Professional Documents
Culture Documents
SBK3013 / TBK4013
2011
DEPARTMENT OF BIOLOGY
DR SHAKINAZ DESA
Laboratory safety will be explained during the first practical session.
Guide Title
2 Protein assay
3 Enzyme experiment
4 Experiment on vitamin C
1. Practical no. :
2. Experiment title - write a title that present your experiment, for example: The
determination of two protein sample using Biuret and Lowry assay
3. Abstract - summary of the experiment which should include objectives, brief
introduction, brief methods, analyzed results, conclusion.
4. Introduction – highlight on the main subject of interest
5. Materials and methods – do not copy from the manual. Write in your own
sentences. You may use diagrams, flow chart etc.
6. Results – analyzed the data and you may present the results in form of charts,
table, figure, and photo.
7. Discussion – answer the questions of what, why, when, where, how.
8. References – state author, book/journal, year, page number(s), and publisher.
All reports must be submitted in two weeks after the practical. Your instructor will
inform you the suitable due date.
23
GUIDE 1: ACID BASE EXPERIMENT
We can measure the pH by emerging the tip of pH meter into the solution
and read the value in the recorder. The higher concentration of H+ , the
lower the pH value.
Objective
Materials
24
Methods
Data analysis
Materials
i. 0.1 M HCl solution (add 8.3 mL of concentrated HCl solution to
enough deionized or distilled water to make 1.0 L of solution)*
ii. 0.1 M NaOH solution (dissolve 4.0 g NaOH in enough deionized or
distilled water to make 1.0 L of solution)*
iii. 2-propanol (isopropyl alcohol) /acetone /deionized or distilled water
iv. Fruits/vegetables/flowers (dark colors are preferred).
25
Caution should be used when working with solutions of hydrochloric acid
and sodium hydroxide. Both can be irritating to the skin. Goggles must be
worn throughout the experiment. If it is necessary to heat the extract in
order to concentrate the indicator, do not heat those containing the
flammable solvents, 2-propanol or acetone, over an open flame. Use a
water bath or hot plate.
For flowers, only a few petals are normally needed. For fruits and
vegetables, finely chopped pieces should be used. Use only that portion
of the fruit or vegetable that is most pigmented.
4. Filter and collect the filtrate from the macerated plant samples.
26
b. Transfer 1.0 mL of 0.1 M acid from test tube #1 to test tube #2
and mix thoroughly. (pH = 2)
c. Transfer 1.0 mL of acid solution from test tube # 2 to test tube
#3 and mix thoroughly. (pH = 3)
d. Continue making the serial dilutions by transferring 1.0 mL of
the most recently diluted acid solution to the next test tube
until six acid solutions of pH 1 to 6 have been prepared. Be
sure to mix each thoroughly before the transfer.
4. Add 10.0 mL distilled or deionized water to test tube #7. (pH = 7)
5. Prepare solutions of base in the following manner:
a. Place 10.0 mL of 0.1 M NaOH in test tube #13. (pH = 13)
b. Transfer 1.0 mL of 0.1 M NaOH from test tube #13 to test tube
#12 and mix thoroughly. (pH = 12)
c. Continue making serial dilutions of the base going from pH 12
down to pH 8 by transferring 1.0 mL of the most recently
diluted basic solution to the next test tube and mixing
thoroughly each time.
6. Label the wells of a spot plate from 1 to 13. Transfer a few drops of
each of the solutions prepared in steps 3, 4, and 5 to the
corresponding well in the spot plate.
7. Add a drop or two of the flower/fruit/vegetable extract indicator to
each well. Observe the pH at which the indicator changes color.
Once the pH ranges of the indicators have been determined, they can be
used in acid-base titrations or to test the pH of household chemicals.
27
GUIDE 2: PROTEIN EXPERIMENT
1. Protein Assays
Procedure:
a) Biuret assay:
Biuret reagent:
i. Add, with stirring, 300 mL of 10% (w/v) NaOH to 500 mL of a solution
containing 0.3% copper sulfate pentahydrate and 1.2% sodium
potassium tartarate, then dilute to one liter.
ii. The reagent is stable for a few months but not a year.
iii. Adding one gram of potassium iodide per liter and storing in the dark
makes it last indefinitely.
A540: Measure the absorbance of 1 mL at 540 nm. Plot the standard curve.
28
b) Lowry assay:
All series should include a zero protein (water) tube (reagent blank).
Test Sample:
Test sample can be sample with protein. Be creative in setting an
experiment.
Substitute the protein in both tests with a test sample.
