Experiment Theory

8/14/2019 Experiment Theory

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Experiment 1

Conduct a qualitative test for: starch, fat, a reducing sugar, a protein

Theory

In order to test for the presence of starch, a solution of Iodine is dropped onto the suspected starch

containing moiety, e.g. potato. If a colour changes from deep red to purple/black occurs, this indicatesthe presence of starch. The reason for this change is that the iodine molecules non-covalently interact

with the long starch molecules and this alters the colour obtained.The simplest test for the presence of fat is to rub the suspected fat containing moiety on a piece of

brown paper. If a translucent spot occurs where the moiety has touched the paper, this shows that fat is

present.Some sugars such as glucose are capable of reducing other compounds and are called reducing sugars.

When reducing sugars are mixed with Benedicts reagent and heated, a reduction reaction causes the

Benedicts reagent to change colour. The colour varies from green to dark red, depending on the amountof and type of sugar.

The copper atoms of biuret solution (CuSO4 and KOH) will react with peptide bonds, producing a

colour change. A deep violet or blue colour indicates the presence of proteins and a lighter violet

colour indicates the presence of peptides.

Experiment 3

Identify and use various apparatus required for collection methods in an

ecological study

Theory

This experiment introduces the student to the basic ideas of understanding about our environment. Itshows student how to observe and capture small animals with the use of beating trays, nets and pooters.

It also helps in class bonding and is a welcome change to carrying out experiments in the laboratory.

Experiment 6

Be familiar with the use of the light microscope

Theory

A light microscope works very much like a refracting telescope, but with some minor differences. Let's briefly review how a telescope works. A telescope must gather large amounts of light from a dim,

distant object; therefore, it needs a large objective lens to gather as much light as possible and bring it

to a bright focus. Because the objective lens is large, it brings the image of the object to a focus at somedistance away, which is why telescopes are much longer than microscopes. The eyepiece of the

telescope then magnifies that image as it brings it to your eye.



Diagram of a typical student light microscope, showing the parts and the light path

In contrast to a telescope, a microscope must gather light from a tiny area of a thin, well-illuminated

specimen that is close-by. So the microscope does not need a large objective lens. Instead, the objectivelens of a microscope is small and spherical, which means that it has a much shorter focal length on

either side. It brings the image of the object into focus at a short distance within the microscope's tube.The image is then magnified by a second lens, called an ocular lens or eyepiece, as it is brought to

your eye.

Experiment 7

Preparation and examination of one animal and one plant cell (stained and

unstained)

Theory

Animal Cell Plant cell

Experiment 8

Investigate the effect of the pH on the rate of one of the following enzymes:

amylase, catalase, pepsin

Theory

An enzyme is a biological catalyst, usually speeding up the rate at which chemical reactions within the body occur. They are protein in nature and their performance is affected by a variety of factors.

Enzymes are affected by changes in pH. The most favourable pH value - the point where the enzyme is

most active - is known as the optimum pH. This is graphically illustrated below.



Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH isalso a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH

optimal stability.

The optimum pH value will vary greatly from one enzyme to another, as shown below:

In addition to temperature and pH there are other factors, such as ionic strength, which can affect the

enzymatic reaction. Each of these physical and chemical parameters must be considered and optimised

in order for an enzymatic reaction to be accurate and reproducible.

Experiment 9

Investigate the effect of temperature on the rate of one of the following enzymes:

amylase, catalase, pepsin

Theory

Like most chemical reactions, the rate of an enzyme-catalysed reaction increases as the temperature is

raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to

100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to20% in the results. In the case of enzymatic reactions, this is complicated by the fact that many



enzymes are adversely affected by high temperatures. As below, the reaction rate increases with

temperature to a maximum level, then abruptly declines with further increase of temperature. Becausemost animal enzymes rapidly become denatured at temperatures above 40·C, most enzyme

determinations are carried out somewhat below that temperature.

Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes

at 5·C or below is generally the most suitable. Some enzymes lose their activity when frozen.

Experiment 10

Investigate the effect of heat denaturation on the activity of one enzyme

Theory

Enzymes Practically all of the numerous and complex biochemical reactions that take place in animals, plants, and

microorganisms are regulated by enzymes. Most enzymes are Proteins. Each enzyme is able to promote onlyone type (or a small number) of chemical reaction. Enzymes can be classified into several broad categories,

such as hydrolytic, oxidizing, and reducing, depending on the type of reaction they control. In an enzyme

catalyzed reaction, the compounds on which the enzyme acts are called substrates and the resulting

compounds are called products.

