Lab Manual Biology Final After Sections Modifications

Laboratory safety practices
Biology Laboratory Manual
1. LABORATORY SAFETY PRACTICES

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.
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 MUST be 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.
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.
Fig. 1: important signs
NEVER
1. Never enter the laboratory unless a teacher is present or without permission.
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.
Never dispose 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.
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.
How to handle chemicals:

7
The National Fire Protection Association (NFPA)
hazard identification system uses a
color-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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Questions:
Students name: _________________________

ID: ________
Lab. Instructors name: __________________________________________
Lab. Teaching assistants name (1): ________________________________
Laboratory number: ____________________________________________
Laboratory section number: ______________________________________
1- What is the difference between fume hood and safety cabinet?
2- In your Biology lab, you found the following signs on chemical

containers, explain what each sign means:
3- What does each of the following hazard sign refer to?
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4- What does each of the following signs indicates?
11
The Scientific Method
12
2-
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.
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.
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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
Students name: _________________________

ID: ________
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.
16
Biologically Important Molecules
17
3- Biologically Important Molecules
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.
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).
HO
HO
Dehydration Synthesis
HO
Hydrolysis
2H2O
2H2O
HO
Figure 1. Dehydration synthesis and hydrolysis of a polymer

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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.
CH2O
O
OH
HO
HO
OH
Glucose
CH2O
CH2O
O
OH
O
HO
HO
OH
CH2O
OH
Sucrose
CH2O
OH
O
OH
O
O
OH
O
OH
OH
O
O
CH2O
O
OH
CH2O
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 of
Benedicts 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:
Students name: _________________________

ID: ________
Table1. Color reaction for Benedicts test for reducing sugar and Iodine test for starch.
Tube
Solution
Benedicts Color
Iodine Color
Reaction
Reaction
10 drops potato juice
.................................
................................
10 drops onion juice
.................................
................................
10 drops sucrose solution
.................................
................................
10 drops distilled water
.................................
................................
10 drops glucose solution
.................................
................................
10 drops fructose solution
.................................
................................
10 drops starch solution
.................................
................................
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
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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:
Students name: _________________________

ID: ________
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?
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Proteins
Proteins are made up of amino acids. Each amino acid has an amino group (NH2), 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.
H2N
OH
H2N
CH3
OH
H2N
CH
CH3
OH
CH
CH3
CH2
CH3
CH3
Alanine
Valine
Leucine
Figure 3. Examples of amino acids. Each amino acid has a caboxyl group
(-COOH), an amino group (-NH2), and unique side chains (colored).
OH H
OH
R
H2O
OH
Figure 4. 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.
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Biuret test for protein (Material and methods):

1. Label five test tubes (1-5)
2. Add to each test tube the materials to be tested as indicated in table 2.
3. Add 2 mL of 2.5% sodium hydroxide (NaOH) each test tube.
3. Add few drops of Biuret reagent to each test tube and mix.
4. Record the colour of each test tube in table2.
Table2. Colour reaction for Biuret test for protein
Tube
Solution
Colour
2 ml egg albumen
.......................................................
2 mL honey
.......................................................
2 ml distilled water
.......................................................
2 mL amino acid solution
.......................................................
2 mL apple juice
.......................................................
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Questions:
Students name: _________________________

ID: ________
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
H2N
CH3
OH
H2N
CH3
OH
H2N
CH
CH3
CH
CH3
CH2
CH3
Alanine
Valine
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Leucine
OH
Lipids
Lipids are a group of naturally occurring molecules that include fats, waxes, sterols, fatsoluble 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
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 mL salad oil + Sudan IV
...........................................
1 mL honey + Sudan IV
...........................................
1 mL distilled water + Sudan IV
...........................................
1 mL lipid solution + Sudan IV
...........................................
1 mL salad oil + water
...........................................
27
28
Questions:
Students name: _________________________

ID: ________
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
29
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

