Lab Manual Biology Final After Sections Modifications

8/10/2019 Lab Manual Biology Final After Sections Modifications

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Biology Laboratory Manual Laboratory safety practices


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1.LABORATORY SAFETY PRACTICES

Important notes in biology laboratory session:

1. All staff and students working in laboratories share the responsibility of safety.

2.No food, drinks or smoking are allowed in the lab.

3. Safety glasses or goggles should be worn in all laboratories when needed.

4. Protective clothing should be worn as specified.

5. Always use gloves when conducting an experiment.

6. Always use mask when dealing with evaporative chemicals

7. Contact lenses, open shoes and sandals are not recommended in laboratories.

8. Long hair must be tied back during laboratory sessions.

9. All work areas MUSTbe kept clean and organized. Separate containers are to be

used for paper and broken glassware.

10.Students should report any accident to supervisor, demonstrator, instructor or

laboratory technician.

11.Notify the instructor if there is a spill of chemicals or broken glass.

12.Follow all the instructions carefully.

Objectives

By the end of the exercise, students should be able to:

1- Learn how to protect themsleveswhenconducting an experiment.

2- Act appropriately when an accident happens.

3- Understand how to handle chemicals, hazardous material and

waste.


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13.Everyone in the laboratory should know where the exits are, the locations of safety

showers, eye-baths, fire extinguishers, and what to do if the evacuation alarm (fire

alarm bell) rings and be familiar with their operation, as shown in the signs below.


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Fig. 1: important signs

1. Never enter the laboratory unless a teacher is present or without permission.

NEVER

2. Never eat or drink in the lab.

3. Never taste or sniff any chemicals or substances you are working with.

4. Never use your mouth for pipetting substances, use a rubber suction bulb or

special pipet filler.

5. Never handle broken glass with bare hands.

6. Never pour chemicals down the drain without permission.

7. Never operate lab equipment without permission.

8. Never perform your own experiments unless given permission.

9. Never leave any heated materials unattended.

10.Never place flammable substances near heat.

11.Never engage in childish antics such as horseplay or pranks.

12.Never run or play in the laboratory.

13.Never remove anything from the laboratory without your instructor's permission.

14.Never use your bare hands to transfer chemicals. Use a spatula instead.

15.Never leave experiments unattended.

Biology Lab Rules:

1. Before you enter a biology lab, you should be prepared for and knowledgeable about

any lab exercises that are to be performed. Read your lab manual to know exactly

what you will do.

2. Before you enter a biology lab, wear your lab coat.


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3. Wear proper clothing and shoes; some chemicals have the potential to damage

clothing. Always wear proper clothes and keep your coat in the laboratory. Wear

shoes that can protect your feet in case something gets broken. Sandals or any type of

open-toed shoes are not recommended.

4. When working in a biology lab, make sure you keep your area organized. If you

happen to spill something, ask for assistance when cleaning it up. Also remember to

clean your work area and wash your hands when you are finished.

5. An important biology lab safety rule is to be careful. You may be working with

chemicals, flames, glass or sharp objects.

6. Keep your area organized and clean during and after every laboratory session.

Neverdispose hazardous and sharp wastes in the in the regular trash containers.

Containers designated for the disposal of sharp wastes (scalpel blades, needles;

dissection pins.etc.) and containers designated for biological wastes (animals and

plants...etc) are present in each laboratory. (Fig. 2)

Laboratories fume hoods:

Fume hoods are installed in laboratories to protect students from hazardous

vapours generated by laboratory experiments. Fume hoods are not the same as biosafety

cabinets. Laboratory hoods and biosafety cabinets (or tissue culture hoods), although

similar in appearance, are different devices. Biosafety cabinets are designed for

protection against exposure to biological materials and for protection against

contamination of biological specimen, and typically offer no protection against

chemical vapours.


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Fig. 2: Types of hazards.

General Laboratory Safety Guidelines

1. Do not mix any chemicals except as instructed. Do not do unauthorized

experiments.

2. NO chemicals are to be flushed down a drain unless specifically instructed to do

so by the lab procedure.

3. Wash your hands before leaving the laboratory.

4. Clean up broken glass immediately. DISPOSE IN SPECIFIED "BROKEN

GLASS" CONTAINER ONLY.

5. Clean up solid and liquid spills immediately, but only after checking with your

laboratory instructor about possible safety hazards.

6. Take containers to the stock of chemicals. Do not bring stock chemicals to your

laboratory table.

7. Read the label on chemical bottles carefully. Insure that you have the correct

chemical.

8. Do not insert a pipet or medicine dropper into a stock bottle. Avoid contamination

by pouring a small quantity into a flask or beaker before taking a sample.


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9. Use special care when handling stoppers or tops of bottles so as not to pick up

contamination.

10.Take no more of a chemical than an experiment requires.

11.Never return as unused chemical to a stock bottle. Dispose of it as waste.

12.Set up your glassware and apparatus away from the front edge of your laboratory

bench.

13.Follow any other housekeeping, safety, or disposal rules given by your instructor.

In the laboratory, be familiar with:

1. Emergency shower and eye wash station.

2. Fire extinguisher (s).

3. Fire blanket.

4. Exits from the room.

5. Fire escape route.

6. Fire alarm boxes.

7. Container for broken glass.

8. Electrical power cut off switch (es).9. First aid box.

10. Biohazards waste container.


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How to handle chemicals:

The National Fire Protection Association (NFPA) hazard identification system uses acolor-coded diamond to represent four different hazards.

The different colours represent three different types of hazards that may be associated

with chemicals:

Blue: indicates health hazard.

Red: indicates flammability.

Yellow: indicates (radioactivity) reactivity.

White: represents other hazards such as if a chemical reacts violently with water () or is

an oxidizer () as shown in fig. 3

The numbers in the blue, red and yellow diamonds are used to indicate the severity of

the hazard for that

category:

0 = no or minimal hazard

1 = slight hazard

2 = moderate hazard

3 = serious hazard

4 = extreme hazard

Fig. 3: color-coded diamond

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Biology Laboratory Manual The Scientific Method


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2- The Scientific Method

Introduction

Science is based on empirical evidence and numerical measurements. Sometimes you

may resort to guessing when you are performing a scientific experiment; however, this

is not an accepted scientific approach to solving problems. Although some prominent

scientific findings were discovered initially by chance/mistake during conducting an

experiment, they had to be confirmed by adapting the scientific method. Applying the

scientific method normalizes any intuition or bias towards certain results. The use of a

common scientific method unifies the way scientists conduct their experiments and

helps objective comparison of the results performed in different laboratories.

The main steps followed to conduct scientific inquiry include:

Make an observation.

Formulate a question.

Set a hypothesis and make a prediction.

Execute experiments.

Collect, analyze, and challenge data to the hypothesis.

Draw a conclusion.

Objectives

By the end of this section , students should be able to:1- Understand the logic behind implementing the scientific method.

2- Determine how to formulate a scientific question.

3- Appreciate the value of applying the scientific method.

4- Differentiate between a hypothesis and a theory.

5- Apply the scientific method on an experimental example.

6- Value the importance of precision when conducting a scientific experiment.


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1.Make an observation

Observation is the driving force for scientists to start a new scientific research. Good

observations are usually made by talented and knowledgeable scientists, but can also be

made by anyone who watches carefully. Remember that Newton was not the first

person to watch an object falls; however, he was the first one to be inspired by this

incident to formulate the theory of gravitation. Observation should be objective and not

subjective. Observation should be verified by others.

