Special Requirements
1. Incubation. Incubation is required for Enzyme Function of Proteins and for Starch Composition.
2. Fresh material. The potato and onion for Carbohydrates (LM page 34) and the materials for Testing
the Chemical Composition of Everyday Materials and an Unknown (LM page 41) can be obtained
locally, close to time of use.
Notes
1. Test tubes. The exercises in this laboratory require students to add solutions to test tubes. As an
expedient, the students are asked to mark off the tubes at various centimeter levels with a ruler and
then to fill to these marks. You may prefer to have students use a standard method of measuring
volume, such as with a graduated cylinder or a pipette. Most experiments use the standard size test
tube. A few experiments require the large size test tube. Mini test tubes can be substituted for most
laboratory exercises as long as the total volume in a given tube does not exceed 9 cm. This will
reduce the volume of reagents used by approximately one-third.
Test tube sizes/volumes are as follows:
Mini
13 100 mm (Carolina 73-0008)
1 cm = 1.0 ml
Standard
16 150 mm (Carolina 73-0014)
1 cm = 1.5 ml
Medium large
20 150 mm (Carolina 73-0019)
1 cm = 2.4 ml
Large
25 150 mm (Carolina 73-0025)
1 cm = 4.0 ml
2. Students results. If students results are not as expected, you may wish to test the reagents.
3. Chemical solutions. All chemicals for this laboratory can be purchased from Carolina Biological
Supply. Information in the Materials and Preparations section may be helpful to those who wish
to prepare their own.
4. An incubator is recommended for some tests. However, a hot water bath (hot plate, beaker of
water, Celsius thermometer, test-tube clamp, beaker clamps, boiling chips) will also work.
wax pencils
rulers, plastic millimeter
test tubes and racks
water, distilled
boiling chips, pumice (Carolina 84-8278, -8280)
13
3.1
Biuret reagent. 30 ml per student group should be sufficient (using standard test tubes for all procedures).
If you buy prepared biuret, use only ten to fifteen drops; otherwise, the solution will be too dark, or dilute
to a 10% solution (10 ml biuret with 90 ml distilled water). To prepare your own biuret reagent, maintain
separate stock solutions of 3% copper sulfate3 g of copper sulfate (cupric sulfate, Carolina 85-6550) per 100
ml of distilled water and 10% potassium hydroxide or sodium hydroxide100 g of potassium hydroxide
(Carolina 88-3488) or sodium hydroxide pellets (Carolina 88-9470) per 1,000 ml of distilled water). Adding
five drops of copper sulfate solution and ten drops of potassium hydroxide solution to each experimental
tube produces more consistent results. Biuret reagent should be prepared fresh for each lab.
1% albumin solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). Prepare pH 7 buffer solution (Carolina 84-9380) as per directions on the vial, and dissolve 1 g of albumin per 100 ml of buffer. Allow time for precipitation to occur, and then decant. Also, check pH with indicator paper, and adjust to pH 7 with dilute acid or base. Swirl the stock, before distributing it to students.
12% pepsin solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). To make a 1% solution, dissolve 1 g of pepsin in 100 ml of distilled water. Stir to dissolve.
12% starch solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). Care must be taken in preparing this solution. To make a 1% solution, dissolve 1 g of starch in a small
amount of cold water to form a pourable paste. Add this to 100 ml of boiling distilled water, while stirring,
and mix a few minutes. Cool. Add a pinch of sodium chloride (NaCl). If refrigerated, this solution will last
for several weeks; otherwise, a fresh supply should be prepared each day.
0.2% hydrochloric acid (HCl). 20 ml per student group should be sufficient (using standard test tubes for
all procedures). Add 2.3 ml of concentrated HCl to 100 ml of distilled water.
3.2 Carbohydrates (LM pages 3437)
_____
1% starch solution (Carolina 89-2530), see prep notes above
_____
iodine-potassium-iodide (IKI) solution, premade (Carolina 86-9051, -9053, -9055),
or see Laboratory 2 for preparation instructions from
potassium iodide (KI) and iodine (I) crystals
_____
dropper bottles, one per student group (Carolina 71-6525)
_____
razor blades, single-edged
_____
cutting board for potato, onion
_____
potato, fresh
_____
microscopes, compound light
_____
lens paper
_____
slides
_____
coverslips
_____
onion, fresh
_____
paper towels
_____
boiling water bath:
_____ hot plate
_____ large beakers
_____ boiling chips, pumice (Carolina 84-8278, -82800
_____ beaker clamps
_____ thermometer, organic based, 50150C range (optional)
_____
1% glucose (dextrose) solution (Carolina 85-7430)
_____
onion juice from fresh onion
Note: Materials and Preparations instructions are grouped by exercise. Some materials may be used in more than one exercise.
