La Salle University
Ozamiz City
SY 2022-2023
Second Semester
College of Arts and Sciences
Science Department
Biochemistry
Laboratory Manual
Ms. Kissy Jean Paredes
Instructor
TABLE OF CONTENTS
TITLE PAGE
1
LABORATORY ACTIVITY 1
Laboratory Apparatuses and Techniques
3
EXPERIMENT 1
The Microscope and Cells
5
EXPERIMENT 2
Water: Its Properties and Purification &
Preparation of Buffers
7
EXPERIMENT 3
Isolation and Purification of Proteins
& Denaturation of proteins
10
EXPERIMENT 4
Color Reaction for Amino Acids and Proteins
14
EXPERIMENT 5
Analysis of Carbohydrates
18
EXPERIMENT 6
Test for Lipids
20
EXPERIMENT 7
Nucleic Acids: DNA and RNA
23
EXPERIMENT 8
Qualitative Analysis of Urine
26
Activity No. 1: Laboratory Apparatus & Techniques
A. Safety in the Laboratory
The concern for laboratory safety can never be overemphasized. You must
always be aware that chemicals used in the laboratory are potentially toxic,
irritating, and flammable. However, such chemicals are a hazard only
when they are mishandled or improperly disposed of. It is my experience
as a lab instructor for 17 years that accidents happen least often to those
who come to each lab session mentally prepared and with a complete
understanding of the experimental procedures to be followed. Because
dangerous situations can develop unexpectedly, though, you must be
familiar with general safety practices, facilities, and emergency actions.
Carelessness on the part of one person can often cause injury to others.
As an adult, please be advised that you must be accountable. Ask the
elderly, if possible, to help you. Keep in mind and always observe the
following:
1. Use your common sense.
2. Read the laboratory instructions/procedures carefully before
starting an experiment.
3. Never perform unassigned experiments or work alone. Somebody
older must be present when working on the experiments. Never work
with a child.
4. Never play, eat, drink, or smoke while working.
5. Read the labels of bottles twice to be sure you use the right
chemical for the experiment. Don’t worry. Such chemicals are just in
your home.
6. Always follow instructions.
7. Wear safety goggles/face shields, laboratory gowns/aprons, and
gloves when doing any experiments.
8. Always wear shoes to protect your feet from chemical spills.
9. Please be very careful in using open flames.
10. Keep all books and none essential items away from the working
area.
11. Tie back long hair and restrict any loose clothing.
12. Never use broken or cracked glassware.
13. Report all injuries or accidents to somebody elder than you
immediately.
14. Dispose of any waste materials in appropriate containers.
15. Close gas cocks, faucets, and switch off the light and ceiling fans
before leaving your make-shift laboratory room.
16. Wash your hands after every experiment.
17. Always keep your working area clean and dry.
B.
FIRST AID IN THE LABORATORY
The following are common laboratory accidents that need first aid
treatments:
1. Chemicals in the eye. Hold an eye open and immediately flush
with water. Continue for at least five minutes.
2. Chemical spilled on skin. Flush with a large amount of water for
five to twenty minutes.
3. Severe bleeding. Apply pressure or tourniquet.
4. Minor burns. Apply burn ointment.
To reiterate, please use your common sense. Be vigilant and follow these
safety measures for a successful laboratory experiment at home.
C. HAZARDS IN THE LABORATORY – Important Terms
• Severe Toxicity- adverse effects of a substance that result either from a
single exposure or multiple exposures in a short space of time (usually
less than 24 hours)
• Irritant – causes redness, inflammation
• Corrosive – “eats away” tissue gradually
• Carcinogenic – causes cancer
• Flammable –easily set on fire
•
Biohazard –substances that pose a threat to the health of living
organisms, primarily that of humans.
D. Personal Protective Equipment (PPE)
Personal Protective Equipment (PPE) refers collectively to equipment
such as safety goggles, lab coats, gloves, protective shoes, respiratory
protective equipment, ear defenders, and similar equipment used to
protect the person during their work.
Laboratory Apparatus and Techniques
View the videos below to know the apparatus and techniques used in the
laboratory:
Process Questions:
1. What is the importance of knowing different laboratory techniques?
2. Give at least three(3) potential chemical hazards in the lab.
Experiment 1
The Microscope and Cells
I. Introduction
II. Objectives
At the end of the experiment, the students should be able to: (refer to the
learning outcomes)
1.
2.
III. Apparatus& Chemicals
IV. Procedures:
V. Data & Results
A. Parts of the compound microscope
Write the correct label for each part of the microscope shown below:
B. Viewing Specimen under the Microscope
Specimen
1. Cut letter “e”
2. Transparent ruler
3. Thread
4. Bacteria
Description
Sketch the Specimens
at low and high power
5. Cheek cells
6. Section
tissue
of
lung
VI. Analysis (Discussion)
Discuss the concepts of how to use microscopes and how they work.