2.Ask a question

Observation leads to a question. The question is usually novel, and should have not

been tackled before by other scientists. Formulation of a good question is not an easy

task. Before you ask the question, you should have a good knowledge of what has been

done in the field. Literature search on the subject ensures that your idea is novel, and

provides you with a good understanding of previous findings in the specific field

3.Formulate a hypothesis and make a prediction

Before you conduct your experiment you need to restate the question to form a clear

hypothesis. A good hypothesis should be based on available observations, and should

be testable. It should be falsifiable (can be proven right or wrong). Many predictions

can be suggested to test one hypothesis.

4.Execute experiments

Experiments are performed to validate a hypothesis. The results of the conducted

experiments should agree with or refute the hypothesis. Experiments that agree with the

hypothesis and do not contradict it do not necessarily prove that the hypothesis is true,

but increase the confidence in the hypothesis. Many parameters need to be taken into

consideration when conducting an experiment, such as using the appropriate controls,

repeating the experiment, and using one variable at a time. One of the most important

parameters in judging the validity of experiments is reproducibility.


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5.Collect, analyze, and challenge data to the hypothesis

Raw data are collected from experiments and should be subjected to scrutiny and

statistical analysis before formulating them in the form of tables or figures. Experiments

may support or refute the hypothesis. If there is a lack of confidence, other experiments

need to be performed to test another prediction for the same hypothesis. To increase

confidence, each prediction should be tested by several experiments.

6.Draw a conclusion

Based on the collected data and their interpretation, a conclusion can be drawn to

support or refute the hypothesis. If data support the hypothesis, then the hypothesis is

valid, and can be considered by other scientists for further investigation. However, if

the data do not support the hypothesis, you need to re-examine your original hypothesis,

observation, or experiments.


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Questions

1-What is the difference between a hypothesis and a theory?

2-Can a hypothesis be false? Explain

3-What does significant difference means when you analyze your data?

4-Is evolution a theory or a fact? Support your selection with evidence.

5-Give an example and brief description of a theory that cannot be proven to be a fact.

6-What measures should you take to improve your ability to conduct the scientific

method?

7-Can cultural bias affect the scientific method? Explain

8-Two scientists in different labs examined the same hypothesis; both found out that

the hypothesis is true. Did both scientists conduct same experiments? Explain your

answer.

Students name: _________________________ ID: ________

Lab. Instructors name: __________________________________________Lab. Teaching assistants name (1): ________________________________

Lab. Teaching assistants name (2): ________________________________

Laboratory number: ____________________________________________

Laboratory section number: ______________________________________


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Biology Laboratory Manual Biologically Important Molecules


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3- Biologically Important Molecules

Introduction

The most common four major classes of organic compounds found in the living

organism are carbohydrates, proteins, lipids, and nucleic acids. The macromolecules

(polymers) of each class are formed by covalently bonding one or subunits (monomer)

together in a dehydration synthesis reaction. This is an energy-requiring process in which

two subunits are bonded covalently and a molecule of water is removed. Breaking the bond

between these two subunits is an energy-releasing process (hydrolysis) that requires

addition of water (Figure 1).

Objectives

By the end of the experiment, the student should be able to:

1- Describe the basic structure and properties of each of the biologically

important molecules.

2- Perform tests to detect the presence of carbohydrates, lipids, proteins, and

nucleic acids in known samples.

3- Recognize the importance of positive and negative control in a biochemical

test.

HHOHHO HOH

HHO

2H2O 2H

2O

Deh dration S nthesis H drol sis

Fi ure 1. Deh dration s nthesis and h drol sis of a ol mer


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Identification the major organic compounds

There are several tests to identify the major types of organic compounds. Each of these

tests includes an unknown solution, and positive and negative control solutions. An

unknown solution may or may not contain the substance that is being tested

A positive control solution contains the substance for which you are testing and shows

what a positive test looks like. A negative control does not contain the substance that you

are testing for and demonstrate what a negative result looks like.

OH

OHHO

CH2O

O

HO

OH

OHHO

CH2O

O

HO

OH

CH2O

O

CH2O

O

OH

OH

CH2O

OOOH

O

OH

CH2O

OHO

OH

CH2O

O

Glucose

Sucrose

Polymers of monosaccharides (Polysaccharides)

Figure 2. Examples of monosaccharides (glucose),

disaccharides sucrose


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I. Carbohydrates

Carbohydrates are molecules made up of C, H, and O in ratio of 1:2:1. For more complex

carbohydrate, this ratio breaks down but it holds for simple carbohydrates as

monosaccharaides. Carbohydrates could be monosaccharide, disaccharide, or

polysaccharide. Examples of mMonosaccharaides (or simple sugars) include glucose and

fructose. Disaccharides are paired monosaccharide (e.g. sucrose, maltose, and lactose).

Polysaccharides are more than two saccharides linked together (e.g. starch, glycogen, and

starch) (Figure 3).

Monosaccharaides, such as glucose and fructose that have free aldehyde (-CHO) or ketone

(-C=O) groups, are reducing sugars. Theses reducing groups reduce weak oxidizing agents

such as the copper in Benedict reagent. Benedict reagent is a copper citrate alkaline

solution. It contains copper in the oxidized form (Cu2+) which develops the blue color of

the reagent. When a solution containing a reducing sugar and Benedicts reagent is heated,

the reducing sugar reduces cupric ions (Cu2+) to cuprous oxide changing the solution color

from blue to green, orange, reddish orange, or brick-red (this depends on the amount of

reducing sugar). All monosaccharide and some disaccharide are reducing sugars, while

others are not reducing sugars.

Benedicts test for reducing sugar:

Material and methods:

1.Number seven test tubes (1-7)

2. Add to each test tube the materials to be tested as indicated in table 1. Add 2 mL ofBenedicts solution to each test tube and mix.

3. Place all tubes in a boiling water-bath for three minutes. Observe the change in color

during this time.

4. After three minutes, take the tubes out from the water-bath and cool them to room

temperature.

5. Record the color of each test tube in table1.


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Questions:

Table1. Color reaction for Benedicts test for reducing sugar and Iodine test for starch.

1. Which groups of a glucose molecule is involved in forming a polysaccharide?

2. Are all monosaccharaides and disaccharides considered reducing sugars?

3. Which of the following is a reducing sugar: sucrose or glucose?

4. In Benedicts test, which solution is a positive control and which is a negative

control?

5. Which juice contains more reducing sugars: onion juice or potato juice

Tube SolutionBenedicts Color

Reaction

Iodine Color

Reaction

1 10 drops potato juice ................................. ................................

2 10 drops onion juice ................................. ................................

3 10 drops sucrose solution ................................. ................................

4 10 drops distilled water ................................. ................................

5 10 drops glucose solution ................................. ................................

6 10 drops fructose solution ................................. ................................

7 10 drops starch solution ................................. ................................

Students name: _________________________ ID: ________

Lab. Instructors name: __________________________________________






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Biologically Important MoleculesBiology Laboratory Manual

Starch

Iodine (Iodine-potassium iodide, I2KI) staining is used to distinguish starch from

monosaccharaides, disaccharide, and other polysaccharides. Starch is a coiled polymer of

glucose where iodine interacts with these coiled molecules and becomes bluish black.