The enzyme Catalase carries out the following reaction:

Put in words, Catalase takes two molecules of hydrogen peroxide and converts them to two water molecules plus a molecule of oxygen gas. Hydrogen peroxide is a toxic molecule (that's why we use it to kill Bacteria).

Hydrogen peroxide is also created in our bodies during normal metabolic events and catalase is present in the

peroxisomes of nearly all human cells. There, it serves to protect the cell from any toxic effects by catalyzingthe decompostion of H2O2 without the production of Oxygen free radicals.

The Catalase protein exists as a dumbbell-shaped tetramer of four identical subunits (220,000 to 350,000 kD).

Each monomer contains a heme prosthetic group at the catalytic center. This is the same type of Heme group

as found in Hemoglobin. In the middle of each heme group sits an iron atom. Catalase uses the iron atom tohel it break the bonds in the two molecules of h dro en eroxide shiftin the atoms around to release two



molecules of water and a molecule of oxygen gas.

Temperature can directly affect the rate of an enzymatic reaction. Firstly, all chemical

reactions are affected by temperature,

according to the laws of thermodynamics.

The increased molecular motion that

occurs as a result of increased temperature,makes collisions between the Enzyme and

substrate more likely, and therefore thereaction will occur at a greater rate.

So generally, as temperature increases so

does the rate of reaction. However onemust also bear in mind that high

temperatures can cause thermal

denaturation of the enzyme and freezing

may also damage an enzyme. Denaturation

is a change in structure of an Enzyme. Anenzyme's function is related to its 3-

dimensional structure. This structure can

be altered by heat, thus causing the enzymeto lose function.

Enzyme activity can also be affected by

pH. We will take advantage of this fact to

"Stop" our reactions in order to takemeasurements. When you add the

sulfuric acid to your catalase reactions you lower the pH below the range where

catalase is functional, and the reaction

stops. In the case of catalase, the optimum

pH is approximately pH 7.0. That is,

catalase works best at a neutral pH. If thesolution is too acidic (low pH value) or too

basic (high pH value) the catalase is inactive - no longer functions as an enzyme.

Summary Enzymes are Proteins that catalyze biochemical reactions. In these reactions, the compounds on

which the enzyme acts are called substrates and the resulting compounds are called products. Both

temperature and pH can affect the rate of the reaction catalyzed by an Enzyme.

Experiment 11

Prepare one enzyme immobilisation and examine its applications

Theory

In an enzyme technology process the enzyme is usually a high-cost item. This is compounded by thefact that catalytic activity is wasted at the end of the process, in fact, it is frequently the case that one

has to spend money removing (i.e. wasting) the enzyme protein from the product stream.Immobilisation leads to ease of removal from the product stream, re-use of the enzyme and lower costs.

Use of immobilised enzymes often facilitates the development of a continuous reactor, enhancing

productivity.

There is four commonly used generic approaches to enzyme immobilisation: Adsorption, covalentcoupling, entrapment and membrane incorporation. In this experiment we will use the method of

entrapment.



Enzymes can be trapped in the pores of gels or fibres. This is very convenient if the enzyme acts on

low molecular weight substrates as very high loadings can often be achieved - over 1g protein/g matrix.Entrapment can be purely physical or can also involve covalent coupling, for example it is possible to

react surface lysine residues with acryloyl chloride (CH2=CH-CO-Cl) and co-polymerise it into a

polyacrylamide gel. The most common entrapment method is the formation of calcium alginate beads.

The enzyme is mixed with sodium alginate, an acidic polysaccharide, and the mixture is dropped into a solution of calcium chloride. The calcium ions replace the sodium ions and cross-link the

polysaccharide. This results in the production of insoluble calcium alginate beads containing trapped enzymes.

Experiment 12

Investigate the influence of light intensity or carbon dioxide on the rate of

photosynthesis

Theory

Two of the main factors affecting the rate of photosynthesis are the light intensity and the presence o

carbon dioxide.

