJJJJJJJJJJJJJJJJ

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ


2. hat is thea) independent variable in this experimentC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

b) dependent variable in this experimentC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

. hat is the control treatmentC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

E#erc se *+* - " gments n "hotosynthes s+

A variety of pigments besides the chlorophylls are found in plants. A pigment is asubstance that absorbs light. If a pigment absorbs all wavelengths of visible light, itappears blac%. ;ellow pigments, for example, reflect light wavelengths in the yellow

portion of the visible light spectrum, green reflects in the green portion, etc.>ome colors are produced by only one pigment, but an even greater diversity of colorscan be produced by the cumulative effects of different pigments in cells. ;ellow, orange,and bright red colors are produced by carotenoids, also found in chloroplasts. lue,violets, purples and dar% reds are usually produced by a group of water soluble pigments,the anthocyan ns , that are located in cell vacuoles and do not contribute to

photosynthesis. Additional colors may be produced by mixtures of these pigments incells.

or%ing in teams of ", you will use the I 2PI test for starch to determine which pigment(s)in Coleus leaf support photosynthesis. efore beginning the experiment, examine yourColeus leaf and hypothesi/e about the location of photosynthesis based on leaf colors.

ypothes s:

Mater als :o Coleus plant with multicolored leaves.o 1 1*** m5 bea%er with ** m5 of water o 1 "** m5 bea%er with 2** m5 of &thanolo Dropper bottle of I 2PI solutiono 'etri plateso >cissorso 'lastic wash bottled water

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"rocedure :1. 7emove a multicolored leaf from a Coleus that has been in strong light for several

hours.2. In -able .$, list the colors of your leaf, predict the pigments present to create that

color and predict the results of the I 2PI starch test in each of the leaf.. >%etch the leaf outline in the next page, mapping the color distribution before the I 2PItest.

". >et up the boiling alcohol bath. 'lace a 1*** m5 bea%er with ** m5 of water on thehot plate. arefully place the "** m5 bea%er containing 2** m5 of *+ alcohol(ethanol)into the larger bea%er of water. -urn on the hot plate and bring both bea%ersto a boil. AdEust the temperature to maintain slow boiling. Caut on@@ Ethanol sh ghly .lamma(le@ Do not place the (ea$er o. alcohol d rectly on the hot plate@

#. arefully drop the leaf, using forceps, into the boiling alcohol solution to extract the pigments.

$. hen the leaves are almost white, use forceps to remove them from the alcohol, and

place them in separate 'etri plates. 7inse each leaf with distilled water and addenough distilled water to Eust cover the leaf. -urn off the hot plates if all teams arefinished with boiling their leaves.

K. Add a few drops of I 2PI solution to the water until a pale amber color is obtained.I2PI reacts with starch to produce a blue blac% color.

. ait about # minutes and s%etch each leaf in the next page, showing which areas ofthe leaf tested positive for starch.

1+ Record the results o. the I , ?I test n Ta(le *+=+

-able .$:'igments in photosynthesis

olor 'igments'redicted 'resence

of >tarch(3 or )

Actual results of >tarch 'resence

(3 or )


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. >%etch the Coleus leaf as instructed in steps and of the procedure.

efore the I 2PI -est After the I 2PI -est

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B. . -he following section is done in preparation for lab 1* only if the@irtual ly 5ab computer simulation will not be used. &ven so, youare advised to read the section to familiari/e yourself with what isinvolved when dealing with the life specimen.