There is no interaction between carbohydrates that are not coiled and Iodine. Therefore, a

bluish-black color is a positive test for starch and a yellowish-brown (Iodine color) is a

negative test for starch.

Iodine test for starch, material and methods:

1. Number seven test tubes (1-7)

2. Add to each test tube the materials to be tested as indicated in Table 1. Add 2 mL of

Benedicts solution to each test tube and mix.

3. Add five to seven drops of iodine to each test tube.

4. Record the colour of each test tube in table1.

Questions:


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1. In Iodine test, which solution is a positive control and which is a negative control?

2. Which solutions contain starch?

3. Which of the following is a reducing sugar sucrose or glucose?

4. Does Iodine stain monosaccharaides or polysaccharides?

Students name: _________________________ ID: ________



Lab. Teaching assistants name (2): ________________________________Laboratory number: ____________________________________________



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Proteins

Proteins are made up of amino acids. Each amino acid has an amino group (NH 2), a

carboxyl group (-COOH), and variable side chain (Figure 3). The peptide bond (C-N)

between two amino acids is formed through dehydration synthesis, linking a carboxyl

group of one amino acid to an amino group of the other (Figure 4). This peptide bond could

be detected by Biuret reagent (1% solution of CuSO4) where Cu2+

complexes with the

peptide bond producing violet color. A Cu2+

must complex with at least four peptide to

produce this violet color. The color intensity correlates with the reacted number of peptide

bonds.

N

R

H O

OHC C

H

HOHN

R

H O

C C

H

H

N

R

H O

OHC C

H

N

R

H O

C C

H

H

H2O

Figure 4. The peptide bond (C-N)between two amino acids is formedthrough dehydration synthesis, linking a carboxyl group of one amino acid

to an amino group of the other.

H2N

+

CH3

H O

OHC C

Alanine

H2N

+

CH

H O

OHC C

CH3

CH3

Valine

H2N

+

CH

H O

OHC C

CH2CH

3

CH3

Leucine

Figure 3. Examples of amino acids. Each amino acid has a caboxyl group

(-COOH), an amino group (-NH2), and unique side chains (colored).


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Questions:

1. Do free amino acids have peptide bonds?

2. Which of the solutions is a positive control?

3. After carrying out the test, which solution seems to have more protein?

4. Does Iodine stain monosaccharides or polysaccharides?

5. Circle and label the carboxyl groups and reactive amino groups in the amino acids

shown below

Students name: _________________________ ID: ________






H2N

+

CH3

H O

OHC C

Alanine

H2N

+

CH

H O

OHC C

CH3

CH3

Valine

H2N

+

CH

H O

OHC C

CH2CH

3

CH3

Leucine


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Lipids

Lipids are a group of naturally occurring molecules that include fats, waxes, sterols, fat-

soluble vitamins, glycerides, and others. Generally, lipids dissolve in non-polar solvents

such as acetone, ether, or methanol but not in polar solvents such as water. Lipids start out

as triglycerides that consist of a glycerol and three fatty acids (Figure 5). An ester linkage

is formed when a hydroxyl group of glycerol links with the carboxyl group of a fatty acid.

Fatty acids are either saturated or unsaturated (contain a double bond between carbon

atoms). To test presence of lipid, Sudan IV solution (fat-soluble dye) is used. This test is

based on the ability of lipid to absorb pigments in the Sudan IV solution.

Sudan IV test for lipids

1. Label five test tubes (1-5)

2. Add to each test tube the materials to be tested as indicated in table 3.

3. Add five drops of Sudan IV to first four tubes and five drops of water to the fifth tube

and mix.

4. Record the color of each test tube in Table 3.

Table3. Color reaction for Sudan IV test for lipids.

Tube Solution Colour

1 1 mL salad oil + Sudan IV ...........................................

2 1 mL honey + Sudan IV ...........................................

3 1 mL distilled water + Sudan IV ...........................................

4 1 mL lipid solution + Sudan IV ...........................................

5 1 mL salad oil + water ...........................................


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Questions:

1. How ester linkage is formed in triglycerides?

2. Which of the used solutions is a positive control? Which is a negative control?

3. Which solution contains more lipids?

4. Does Sudan IV stain monosaccharaides or polysaccharides? Explain

5. Is salad oil is soluble in polar solvents? Explain

Students name: _________________________ ID: ________







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II.Nucleic acids

Nucleic acids include DNA (deoxyribonucleic acid) and RNA ((ribonucleic acid), the

former contains deoxyribose sugar whereas the latter contains ribose sugar (Figure 6). To

test the presence of DNA, Dische diphenylamine reagent is used. In Dische diphenylamine

test, under acidic conditions, deoxyribose is converted to a molecule that binds

diphenylamine and form a blue complex. The intensity of the color increases with

increasing the detected amount of DNA.

Sudan test for Dische diphenylamine test

1. Label five test tubes (1-5)

2. Add to each test tube the materials to be tested as indicated in table 4.

3. Add Dische diphenylamine reagent to all tubes and mix.4. Heat the tubes by placing then in a boiling ware bath for 10 minutes and then place the

tubes in ice bath.

5. Record the color of each test tube in table 4.


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Table 4. Color reaction for Dische diphenylamine test for DNA.

Tube Solution Color

1 2 mL DNA solution ...................................................

2 2 mL RNA solution ..................................................

3 1 mL DNA solution, 1 mL water ..................................................

4 1 mL RNA solution, 1 mL water ..................................................

5 2 distilled water ..................................................


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Biology Laboratory Manual Water Treatment


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4-Water Treatment

Decomposition of Organic Substances in Water

Introduction:

Water covers over 70% of the earths surface and is considered a very important resource

for people and the environment. One way of judging water quality is to determine the

amount of oxygen dissolved in the water. Clean water usually has high oxygen content.

Polluted water usually has low oxygen content because organisms in the water use oxygen

as they decompose. Water pollution has many dangerous effects in drinking water, oceans,

rivers, and lakes. Practically all types of water pollution are deleterious to the health of

humans and animals. Health damage caused by water pollution may not appear

immediately but can be harmful after long term exposure. Forms of pollutants include: (a)

Heavy metals that come from industrial wastes and accumulate in lakes and rivers. They

are toxic to marine life and are transmitted to humans through diet. (b) Microbial

pollutants from sewage cause infectious diseases of contaminated drinking water. This is

considered a major problem in the developing countries where diseases like Cholera and

Typhoid are the primary cause of infant mortality. (c) Organic waste: aerobic algae

increased due to organic matter and nutrients and as a result oxygen is depleted from

water, which results in suffocation of fish and other aquatic organisms.

The amount of organic material that can decay in the sewage is measured by the

biochemical oxygen demand. BOD is the amount of oxygen needed by micro-organisms

to decompose the organic substances. Therefore, the more organic material there is in the

sewage, the higher the BOD. Dissolved oxygen is an important factor that determines the

ObjectivesBy the end of the experiment, the student should be able to:

2. Determine the oxygen content of clean water and waste water.

3. Predict the pollution difference between clean and waste water.


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quality of water in lakes and rivers. The higher the concentration of dissolved oxygen, the

better the water quality.