VII. QUESTIONS
1. Refer to Part V. A; In tabulated form, list the parts of the microscope
and give their functions/uses.
2. What objective lens you should have in place to begin looking at your
specimen? Explain why.
3. What happens if you try to use the coarse adjustment when the 10X lens
is in place?
4. How would you compare the 4x and 10x objectives in terms of
differences in magnification and resolution??
5. Assuming the light is on and the oculars are in place, what are the next 2
steps?
6. Give at least two precautionary measures in the use of the microscope.
VIII. Conclusion
IX. References
X. Documentation
Experiment 2
Water: Its Properties and Purification & Preparation of Buffers
I. Introduction
II. Objectives
At the end of the experiment, the students should be able to: (refer to the
learning outcomes)
1.
2.
III. Apparatus& Chemicals
IV. Procedures:
V. Data & Results
I. Water: Its Properties and Purification
A. Properties of Water
Properties
Observation
Cohesion
Hydrates
Pentahydrate)
(Copper
Sulfate
B. Impurities in seawater
Impurities
1. Sodium ion (Na+)
2. Chloride ion (Cl-)
Observation
C. Methods of purifying impure water
Methods
Observation
1. Sedimentation
2.Decantation
3.Filtration
4.Distillation
D. Hardness of water
Descriptions:
II. Preparation of Buffers
A.
Descriptions (Remarks)
1. pH and volume of buffer chosen:
2.Give the formula of the acid and
salt used to prepare the buffer
solution
3.Calculation of the salt/acid ratio
of the buffer:
4. What is the pH of the buffer
solution used using the pH meter?
5. Is the experimental pH gives
greater than, lesser than, or equal to
the desired pH?
6. If the experimental pH obtained
is greater than the desired pH, what
substance (acid or salt) is added to
adjust the experimental pH to the
desired pH?
B. Effect of Buffer
Remarks
Give the pH changes before and after
putting acid or base to the buffer
solution
VI. Analysis (Discussion)
In the first section, discuss the properties and purification of water,
and in the 2nd section discuss the concepts on the preparation of buffers.
VII. QUESTIONS
Answer questions 1 -4 based on your experimentation at home.
1. How does it differ?
2. What variations do coins have that allow one to hold more or less
than the other?
3. Do all pennies hold the same amount of water? Explain
4. What other things besides size affect the number of drops?
5. Why do you think rainwater is soft
Qs 6-10, are for buffers:
6. Identify the two components of an acidic buffer solution and
explain the function of each component. Give also the criteria of a
classic buffer system: acidic buffer or alkaline buffer.
7. Write the chemical equilibrium equation representing the sodium
phosphate buffer system.
8. Explain how water reacts when acid is added, and when the base is
added.
9. Explain how one component of a buffer system reacts when acid is
added, and the other component reacts when the base is added.
10. Based on the experiment, what can you say about buffers and pH
changes?
VIII. Conclusion
IX. References
X. Documentation
Experiment 3
Isolation and Purification of Proteins & II) Denaturation of Proteins
I. Introduction
II. Objectives
At the end of the experiment, the students must have: (refer to the learning
outcomes)
1.
2.
III. Apparatus& Chemicals
IV. Procedures:
V. Data & Results
I. Isolation and Purification of Casein and Albumin
A. Casein Isolation
Appearance before isolation
Appearance after isolation
B. Albumin Isolation
Appearance before isolation
Appearance after isolation
II. Denaturation of proteins
A. Experiment at Home
Sample
Added
Observation
1
Heat
2
NaCl
3
NaHCO3
4
Lemon juice or Vinegar
5
Rubbing alcohol
6
Tap water
7
AgNO3
B. Experiment in the Lab
1. Heat effect
Albumin
Casein
Saliva
1
2
2. pH Effects
Effect
Albumin
Medium (pH)
Water
(control)
(pH
7)
10 % NaOH (pH 14)
on
Effect on Casein
Semen
Food sample
5 % NaHCO3 (pH 9)
2% HCl (pH 2)
3. Effects of ethanol, lead (II) nitrate, silver nitrate, and tannic acid
Agent
Effect on Albumin
Effect on Casein
None (Control)
95 % ethanol
Pb2+
Ag+
Tannic acid
VI. Analysis (Discussion)
Discuss the principles of isolation of proteins and denaturation of
proteins.
VII. QUESTIONS
1. Of the methods you tested, which would be more likely to be used in
the food industry?
2. In your own words, explain the effect of heat on the solubility of
albumin.
3. Is casein more soluble in base or in acid? Why?
4. When milk sours, lactic acid is produced, and a white precipitate forms.
What is this precipitate? Explain what is happening.