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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Tube
Solution
Color
2 mL DNA solution
...................................................
2 mL RNA solution
..................................................
1 mL DNA solution, 1 mL water
..................................................
1 mL RNA solution, 1 mL water
..................................................
2 distilled water
..................................................
Table 4. Color reaction for Dische diphenylamine test for DNA.
31
Water Treatment
32
Water Treatment
4-Water Treatment
Decomposition of Organic Substances in Water
Objectives
2. Determine the oxygen content of clean water and waste water.
3. Predict the pollution difference between clean and waste 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
33
Water Treatment
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, it
gives 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.
Materials:
1. Precision Balance.
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.
Experimental procedure:
1- Read and follow the lab safety form.
2- Fill 4 culture vessels with tap water,
leave about 1 finger length below to
edge of the vessel.
34
Water Treatment
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.
35
Water Treatment
Observations:
Record the measurements of the level of oxygen and temperature in the following
table:
Oxygen Level
24hr
48hr
72hr
24hr
48hr
72hr
Vessel 1
Temperature
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.
36
Water Treatment
Questions:
Students name: _________________________

ID: ________
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?
37
Frog Dissection
38
Frog Dissection
5- Frog Dissection:
Digestive, Reproductive and Nervous
Organs Identification
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.
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 small
invertebrates, 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.
39
Frog Dissection
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
as buccal pumping. Its lungs are similar to those of humans but the chest muscles are not
involved in respiration, and there are no ribs or diaphragm 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-chambered hearts,
feature
they
share
with lizards.
http://en.wikipedia.org/wiki/Frog - cite_note-Kimball-53. Oxygenated blood from the

lungs and de-oxygenated blood from the respiring tissues enter the heart through
separate atria. When these chambers contract, the two blood streams pass into a
common ventricle before being pumped via a spiral valve to the appropriate vessel,
the aorta for oxygenated blood and pulmonary 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.
40
Frog Dissection
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 two testes are attached to the kidneys and semen passes into the
kidneys through fine tubes called efferent 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 the cerebellum,
and
the Medulla
oblongata regulates
respiration,
digestion
and
other
automatic
functions. The relative size of the cerebrum in frogs is much smaller than it is in humans.
Frogs have ten pairs of cranial nerves which pass information from the outside directly to
the brain, and ten pairs of spinal nerves which pass information from the extremities to the
brain through the spinal cord. By contrast, all amniotes (mammals, birds and reptiles) have
twelve pairs of cranial nerves.
41
Frog Dissection
The first system to be observed will be the muscular system to recognize the major ventral
muscles.
42
Frog Dissection
The second system uncovered will be the digestive tract and the different organs should be
identified by the end of the session.
Liver
Heart
Spleen
Right lung
Left lung
Pancreas
Stomach
Fat bodies
Gall bladder
Large intestine
Duodenum
Urinary bladder
43
Frog Dissection
The last system will be the nervous system and similarly, the student will be familiar with
the different components of this system.
Brachial plexus
Spinal cord
Sciatic nerve
Femoral nerve
Iliohypogastric nerve
Equipment:
Dissecting board
Dissecting kit (scissors, blunt tipped forceps, and pins)
Saline solution
Object: Anesthetized frog
44
Frog Dissection
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.
45
Frog Dissection
Questions:
Students name: _________________________

ID: ________
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. 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
46
(3 pts)
Frog Dissection
47
Frog Dissection
6- Muscle Physiology:
Effect of Acetylcholine on the Frogs Rectus Abdominis
Muscle
Objectives
1. Dissect the muscle of the frog
2. Correctly handle the hanging device
3. Observe the effect of a muscle stimulant
4. Observe the effect of different concentrations of the drug used
Introduction:
Rectus abdominis muscle is a paired muscle running vertically on each side of the
anterior wall.
Rectus abdominis is a long flat muscle, which extends along the whole length of the
front of the abdomen.
It extends from pubic symphysis to the xiphistretum & lower costal cartilages (5-7)
superiorly.
48
Frog Dissection
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 of Nicotinic 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.
The Rectus Muscle of frog is used in the screening of parasympatholytic agents.
It is also useful for bioassay of Acetylcholine.
Bioassay of D-Tubocurarine.
49
Frog Dissection
Equipment:
Dissecting board
Dissecting kit (scissors, blunt tipped forceps, pins)
Cotton
Ringers solution
Kymograph
Organ bath
Acetylcholine solution
Syringes
Test tubes
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. Make a vertical cut on the medial line of the abdominal muscles
6. Make to horizontall cuts along the rectus abdominis muscle
7. Make a last vertical cut along the muscle to extract it
8. The preparation is suspended in a glass organ bath.
9. One end of the muscle is tied to a hook attached to the aeration tube while the other end
is attached to the needle.
10. The organ bath is filled with ringer solution.
11. It is important to leave the lever just touching the drum during the whole experiment.
Why?? Changes in friction between the lever & the kymograph affect the magnitude of the
response.
12. Adjust the kymograph on 1revolution/96min.
13. Add the drug on the organ bath.
14.
Observe the curve drawn on the kymograph.
50
Frog Dissection
Questions:
Students name: _________________________

