The Floating Leaf Disk\u00a0Assay for Investigating
\nPhotosynthesis
\n(A resource page)
\nBrad Williamson<\/h4>\nIntroduction:<\/h5>\n
Trying to find a good,\u00a0quantitative procedure that\u00a0students can use for exploring photosynthesis is a challenge. The\u00a0standard procedures\u00a0such as counting\u00a0oxygen bubbles generated by an elodea stem tend to not be\u00a0\u201cstudent\u201d proof or\u00a0reliable. This is a\u00a0particular problem\u00a0if your laboratory instruction emphasizes student-generated questions.\u00a0Over the years, I have found the floating leaf disk assay technique\u00a0to be reliable and understandable to students. Once\u00a0the students are familiar with the technique they can readily design\u00a0experiments to answer their own questions about\u00a0photosynthesis.<\/p>\n<\/div>\n
<\/p>\n
The biology behind the procedure:<\/h5>\n
Leaf disks float, normally. \u00a0When the air spaces are infiltrated with solution the overall density of the leaf disk increases and the disk sinks. The infiltration solution includes a small amount of Sodium bicarbonate. Bicarbonate ion serves as the carbon source for photosynthesis. \u00a0As photosynthesis proceeds oxygen is released into the interior of the leaf which changes the buoyancy–causing the disks to rise. \u00a0Since cellular respiration is taking place at the same time, consuming oxygen, the rate that the disks rise is an indirect measurement of the net rate of photosynthesis.<\/p>\n
![\"Basis](\"..\/Images\/basis.jpg\")
<\/p>\n
Materials:<\/h5>\n\n- Sodium\u00a0bicarbonate (Baking soda)<\/li>\n
- Liquid\u00a0Soap<\/li>\n
- Plastic syringe (10 cc or\u00a0larger)\u2014remove any needle!<\/li>\n
- Leaf material<\/li>\n
- Hole punch<\/li>\n
- Plastic cups<\/li>\n
- Timer<\/li>\n
- Light source
\n![\"Materials](\"..\/Images\/mater.jpg\")
<\/h2>\n<\/li>\n<\/ul>\nOptional:<\/h5>\n\n- Buffer\u00a0Solutions<\/li>\n
- Colored\u00a0Cellophane or filters<\/li>\n
- Leaf\u00a0material of different ages<\/li>\n
- Variegated\u00a0leaf material<\/li>\n
- Clear Nail\u00a0polish<\/li>\n<\/ul>\n
Procedure:<\/h5>\n\n- Prepare\u00a0300 ml of bicarbonate solution for each\u00a0trial.<\/li>\n<\/ul>\n
![\"Bicarb](\"..\/Images\/bicarb.jpg\")
<\/small><\/div>\n <\/p>\n
<\/p>\n
<\/p>\n
\n- The\u00a0bicarbonate serves as an alternate\u00a0dissolved source\u00a0of carbon dioxide for photosynthesis. Prepare a 0.2% solution. (This is\u00a0not very\u00a0much it is only about 1\/8 of a teaspoon of baking soda in 300 ml of\u00a0water.)<\/li>\n<\/ul>\n
\n- Add 1 drop of dilute\u00a0liquid soap to this\u00a0solution. The soap wets the\u00a0hydrophobic surface of the\u00a0leaf allowing the solution to be drawn into the leaf.\u00a0 It\u2019s\u00a0difficult to quantify this since liquid\u00a0soaps vary in concentration. Avoid\u00a0suds. If your\u00a0solution generates suds then dilute\u00a0it with more bicarbonate solution.<\/li>\n<\/ul>\n
<\/p>\n
<\/p>\n
![\"soap\"](\"..\/Images\/addsoap.jpg\")
<\/small><\/div>\n <\/p>\n
\n- Cut 10 or more\u00a0uniform leaf disks for each trial.<\/li>\n<\/ul>\n
<\/p>\n
![\"Leaf](\"..\/Images\/leafdisk.jpg\")
<\/small><\/div>\n <\/p>\n
<\/p>\n
\n- Single hole\u00a0punches work well for this but stout plastic\u00a0straws will work as well.<\/li>\n