Organic substances which end up in natural water bodies as waste water are broken down

into simpler forms of matter by native microorganisms. Bytime, oxygen is consumed as

micro-organisms use it in their metabolism to decompose the organic substances in water

and the organically fixed carbon content eventually eliminated from the water through

respiration. However, due to the decrease of dissolved oxygen non-poisonous organic

waste can threaten animal life in water and most of the animals die and creates more

organic matter for the bacteria to decompose. In fact, if the oxygen level drops to zero, the

water will become septic and when organic compounds decompose without oxygen, itgives rise to the undesirable odours usually associated with septic or putrid conditions.

Accordingly, adding oxygen is useful in maintaining aerobic conditions in the biological

purification steps of a wastewater treatment.

1. Precision Balance.

Materials:

2. Oxygen ECO-Test.3. Culture vessel: Cylindrical glass vessel that can be adjusted using different

cover on a variety of applications, e.g. breeding glass, moist chamber for

incubation, osmosis chamber and assimilation chamber.

4. Thermometer.

5. Spoon, w. spatula end, 18 cm,

plastic.

6. Glass rod.

1- Read and follow the lab safety form.

Experimental procedure:

2- Fill 4 culture vessels with tap water,

leave about 1 finger length below to

edge of the vessel.


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3- In 3 of the culture vessels, add 0.5g food for aquarium fish.

4- Mark vessel number # 4 as control, without adding food.

5- Stir the vessels content, and measure the temperature in each vessel as well as the

level of oxygen.

6- Cover the surface area with polyethylene coated filter paper.

7- Pour 1ml water sample into one of the measuring glasses and place it in position A

in the comparator.

8- Rinse the oxygen reaction bottle several times with the water to be tested and fill

until overflows without air bubbles.

9- Add 5 drops of oxygen 1.

10-Add 5 drops of oxygen 2, close the bottle with the stopper (avoid air bubbles) and

mix by shaking.

11-After 1 minute, add 12 drops of oxygen 3, close the bottle and shake well until the

deposit is dissolved.

12-Pour 1 ml of the resulted reaction into the second measuring glass and place it on

position B in the comparator.

13-Slide the comparator until the colours match in the inception hole on top. Check the

measurement reading in the recess on the comparator reed. Mid values can be

estimated.

14-After use rinse out the oxygen reaction bottle and both measuring glasses

thoroughly and seal them.

15-Record the measurement results.

16-Repeat these measurements after 24, 48 and 72 hours in the same manner. The

water temperature should stay as constant as possible during that time.

17-From the three results, you get in the vessels with food addition, calculate the

average value. This value is compared to the results from the control vessel.

18-When you have completed your experiment, dispose of materials as directed by

your instructor.


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Observations:

Record the measurements of the level of oxygen and temperature in the following

table:

Oxygen Level 24hr 48hr 72hr

Vessel 1

Temperature 24hr 48hr 72hr

Vessel 1

Results:

You will then notice that the oxygen level sinks from day to day, as the microorganisms

that have been introduced with the water and the fish are mineralizing the fish food. In the

control vessel the oxygen level stays unchanged. After a while, the water cloudiness

decreases as more fish food is decomposed.


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Questions:

1. List three ways in which water is polluted and how can we prevent them?

2. Explain why the water samples have different concentrations of dissolved oxygen?

Students name: _________________________ ID: ________

Lab. Instructors name: __________________________________________Lab. Teaching assistants name (1): ________________________________





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Biology Laboratory Manual Frog Dissection


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Objectives


1. Handle laboratory animals.

2. Become proficient in dissecting the frog.

3. Identify the different organs of the frog.

4. Find the relations between the organs of the different systems.

5- Frog Dissection:

Digestive, Reproductive and Nervous

Organs Identification

Introduction

The frog is a well-known and established biological system for dissection and research. An

adult frog is generally characterized by a stout body, protruding eyes, cleft tongue, limbs

folded underneath and the absence of a tail. Besides living in fresh water and on dry land,

the adults of some species are adapted for living underground or in trees. The skin of the

frog is glandular, with secretions ranging from distasteful to toxic.

Frogs typically lay their eggs in water. The eggs hatch into aquatic larvae, called tadpoles,

which have tails and internal gills. They have highly specialized rasping mouth parts

suitable for herbivorous, omnivorous or planktivorous diets. The life cycle is completed

when they metamorphose into adults. A few species deposit eggs on land or bypass the

tadpole stage. Adult frogs generally have a carnivorous diet consisting of smallinvertebrates, but omnivorous species exist and a few feed on fruit. Frogs are extremely

efficient at converting what they eat into body mass, which makes them an important food

source for predators. Frogs are a keystone group in the food web dynamics of many of the

world's ecosystems. The skin is semi-permeable, making frogs susceptible to dehydration,

so they either live in moist places or have special adaptations to deal with dry habitats.


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Frogs produce a wide range of vocalizations, particularly in their breeding season, and

exhibit many different kinds of complex behaviours to attract mates, to fend off predators

and to generally survive.

Respiration and circulation

The skin of a frog is permeable to oxygen and carbon dioxide, as well as to water. There

are blood vessels near the surface of the skin and when a frog is underwater, oxygen

diffuses directly into the blood. When not submerged, a frog breathes by a process known

asbuccal pumping.Its lungs are similar to those of humans but the chest muscles are not

involved in respiration, and there are noribsordiaphragm to help move air in and out.

Instead, it puffs out its throat and draws air in through the nostrils, which in many species

can then be closed by valves. When the floor of the mouth is compressed, air is forced into

the lungs. The Borneo flat-headed frog (Barbourula kalimantanensis) was first discovered

in a remote part of Indonesia in 2007. It is entirely aquatic and is the first species of frog

known to science that has no lungs.

Frogs have three-chamberedhearts, a feature they share withlizards.

http://en.wikipedia.org/wiki/Frog - cite_note-Kimball-53.Oxygenated blood from the

lungs and de-oxygenated blood from therespiring tissues enter the heart through

separateatria. When these chambers contract, the two blood streams pass into a

commonventriclebefore being pumped via a spiral valve to the appropriate vessel,

theaorta for oxygenated blood andpulmonary artery for deoxygenated blood. The ventricle

is partially divided into narrow cavities which minimizes the mixing of the two types of

blood. These features enable frogs to have a higher metabolic rate and be more active than

would otherwise be possible.

Digestion and excretion

Frogs have Maxillary teeth along their upper jaw which are used to hold food before it is

swallowed. These teeth are very weak, and cannot be used to chew or catch and harm agile

prey. Instead, the frog uses its sticky, cleft tongue to catch flies and other small moving

prey. The tongue normally lies coiled in the mouth, free at the back and attached to the

mandible at the front.
http://en.wikipedia.org/wiki/Buccal_pumpinghttp://en.wikipedia.org/wiki/Ribhttp://en.wikipedia.org/wiki/Ribhttp://en.wikipedia.org/wiki/Ribhttp://en.wikipedia.org/wiki/Diaphragm_(anatomy)http://en.wikipedia.org/wiki/Hearthttp://en.wikipedia.org/wiki/Lizardhttp://en.wikipedia.org/wiki/Frog#cite_note-Kimball-53http://en.wikipedia.org/wiki/Frog#cite_note-Kimball-53http://en.wikipedia.org/wiki/Respiration_(physiology)http://en.wikipedia.org/wiki/Atrium_(anatomy)http://en.wikipedia.org/wiki/Ventricle_(heart)http://en.wikipedia.org/wiki/Aortahttp://en.wikipedia.org/wiki/Pulmonary_arteryhttp://en.wikipedia.org/wiki/Pulmonary_arteryhttp://en.wikipedia.org/wiki/Aortahttp://en.wikipedia.org/wiki/Ventricle_(heart)http://en.wikipedia.org/wiki/Atrium_(anatomy)http://en.wikipedia.org/wiki/Respiration_(physiology)http://en.wikipedia.org/wiki/Frog#cite_note-Kimball-53http://en.wikipedia.org/wiki/Lizardhttp://en.wikipedia.org/wiki/Hearthttp://en.wikipedia.org/wiki/Diaphragm_(anatomy)http://en.wikipedia.org/wiki/Ribhttp://en.wikipedia.org/wiki/Buccal_pumping


