General Laboratory Safety Rules

1. Never eat or drink in the lab .Do not bring food or drink s in to the lab.
2. Do not chewing gum ,do not smoking
3. Do not do unauthorized experiments and do not mix any chemicals except as instructed.
4. Wear all safety equipment’s required by the lab procedure or your instructor.
5. Wear lab coat or apron, safety glasses or eye goggle and gloves when handling chemicals
or body fluids.
6. Tie or pin up long hairs back to keep it out of flames or harmful I liquids.
7. Do not wear loose or flowing clothing, sandals, open toe shoes, dangling jewelry in the
labo4ratory.Roll up loose sleeves.
8. Do not taste any chemicals and do not smell chemicals directly.
9. Do not pipette solutions by mouth .Use a rubber suction bulb or special pipette filter.
10. Never return as unused chemicals to a stock bottle. Dispose of it as a waste.
11. Report broken glasses immediately to your instructor and always dispose of broken
glasses in its designated container.
12. Clean up your working stations and wash your hands before leaving the lab room.
13. Then work with the microorganisms, always treat them as if it had the po9tential to cause
disease prevent spills and avoid dire ct contact, especially open sores.
14. Dispose of all potentially contaminated objects in a biohazard bag, a container fi9lled
with a bleach solution, as described by your instructor.
15. Spray and wipe the working areas with 10% chlorine bleach solution before and after lab.
16. Wash your hands after handling a biohazard.
17. Never “horse round “or practical jokes in the laboratory.
Laboratory Report Writing Formant

The cover page

The cover page is the first page of the report that includes the name of the institution, college,
department, course name, course code, and experiment no., title of the experiment, group,
section, name and ID Number of the students, submission date and submission to, place and year
of the experiments.

The body
 Title Page: The title describes the focus of the topic
 Objective: Aim or goal of the experiment under investigation.
 Introduction: The introduction provides the reader with background information about
the problem and provides the rationale for conducting the research. The introduction
should incorporate and cite outside sources. You should avoid using websites and
encyclopedias for this background information. The introduction should start with more
broad and general statements that frame the research and become more specific, clearly
stating your hypotheses near the end.
 Materials and Methods: The methods section describes how the study was designed to
test your hypotheses. This section should provide enough detail for someone to repeat your
study. This section explains what you did. It should not be a bullet list of steps and
materials used; nor should it read like a recipe that the reader is to follow. Typically this
section is written in first person past tense in paragraph form since you conducted the
experiment.
 Results: This section provides a written description of the data in paragraph form. What
was the most reaction? The least reaction? This section should also include numbered
graphs or tables with descriptive titles. The objective is to present the data, not interpret
the data. Do not discuss why something occurred, just state what occurred.
 Discussion: In this section you interpret and critically evaluate your results. Generally, this
section begins by reviewing your hypotheses and whether your data support your
hypotheses. In describing conclusions that can be drawn from your research, it is important
to include outside studies that help clarify your results. You should cite outside resources.
What is most important about the research? What is the take-home message? The
 discussion section also includes ideas for further research and talks about potential sources
of error. What could you improve if you conducted this experiment a second time?
 Conclusion: is the brief summary /generalization/short description of the practical
activities.
 Reference: You will need to include a Reference List at the end of your report: a list of
all the references you have cited in the report (e.g. textbooks, journals, lab manuals, web
sites). If you have read a reference but haven’t cited it in the report, you do not include it
here. However, if you are asked to provide a Bibliography, you include everything you
read, whether you have cited it or not. You need to check with your lecturer or
demonstrator regarding the referencing style requirements in your department.
 Appendices: You won’t always have to have an Appendix in your report, but if you
have some detailed information or raw data that you want to include, you can include this
at the very end of the report
Laboratory session ≠ 1

Microscope
A microscope is an instrument that is used to magnify small objects. Some microscopes can even
be used to observe an object at the cellular level, allowing scientists to see the shape of a cell, its
nucleus, mitochondria, and other organelles. While the modern microscope has many parts, the
most important pieces are its lenses. It is through the microscope’s lenses that the image of an
object can be magnified and observed in detail. A simple light microscope manipulates how light
enters the eye using a convex lens, where both sides of the lens are curved outwards. When light
reflects off of an object being viewed under the microscope and passes through the lens, it bends
towards the eye. This makes the object look bigger than it actually is.