ID: ________
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)
51
Photosynthesis
52
Photosynthesis
7- Photosynthesis
Bubble-counting method
Objectives
4. Use the bubble counting method by counting the oxygen bubbles
that are released by a water plant
5. Measure the photosynthesis rate as a function of light intensity
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 lower
epidermis. Their function is to allow for air exchange: they let CO2 in and O2 out. 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
53
C6H12O6 + 6O2
Photosynthesis
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 CO2 to 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 CO2 and 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 betacarotene 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 called ATP
(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
54
Photosynthesis
Material:
1. Software Cobra4.
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.
Experimental Procedure:
1. Read and follow the lab safety form.
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.
55
Photosynthesis
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.
56
Photosynthesis
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.
Notes
-
When the light intensity is higher (e.g. when the lamp is positioned very close to the
waterweed), 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.
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).
57
Photosynthesis
Questions:
Students name: _________________________

ID: ________
1. What is photosynthesis?
2. What are the requirements of photosynthesis?
3. What evidence do you have that plants need light for photosynthesis.
4. Why is chlorophyll efficiency in absorbing light energy important for photos
58
Ionic Permeability of The cell membrane
59
9- Ionic Permeability of
The Cell Membrane
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
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
60
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
61
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 the universal 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.
62
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
63
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).
64
For the sodium hydroxide experiment: The pH-time-curves should be similar to the one
in the Fig. 4. Due to the release of the OH- in the water the pH will increase. The speed
of changes in the pH can be evaluated when you select Survey tab. In Fig. 4, the
change speed of the pH is 7.40 pH/ 180 s) =0.041 pH/s).
Record your data.

Note that using none distilled water or boiling water may affect your data.
65
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66
Questions:
Students name: _________________________