5. Did you observe any differences in the solubility of casein and albumin
at pH 2? at pH 9? at pH 14?
From your observations, which is more affected by pH, casein or albumin?
6. What kinds of disruptions to the native conformation occur in each of
the following? In other words, are hydrogen bonds, disulfide linkages, etc.
disrupted?
Albumin is heated.
Alcohol is added to egg white.
Metal ions are added to albumin.
VIII. Conclusion
IX. References
X. Documentation
Experiment 4
Color Reactions of Amino Acids & Proteins
I. Introduction
II. Objectives
At the end of the experiment, the students should be able to: (refer to the
learning outcomes)
1.
2.
III. Apparatus& Chemicals
IV. Procedures:
V. Data & Results
Color Reactions for Amino Acids & Proteins
1. Biuret Test
Samples
Color of
solution
the
original
Color
after
NaOH/CuSO4 (Biuret
reagent)
2. Ninhydrin Test
Samples
Color of original Color with Ninhydrin Color after
solution
reagent)
minutes
10
3. Xanthoproteic Test
Color before adding NaOH
Color
NaOH
Color Observation
Remarks
Samples
4. Millon’s Test
Samples
after
adding
5. Hopkin’s Cole Test
Samples
Color Observation
Remarks
6. Sakaguchi’s Test
Samples
Color Observation
Remarks
7. Sulfur Test
Samples
Color Observation
Remarks
VI. Analysis (Discussion)
Discuss the principles of the color test for amino acids and proteins.
VII. QUESTIONS:
1. Write the reactions involved in each test.
2. Do all protein samples react positively with the Biuret test? Justify your
answer.
3. What is a peptide? State the psychological significance of breaking
down proteins into peptide.
VIII. Conclusion
IX. References
X. Documentation
Experiment 5
Qualitative Test for Carbohydrates
I. Introduction
II. Objectives
At the end of the experiment, the students should be able to: (refer to the
learning outcomes)
1.
2.
III. Apparatus& Chemicals
IV. Procedures:
V. Data & Results
(Note: Indicate the color of solution or precipitate and include (+)/(-) for
the results)
A. General Test for Carbohydrates
Sample Solutions Solubility
Molisch’s Test
B. Iodine Test for Polysaccharides
Sample Solutions
Food Samples
·
Dairy
Color Response to Iodine
·
Fruit juice
·
Cereals
·
Crackers
·
Cookies
C. Qualitative Test for Sugars
Sample
Solutions
Benedict’s
Barfoed’s
Seliwanoff’s
Bial’s
·
VI. Analysis
Discuss the principle involved in each test for carbohydrates.
VII. Answers to Questions
1. Give at least three clinical correlations to the chemical test for sugars.
2. Compare the results you obtained for the iodine test of starch to the
iodine test of hydrolyzed starch. Explain your results.
3. What is meant by the term “reducing sugar”?
4. What is the purpose of testing water in the specific test for sugars?
5. An unknown carbohydrate gave a purple junction when tested with the
Molisch reagent, turned red when reacted with Seliwanoff’s reagent, and
quickly gave a red precipitate when reacted with Barfoed’s reagent. What
conclusions can be made about this carbohydrate?
6. What test could be used to differentiate between sucrose and lactose?
glucose and starch? glucose and fructose? Explain.
VIII. Conclusion
IX. References
X. Documentation
Experiment 6
Tests for Lipids
I. Introduction
II. Objectives
At the end of the experiment, the students must have: (refer to the learning
outcomes)
1.
2.
III. Apparatus& Chemicals
IV. Procedures:
V. Data & Results
(Note: Indicate the color of solution or precipitate and include (+)/(-) for
the results)
Part I. Qualitative Analysis of Lipids
I. Physical Properties
A. Solubility
Use the letter ‘S’ for soluble, ‘PS’ for partially soluble, and ‘I’
for insoluble.
Solubility
Solubility
in Water
in Organic Solvent (Chloroform)
Samples
B. Transparency Test
Samples
Translucent Spot Result Remarks
Part II. Identifying Lipids Using Chemical tests
Samples:
Tests
A. Acrolein
B. Unsaturation
C. Rancidity
D. Saponification
E. Cholesterol
1. Liebermann–Burchard
2. Salkowski
Part III.