What separates a basic microscope from a powerful machine used in a research lab? Two
parameters are especially important in microscopy: magnification and resolution.

 Magnification is a measure of how much larger a microscope (or set of lenses within a
microscope) causes an object to appear. For instance, the light microscopes typically used
in high schools and colleges magnify up to about 400 times actual size. So, something
that was 1 mm wide in real life would be 400 mm wide in the microscope image.

 The resolution of a microscope or lens is the smallest distance by which two points can
be separated and still be distinguished as separate objects. The smaller this value, the
higher the resolving power of the microscope and the better the clarity and detail of the
image. If two bacterial cells were very close together on a slide, they might look like a
single, blurry dot on a microscope with low resolving power, but could be told apart as
separate on a microscope with high resolving power.
Laboratory session ≠ 1 Part One

Handling of Microscope
1. Turn the revolving turret so that the lowest power objective lens (eg. 4x) is clicked into
position.

2. Place the microscope slide on the stage and fasten it with the stage clips.

3. Look at the objective lens and the stage from the side and turn the focus knob (4) so the
stage moves upward. Move it up as far as it will go without letting the objective touch the
coverslip.

4. Look through the eyepiece and move the focus knob until the image comes into focus.

5. Adjust the condenser and light intensity for the greatest amount of light.

6. Move the microscope slide around until the sample is in the centre of the field of view
(what you see).

7. Use the focus knob (4) to place the sample into focus and readjust the condenser (7) and
light intensity for the clearest image (with low power objectives you might need to reduce
the light intensity or shut the condenser).

8. When you have a clear image of your sample with the lowest power objective, you can
change to the next objective lenses. You might need to readjust the sample into focus
and/or readjust the condenser and light intensity. If you cannot focus on your specimen,
repeat steps 3 through 5 with the higher power objective lens in place. Do not let the
objective lens touch the slide!

9. When finished, lower the stage, click the low power lens into position and remove the
slide.
Laboratory session ≠ 1 Part Two

Preparing wet Mount Slide

Objective:

 To learn how to wet mount a specimen

 To learn why a specimen might be wet mounted

Wet mounts are commonly used in the science of Biology. This is an important skill to develop
in order to be able to study specimens with a Microscope. This skill is only developed with
practice and by following precise instructions. Before you start building your slides, make sure
you have everything you will need, including slides, cover slips, droppers or pipets and any
chemicals or stains you plan to use. You will be using two main types of slides, 1) the common
flat glass slide, and 2) the depression or well slides. Well slides have a small well, or indentation,
in the center to hold a drop of water or liquid substance. They are more expensive and usually
used without a cover slip. Standard slides can be either plastic or glass and are 1 x 3 inches (25 x
75 mm) in size and 1 to 1.2 mm thick. Wet slides will use a cover slip or cover glass, a very thin
square piece of glass (or plastic) that is placed over the sample drop. Without the cover in place,
surface tension would cause the droplet to bunch up in a dome. The cover breaks this tension,
flattening the sample and allowing very close inspection with minimal focusing. The cover also
serves to protect the objective lens from interfering with the sample drop.

Materials Required

 A compound microscope

 Slide

 Cover slip

 Water

 Dropper
  Tweezers

 Specimen (some thread fibers will do fine)

Procedure

1. Place a drop of water on the center of a clean dry slide

2. Using the tweezers, place the specimen in the middle of the drop.

3. While holding the cover slip upright, carefully place one edge of the cover slip next to the
water.

4. Slowly lower the upper edge of the cover slip onto the water. The objective is to
minimize or eliminate air bubbles under the cover slip. You might find it helpful to use
one toothpick to hold the lower edge in place, while using another to carefully lower the
slip into place.

5. An absorbent towel can be placed at the edge of the cover slip to draw out some of the
water, further flattening the wet mount slide.
Laboratory session ≠ 2

Observing Cellular Structure

Objectives:
Students will discover that onions are made up of cells.

 Observe plant cell (onion) under a microscope.

 Discover that their skin is made up of cells.
 Observe animal cell (cheek) under a microscope.