E#erc se *+ - "reparat on .or 0a( A8

rosophila occurs naturally on rotting fruits6 both the adults and larvae feed on plant sugars and on the wild yeasts that grow in rotting fruit. -he female lays eggs on thisfruit. rosophila melanogaster is an insect that has a complete metamorphosis. -hismeans that it goes through " readily identifiable stages in its life cycle (see igure .$).-he adult female fly lays its eggs in the soft medium. -he eggs are white, very small, andhave two prong li%e proEections extending from one end. In about 2 days the eggs hatchand the larvae appear as wormli%e creatures that wor% their way through the mediumeating constantly. During this stage they molt twice. -here are, therefore, larval stagesseparated by 2 molts6 these periods of larval growth are called nstars . -he total larval

period lasts about days. -he larvae finally move onto the side of the vial and formpupae (the pupa is immobile, non feeding and protected by a case). During the pupalstage of development the larval tissues reorgani/e to form adult flies. -he pupal casesdar%en as they mature, and in about " days, the adults hatch. ?ost of the flies hatchduring the early morning (approximately $ A.?.) and are capable of mating within 1*hours. -hus any female left in a vial of hatching flies longer that this period of time will

probably have mated. In order to obtain virgin female flies, you must remove the femalesand isolate them in a new vial of medium within 1* hours.

igure .$: 5ife cycle of rosophila . 5etters a, b, and c indicate features found in theadult male only.

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Mater als:o Instant rosophila

medium and water o ulture vials and plugso Dissecting microscopeo lynap

o rosophila melanogaster cultures: dihybrid flies, bothmale and female

o x # white cardo >mall paint brusho ly morgue

"rocedure:

)" 'reparing the fly medium: btain a vial and add one scoop of fresh fruit flymedium and add 1# m5 of distilled water. 5et dry before you add the flies.

!" 'erforming your cross: btain male and female flies from the 1 generationthat have been anestheti/ed with lynap. ?ale flies can be differentiated fromfemales by their smaller si/e, wider abdominal bands, and blunt abdomen (see

igure .K).

%" 'lace your flies in a vial (on its side) containing fresh medium and close with thefoam and lid provided in the supply counter. -his is the 1 cross and will producethe 2 generation. -he 1 generation is hetero/ygous for two traits: wing shapeand eye color ( *s+ x *s+ ). P&&' @IA5 B I-> >ID& LB-I5 A55 5I&>A7& A AP& 7 -4&; I55 -L P IB -4& ?&DIA ABD DI&.

-" 5abel the vial with the following information: cross, date, your name, lab section.;our cultures should be grown at home at 2* = 22 V . o not lea e in your car"

." 7elease the parents after " = # days, when larvae are seen in the medium./" ring bac% to lab two wee%s from today (i.e. the day you crossed them in lab).

igure .K: ?ale and emale fruit flies. ompare the si/e, shape of the abdomen, and the strip pattern of the male and female flies.

1c$no%ledgments:>pecial than%s are given to the late 'rofessor ernard rit/e for allowing us to include the

extraction of chlorophyll techni0ues, &xercise .2, and the -hin 5ayer hromatography techni0ue used in&xercise . .

Re.erences:Dic%ey, Wean. 199#. Laboratory 0n estigations for Biology . enEamin! ummings

'ublishing. (&xercise .K)4elms, D. 7. 199". Biology in the Laboratory . 2 nd ed. orth 'ublishers. (&xercise .#)?organ, W. D., and arter, ?. &. 199 . 0n estigating Biology . enEamin! ummings

'ublishing. (&xercise .$)

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Appendix .A

Data from a previous lab.

Absorbance of 'hotosynthetic 'igments

3a2elengthnm; Chlorophyll a Chlorophyll b


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Appendix .

Data from a previous lab:

Absorbance of 'hotosynthetic 'igments3a2elengthnm; Chlorophyll

a Chlorophyll b


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Appendix .



a Chlorophyll b


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Appendix .D



a Chlorophyll b


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RE"ORT 0a( A*Date : FFFFFFFFFFFFFFFFFFF

Bame: JJJJJJJJJJJJJJJJJJJJJJJJJJ "hotosynthes s

&xercise .1 = >tructure of a 5eaf 1. (a) hat is the function of the cuticleC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

(b) hy is it thic%er on the upper surfaceC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

2. 4ow many cells thic% is the upper epidermisC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

(a) Does this layer contain chloroplastsC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

. (a) hat photosynthetic reaction occurs in the chloroplastsC JJJJJJJJJJJJJJJJJJJJJ