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It can be shot out and retracted at great speed. Some frogs have no tongue and just stuff

food into their mouths with their hands. The eyes assist in the swallowing of food as they

can be retracted through holes in the skull and help push food down the throat. The food

then moves through the oesophagus into the stomach where digestive enzymes are added

and it is churned up. It then proceeds to the small intestine (duodenum and ileum) where

most digestion occurs. Pancreatic juice from the pancreas, and bile, produced by the liver

and stored in the gallbladder, are secreted into the small intestine, where the fluids digest

the food and the nutrients are absorbed. The food residue passes into the large intestine

where excess water is removed and the wastes are passed out through the cloaca.

Reproductive system:

In the male frog, the twotestesare attached to the kidneys andsemenpasses into the

kidneys through fine tubes calledefferent ducts. It then travels on through the ureters,

which are consequently known as urinogenital ducts. There is no penis, and sperm is

ejected from the cloaca directly onto the eggs as the female lays them. The ovaries of the

female frog are beside the kidneys and the eggs pass down a pair of oviducts and through

the cloaca to the exterior.

Nervous system

The frog has a highly developed nervous system that consists of a brain, spinal cord and

nerves. Many parts of the frog's brain correspond with those of humans. It consists of two

olfactory lobes, two cerebral hemispheres, a pineal body, two optic lobes, a cerebellum and

a medulla oblongata. Muscular coordination and posture are controlled by thecerebellum,

and theMedulla oblongataregulates respiration, digestion and other automatic

functions. The relative size of thecerebrumin frogs is much smaller than it is in humans.

Frogs have ten pairs ofcranial nerveswhich pass information from the outside directly to

the brain, and ten pairs ofspinal nerveswhich pass information from the extremities to the

brain through the spinal cord. By contrast, allamniotes(mammals, birds and reptiles) have

twelve pairs of cranial nerves.
http://en.wikipedia.org/wiki/Testishttp://en.wikipedia.org/wiki/Testishttp://en.wikipedia.org/wiki/Testishttp://en.wikipedia.org/wiki/Semenhttp://en.wikipedia.org/wiki/Semenhttp://en.wikipedia.org/wiki/Semenhttp://en.wikipedia.org/wiki/Efferent_ductshttp://en.wikipedia.org/wiki/Efferent_ductshttp://en.wikipedia.org/wiki/Efferent_ductshttp://en.wikipedia.org/wiki/Cerebellumhttp://en.wikipedia.org/wiki/Cerebellumhttp://en.wikipedia.org/wiki/Cerebellumhttp://en.wikipedia.org/wiki/Medulla_oblongatahttp://en.wikipedia.org/wiki/Medulla_oblongatahttp://en.wikipedia.org/wiki/Medulla_oblongatahttp://en.wikipedia.org/wiki/Cerebrumhttp://en.wikipedia.org/wiki/Cerebrumhttp://en.wikipedia.org/wiki/Cerebrumhttp://en.wikipedia.org/wiki/Cranial_nerveshttp://en.wikipedia.org/wiki/Cranial_nerveshttp://en.wikipedia.org/wiki/Cranial_nerveshttp://en.wikipedia.org/wiki/Spinal_nerveshttp://en.wikipedia.org/wiki/Spinal_nerveshttp://en.wikipedia.org/wiki/Spinal_nerveshttp://en.wikipedia.org/wiki/Amnioteshttp://en.wikipedia.org/wiki/Amnioteshttp://en.wikipedia.org/wiki/Amnioteshttp://en.wikipedia.org/wiki/Amnioteshttp://en.wikipedia.org/wiki/Spinal_nerveshttp://en.wikipedia.org/wiki/Cranial_nerveshttp://en.wikipedia.org/wiki/Cerebrumhttp://en.wikipedia.org/wiki/Medulla_oblongatahttp://en.wikipedia.org/wiki/Cerebellumhttp://en.wikipedia.org/wiki/Efferent_ductshttp://en.wikipedia.org/wiki/Semenhttp://en.wikipedia.org/wiki/Testis


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The first system to be observed will be the muscular system to recognize the major ventral

muscles.


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The second system uncovered will be the digestive tract and the different organs should be

identified by the end of the session.

LiverHeart

Spleen

Right lungLeft lung

Pancreas

Fat bodiesStomach

Gall bladder

Duodenum

Large intestine

Urinary bladder


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The last system will be the nervous system and similarly, the student will be familiar with

the different components of this system.

Equipment:

Dissecting board

Dissecting kit (scissors, blunt tipped forceps, and pins)

Saline solution

Object: Anesthetized frog

Brachial plexus

Spinal cord

Sciatic nerve

Femoral nerve

Iliohypogastric nerve


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Procedures:

1.Lay the frog on its dorsal side on the dissecting board. Pin the arms and legs with the

pins into the board.

2.Using the forceps, pull the skin at the V between the legs then make a cut with the

scissors. Continue cutting the skin along the median line till reaching the head.

3.Make horizontal cuts over the arms and legs.

4.Pull the skin to both sides of the frog and pin it to the board.

5.After the observation of the muscles, cut them off with scissors without injuring other

organs.

6.The digestive system appears.7.Identify the liver, stomach, gall bladder, pancreas, intestine and urinary bladder.

8.Remove all the previously observed organs to inspect the nervous system.

9.Identify the spinal cord, brachial plexus femoral nerve and sciatic nerve.


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Questions:

1.Dissect a frog to demonstrate either the digestive or the nervous system (3 pts)

2.Fill the spaces on the frog scheme (digestive or nervous system). (3 pts)

3.Which nerves are involved in::

The movement of the arms?

The movement of the legs?

The movement of the bowels?

What are the yellow (fat) bodies used for

Students name: _________________________ ID: ________







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It has several sources for arterial blood supply.

Inferior epigastric artery & veins.

Superior epigastric artery.

Numerous small segmental contributions coming from lower inter costal arteries.

The rectus abdominis muscle of frog mainly rich ofNicotinic receptor.

They mainly respond to the acetylcholine released from motor neuron terminal.

It is an important postural muscle.

Helps to flexing lumbar spine.

Plays an important role in breathing

It helps to keep internal organ intact.

TheRectus Muscle of frog is used in the screening of parasympatholytic agents.

It is also useful for bioassay of Acetylcholine.

Bioassay of D-Tubocurarine.