Introduction
Ever since the first microscope was used, biologists have been interested in studying the cellular
organization of all living things. After hundreds of years of observations by many biologists, the
cell theory was developed. The cell theory states that the cell is the structural and functional unit
of living things. Eukaryotic cells contain structures called organelles that carry out life processes.
Eukaryotic cells can be classified by the types of organelles they contain. In plant and animal
cells, similarities and differences exist because of varied life functions. In this investigation, you
will compare the structures of typical plant cells, onion (Allium) and a typical animal cell, a
cheek cell.

Materials Required
 microscope

 two glass slides

 iodine stain

 purple stain

 two cover slips

 an onion

 a toothpick
Procedures

Plant cell

1. Peel a translucent piece of tissue from the onion. (The smaller the piece the better.)
Translucent means that you can see light through the specimen, but it is not transparent.
2. Place the piece of onion on a glass slide and add a drop of the iodine solution. Cover the
slide with a cover slip using your best wet-mount making techniques.
3. Observe the onion cell under both low and high power. Make a drawing of one onion
cell, labeling all of its parts as you observe them.

Animal cell

1. To view cheek cells, gently scrape the inside lining of your cheek with a toothpick.
2. Gently roll and rub the toothpick onto the top of a glass slide in an area that will be
visible through the microscope.
3. Add a drop of purple stain (specific for animals) and cover with a cover slip.
4. Observe the cheek cells under both low and high power of your microscope. Draw a
diagram of one cheek cell and label the parts.
Laboratory session ≠ 3

Diffusion and Osmosis across a Semipermeable Membrane

Part One: Diffusion

Objective
 Describe diffusion and the factors that affect diffusion across the cell membrane.

Introduction

Diffusion is a passive process of transport. A single substance tends to move from an area of
high concentration to an area of low concentration until the concentration is equal across a space.
You are familiar with diffusion of substances through the air. For example, think about someone
opening a bottle of ammonia in a room filled with people. The ammonia gas is at its highest
concentration in the bottle; its lowest concentration is at the edges of the room. The ammonia
vapor will diffuse, or spread away, from the bottle; gradually, more and more people will smell
the ammonia as it spreads. Materials move within the cell ‘s cytosol by diffusion, and certain
materials move through the plasma membrane by diffusion. Diffusion expends no energy. On the
contrary, concentration gradients are a form of potential energy, dissipated as the gradient is
eliminated. Because of its structure, the cell membrane is a semipermeable membrane. This
means that some substances can easily diffuse through it, like oxygen, or carbon dioxide. Other
substances, like glucose or sodium ions, are unable to pass through the cell membrane unless
they are specifically transported via proteins embedded in the membrane itself. Whether or not a
substance is able to diffuse through a cell membrane depends on the characteristics of the
substance and characteristics of the membrane. In this lab, we will make dialysis tubing “cells”
and explore the effect of size on a molecule’s ability to diffuse through a “cell membrane.”
Materials Required

 Food coloring (red & blue)

 2 clear glass cups of the exact same size and shape
 2 differently shaped clear containers (narrow and wide), preferably about the same size

Procedure
1. Fill one glass cup with hot water. Fill the second glass cup with cold water.

2. Drop 1-2 drops of red food coloring in the hot cup, and 1-2 drops of blue food coloring in
the cold in the cold one.

3. Watch and wait for color to disperse entirely.

Questions

1. . In which beaker diffusion is faster?

2. . What other factors do you think affect the rate of diffusion?
3. . Can you mention areas (biological processes) where diffusion is important in the body
of human beings?
Part two: Osmosis and the Cell Membrane

Demonstrating Osmosis Using Potato cups as Osmometer

Objective
 Observe the effects of different concentrations of salt solutions on potato cores.
 Infer the relationship between weight loss and rate of osmosis.

Introduction

Osmosis is the movement of water across a semipermeable membrane (such as the cell
membrane). The tonicity of a solution involves comparing the concentration of a cell’s
cytoplasm to the concentration of its environment. Ultimately, the tonicity of a solution can be
determined by examining the effect a solution has on a cell within the solution. By definition, a
hypertonic solution is one that causes a cell to shrink. Though it certainly is more complex than
this, for our purposes in this class, we can assume that a hypertonic solution is more
concentrated with solutes than the cytoplasm. This will cause water from the cytoplasm to leave
the cell, causing the cell to shrink. If a cell shrinks when placed in a solution, then the solution
is hypertonic to the cell. If a solution is hypotonic to a cell, then the cell will swell when placed
in the hypotonic solution. In this case, you can imagine that the solution is less concentrated than
the cell’s cytoplasm, causing water from the solution to flow into the cell. The cell swells!
Finally, an isotonic solution is one that causes no change in the cell. You can imagine that the
solution and the cell have equal concentrations, so there is no net movement of water molecules
into or out of the cell.