ID: ________
1- Compare the data of the hydrochloric acid experiment with the data in Fig.3.
2- Compare the data of the sodium hydroxide experiment with the data in Fig.4.
3- What are the differences between diffusion, osmosis, and active transport?
4- Why the data are different when comparing the use of the distilled water with the tab
water?
5- Boiling water increases carbon dioxide dissolved in water. How can this affect the pH
measurements?
Take home exam to be ready by the next lab
6- From the obtained data which of these 2 ions (H+ or OH-) passed through the dialysis
membrane faster and why?
7- What is the ion channel? Give an example with a brief description.
8- Describe the cell membrane structure and cite your references (has to be article source
not Wikipedia source).
9- Compare the cell membrane structure and function of eukaryotic cells with prokaryotic
cells.
10- There are two methods of active transportation. Mention them and give a brief
description for each.
67
Microscopy
68
Microscopy
8- Microscopy
Objectives
1. Identify the parts of a compound light microscope
2. Practice carrying and using a microscope to competently examine biological
specimens
3. Determine the diameter of the field of view for the various objective lens of a
microscope
Introduction
The microscope is an essential tool in microbiology to see the invisible microorganisms to
the unaided eye. Robert Hooke, the English biologist who observed algae and fungi in the
1660s, is one of the first to use a microscope to observe microorganisms. There are many
types of microscopes, the most common one is the light microscope, the most well-known
and well used research tool in biology, which uses light to image the sample. Other types
of microscopes are the electron microscope and the various types of scanning probe
microscope. Light microscopes have improved our knowledge in basic biology,
biomedical research and material science. Special techniques and optics have been
developed to study the structures and biochemistry of living cells.
The challenge of viewing small microorganisms lies in getting enough magnification, the
most challenges aspects in microscopy are:
Obtaining sufficient contrast
Finding the focal plane
Obtaining good resolution
Recognizing the subject when one sees it
The microscope is a precise piece of equipment that should be handled with special care.
A microscope may be seriously damaged if dropped or bumped against a hard object.
69
Microscopy
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.
70
Microscopy
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.
71
Microscopy
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 the total 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 = MagObj X 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).
72
Microscopy
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 three
cell 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.
73
Microscopy
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 the63lens 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.
74
Microscopy
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.
75
Microscopy
7. Bring the mounted material into the sharpest possible focus by turning the fine
adjustment wheel about one quarter of a turn. Prepare the slide so the magnification
may be increased by placing the material to be observed in the centre of the field of
view. If the material is not in the centre, it may be lost when switching to a higher
power because the field gets smaller.
8. Adjust the inter-pupillary distance.
a. Pull the oculars apart to the maximum distance.
b. While looking through the oculars slowly, move the oculars together until the two
images form into one image.
c. Pull the oculars apart until the images split into two parts again. Return to the best
position. NOTE: Errors in this adjustment generally produce eye strain in one eye
within 10 minutes.
9. Adjust the dioptre.
a. With the inter-pupillary adjustment properly adjusted, the dioptre adjustment
becomes much easier. While the left eye is closed, carefully use the fine focus to
provide the best possible images to your right eye.
b. Without touching the fine focus, open the left eye, close the right eye and rotate the
dioptre adjustment slowly from one extreme to the other. Somewhere between the
extremes there will be a best place.
c. Students generally notice no difference when first attempting the adjustment. Be
assured there is a best place. NOTE: Errors in dioptre adjustments generally
produce nebulous headaches within 20 to 30 minutes.
10. Progress from the 4X to the 10X objective. Rotate the nosepiece until the 10X
objective clicks into place. To focus, ONLY the fine adjustment wheel needs to be moved.
76
Microscopy
11. Follow the same procedure when shifting from the 10X to the 40X objective.
PROCEDURE 2:
Coloured Thread Slide
1. using the thread slide, determine the spatial relationships such as the depth of field.
The depth of field is the thickness of the specimen that may be seen in focus at any
one time.
a. Rotate the 4X objective into position and place the thread slide on the stage. Centre the
slide so that the region where the threads cross is in the centre of the stage opening.
Try to focus all threads at the same time.
b. Rotate the 10X objective into position and focus on the cross. Can you focus on all the
threads at the same time?
c. Focus upward until the threads are just out of focus. Slowly focus down using the fine
adjustment. Which thread comes into focus first?
d. Is this thread lying under or over the other threads? Record your findings on the lab
report.
PROCEDURE 3:
Letter e Slide
1. Look at the letter e slide. Move the slide slowly to the right. In which direction does
the image in the ocular move? Is the image in the ocular inverted relative to the specimen
on the stage?
PROCEDURE 4:
Preparing Wet Mounts
Wet mounts specimens are those that you make fresh in lab. The specimen is placed in a
drop of water or stain on a microscope slide and then covered by a thin piece of glass or
plastic called a cover slip. Wet mounts are useful for speed and easy preparation, but they
do not usually allow great detail to be observed.
77
Microscopy
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 onion
skin (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.
78
Microscopy
7. Wash wet mount slides and throw away cover slips

Observations and results
Fill out this table
Low power
Medium
power
Magnification of
objective lens
Total magnification
Field size (diameter)
79
High power
Oil immersion
Microscopy
Questions
Students name: _________________________

ID: ________
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
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Bacterial Staining
Bacterial Staining
10. Bacterial Staining