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Questions:

1- Dissection and setting up the experiment. (2pts)

2- Resulting chart.(2pts)

3- What is the effect of Acetylcholine on the muscles? (1pt)

4- What is the function of the Rectus abdomens muscle?(1pt)

5- If the frog didnt have this muscle, what would happen to it?(2pts)

Students name: _________________________ ID: ________







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Biology Laboratory Manual Photosynthesis


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7- Photosynthesis

Bubble-counting method

Introduction:

Photosynthesis is the process by which light energy is converted to chemical energy. It

occurs in plants and some algae. Photosynthesis requires light energy, CO2, and H2O to

make sugar. This takes place in the chloroplasts, using the chlorophyll. Photosynthesis

takes place primarily in plant leaves. The parts of a typical leaf include the upper and lower

epidermis, the mesophyll, the vascular bundles, and the stomata. Photosynthesis does not

occur in the upper and lower epidermal cells due to the absence of chloroplasts. Their

function primarily is protection of the rest of the leaf. The stomata are holes in the lowerepidermis. Their function is to allow for air exchange: they let CO2in and O2out. The

vascular bundles or veins in a leaf are part of the plant's transportation system, moving

water and nutrients around the plant as needed. The mesophyll cells have chloroplasts and

this is where photosynthesis occurs.

Chlorophyll looks green since it absorbs red and blue light, making these colours

unavailable to be seen by our eyes. The green light finally reaches our eyes, making

chlorophyll appear green. However, it is the energy from the red and blue light that are

absorbed that is, thereby, able to be used to do photosynthesis. The green light cannot be

absorbed by the plant, and thus cannot be used to do photosynthesis.

The overall chemical reaction involved in photosynthesis is:

6CO2+ 6H2O + light energy C6H12O6+ 6O2

Objectives


4. Use the bubble counting method by counting the oxygen bubbles

that are released by a water plant

5. Measure the hotos nthesis rate as a function of li ht intensit


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Photosynthesis has two parts (1) The Calvin Cycle (light-independent reactions) (2) the

Light Reaction (light-dependent reaction). The Calvin Cycle takes place in the stroma

within the chloroplast, and converts CO2to sugar. This reaction does not need light directly

in order to occur, but it does need the products of the light reaction (ATP and another

chemical called NADPH). Each Calvin Cycle fixes one CO2and produces one sixth of a

glucose molecule and it takes six coordinated Calvin Cycles to produce one whole glucose

molecule.

The light reaction occurs in the thylakoid membrane of the chloroplast and converts light

energy to chemical energy. Chlorophyll molecules and several other pigments such as beta-

carotene are embedded in the thylakoid membranes and are involved in the light reaction.There are two kinds of chlorophyll; chlorophyll a and chlorophyll b. The pigments can

absorb light and pass its energy to the central chlorophyll molecule to do photosynthesis.

The energy harvested through the light reaction is stored by forming a chemical calledATP

(adenosine triphosphate). The production of ATP, using the energy of light, is called

photophosphorylation. ATP is made of the nucleotide adenine bonded to a ribose sugar,

and that is bonded to three phosphate groups. This molecule is very similar to the building

blocks for our DNA.

Figure1. The structure of ATP molecule
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1. Software Cobra4.

Material:

2. Cobra4 Wireless-Link.

3. Cobra4 Sensor-Unit Weather.

4. Cobra4 Wireless Manager.

5. Ceramic lamp socket E27.

6. Lab jack, 160 x 130 mm.

7. Holder for Cobra4 with support rod.

8. Support base variable.

9. Boss head.

10.Support rod, stainless steel.

11.Filament lamp, 220V/120W.

12.Beaker 1000 ml.

13.Beaker 250 ml.

14.Test tubes.

15.Rural.

1. Read and follow the lab safety form.

Experimental Procedure:

2. Cut off one stem of the waterweeds plant

and place it into a test tube then into a 250

ml beaker (filled with mineral water), with

the cut facing upwards.

3. Attach a weight to the plant in order to

prevent it from floating.

4. Fasten the lamp on one side of the beaker and fasten the Cobra4 Wireless-Link with the

Cobra4 Sensor-Unit Weather horizontal on the other side. At the beginning, the

distance between the lamp and module should be approximately 50 cm.

5. Place a water-filled 1000 ml beaker as a heat filter between the lamp and the 250 ml

beaker.


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6. Plug the Cobra4 Wireless Manager into the USB port of the PC.

7. Start the software measure Cobra4. The measuring instrument will be automatically

identified.

8. Load the experiment Photosynthesis (bubble counting method) (Experiment > Open

experiment). The software will now load all of the necessary pre-set values for

recording a measurement.

9. At first, the carbon dioxide bubbles up and out of the stem and the water itself also

bubbles strongly (ensure that the beaker is not contaminated!). This is why the actual

measurement should not be started until a few minutes later. Then, for one minute,

count the oxygen bubbles that are released at the end of the stem and note the values on

a piece of paper. Furthermore, note the light intensity values in lux.

10.Push the lamp approximately 10 to 15 cm closer to the object and wait approximately

one minute until the plant has adapted to this new condition.

11.Repeat the measurement, which is described above, until the lamp is located directly in

front of the 1000 ml beaker. Please note: the measurements should be performed as

quickly as possible, since the mineral water is continuously losing CO2.

12.If the number of bubbles decreases even though the light intensity increases, then the

mineral water should be replaced.

13.After the end of the measurement, the values can be displayed in a graphical form. For

this purpose, enter the Light intensity in E/lx in X-Data under Measurement >

Enter data manually. Enter the number of bubbles which you have counted in

Number of bubbles/min. under Measurement channels. OR, use the provided Excel

sheet to create a graph.

14.When you have completed your experiment, dispose of materials as directed by your

instructor.


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Observations and results

Distance Oxygen Bubble The Light Intensity

50 cm

40 cm

30 cm

20 cm

10 cm

0 cm

The photosynthesis rate, which is measured based on the oxygen released, increases nearly

linearly as a function of the light intensity. This is due to the fact that under conditions with

reduced light intensity, light is the limiting factor of photosynthesis.

- When the light intensity is higher (e.g. when the lamp is positioned very close to thewaterweed), other factors, e.g. the available carbon dioxide, play the limiting role. In

this case, the photosynthesis rate does not increase linearly as a function of the light

intensity. Instead, it tends to the saturation value.

Notes

- The influence on the photosynthesis rate can also be proven by reducing the carbon

dioxide content of the water (use tap water instead of mineral water).


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Biology Laboratory Manual Ionic Permeability of The cell membrane

9- Ionic Permeability of

The Cell Membrane

Introduction

Cells are active in exchanging molecules to sustain their viability. Some molecules do

not exert effort to enter cells but others need energy and sophisticated approaches.

Molecules can enter cells either by passive or active transportation. Active transportation

needs energy because molecules are transporting against concentration gradient.

There are several factors that affect molecule movement through the cell membrane; these

include molecules size and concentration inside and outside the cells. Oxygen and Carbone

dioxide are examples of simple diffusion where they can pass through cell membranes

without the need for energy.

Glucose molecules do not need energy to move through the cell membrane, but they

have to move through membrane channels using a process called facilitated diffusion.

Molecules that move by facilitated diffusion move according to their concentration

gradients, from higher concentration to lower concentration until they reach equilibrium.

For example, if the concentration of glucose outside the cell is higher than that inside the

cell, the glucose molecules will move from outside to the inside of the cell.