Material Required

 Potato
 Cork borer
 Knife or razor blade ·
 Beakers,
 Salt solutions (1%, 3%, 5%) ·
  Distilled water ·
 Balance ·

Procedure
1. Label 4 containers with your name and the following: distilled water, 1% salt, 3%
salt, and 5% salt.
2. Using the cork borer, make 12 cylinders from your potato. Trim them with a knife
until they are 3 cm long. Caution: be very careful with the cork borer and knife.
Always cut away from yourself. Make sure there is no peel left on the core. (If a
cork borer is not available, you may also cut strips of potato using a knife. Try to
make them all the same width.)
3. Place three cores in each of the containers and cover them temporarily. When you
are ready, remove the cores and find the mass of each group of three using the
balance. Mass all three of them together, not separately. You should mass them to
the nearest 0.1 g. Record your data in the data table.
4. Immediately return the cores to the correct container and cover them with the
correct solution. Place a lid on each container and set them aside for 24 hours.
5. After 24 hours, remove each set of three cores from their containers. Briefly blot
them with a paper towel to remove excess water. Quickly find the mass of each
group of 3 and record the mass in the data table.
6. Make observations of the texture, color and flexibility of the cores. Record these
observations in the data table.
7. Determine the change in mass by subtracting.

Question
1. What is plasmolysis?
2. What is the significance of osmosis?
Laboratory session ≠ 4

Test for carbohydrates

Carbohydrates are the most abundant macromolecules on earth, and the source of immediate
energy needs in living systems. Carbohydrates also participate in defining the structure of cells
and living systems. There are 3 general chemical grouping for carbohydrates: monosaccharaides,
disaccharides, and polysaccharides.

Monosaccharaides, also referred to as simple sugars, are made up of a single sugar molecule. The
major example of a monosaccharide is glucose (C6H12O6). Other monosaccharaides include
isomers of glucose, such as fructose and galactose. Monosaccharaides are transported in the
blood of animals, broken down to produce chemical energy inside the cell, and can also be found
within other macromolecules, such as nucleic acids.

Disaccharides are composed of two single monomers of sugar linked together. Examples of
disaccharides are maltose (glucose + glucose) and sucrose (glucose + fructose). Disaccharides
are broken down into their subunits for use inside living systems.

Polysaccharides are polymers, or long chains of sugar monomers linked together, and are stored
inside the cell for future energy use. In plants, the major storage polysaccharide is starch, while
in animals it is glycogen. Plants also contain cellulose, which is the most abundant of all
carbohydrates. Cellulose is found in the plant cell wall, where it provides structure and support to
the plant cell.
Laboratory session ≠ 4 Part One

Tests for reducing sugar/benedicts test/

Objective

 To test the presence of reducing sugar in a given food sample

Introduction

Benedict’s test is used to test for simple carbohydrates. The Benedict’s test identifies reducing
sugars (monosaccharide’s and some disaccharides), which have free ketone or aldehyde
functional groups. Benedict’s solution can be used to test for the presence of glucose in
urine. Some sugars such as glucose are called reducing sugars because they are capable of
transferring hydrogens (electrons) to other compounds, a process called reduction. When
reducing sugars are mixed with Benedict’s reagent and heated, a reduction reaction causes the
Benedicts reagent to change color. The color varies from green to dark red (brick) or rusty-brown,
depending on the amount of and type of sugar. Benedict’s quantitative reagent contains potassium
thiocyanate and is used to determine how much reducing sugar is present. This solution forms a
copper thiocyanate precipitate which is white and can be used in a titration. The titration should
be repeated with 1% glucose solution instead of the sample for calibration

Materials Required

 Glucose
 Benedict’s solution
 Test tube, beaker, sprit lamp/hot water bath, stirrer
 Test tube rack, spatula, and dropper
Procedure

1. Approximately 1 ml of sample is placed into a clean test tube.

2. 2 ml (10 drops) of Benedict’s reagent (CuSO4) is placed in the test tube.
3. The solution is then heated in a boiling water bath for 3-5 minutes.
4. Observe for color change in the solution of test tubes or precipitate formation.
Laboratory session ≠ 4 Part Two

Test for non- reducing sugars/acid hydrolysis test/

Objective
 To carry out food test for non-reducing sugar in a given food sample.