- Choice of the\u00a0leaf material is perhaps the most critical\u00a0aspect of this procedure.The\u00a0leaf\u00a0surface should be smooth and not too thick. Avoid plants with hairy\u00a0leaves. Ivy, fresh spinach, Wisconsin Fast Plant\u00a0cotyledons–all work well. Ivy seems to provide very consistent\u00a0results.Many different plant leaves work for this lab. \u00a0My classes have\u00a0found that in the spring, Pokeweed may be the best choice.<\/li>\n
- Avoid major\u00a0veins.<\/li>\n<\/ul>\n
\n- Infiltrate the\u00a0leaf disks with sodium bicarbonate\u00a0solution.<\/li>\n<\/ul>\n
\n- Remove\u00a0the piston or plunger and place the leaf disks\u00a0into the syringe barrel. Replace the plunger being careful not to crush\u00a0the leaf disks. Push on the plunger until only a small volume\u00a0of air and leaf disk remain in the barrel (< 10%).<\/li>\n<\/ul>\n\n
![\"Disks](\"..\/Images\/disksyr.jpg\")
<\/small><\/p>\n<\/div>\n\n- Pull a small volume of Sodium bicarbonate solution into the syringe. \u00a0Tap the syringe to suspend the leaf disks in the solution.<\/li>\n
- Holding\u00a0a finger over the syringe-opening, draw back on\u00a0the plunger to create a vacuum.\u00a0 Hold\u00a0this vacuum for about 10 seconds. \u00a0While\u00a0holding the vacuum, swirl the leaf disks to suspend them in the\u00a0solution.\u00a0 Let off\u00a0the vacuum.\u00a0 The\u00a0bicarbonate solution will infiltrate the\u00a0air spaces in the leaf causing the disks to sink.\u00a0You will probably have to repeat this
\nprocedure 2-3 times in order to get the disks to sink.\u00a0If you\u00a0have difficulty getting your disks to sink after about 3 evacuations,\u00a0it is usually because there\u00a0is not enough soap in the solution. \u00a0Add a few more drops\u00a0<\/span>of soap<\/span>.<\/li>\n<\/ul>\n <\/p>\n
\n![\"Evacuating](\"..\/Images\/vacuum.jpg\")
\n<\/small><\/p>\n
<\/p>\n
<\/p>\n
<\/p>\n<\/div>\n
![\"Sunk\"](\"..\/Images\/sunk.jpg\")
<\/div>\n <\/p>\n
\n- Pour the disks\u00a0and solution into a clear plastic\u00a0cup.\u00a0 Add\u00a0bicarbonate solution to a depth\u00a0of about 3 centimeters.\u00a0 Use\u00a0the same\u00a0depth for each trial.\u00a0 Shallower\u00a0depths\u00a0work just as well.<\/li>\n<\/ul>\n
<\/p>\n
\n![\"Start\"](\"..\/Images\/start.jpg\")
<\/small><\/p>\n<\/div>\n
<\/p>\n
\n- For a control\u00a0infiltrate leaf disks with a solution of only\u00a0water with a drop of\u00a0soap–no bicarbonate.<\/li>\n<\/ul>\n
\n- Place\u00a0under the\u00a0light source and start the timer. \u00a0At the end of each minute,\u00a0record the number of floating disks. Then swirl the disks to dislodge\u00a0any that are stuck against the sides of the cups. Continue until all of\u00a0the\u00a0disks are floating<\/span>.<\/li>\n<\/ul>\n\n
![\"Rising\"](\"..\/Images\/rising.jpg\")
<\/small><\/p>\n<\/div>\n
\u00a0<\/span><\/small><\/p>\nData Collection and Analysis<\/h5>\n
These data are from a investigation using grape ivy leaf disks.<\/p>\n
\n
<\/h2>\n<\/li>\n<\/ul>\nOptional:<\/h5>\n\n- Buffer\u00a0Solutions<\/li>\n
- Colored\u00a0Cellophane or filters<\/li>\n
- Leaf\u00a0material of different ages<\/li>\n
- Variegated\u00a0leaf material<\/li>\n
- Clear Nail\u00a0polish<\/li>\n<\/ul>\n
Procedure:<\/h5>\n\n- Prepare\u00a0300 ml of bicarbonate solution for each\u00a0trial.<\/li>\n<\/ul>\n<\/small><\/div>\n