The movement of water through the cell membrane is called Osmosis. Water moves

from lower solutes content to higher solute content. If the cytoplasmic solution of a cell has

solute concentration equal to the extracellular solution, the cell will be isotonic to the

extracellular solution. However, if the cytoplasmic solution has lower solute concentration

Objectives

By the end of the experiment, the student should be able to do the following:

1- Differentiate between diffusion, osmosis and active transport

2- Explain factors affecting diffusion rate

3- Appreciate the complexity of cell membranes

4- Identify the differences between the natural and artificial membranes

5- Explain how active transportation works


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than the extracellular solution, the cell will be hypotonic. If the cytoplasmic solute

concentration is higher than that on the outside of the cell, it will be hypertonic. Water

moves from higher water concentration to lower water concentration, i.e, from lower solute

content to higher solute content.

Molecules move by active transportation against a concentration gradient. Cells use

active transportation to build up more molecules even if their concentration inside the cell

is higher than outside the cell. During active transportation, molecules use ATP as a source

of energy to push molecules inside cells against their concentration gradient. This active

transportation is achieved via channels called pumps, such as the sodium-potassium pump

and chloride pump. Large molecules cannot pass through channels but they can use

vesicle-mediated transport to move in and outside cells. In this experiment, selective

permeability of an artificial membrane to H+and OH

-ions will be examined.

Experimental Materials

1. Two dialysis tubes (15 cm each)

2. Disposable gloves

3. Two pieces of dialysis clips

4. Beaker (1000 ml)

5. Two Beakers (250 ml)

6. Two Beakers (50 ml)

7. Washing bottle (500 ml filled with water)

8. Graduated cylinder (25 ml)

9. Funnel

10.Two universal clamps

11.Two boss head clamp holders

12.Mini magnetic stirrer

13.Magnetic stirring bar

14.Separator for magnetic bars

15.Retort stand

16.pH electrode

17.Cobra4 wireless-link


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18.Cobra4 Sensor-Unit pH

19.Hydrochloric acid (1mol/l)

20.Sodium hydroxide (1mol/l)

21.Buffer solution tablet (pH 4.00)

22.Buffer solution tablet (pH 10.00)

Experiment Procedures:

1- Connect one of theuniversal clamps to the retort stand by a boss head clamp holder as

shown in Fig. 1.

Fig. 1: expermintal set

2- Use the universal clamp to hold the pH electrode.

3- Connect the Cobra4 Sensor-Unit pH to the Cobra4 wireless-link

4- Connect the pH electrode to the Cobra4 Sensor-Unit pH

5- Plug the Cobra4 wireless manager into the USB port of the PC. Make sure that the

software measure Cobra4 can detect your cobra4 devices

6- Adjust the measurement data as follow.


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a- Open the Navigator menu

b- Click the General configuration tab

c- Set the measurement duration to 200 s under end of measurement.

d- Right-click the diagram and set the pH range to 1-12 under display option.

Alternatively, you can simply load the experiment ionic permeability of the cell

membrane (experiment >open experiment). The software will now load all of the

necessary preset values for recording a measurement.

7- Insert a magnetic stirrer bar to a 50 ml beaker and place it on the mini magnetic stirrer.

8- Measure 20 ml water by the graduated cylinder and pour it inside the beaker.

9- Add one buffer solution tablet (pH 4.00) to the solution and dissolve it by stirring.

(Slowly turn on the magnetic stirrer to avoid water splash)

10-Immerse the electrode into the buffer solution without touching the magnetic bar

(electrode may break if touches the magnetic bar).

11-Wait until the pH reaches 4.00. To calibrate the pH electrode, select the calibration

tab in the channel pH / potential pH window. If the electrode has been already

calibrated, new calibration is not necessary.

12-Repeat steps 3-7 but use the buffer solution tablet (pH 10.00) instead.

13-Soften the Dialysis bags with distilled water


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14-Seal both dialysis tubes at one end with the dialysis clips.

15-Place one of the dialysis bags into a 250 ml beaker and fill it with 15 ml of hydrochloric

acid (1 mol/l) using the graduated cylinder.

16-Seal the tube with a dialysis clip.

17-Clean the tube from outside by distilled water using the wash bottle and collect the

drain in the 1000 ml beaker.

18-Make sure to clean up the 250 ml beaker from any spill of hydrochloric acid.

19-Add 150 ml distilled water into the 250 ml beaker and place the 250 ml beaker on the

mini magnetic stirrer and set the stirrer to medium speed.

20-Start the measurement. After approximately 20 s submerge the dialysis bag filled with

hydrochloric acid into the beaker.

21-The time course of the experiment will be observed on the screen up to 200 s.

22-After the experiment, the data can be saved using the save option in the window data

processing

23-Use the open measurement tab, search for your saved file and open it.

24-Use the adjoining tool to survey distances within the diagram and determining the X

and Y values.

25-Repeat steps 15 22 but use sodium hydroxide solution (1mol/l) instead. Make sure to

rinse with distilled water or use a new 250 ml beaker, pH electrode, and a magnetic

stirring bar.

26-Repeat steps 15-22 and 23 but use tap water instead.

Observations

For the hydrochloric acid experiment, the pH-time-curves should be similar to the one

in the Fig. 3. Due to the release of the H+in the water the pH will decrease. The speed

of changes in the pH can be evaluated when you select the Survey tab. In Fig. 3, the

change speed of the pH is 2.46 pH/ 183 s) =0.013 pH/s).


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Biology Laboratory Manual Microscopy


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The student should report immediately to the instructor any defects that might occur to his

or her microscope. The microscope should always be carried with both hands, one under

the base and the other on the arm.

The Light Microscope

The following are the parts of the microscope that the student should be familiar with.

Refer to Figure 1 to aid in locating these parts on your microscope.

1. The OCULAR (eyepiece) contains the upper most lenses of the microscope. Its

function is to magnify. The part may be loose, but it should never be removed from the

microscope as such practice will allow dust to get inside the instrument. As you look

through the ocular, you may notice a solid line; this is a POINTER. Never attempt to

clean the inner part of the ocular or you will remove the pointer.

2. The BODY TUBE connects the ocular to the nosepiece. This is a tube through which

light rays pass between the upper and lower lenses.

3. The NOSEPIECE is a rotating disc on which the objectives are mounted. When

moving the nosepiece, your fingers should be placed on the disc and not the objectives.

4. There may be three or four OBJECTIVES of different lengths and magnifying powers

attached to the nosepiece of your microscope. These objectives, together with the

ocular, magnify the size of the objects that you are observing. Remember, the shorter

the objective the lower the power of magnification.


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Figure 1

5. The ARM supports the above parts. This is one of two structures that should be held

when carrying the microscope.

6. The STAGE is the platform with a mechanical stage for holding the slides in place.

Note the circular opening in the centre of the stage which allows light to pass through.

The object which is to be viewed should be cantered over this opening.

7. The ILLUMNATOR is a small lamp located directly beneath the stage. Electrical

outlets are located on the tables.

8. The DIAPHRAGM may be an iris or rotating disc, depending on the kind of

microscope.


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It is located below the stage. The diameter of the diaphragm may be controlled by

either a lever on the iris or by rotation of the disc. Various objects can be seen well

under certain light conditions. When using the highest magnification, more light is

needed than when using lower magnification.