Introduction
Disaccharides are compound sugars formed when two monosaccharide molecules combine.
Disaccharides are found in sugar cane (sucrose), malt (maltose), and milk (lactose). Some
disaccharides are reducing sugars (lactose and maltose), while others are non-reducing sugars
(sucrose). Non-reducing agents don't have free ketone or aldehyde groups, and therefore contain
an acetal instead of a hemiacetal. An acetal has two O-R groups, one –R group and a –H atom
attached to the same carbon. (The key difference between an acetal and a hemiactal is that in a
hemiacetal, an –OH group replaces one of the –OR acetal groups.) A sugar without a hemiacetal
is non-reducing because it does not behave as a reducing agent toward oxidizing metal salts.
Sucrose is one example of a non-reducing sugar.

Material Required
 Sucrose
 Sodium hydroxide, HCl
 Benedict solution, test tube, rack, stirrer, beaker,
 Dropper, hot plate/sprit lamp, water bath.

Procedure

1. Add 2ml of sucrose solution in both test tubes.

2. Add 1ml of HCl solution in test tube one and heat for one minute in a water bath.

3. Add 1ml of sodium hydroxide solution in test tube one containing the acid.

4. Add 2ml of benedict’s solution in both test tubes and boil in a water bath 2 -5 minutes.

5. Observe and record the expected results.

Laboratory session ≠ 4 Part Three

Test for starch/Iodine test/

Objective

Introduction
Starch is a carbohydrate and exists in two types of molecules: amylose (linear) and amylopectin
(branched). Most starch contains a mixture of these two molecules, generally with more
amylopectin (65% to 85%). The reaction between amylose (even though it is often present in
lesser amounts) and iodine is said to account for the intense color change seen. Many details of
the reaction of iodine with starch are unknown, but one explanation is that when a solution of
diluted iodine is added to starch, an intensely colored starch-iodine complex forms. Amylose
molecules consist of single, mostly unbranched chains of glucose molecules, shaped like a
spring. It is speculated that the iodine (in the form of I5- ions) gets stuck in the coils of the beta
amylose molecules (soluble starch). The starch forces the iodine into a linear arrangement in the
middle groove of the amylose coil. There is some transfer of charge between the starch and the
iodine. This changes the electron arrangements and hence the spacing between energy levels.
The new spacing absorbs visible light selectively and gives the complex its intense blue color.

Materials Required
 Starch
 Potassium Iodide/Iodine/
 Test tube, dropper, spatula, and stirrer.

Procedure:
1. Add 2ml of starch solution in a test tube.

2. Add 2ml of water in another test tube.

3. Add 2-3 drops of Iodine/KI/ solution and shake well the mixtures.

4. Observe the changes and record the changes that you are observed.
Laboratory session ≠ 5

Test for Protein (Biuret Test)

Objective

 To test the presence of proteins in a given food samples.

Introduction

Proteins are polymers of amino acids. Amino acids are small molecules that contain an amine (-
NH2), a carboxyl acid (-COOH), and a side chain (R). There are twenty naturally occurring
amino acids, and each amino acid has a unique side-chain or R-group. Amino acids are
connected by peptide bonds to form protein polymers. This gives rise to different levels of
structure for proteins. The primary (1°) structure of a protein is a made up of a string of amino
acids connected via peptide bonds. The secondary (2°) structure of a protein is formed by the
coiling and folding of the 1° structure, due to hydrogen bonding. At this level, structures called
alpha-helices and beta-sheets are visible. The tertiary (3°) structure of a protein is formed by
interactions between the components of the 2° structure. Some proteins have quaternary (4°)
structure, which includes the assembly of multiple individual subunits to form the functional
protein.

Material Required
 Egg albumin
 NaOH solution
 Test tube, CuSO4 solution, beaker, spatula, and stirrer.