<\/p>\n
<\/p>\n
<\/p>\n
\n- The\u00a0bicarbonate serves as an alternate\u00a0dissolved source\u00a0of carbon dioxide for photosynthesis. Prepare a 0.2% solution. (This is\u00a0not very\u00a0much it is only about 1\/8 of a teaspoon of baking soda in 300 ml of\u00a0water.)<\/li>\n<\/ul>\n
\n- Add 1 drop of dilute\u00a0liquid soap to this\u00a0solution. The soap wets the\u00a0hydrophobic surface of the\u00a0leaf allowing the solution to be drawn into the leaf.\u00a0 It\u2019s\u00a0difficult to quantify this since liquid\u00a0soaps vary in concentration. Avoid\u00a0suds. If your\u00a0solution generates suds then dilute\u00a0it with more bicarbonate solution.<\/li>\n<\/ul>\n
<\/p>\n
<\/p>\n
<\/small><\/div>\n <\/p>\n
\n- Cut 10 or more\u00a0uniform leaf disks for each trial.<\/li>\n<\/ul>\n
<\/p>\n
<\/small><\/div>\n <\/p>\n
<\/p>\n
\n- Single hole\u00a0punches work well for this but stout plastic\u00a0straws will work as well.<\/li>\n
- Choice of the\u00a0leaf material is perhaps the most critical\u00a0aspect of this procedure.The\u00a0leaf\u00a0surface should be smooth and not too thick. Avoid plants with hairy\u00a0leaves. Ivy, fresh spinach, Wisconsin Fast Plant\u00a0cotyledons–all work well. Ivy seems to provide very consistent\u00a0results.Many different plant leaves work for this lab. \u00a0My classes have\u00a0found that in the spring, Pokeweed may be the best choice.<\/li>\n
- Avoid major\u00a0veins.<\/li>\n<\/ul>\n
\n- Infiltrate the\u00a0leaf disks with sodium bicarbonate\u00a0solution.<\/li>\n<\/ul>\n
\n- Remove\u00a0the piston or plunger and place the leaf disks\u00a0into the syringe barrel. Replace the plunger being careful not to crush\u00a0the leaf disks. Push on the plunger until only a small volume\u00a0of air and leaf disk remain in the barrel (< 10%).<\/li>\n<\/ul>\n\n
<\/small><\/p>\n<\/div>\n\n- Pull a small volume of Sodium bicarbonate solution into the syringe. \u00a0Tap the syringe to suspend the leaf disks in the solution.<\/li>\n
- Holding\u00a0a finger over the syringe-opening, draw back on\u00a0the plunger to create a vacuum.\u00a0 Hold\u00a0this vacuum for about 10 seconds. \u00a0While\u00a0holding the vacuum, swirl the leaf disks to suspend them in the\u00a0solution.\u00a0 Let off\u00a0the vacuum.\u00a0 The\u00a0bicarbonate solution will infiltrate the\u00a0air spaces in the leaf causing the disks to sink.\u00a0You will probably have to repeat this
\nprocedure 2-3 times in order to get the disks to sink.\u00a0If you\u00a0have difficulty getting your disks to sink after about 3 evacuations,\u00a0it is usually because there\u00a0is not enough soap in the solution. \u00a0Add a few more drops\u00a0<\/span>of soap<\/span>.<\/li>\n<\/ul>\n <\/p>\n
\n
\n<\/small><\/p>\n
<\/p>\n
<\/p>\n
<\/p>\n<\/div>\n
<\/div>\n <\/p>\n
\n- Pour the disks\u00a0and solution into a clear plastic\u00a0cup.\u00a0 Add\u00a0bicarbonate solution to a depth\u00a0of about 3 centimeters.\u00a0 Use\u00a0the same\u00a0depth for each trial.\u00a0 Shallower\u00a0depths\u00a0work just as well.<\/li>\n<\/ul>\n
<\/p>\n
\n<\/small><\/p>\n<\/div>\n
<\/p>\n
\n- For a control\u00a0infiltrate leaf disks with a solution of only\u00a0water with a drop of\u00a0soap–no bicarbonate.<\/li>\n<\/ul>\n