Objectives
1. Differentiate between gram positive and gram negative bacteria
2. Explain the role of each stain in bacterial staining process
3. Explain the difference between the cell wall of gram positive and negative bacteria
4. Discuss the importance of identifying the type of bacteria for treatment purposes
5. Learn how to make a bacterial smear
6. Identify other staining techniques
Introduction
Bacterial staining is one of the most common techniques in microbiology. It is of great
value to identify the type of bacteria in a sample for many purposes. The reflective index of
the bacteria is close to water therefore it is difficult to distinguish bacteria under the
microscope without staining them. There are two main staining methods: the simple
staining and the differential staining. Simple staining provides basic information about the
bacteria such as shape (round or rod) and size. Differential staining provides more details
about the bacteria based on their cell wall structure. One of the most used bacterial staining
techniques is Gram stain, which is used to distinguish between the gram positive and the
gram negative bacteria based on their cell wall staining. Gram positive bacteria retain
stains due to their cell wall structures that have higher peptidoglycan and lower lipid than
gram negative bacteria. Crystal violet, which is the dye used in Gram stain penetrates
bacterial cell walls. Iodine is used as mordant to fix the dye by forming crystal violetiodide complex. Decolorizer solution (a mixture of ethanol and acetone) dissolves the lipid
layer from the cell walls of the gram negative bacteria, which leads to leach of the crystal
violet dye. In contrast, a decolorizer solution dehydrates Gram positive bacteria and close
the cell wall pores as the bacteria shrink therefore they retain the crystal violet dye and its
83
Bacterial Staining
purple color. A counterstaining technique by basic fuchsin or safranin is applied to give the
decolorized gram negative bacteria their pink colors.
Materials and methods
1. Glass slides
2. Slide cover slips
3. 0.85% NaCl, sterile
4. Pasteur pipettes
5. Rubber bulbs
6. Wood applicator sticks, sterile
7. Microbiological loops
8. Inoculating needles
9. Cotton swaps
10. Bunsen burner
11. Immersion oil
12. Electric slide warmer
13. Slide forceps
14. Escherichia coli
15. Staphylococcus epidermidis or Staphylococcus aureus
16. Gram's crystal violet Solution
17. Gram's iodine Solution
18. Gram's Decolorizer Solution
19. Gram's safranin Solution
20. Basic Fuchsin
21. Kimiwipe
22. Bibulous paper
23. Malachite green stain
24. Light Microscope
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Bacterial Staining
Experimental Procedure
1- Wear a pair of gloves and pick two glass slides; make sure that glass slides are clean
from any dust or finger prints. If necessary, clean slides by ethanol and wrap them with
kimwipe or Bibulous papers.
2- Let slides air dry. Always touch slides from their ends.
3- Add a drop of water in the middle of one of the slides and touch a mixture of bacteria
(Escherichia coli, Staphylococcus epidermidis, and Staphylococcus aureus) with the
bacterial loop and mix it with the drop of water.
4- Form a bacterial smear by spreading the bacterial sample on the middle of the slide. A
good smear is a thin, and forms a uniform monolayer which does not block the light.
5- Allow the smear to air dry or you can dry it faster by placing the slide gently on the
electric slide warmer (set on 60C).
6- Use a clean cotton swab to obtain the sample. Wipe the inside of your mouth starting by
your cheek, then under and over your tongue. Make a smear on the second slide by
moving the swab while rotating it over the middle of the slide.
7- Air-dry the second slide.
8- After smears are dried pass them briefly on a flame 2 to 3 times to fix the bacteria on
the slides. Avoid overheating.
9- Cover the smear with crystal violet stain for 1 min.
10- Gently wash the slide with water (do not apply water directly on the smear but apply it
at one end of the slide and allow water to flow gently over the smear).
11- Briefly wash the smear with Gram's iodine Solution (mordant) then cover the smear
with Gram's iodine Solution for 1 min.
12- Gently wash the slide with water (Do not apply water directly on the smear but apply it
at one end of the slide and allow water to flow gently over the smear)
13- Wash by applying Gram's Decolorizer Solution at the end of the slide and allow it to
flow over the smear. Stop when you have a clear runoff. The time of decolorization will
vary based on the thickness of the smear.
14- Gently wash the slide with water (Do not apply water directly on the smear but apply it
at one end of the slide and allow water to flow over gently the smear).
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Bacterial Staining
15- Cover the smear with Gram's safranin Solution for 1 min.
16- Gently wash the slide with water.
17- Blot the slide with bibulous paper to get rid of any excess water.
18- Examine the slide under microscope and record your data.
Observations
Draw the specimen under microscope and determine its type:
1-
Name of bacteria:---------------------------Type of bacteria:-----------------------------
2-
Name of bacteria:---------------------------Type of bacteria:-----------------------------
1- Evaluate the quality of your sample. A good sample should have proper bacterial
distribution (monolayer with limited clumps).
2- Bacteria with purple colour are Gram positive while bacteria with pink or red colour are
Gram negative.
3- Observe that the swipe pepared from the inside of your mouth will show fewer numbers
of bacteria and more epithelial cells.
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Bacterial Staining
Questions
Students name: _________________________