Procedure
1. Add 2ml of egg albumin solution in a test tube and 2ml of water in another test tube.
2. Add 2ml of NaOH solution in both test tubes
3. Add carefully up to 10 drops of 1% CuSO4 solution drop by drop. Leave the contents for
10-15 minutes at room temperature.
Laboratory session ≠ 6

Test for Lipids (Ethanol Emulsion Test)

Objective
 To test the presence of fats and oils in a given feed sample

Introduction
Lipids are nonpolar macromolecules; thus they are insoluble in water. They include oils and fats,
phospholipids, and steroids. Fats and oils are triglycerides, composing of one glycerol and 3 fatty
acids. A fatty acid is a long chain of carbon-hydrogen (C-H) bonds, with a carboxyl group (-
COOH) at one end. Fatty acids can be classified as saturated or unsaturated. Fatty acids are
saturated when they do not contain any double bonds between the carbons and unsaturated when
they contain double bonds. An example of a saturated fat is butter, while an example of an
unsaturated fat is vegetable oil. The Ethanol Emulsion Test is a food test which determines the
presence of a broad group of naturally occurring compounds known as lipids. Lipids consist
of fats and oils. Other lipid tests include the Grease Spot Test and the Sudan Stain Test. The
Grease spot test is performed on fats - lipids which are solid at room temperature. Sudan stain
colours lipids red, but is a less common bench reagent than ethanol. The Ethanol Emulsion Test
is the most common test.

Materials Required

 Clean, dry test tubes

 Cooking oil or cooking fat

 Ethanol
Procedure
1. Place a sample of ethanol in a dry test tube to a depth of about 2 cm

2. Place a small sample of oil/cooking fat or a food sample in a dry test tube and add a similar
amount of ethanol

3. Shake the tube to dissolve any lipid in the ethanol.

4. Take two more test tubes and about half fill each with water.

5. Carefully pour the contents of the tube containing the oil fat or food sample into one of the
tubes containing water.

6. Pour about 2 cm³ pure ethanol into the other tube containing water and compare the two.
Laboratory session ≠ 7

Factors affecting enzyme activity

Objective
 To explore the effect of temperature, pH, and enzyme concentration on the rate of a
reaction.

Introduction
Enzymes speed the rate of chemical reactions. A catalyst is a chemical involved in, but not
consumed in, a chemical reaction. Enzymes are proteins that catalyze biochemical reactions by
lowering the activation energy necessary to break the chemical bonds in reactants and form new
chemical bonds in the products. Catalysts bring reactants closer together in the appropriate
orientation and weaken bonds, increasing the reaction rate. Without enzymes, chemical reactions
would occur too slowly to sustain life. The functionality of an enzyme is determined by the
shape of the enzyme. The area in which bonds of the reactant(s) are broken is known as the
active site. The reactants of enzyme catalyzed reactions are called substrates. The active site of
an enzyme recognizes, confines, and orients the substrate in a particular direction. Enzymes are
substrate specific, meaning that they catalyze only specific reactions. For example, proteases
(enzymes that break peptide bonds in proteins) will not work on starch (which is broken down by
the enzyme amylase). Notice that both of these enzymes end in the suffix -ase. This suffix
indicates that a molecule is an enzyme. Environmental factors may affect the ability of enzymes
to function. You will design a set of experiments to examine the effects of temperature, pH, and
substrate concentration on the ability of enzymes to catalyze chemical reactions. In particular,
you will be examining the effects of these environmental factors on the ability of catalase to
convert H2O2 into H2O and O2.

Materials Required
Part 1: Observing the Effects of Catalase

Procedure
1. Obtain two test tubes and label one as A and one as B.
2. Use your ruler to measure and mark on each test tube 1 cm from the bottom.
3. Fill each of two test tubes with catalase (from the potato) to the 1 cm mark
4. Add 10 drops of hydrogen peroxide to the tube marked A.
5. Add 10 drops of distilled water to the tube marked B.
6. Wait 60 seconds and measure the height of any bubbling you observe.