1. Mass of a dry and clean 125-mL Erlenmeyer
=_____________________g
2. Mass of the 125-mL Erlenmeyer flask plus potato
=_____________________g
3. Mass of potato chips = #2 - #1 =_____________________g
flask
chips
4. Mass of dry and clean 150-mL beaker =_____________________g
5. Mass
of
the
150-mL
beaker
plus
lipid
extracted
=_____________________g
6. Mass of lipid extracted = #5 - #4 =_____________________g
7. % of lipid in potato chips = (mass of lipid extracted / mass of chips) x
100 = (#6 / #3) x 100 = _____________% = Experimental value
8. Calculate the % of lipid in potato chips from the Nutritional label
= (grams of fat per serving/ serving size in grams) x 100 =
_____________%= Theoretical value
9. Calculate the % error = (Difference between theoretical and
experimental values/ theoretical value) x 100 = (Difference between step-7
and step-8÷step-8) x 100=_______%
VI. Analysis
Discuss the principle involved in each test of lipids.
VII. Answers to Questions
1. What do “LDL” and “HDL” stands for? Explain their importance in
biological systems.
2. Show the chemical reaction between acetic anhydride and cholesterol.
3. How can you tell that the dark wet spot on the paper towel is fat and not
water?
4. Rank from most to least the percentage of lipid extracted from your food
samples (if there’s any). Look at the Nutrition Facts label on the packages
of all the foods and rank them. Did your ranking agree with the ranking of
the product labels?
VIII. Conclusion
IX. References
X. Documentation
Experiment 7
Nucleic Acids: DNA & RNA
I. Introduction
II. Objectives
At the end of the experiment, the students should be able to: (refer to the
learning outcomes)
1.
2.
III. Apparatus& Chemicals
IV. Procedures:
V. Data & Results
(Note: Indicate the color of solution or precipitate and include (+)/(-) for
the results)
I. Extraction of DNA from cheek cells
Description
Extracted DNA
II. Isolated RNA from yeast
Theoretical results
Experimental results
RNA
yeast
from
(filtrate)
III. Qualitative Tests for Nucleic Acids
Tests
Results
Solubility:
Test for Nucleoproteins:
Mild Acid Hydrolysis:
A. Test for the presence of Purines:
B. Test for the presence of Pentose:
C. Test for the presence of Phosphate:
VI. Analysis
Discuss the principle involved in the extraction and isolation of
DNA & RNA.
VII. Answers to Questions
1.Why are we using dishwashing liquid? What does it do to the cells?
2.Why did we use the pineapple juice?
3. Why did the isopropyl alcohol need to be cold?
4. What role does salt play?
5. What is the purpose of using 10% NaOH in RNA hydrolysis?
VIII. Conclusion
IX. References
X. Documentation
Experiment 8
Qualitative Analysis of Urine (Urinalysis)
I. Introduction
II. Objectives
At the end of the experiment, the students should be able to: (refer to the
learning outcomes)
1.
2.
III. Apparatus& Chemicals
IV. Procedures:
V. Data & Results
A. Physical Properties of Urine
Physical Properties
Urine Sample
Color
Appearance (Turbidity)
Odor
pH
Specific gravity (state the normal
values)
B. Normal Organic/Inorganic Constituents
(Note: Indicate the color of solution or precipitate and include (+)/(-)
for the results)
Tests
Results
1. Urea
2. Uric Acid
3. Creatinine
4. Chloride
5. Phosphate
C. Pathological (Abnormal) Organic Constituents
Urine Sample
Tests
Color
1. Ketone
2. Glucose
3. Albumin
4.
A.
Pigment
Bile
a.
Gmelin’s
b.
Fouchets Test
B. Bile Acids and
Bile Salt
a. Pettenkofer's
b. Hays
5. Blood
Unknown Urine Sample
(+ or -)
Color
(+ or -)
VI. Analysis
Discuss the principle involved in urinalysis.
VII. Answers to Questions
1. Why it is important to refrigerate a urine sample if the analysis cannot
be performed immediately after collecting it?
2. Examine the following urinalysis data from Patient's A and B and
answer questions that follow.
Normal range
Patient A
Patient B
color
pale yellow
dark yellow
medium yellow
appearance
clear
cloudy
clear
pH
4.5 - 8.0
6.0
7.2
specific gravity
1.001 - 1.030
1.015
1.03
blood
none
+++
none
sodium
40-220 mEq/L
340 mEq/L
140 mEq/L
glucose
none
none
none
ketone bodies
none
none
none
creatinine
150 mg/dL
180 mg/dL
150 mg/dL
ammonia
50 - 70 mg/dL
90 mg/dL
65 mg/dL
Patient A is a 45-year-old man that came to his physician after spending a
second sleepless night with excruciating lower abdominal pain. The pain
seemed to come in waves and was unrelieved by analgesics or lying or
standing in any position. He had not experienced any similar pain before.
On physical examination, there are no abnormal findings.
3. What abnormal findings are present on Patient A's urinalysis chart?
4. Based on these abnormalities what do you think could be the reason for
Patient A's pain?
5. Can you observe anything abnormal in Patient B's urinalysis chart?
VIII. Conclusion
IX. References
X. Documentation