The use of Elodea (pond weed) under water can show how the rate is influenced by these factors. Theoxygen gas evolved during this set-up can be seen as bubbles and the faster the evolution of the

bubbles the faster is the rate of photosynthesis. The distance of a lamp from the experimental set-up the

slower the evolution of oxygen, hence, the slower the rate of photosynthesis. This is due to the fact thatthe light intensity reaching the pond weed has decreased.

Dissolution of sodium bicarbonate leads to the formation of carbon dioxide and the more carbon

dioxide the faster the rate of photosynthesis.

Experiment 13

Prepare and show the production of alcohol from yeast

Theory

This experiment is a practical application of industry. This reaction is carried out in Industry on a large

scale. It is the way by which alcoholic drinks are made. There are two tests for alcohol in this

experiment: The iodoform test which uses potassium iodide and the jones reagent which uses chromic

acid (sodium dichromate and sulphuric acid).



Experiment 14

Conduct any activity to demonstrate osmosis

Theory

Osmosis can be defined as the passage of water from a region of high water concentration through asemi-permeable membrane to a region of low water concentration. This experiment shows osmosis

taking place across the visking tubing which either expands or shrinks depending on the strength of the

solution.

Experiment 15

Isolate DNA from a plant tissue

Theory

DNA (deoxyribonucleic acid) is the code used within cells to form proteins. It is made up of a sugar

phosphate backbone and four bases, namely, adenine, guanine, cytosine and thymine. DNA is unique to

an individual, hence our DNA makes us all different. It can be used in forensics to identify people andin the future could be a form of identification for us.

(a) DNA Representation

(b) DNA as seen through an electron microscope

Experiment 16

Investigate the growth of leaf yeast using agar plates and controls

Theory

This experiment places a slant on this investigation. The quality of the air in the laboratory is indirectly

tested and can be loosely correlated to the yeast growth.

Members of the genus Sporobolomyces, a genus of basidiomycetous yeasts, form an invisible population on the surface of leaves. They do no harm to the leaves. In fact, sitting on the outside of the

leaf cuticle, they are dependent on tiny amounts of leaf-exudates, plus anything that comes to them

through the air or in the rain.

If there are any pollutants in the air, and most specifically, sulphur dioxide, this will affectSporobolomyces very quickly. Since their cells divide fairly often, this suggests that they could perhaps

be used to monitor short-term changes in air quality.



How can we test air quality with leaf yeasts? One simple technique takes advantage of the fact thatSporobolomyces shoots basidiospores as stated below:

1. Prepare some Petri plates with sterile malt extract .2. Cut the ash leaf into discs (5 mm in diameter) .

3. Attach seven discs by their upper surfaces to the lid of each petri dish with vaseline petroleum

jelly.

4. Incubate the dishes for 24 hours at room temperature with the leaf discs uppermost: thisensures that, when the basidiospores are shot, they will land on the surface of the sterile

medium.

5. Invert the dishes and incubate them at room temperature for three days. At this point you

should find groups of pink yeast colonies at the places where the spores landed on themedium.

6. The correspondence of these groups with the locations of the leaf discs explains why we callSporobolomyces a 'mirror yeast.' The number of colonies will reflect the health of the yeast,and so, indirectly, the quality of the air. Large scale comparative studies have been done by

school children in several European countries, and have established that the lowest numbers of

leaf yeast colonies (usually plotted as the square roots of the median counts), correlate wellwith higher levels of sulphur dioxide pollution.

Experiment 17

Prepare and examine microscopically the transverse section of a dicotyledonous

stem

Theory

Transverse section of angelica stem (Angelica). This transverse stem section displays an organization

that you might find in almost any stem whatsoever, with the exception of monocots and ferns. In all

seed plants except for the monocots, you will find the four zones visible here:

1) epidermis

2) cortex (in many species, as here, the outermost part is a hypodermis)

3) ring of vascular tissues (as here, usually a ring of vascular bundles)

4) pith.

Technically, the vascular tissues are called the stele, and stems with one ring of vascular tissues

surrounding a pith are said to have a eustele. Stems as diverse as slender vines, fat cacti, or as modified

as potato tubers all have this organization, it is just that the various zones might be modified. For example, cacti are so wide because they have an exceptionally thick cortex. Potato tubers on the other

hand have a gigantic pith and almost no wood. This uniformity of stem organization makes it much

easier for us to analyze and understand stem anatomy.





