Methods and Materials
Katlin Daugherty
During our first lab, we performed five carbohydrate tests – Benedict's, Barfoed's, Selivanoff's, Bial's, and an iodine test, as outlined directly in the directions from our lab
book on eight known sugars and one unknown (Malejewski, 2004). In order to adapt the carbohydrate tests to our independent research on apples and pears, we made
apple and pear slurries to use as the basis for testing instead of the eight sugars and one unknown from the week 2. In order to make the apple and pear solutions, we first
peeled, cored, and cut up 2 Macintosh apples and 2 Bartlett pears. Afterwards, using a typical household blender, we blended 195 g of each fruit with 200 mL of distilled
water. Next, we strained the solution through four layers of cheesecloth into a 250 mL beaker. After this, we poured 50 mL of each liquid solution into a centrifuge tube,
and then we centrifuged the solutions for 5 minutes at 4000 rpm's to remove all remaining solids to form the purified slurries. In our independent lab, we followed the lab
protocol from the Lab 1 (Malejewski, 2004), except we combined 1 mL of apple and pear slurry solutions, respectively, with three mL of each reagent, instead of three
mL of sugar/unknown to 3 mL of reagent.
For all of the tests, we performed three replicates using lab protocol and used both negative and positive controls (Malejewski, 2004). Water functioned as an appropriate
negative control for all of the tests. For Benedict and Barfoed's tests, glucose functioned well as a positive control. Fructose, furthermore, functioned as a positive control
for Selivanoff's test. Bial's test requires two positive controls – one for pentose furanoses and another for hexose furanoses. Xylose and sucrose, respectively, worked well
for these controls. Lastly, starch functioned as an appropriate positive control for the iodine test.
In addition to all of the tests in the lab manual, we also performed a test for sweetness on the apple and pear slurries. To accomplish this, we used a CVS/Pharmacy
Complete Blood Glucose Monitoring System to test the glucose levels of two samples of each slurry type. For this experiment, we used water as a negative control and
would have used a pre-measured concentration of glucose water for out positive control if it had been available. To perform this test a drop of solution was placed on a
testing strip, then the strip was inserted into the CVS/Pharmacy Complete Blood Glucose Monitor. Lastly, after performing all of the tests above, we made sure to take
pictures of all of the clearly labeled test tubes from each test for our record.
The next series of tests we ran in the lab dealt with photosynthesis. During the fourth week, we performed paper chromatography, absorbance, and action tests on
spinach-leaf solutions, following exactly the lab protocol from Lab 2 in our lab manuals (Malejewski, 2004). During week 5, we performed all three tests again for our
independent research on leaf solutions from Macintosh apples and Bartlett pears. We made our leaf solutions by substituting apple and pear leaves for the spinach leaves
in the procedure in the lab manual, though we altered the amounts of solution and leaf weight (Malejewski, 2004). We used 11.0 g of apple leaves and 44 mL of
phosphate buffer and 11.0 g of pear leaves and 44 mL of phosphate buffer to make our solutions, respectively. We followed the Lab 2 protocol then, for the paper
chromatography, and the absorption and action spectra (Malejewski, 2004). In the action spectra test a few details were changed, mainly the way the solution was kept
cold during the time it was exposed to the light. To do this the spec tubes were placed in 100 mL beaker and surrounded by ice, making sure that the clear window was
facing out. Another change that was made to the lab manual was that the Terbicel was not added when the boxes were opened; instead it was added when brought over
to the spectrometer for the absorbance readings. As with the carbohydrate lab, we made sure to use appropriate positive spinach and negative water controls, ran three
replicates for the paper chromatography, two for the absorption spectra, and one for the action spectra, and took pictures to record our results.
The third and final series of tests we performed concerned enzymatic activity. During week 6, we followed the lab protocol for Lab 3 to test for the presence of PPO, the
effect of heat and inhibitors, the effect of pH, and the Bradford Assay on potato solutions (Malejewski, 2004). In our independent research we used Bartlett pear and
Macintosh apple solutions for our research, following the same procedure we used to make the apple and pear slurries in the carbohydrate lab. Similarly, we used the lab
protocol for the PPO, effect of heat and inhibitors, pH, and Bradford assay tests using concentrations of 10, 25, and 45 µ L/ µ g, only substituting the fruit for the potato
solutions (Malejewski, 2004). As always, we made sure to use appropriate positive potato and negative water controls, used three replicates for each test, and recorded
our results through photography.