Measure the absorbance and compare the concentration using both
methods.
Questions:
1. Under what conditions would this methodology not be appropriate?
2. Describe (in brief) three alternative methods of determining protein
concentration.
3. What is an "appropriate blank" and why?
29
GUIDE 3: EXPERIMENT ON ENZYME
Enzyme Kinetics
Procedure:
1. Prepare starch solutions from the stock solution (1.0 mg/ml) into
dilutions of 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from
the starch stock solution.
2. Iodine solution is prepared by adding 5 g potassium iodide to 100 ml
water. The dissolved potassium iodide is added with 1 g of iodine and
is allowed to dissolve.
3. Prepare a standard curve of Absorbance (@ 590 nm) vs.
Concentration of a starch/iodine mixture. Use the following table as
guide.
Table 1
Preparation of starch/iodine mixture for standard curve determination
Test 8 ml starch Water Iodine
tube of x mg/ml (ml) (ml)
1 0 9 1
2 0.01 1 1
3 0.025 1 1
4 0.05 1 1 Measure the
5 0.1 1 1 absorbance
6 0.3 1 1 at 590nm
7 0.5 1 1
8 0.7 1 1
9 1.0 1 1
30
2. The effect of substrate concentration
Table 2
Data analysis
Table 3
Data analysis
Plot the Lineweaver-Burke line for the result of 20, 28, 35 and 40°C.
Compare all three plots.
What are the values of Vmax and Km for all plots?
32
4. The effect of pH
Table 4
Data analysis
33
GUIDE 4: EXPERIMENT ON VITAMIN C
Iodine in excess of the vitamin C will react with a starch solution you
will add to the extract to produce a bluish-black color. The addition of a
chemical to measure another chemical is called a titration.
Materials:
2. Starch solution (1%): Mix 1 g starch in 100 ml boiling H2O. Boil for one
minute while stirring. Stir until completely dissolved (this solution will
be cloudy).
3. Iodine solution: Mix 0.6 g potassium iodide in 500 ml H2O. Mix 0.6 g
iodine in 50 ml of ethyl alcohol. These two iodine solutions should be
mixed well before combining. Combine the two iodine solutions and
add an additional 450 ml of H2O.
5. Blender
6. Filter/ cheesecloth
34
Procedure:
Does the way you prepare your food affect the vitamin C available to
be ingested? Vitamin C is a water-soluble vitamin. Would cooking
food by boiling in water affect the vitamin C content? If vitamin C is
lost during the cooking process, where does it go? What types of
experiments could you design to test your hypothesis? You will be
testing your hypothesis to determine if vitamin C content is changed
during cooking or if different ways of food preparation yield different
amounts of vitamin C.
35
i. Food can be prepared according to your creativity. For
example, you can boil or steam or place in a freezer. You
can also prepare the food by different exposing time to
heat etc.
ii. Chop food material into small pieces and place into
blender.
iii. Obtain your data using the same method in previous
section.
iv. Record the volume reading on the burette.
v. Compare the relative amounts of ascorbic acid present in
the samples you are testing.
vi. Compare your results with those of other members of the
class. What do the results show?
Questions:
36
2. Application: Magic Writing
Materials
Beaker
Iodine
Lemon/Lime juice
Notebook paper
Cup
Art brush
Procedure:
STEP B:
STEP D:
RESULTS
37
GUIDE 5: EXPERIMENT USING LIPID
1. Saponification of triglycerides
38
Materials:
Triglyceride sample: e.g. Coconut oil, corn oil, palm oil, margarine, butter
Solvent (1:1 ethanol/ether)
0.5M KOH
Phenolphthalein
0.5M HCL
WARNING: KOH is a very strong corrosive agent and can cause serious
burns on contact. Use appropriate eye / hand protection during this
procedure.
39
8. Calculate the weight (mg) of KOH used to saponify the 1 g sample.
Determine the saponification number for tested samples
9. Obtain the final results from the other groups for each sample and
prepare a table to summarize the results.
Use the following table to assist you to calculate the saponification number
40
2. Application: Making Soap
Materials:
Methods:
41
6. As the crude soap cools, a waxy solid should form. Add to it about
12.5 ml of distilled water and about 50 ml of hot, saturated sodium
chloride solution.
7. Stir the mixture, breaking up lumps with your stirring rod.
8. Decant the wash solution by pouring it through a wire screen, which
will trap small soap particles.
9. Repeat the wash process twice. After the final washing, press the soap
between two sheets of paper towelling to expel as much water as
possible
Questions:
42