\n- Place\u00a0under the\u00a0light source and start the timer. \u00a0At the end of each minute,\u00a0record the number of floating disks. Then swirl the disks to dislodge\u00a0any that are stuck against the sides of the cups. Continue until all of\u00a0the\u00a0disks are floating<\/span>.<\/li>\n<\/ul>\n\n
<\/small><\/p>\n<\/div>\n
\u00a0<\/span><\/small><\/p>\nData Collection and Analysis<\/h5>\n
These data are from a investigation using grape ivy leaf disks.<\/p>\n
- \n
- Buffer\u00a0Solutions<\/li>\n
- Colored\u00a0Cellophane or filters<\/li>\n
- Leaf\u00a0material of different ages<\/li>\n
- Variegated\u00a0leaf material<\/li>\n
- Clear Nail\u00a0polish<\/li>\n<\/ul>\n
Procedure:<\/h5>\n
- \n
- Prepare\u00a0300 ml of bicarbonate solution for each\u00a0trial.<\/li>\n<\/ul>\n<\/small><\/div>\n
<\/p>\n
<\/p>\n
<\/p>\n
- \n
- The\u00a0bicarbonate serves as an alternate\u00a0dissolved source\u00a0of carbon dioxide for photosynthesis. Prepare a 0.2% solution. (This is\u00a0not very\u00a0much it is only about 1\/8 of a teaspoon of baking soda in 300 ml of\u00a0water.)<\/li>\n<\/ul>\n
- \n
- Add 1 drop of dilute\u00a0liquid soap to this\u00a0solution. The soap wets the\u00a0hydrophobic surface of the\u00a0leaf allowing the solution to be drawn into the leaf.\u00a0 It\u2019s\u00a0difficult to quantify this since liquid\u00a0soaps vary in concentration. Avoid\u00a0suds. If your\u00a0solution generates suds then dilute\u00a0it with more bicarbonate solution.<\/li>\n<\/ul>\n
<\/p>\n
<\/p>\n
<\/small><\/div>\n<\/p>\n
- \n
- Cut 10 or more\u00a0uniform leaf disks for each trial.<\/li>\n<\/ul>\n
<\/p>\n
<\/small><\/div>\n<\/p>\n
<\/p>\n
- \n
- Single hole\u00a0punches work well for this but stout plastic\u00a0straws will work as well.<\/li>\n
- Choice of the\u00a0leaf material is perhaps the most critical\u00a0aspect of this procedure.The\u00a0leaf\u00a0surface should be smooth and not too thick. Avoid plants with hairy\u00a0leaves. Ivy, fresh spinach, Wisconsin Fast Plant\u00a0cotyledons–all work well. Ivy seems to provide very consistent\u00a0results.Many different plant leaves work for this lab. \u00a0My classes have\u00a0found that in the spring, Pokeweed may be the best choice.<\/li>\n
- Avoid major\u00a0veins.<\/li>\n<\/ul>\n
- \n
- Infiltrate the\u00a0leaf disks with sodium bicarbonate\u00a0solution.<\/li>\n<\/ul>\n
- \n
- Remove\u00a0the piston or plunger and place the leaf disks\u00a0into the syringe barrel. Replace the plunger being careful not to crush\u00a0the leaf disks. Push on the plunger until only a small volume\u00a0of air and leaf disk remain in the barrel (< 10%).<\/li>\n<\/ul>\n\n<\/small><\/p>\n<\/div>\n
- \n
- Pull a small volume of Sodium bicarbonate solution into the syringe. \u00a0Tap the syringe to suspend the leaf disks in the solution.<\/li>\n
- Holding\u00a0a finger over the syringe-opening, draw back on\u00a0the plunger to create a vacuum.\u00a0 Hold\u00a0this vacuum for about 10 seconds. \u00a0While\u00a0holding the vacuum, swirl the leaf disks to suspend them in the\u00a0solution.\u00a0 Let off\u00a0the vacuum.\u00a0 The\u00a0bicarbonate solution will infiltrate the\u00a0air spaces in the leaf causing the disks to sink.\u00a0You will probably have to repeat this