(b) Is an isolated chloroplast a living unitC JJJJJJ &xplain: JJJJJJJJJJJJJJJJJJJJJ


". (a) Do cells of the lower epidermis contain chloroplastsC JJJJJJJJJJJJJJJJJJJJJJJJ

(b) Are there chloroplasts in the guard cellsC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

(c) 4ow do you thin% guard cells functionC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ


#. 4ow does the mesophyll layer of corn differ from that of the privet ( Ligustrum )C


$. hy would the si/e of the bundle sheath cell be important in cornC


K. hat other structural adaptations can you find in corn that would ma%e it suitable for survival in drierclimatesC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

. Lsing igure .1 as a guide and label the diagram below:

&xercise . : >eparation of 'igments by -hin 5ayerhromatography

1. 4ow many different pigments were there in the extractCJJJJJJJJJJJJJJJJJJJJJJJJJ

2. n the basis of color, try to identify them: JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

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. 7emove the chromatogram, allow it to dry, and then attach to 5ab 7eport sheet on the right.

&xercise ." : >eparation of 'igments by 'aper hromatography

1. ?a%e a diagram of your chromatogram in the diagram provided on the bottom right. Identify and labelthe color of each pigment band.

2. omplete -able .2 below:-able .2: 7 f @alues for 'igments found in hloroplasts

" gment Band D stance .rom Or g n R . Valuearotene

Oanthophyllhlorophyll ahlorophyll b

. hat does a small 7 f value tell you about the characteristics of the moving moleculesC

". hich are more soluble in the chromatography solvent: xanthophylls or chlorophyll aC

#. ould you expect the 7 f value of a pigment to change if we altered the composition of the solventC hyor why notC

$. If yellow xanthophylls were present in the extract, why did the extract appear greenC

K. Is it possible to have an 7f value greater than 1C hy or why notC

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&xercise .# = Determining the Absorption >pectrum for 5eaf'igments

1. omplete -able . :-able . : Absorbance of 'hotosynthetic 'igments

3a2elengthnm; Chlorophyll a Chlorophyll b


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2. Lsing the readings recorded in -able . , plot in the graph below the absorption spectrum for each pigment.

. hich pigment is most important in the process of photosynthesisC JJJJJJJJJJJJJJ >upport your answer with evidence from your results:



". hlorophyll b and the carotenoids are called accessory pigments. Lsing data from your results,speculate about the role of these pigments in photosynthesis:




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&xercise .$ = -he Becessity of arbon Dioxide in 'hotosynthesis

1. omplete -able .# -able .#: olor changes before and after exposure to light

Tu(e Color (e.ore e# posure to l ght Color a.ter e#posure to l ght-ube U1-ube U2-ube U

2. 4ave any color changes ta%en placeC hichC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

. 4as the control tube U changed colorC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

". 4as tube U2 changed colorC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

a. hat does this indicateC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

#. 4as tube U1 with the plant changed colorC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

$. Is there any 2 in the li0uid of tube U1 after exposure to lightC JJJJJJJJJJJJJJ K. hat can you conclude happened to the 2C



&xercise .K = ?easuring 'hotosynthetic 7ate in >pinach 5eafDis%s.

-able .$: 7esults of >ubEecting >pinach 5eaf Dis%s to Different 5ight onditions

0 ght A d s$s .loat ng d s$s .loat ngDar% 7oom 5ightLnder 5amp

1. hat hypothesis is being tested with this experimentC JJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ 2. hat is the

a) independent variable in this experimentC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

b) dependent variable in this experimentC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

. hat is the control treatmentC JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

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&xercise . = 'igments in 'hotosynthesis

1. 7ecord the results of the I 2PI test in -able .K.

olor 'igments

'redicted 'resence

of >tarch(3 or )

Actual results of

>tarch 'resence(3 or )

%etch the Coleus leaf as instructed in steps and # of the procedure.

efore the I 2PI -est After the I 2PI -est

. hich pigments supported photosynthesisC7ecord your results in the RActualsS column of -able .K

lab_8 photosynthesis

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