14
_____
_____
_____
_____
_____
_____
_____
Iodine (IKI) solution. Use one dropper bottle per student group. For ease of comparison, the same amount
should be used each time. Premade iodine-potassium-iodide solution can be purchased, or the ingredients
can be purchased separately as potassium iodide (KI) (Carolina 88-3790,-3792) and iodine (I) (Carolina 868970,-8972). These dry ingredients have a long shelf life and can be mixed as needed, according to the
instructions in Laboratory 2.
Boiling water bath. Place a large beaker of water on a hot plate. Adjust the dial on the hot plate so that the
water is maintained at a gentle rolling boil during the experiment. Thermometers are optional, since students should know that boiling water is 100C.
1% glucose solution. This makes enough for all procedures for 20 student groups. Place 1 g of glucose in 50
ml of distilled water. Stir to dissolve, and bring the volume up to 100 ml.
Onion and potato juice. Onion may be mashed the day before, then stored in a refrigerator overnight.
Potato juice should be extracted as close to time of use as possible.
Benedicts reagent. 50 ml per student group is sufficient. Benedicts reagent can be purchased as a powder
to make 1 liter. Or to make 1 liter, mix 173 g of sodium citrate (Carolina 88-9060) and 100 g of sodium carbonate, anhydrous (Na2CO3) (Carolina 88-8770) with 800 ml of distilled water. Warm this mixture to dissolve; then cool and filter it. Add distilled water to make 850 ml. Then dissolve 17.3 g of copper sulfate
(cupric sulfate, Carolina 85-6550) in 100 ml of distilled water, and stir slowly into the first solution. Add distilled water to make 1 liter. When testing, Benedicts reagent should be boiled approximately 5 minutes or
longer.
1% amylase solution. 20 ml per student group is sufficient. Mix 1 g alpha amylase with 100 ml distilled
water. Keep the alpha amylase powder refrigerated when not in use, and make the solution fresh when
needed.
3.3 Lipids (LM pages 3840)
_____
paper, brown (squares 4 cm 4 cm)
_____
water
_____
vegetable oil
_____
test tubes
_____
Tween (Carolina 20-7861, -7862) or bile salts (oxgall, Wards Biology 38W2179)
_____
medicine droppers, dropper bottles
_____
slides
_____
coverslips
_____
microscopes, compound light
_____
lens paper
_____
adipose tissue slide (Carolina 31-2698, -2728)
_____
everyday materials for unknowns; suggestions: bread crumbs, flour and water mixture,
squeeze bottle of margarine, skim milk, orange juice, syrup from canned fruit
1% Tween or bile solution. Tween is a standard wetting agent. Measure 1 ml of Tween or 1 g of bile salts,
and dissolve in 100 ml of distilled water.
15
EXERCISE QUESTIONS
Contents
Final Color
Conclusions
Distilled water
Blue
No protein is present.
Albumin
Purple
Protein is present.
Pepsin
Purple
Protein is present.
Starch
Blue
No protein is present.
Contents
Final Color
Conclusions
Albumin
Water
HCl* (incubate)
Purple
Albumin
Pepsin
HCl (incubate)
Pinkish-purple to pink
Why were the tubes incubated at 37C rather than at another temperature? This is normal human
body temperature, the optimum for digestion.
Enzymes are specific and speed up only one type of chemical reaction. Considering this, do
you predict that pepsin will break down starch? no Why or why not? Starch does not contain peptide bonds, which are specifically broken by pepsin.
If your results are not as expected, offer an explanation. Answers will vary.
16
Color Change
Conclusions
Tube 1: Starch
Blue-black
Starch is present.
Tube 2: Water
No starch is present.
Potato
Blue-black
Starch is present.
Onion
No starch is present.
Contents
Conclusions
Water
Blue
No sugar is present.
Glucose solution
Orange
Onion juice
Yellow to orange
Potato juice
Yellow
Starch suspension
Color Change
Conclusions
None
No sugar is present.
Green
Maltose is present.
Water
Amylase
2 Starch
Amylase
17
Results
Conclusions
Water spot
Oil spot
Penetrates paper
Substance is an oil.
Contents
Observations
Conclusions
Oil
Water
Oil
Water
Emulsifier
18
Thought Questions
15. Why is it necessary to shake a abottle of salad dressing before adding it to a salad? The oil in salad
dressing is not soluble in water, and shaking mixes the otherwise separate layers.
16. An unknown sample was tested with both biuret reagent and Benedicts reagent. Both tests result
in a blue color. What has been learned? No protein and no sugar were present.