Questions

1. What happened when H2O2 was added to the potato in test tube A?
2. What caused this to happen?
3. What happened in test tube B?
4. What was the purpose of the water in tube B?
Part 2: Observe the Effects of pH

Procedure
1. Mark three test tubes with a wax pencil 3 cm from the bottom and 6 cm from the bottom.
2. Add 3 cm of hydrogen peroxide to each tube.
3. Fill one of the tubes to the 6 cm mark with 5 m HCl.
4. Fill another tube to the 6 cm mark with 5 m NaOH.
5. Fill the third tube to the 6 cm mark with distilled water.
6. Cut three strips of potato that is approximately 3 cm long. The strips should be cut thin
enough to easily fit into a test tube.
7. Place one of the strips of potato into each of the three tubes that you prepared in steps 2-6
above.
8. Write your observation of the amount of bubbling in your notebook. We are only
interested in the amount of bubbling. We are not interested in any change in color or
whether the potato floats or sinks.

Questions
1. At what pH did catalase function best in this experiment?
2. Do your results support your hypothesis? Explain.
3. What can you say about pH and enzyme functioning? Is there a single pH that enzymes
function best at or does it depend on the enzyme?
Laboratory session ≠ 8

Test for Plant Pigment by using paper Chromatography

Objectives

 To distinguish and study the various pigments present in plants through the process of
paper chromatography

Introduction
Plants carry out the process of photosynthesis, during which light energy from the sun is
converted into chemical energy (food). The capturing of light energy is carried out by molecules
known as pigments, which are present within the plant cells. Pigments are chemical compounds,
which are able to reflect only a particular range of wavelengths of visible light. Leaves of plants
primarily contain different types of pigments within their tissues. The four different types of
pigments are listed below in a tabular column along with their colours. The primary pigments in
green plants are chlorophylls, represented by chlorophyll a and b, which appear green. Visible
light, or white light, is made up of the colors of the rainbow. Some of these colors are absorbed
("used") by pigments and others are reflected. Pigments appear the color of the reflected light, so
the chlorophyll pigments do not use the green portion of the spectrum. The other two pigments
are types of carotenoids, which appear yellow, orange, or brown. The top bands of pigments in
the separation are carotenoids called carotenes, most likely beta-carotene, and appear yellowish-
orange. The second type of carotenoid separated in the experiment is xanthophylls, which appear
bright yellowish and are most likely lutein. The "loading line" is the location of the original
pigment line painted on the paper.
Material Required

 Chromatography chamber

 Spinach leaves

 Mortar and pestle

 Scissors

 Ether acetone solvent

 Acetone

 Capillary tube

 Pencil

 Spatula

 Scale

 Filter paper strips

 Stapler

 Thread

 Watch glass

Procedure
1. Take a strip of chromatography paper approximately 18 cm long. One end is blunt and
the other is pointed.
2. With a pencil lightly make a line 2 cm from the pointed end of the paper.
3. Bend the strip of paper at the blunt end and attach it to the small end of the cork with the
push pin. Adjust the length of the paper so that when it is inserted into the test tube, it
will touch the bottom without curling.
4. Place a ruler over the leaf so that is covers the pencil line on either end.
5. Cut up the leaves and fill a mortar up to 2cm depth.
6. Add a pinch of sand and about six drops of propanone from the pipette.
7. Grid the leaves by using pestle for at least three minutes.
8. On a strip chromatography paper, draw a pencil line 2-3cm from the bottom.
 9. Add the plant extract/plant juice/ in a beaker and take the plant juice on to the center of
the line. Keep the spot small as possible.
10. Allow the spot to dry, then add another spot on the top, letting it each one dry before
putting on the next. This is to build up a very concentrated small spot on the paper.
11. Attach the paper to the pencil using sell tape so that when placed in the beaker, the paper
is just clear of its base.
12. Add the alcohol in a beaker and add the paper in the beaker and hang the paper so it dips
in the alcohol. Ensure the alcohol level is below the spot.
13. Avoid moving the beaker in any way once the chromatography paper has started.
14. Leave the experiment until the alcohol has soaked near to the top and then remove the
paper from the beaker.
15. Mark how high the alcohol gets on a paper with a pencil and let the chromatograph to
dry.
16. Identify the pigments/chlorophyll a, chlorophyll b, carotin, and xanthophylls/.

Questions
1. Describe the color of each band?
2. Measure the distance from the first pencil line to the solvent front. Record this value for
each pigment and calculate, the RF values of each pigments.
3. What is a benefit of the pigments in photosynthesis?