9. The CONDENSER is a group of lenses beneath the stage. The condenser causes light

rays from the illuminator to converge on the surface of the microscope slide. For most

microscopic work, it is best to keep the condenser at its highest level. Only rarely is it

desirable to lower it slightly. When the condenser is used at a lowered position, the

resolving power is greatly reduced. There is a small milled wheel just under the stage

that is used to control the position of the condenser.

10. The BASE is the heavy, horseshoe-shaped structure upon which the microscope rests.

This is the other part of the microscope that is held when the microscope is being carried.

11. The COARSE ADJUSTMENT is the large milled wheel on the microscope, which is

used in focusing the lenses.

12. The FINE ADJUSTMENT is the smaller milled wheel on the microscope. The wheel

may be separate from the coarse adjustment wheel on some microscopes, where on

others it is the smaller, outermost portion of a dual wheel assembly.

Resolution and Magnification

In using the microscope it is important to know how much you are magnifying an object.

To compute thetotal magnification of any specimen being viewed multiply the power of

the eyepiece (ocular lens) by the power of the objective lens being used. For example, if

the eyepiece magnifies 10x and the objective lens magnifies 40x, then 10 times 40 gives a

total magnification of 400x (MagTot= MagObjX MagOcu).

The compound microscope has certain limitations. Although the level of magnification is

almost limitless, the resolution (or resolving power) is not. Resolution is the ability to

discriminate two objects close together as being separate (clarity). The human eye can

resolve objects about 100 m apart (note: 1 m = 1 micrometre = 1 millionth of a meter).


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Under ideal conditions the compound microscope has a resolution of 0.2 m, about 500

times the resolving power of the human eye. Objects closer than 0.2 m are seen as a

single fused image.

Resolving power is determined by the amount and physical properties of the visible light

that enters the microscope. In general, the greater the amount of light delivered to the

objective lens, the greater the resolution. The size of the objective lens aperture (opening)

decreases with increasing magnification, allowing less light to enter the objective lens.

Thus, it is often necessary to increase the light intensity at the higher magnifications.

Depth Perception and the Microscope

Any viewed microscopic object has depth as well as length and width. While the lens of

your eye fully adjusts to focus on an object being viewed and provides an image that

allows your brain to develop a three dimensional interpretation, the lenses of a microscope

are focused mechanically and can only see in two dimensions: length and width. For

example, if the specimen you are examining has three layers of cells, you will only be able

to focus on one cell layer at a time. In order to perceive the relative depth of your

specimen, use the fine adjustment knob to focus through the different planes (i.e. the threecell layers) individually to build a three-dimensional picture or interpretation of your

specimen.

The Field of View and Estimating the Size of Specimens

When you view an object under the microscope you will observe that it lies inside a

circular field of view. Each magnification lens has a different sized field of view. If you

determine the diameter of the field of view you can estimate the size of an object seen in

that field. As you increase the magnification, the field of view (and diameter) gets

proportionately smaller. As a consequence, a critter that appears small under scanning

Power (4x) may appear large under high power. The actual size of the critter did not

change, only the space in which you placed it for viewing.


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The Oil Immersion Lens

Although the oil immersion lens (100x) when used properly offers the ability to view

objects at high magnification, the objects viewed in this lab exercise do not warrant its

use. As its name implies, an oil immersion lens requires a drop of immersion oil to be in

contact between the lens and the slide for the lens to function effectively. Since immersion

oil has the same refractive index as glass, it prevents the scattering of light as light passes

from the glass slide to the objective lens (also made of glass).Poor resolution results if

the immersion lens is used without oil since light will be bent (and thus scattered) as it

passes from the slide to air, and then through the objective because air and glass bend light

differently as a result of having different refractive indices.

Materials:

1. Light or compound microscope

2. Prepared slides

3. Letter e

4. Coloured threads

5. Clean microscope slides

6. Cover slips

7. Knife

8. Toothpicks

9. Iodine solution

10.Onion

11.Cutting Board

EXPERIMENTAL PROEDURES:

PROCEDURE 1:

Using the Microscope

When properly used, the microscope should cause no eye strain. Try to keep both eyes

open when working the monocular microscope and use the dominant eye to look through

the ocular.

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If you wear glasses, it will not be necessary to use them with the microscope, since the

microscope automatically corrects for this.

1. Obtain the microscope to which you were assigned from the microscope cabinet. When

carrying the microscope, remember to place one hand under the base and the other on

the arm.

2. Before the microscope is placed on the desk, ample space must be provided for it. All

books, purses and other unneeded paraphernalia should be put away.

3. Place your microscope in front of you in a comfortable working position about one inch

from the tables edge. The nosepiece should have the low-power (4X) objective in

position over the opening in the stage. Make sure the switch is in the off position and

plug the power cord into a suitable grounded electrical outlet. Turn on the illuminator.

4. Obtain a prepared microscope slide. Notice the label which describes the material

mounted on the centre of the slide. Make a gross examination of the slide. (If the slide

is dirty, clean it by rubbing lightly with a soft cloth or paper towel; do not use

expensive lens paper for this purpose.)

5. Secure the slide firmly in the mechanical stage. Be sure to wedge the slide BETWEEN

the stage clips and NOT UNDERNEATH them. Rotate the coarse adjustment knob so

that the low-power objective is about one inch above the stage. While observing from

one side of the microscope (not through the ocular), adjust the slide so that the

embedded material is in the approximate centre of the opening. Make sure the side

with the cover slip is up. Move the low-power objective down as close to the cover

glass as possible without actually touching it.

6. While looking through the eyepiece, move the body tube or stage by turning the coarse

adjustment until you can see the mounted material clearly. If you do not see the

material, re-centre the slide and repeat the procedure. If you turn the coarse adjustment

too rapidly you may go past the point of focus without realizing it.


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a.Make a wet mount of some onion cells using iodine stain.

b.Obtain a clean slide and cover slip.

c.Place a drop of iodine on the center of the slide, then place a single layer of onionskin (skin between the onion layers) in the iodine drop. Be sure to spread it out

evenly so there is no overlap or double layering.

d.Touch the cover slip to one edge of the drop, and gently lower it. (If you drop the

cover slip too quickly, air bubbles will be trapped. You cannot see through an air

bubble).

e.Observe these cells first at 4X, then at 10X and make a sketch of a few cells. Be

careful when working with the stain!

PROCEDURE 5:

Storing the Microscope

1. Turn off the power.

2. Rotate the 4X objective into position.

3. Remove the slide from the stage and clean the stage.

4. Unplug the cord and wrap it around the base of the scope.

5. Replace the dust cover.

6. Return the scope to its numerical cabinet space using two hands; one hand should hold

the arm and the other should support the base. Make sure the oculars face the wall so they

will not be bumped and damaged.


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7. Wash wet mount slides and throw away cover slips

Observations and results

Fill out this table

Low power Medium

power

High power Oil immersion

Magnification of

objective lens

Total magnification

Field size (diameter)


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Questions

1-Does the switch from low power (4x) to high power 40x) change the position of the

image?

2-Why is it necessary to centre your object (or the position of the slide you wish to

view) before changing to high power (40x)?

3-Under high power (40x), is the illumination brighter or less bright than it is with low

power?

4-Is it more desirable to increase or decrease the light when changing to a higher

magnification

Students name: _________________________ ID: ________

Lab. Instructors name: __________________________________________Lab. Teaching assistants name (1): __________

Lab Manual Biology Final After Sections Modifications

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