\nprocedure 2-3 times in order to get the disks to sink.\u00a0If you\u00a0have difficulty getting your disks to sink after about 3 evacuations,\u00a0it is usually because there\u00a0is not enough soap in the solution. \u00a0Add a few more drops\u00a0<\/span>of soap<\/span>.<\/li>\n<\/ul>\n<\/p>\n
\n
\n<\/small><\/p>\n<\/p>\n
<\/p>\n
<\/p>\n<\/div>\n
<\/div>\n<\/p>\n
- \n
- Pour the disks\u00a0and solution into a clear plastic\u00a0cup.\u00a0 Add\u00a0bicarbonate solution to a depth\u00a0of about 3 centimeters.\u00a0 Use\u00a0the same\u00a0depth for each trial.\u00a0 Shallower\u00a0depths\u00a0work just as well.<\/li>\n<\/ul>\n
<\/p>\n
\n<\/small><\/p>\n<\/div>\n<\/p>\n
- \n
- For a control\u00a0infiltrate leaf disks with a solution of only\u00a0water with a drop of\u00a0soap–no bicarbonate.<\/li>\n<\/ul>\n
- \n
- Place\u00a0under the\u00a0light source and start the timer. \u00a0At the end of each minute,\u00a0record the number of floating disks. Then swirl the disks to dislodge\u00a0any that are stuck against the sides of the cups. Continue until all of\u00a0the\u00a0disks are floating<\/span>.<\/li>\n<\/ul>\n\n<\/small><\/p>\n<\/div>\n
\u00a0<\/span><\/small><\/p>\n
Data Collection and Analysis<\/h5>\n
These data are from a investigation using grape ivy leaf disks.<\/p>\n
- Place\u00a0under the\u00a0light source and start the timer. \u00a0At the end of each minute,\u00a0record the number of floating disks. Then swirl the disks to dislodge\u00a0any that are stuck against the sides of the cups. Continue until all of\u00a0the\u00a0disks are floating<\/span>.<\/li>\n<\/ul>\n
- For a control\u00a0infiltrate leaf disks with a solution of only\u00a0water with a drop of\u00a0soap–no bicarbonate.<\/li>\n<\/ul>\n
- Pour the disks\u00a0and solution into a clear plastic\u00a0cup.\u00a0 Add\u00a0bicarbonate solution to a depth\u00a0of about 3 centimeters.\u00a0 Use\u00a0the same\u00a0depth for each trial.\u00a0 Shallower\u00a0depths\u00a0work just as well.<\/li>\n<\/ul>\n
- Remove\u00a0the piston or plunger and place the leaf disks\u00a0into the syringe barrel. Replace the plunger being careful not to crush\u00a0the leaf disks. Push on the plunger until only a small volume\u00a0of air and leaf disk remain in the barrel (< 10%).<\/li>\n<\/ul>\n
- Infiltrate the\u00a0leaf disks with sodium bicarbonate\u00a0solution.<\/li>\n<\/ul>\n
- Cut 10 or more\u00a0uniform leaf disks for each trial.<\/li>\n<\/ul>\n
- Add 1 drop of dilute\u00a0liquid soap to this\u00a0solution. The soap wets the\u00a0hydrophobic surface of the\u00a0leaf allowing the solution to be drawn into the leaf.\u00a0 It\u2019s\u00a0difficult to quantify this since liquid\u00a0soaps vary in concentration. Avoid\u00a0suds. If your\u00a0solution generates suds then dilute\u00a0it with more bicarbonate solution.<\/li>\n<\/ul>\n
- The\u00a0bicarbonate serves as an alternate\u00a0dissolved source\u00a0of carbon dioxide for photosynthesis. Prepare a 0.2% solution. (This is\u00a0not very\u00a0much it is only about 1\/8 of a teaspoon of baking soda in 300 ml of\u00a0water.)<\/li>\n<\/ul>\n
- Prepare\u00a0300 ml of bicarbonate solution for each\u00a0trial.<\/li>\n<\/ul>\n
![\"Graph\"](\"..\/Images\/pgraph.jpg\")
<\/p>\n![\"Inverse\"](\"..\/Images\/pgraphi.jpg\")
<\/p>\n![\"Direct\"](\"..\/Images\/pgraphd.jpg\")
<\/p>\n![\"Photosynthesis](\"..\/Images\/ph_res.jpg\")
<\/p>\n