Experiment 18

Dissect, display and identify an Ox’s or sheeps’s heart

Theory

A central activity in Biology is the art of dissection. The forefathers of modern anatomy dissectedanimals in order to view their inner workings. Below is a picture of dissected sheep’s heart. This

experiment introduces the students to their dissection kit, it uses and its contents.



Experiment 20

Investigate the effect of IAA growth regulator on plant tissue

Theory

Tropisms

A tropism is a growth response of a plant to an external stimulus.

A tropism can be positive or negative.

• Positive: the growth response is in the direction of the stimulus.

• Negative: the growth response is away from the stimulus.

• Phototropism is the growth response of a plant in response to light direction.

• Geotropism is the growth response of a plant in response to gravity.

• Hydrotropism is the growth response of a plant to water.

• Tropisms are adaptive responses; they increase the plant’s chance of survival and

reproduction.

Plant Growth Regulators

Tropisms are controlled and moderated by special chemicals called growth regulators. A plant growth

regulator is an organic substance that is made in tiny amounts by the plant and has very definite

specific effects on tissue metabolism and growth. The target tissue of the growth regulator may be thelocal tissue or tissue in a different part of the plant. The growth regulator affects the cell cycle, cell

enlargement and cell differentiation .Natural plant growth regulators that move to their target are called

plant hormones. The transport of plant hormones to distant targets may be by diffusion, in xylem or in phloem. Auxin and gibberellin are together involved in stem cell elongation – each affecting a different

part of the process. Auxin and cytokinin are together involved in the terminal bud suppressing the

development of lateral buds – this is termed ‘apical dominance’.

Auxin

Functions

• increase the plasticity of plant cell walls for enlargement and shaping.

• influences the expression of specific genes involved in growth.

• role in stimulating cell division.

Effects

• change in growth direction of stem and root,

• apical dominance – prevent lateral bud growth,• fruit development,

Commercial Uses

• Rooting Power: increase the success of stem cutting by promoting extensive early rooting.

• Cytokinin: use in tissue culture to stimulate cell differentiation.

• Ethelene: quick ripening of mature ‘green bananas’ for the market.

• Auxin: as a selective weed killer to reduce competition and so promote crop growth.



Experiment 21

Investigate the effect of water, oxygen and temperature on germination

Theory

Seed Germination

Seed germination is the restart of growth by the plant embryo using the food stored in the seed.

Water, oxygen and a suitable temperature are the major factors for successful germination.

Water

• Plant cells are 90% water.

• Water is essential for increase in cell number during growth.

• Water is needed for food reserve digestion and transport of nutrients to the growing points.

Oxygen

• Oxygen is needed for efficient ATP production from the reserve food.

Suitable Temperature

• Required for optimum enzyme activity and so for optimum growth.

• Some of the enzymes are involved in the digestion of the complex food reserve.

• Other enzymes are involved in growth and ‘housekeeping’ activities.

The experiment to investigate the Effect of Water, Oxygen and Temperature on Seed

Germination can be carried out as below:

(1) Four small clear glass jars kept in darkness.

(2a) Jar A: water-soaked seeds on soaked cotton wool, open to air at 20ºC.

(2b) Jar B: dry seeds on dry cotton wool, open to the air at 20ºC.

(2c) Jar C: 4°C – water-soaked seeds on soaked cotton wool (in a fridge) open to the air.

(2d) Jar D: water-soaked seeds at 20°C in a sealed jar that does not have oxygen in the air –

oxygen removed by pyrogallol or wet iron filings on filter paper.

One week later check the results.

Results

Jar A: Germination occurs as water, oxygen and temperature are present in sufficient quantities for growth to take place.

Jar B: no water / no germination.

Jar C: low temperature / no germination.



Jar D: no oxygen / no germination.

This result concludes that water, suitable temperature and oxygen are all together

required for seed germination.

Experiment 22

Use starch agar or skimmed milk plates to show digestive activity duringgermination

Theory

This experiment investigates whether or not starch breakdown occurs during germination. The

experimental process compares dead seeds with live seeds and the use of iodine as a stain in theexperiment. The results show that the dead seeds still posesses starch whereas the live seeds there are

yellow brown areas showing that starch breakdown has occurred.

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