ID: ________
1- Draw what you have observed under microscope for the bacterial culture sample.
Explain what you see.
.
..................................................................................................................................................
..................................................................................................................................................
..................................................................................................................................................
..................................................................................................................................................
..................................................................................................................................................
..................................................................................................................................................
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Bacterial Staining
2- Draw what you have observed under microscope for the mouth swab sample.
Explain what you see.
................................................................................
................................................................................
................................................................................
................................................................................
................................................................................
................................................................................
................................................................................
................................................................................
................................................................................
...............................................................
..................................................................................................................................................
..................................................................................................................................................
..................................................................................................................................................
..................................................................................................................................................
..................................................................................................................................................
.................................................
3- Explain why Gram negative bacteria is stained in pink or red color.
4- Compare Gram positive and Gram negative bacteria by drawing their cell wall
structures.
5- Mention the critical steps that can affect the quality of Gram staining.
88
Bacterial Staining
Take home exam to be ready by next lab

6- Mention and briefly describe other bacterial staining methods.
7- Give an example and brief description of a Gram negative bacteria that is
pathogenic.
8- Give an example and brief description of a pathogenic Gram positive bacteria.
9- Although epithelial cells form the inside of your mouth do not have a cell wall like
bacteria, they are colored by Gram staining. Explain the reason.
10- Based on bacterial cell wall structure, explain why Gram positive bacteria are more
susceptible than Gram negative bacteria to antibiotics
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Types of Worms and Protists
90
12- Types of worms and Protists

Objectives
1- Identify the common helminthes that cause health problems
2- Identify the common protists that cause health problems
3- Explain the life cycles of common worms and protists
4- Be familiar with the structure of common worms and protists
Worms (Helminthes)
1- Schistosomiasis (Haematobium and Mansoni)
Schistosoma or bilharzia is a parasitic-worm disease. Schistosoma mansoni and S.
haematobium are the main causes of human Schistosomiasis in Egypt. There are 200
million infected people worldwide. Early signs of infection maybe in the form of rashes or
itchy skin. Schistosoma eggs usually travel to specific organs and cause inflammation that
leads to enlarged liver, spleen, and/or bladder.
Children are frequently infected in rural areas with poor sanitation due to swimming in
stagnant water containing infectious cercariae. Symptoms of Schistosomiasis are due to the
interaction of the body to the produced eggs not to the worms themselves. Signs of
infection include anemia, liver, intestine, lungs, and bladder are frequently involved in the
late stage of the disease.
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http://www.smittskyddsinstitutet.se/presstjanst/pressbilder/parasiter/
Life cycle:
Figure 2: Life cycle of Schistosoma
92
The life cycle starts when S. mansoni or S. haematobium eggs are releasedvia faeces or
urine, respectively, in freshwater. The eggs hatch and release miracidia, which require the
presence of particular snails to penetrate their tissue and form sporocysts. Sporocysts
develop to cercariae, which release into water and swim freely until they penetrate the skin
of humans swimming in the water body. Upon penetrating the skin, cercariae lose their
tails (schistosomulae), which start to migrate through several tissues until they reach the
portal blood in the liver and mature into adults. A male and a female S. mansoni are paired
together and migrate to mesenteric venules of rectum where they lay their eggs that shed in
stools. Paired S. haematobium worms on the other hand, migrate to venous plexus of
bladder and their eggs are shed in urine.
2- Ascaris lumbricoides
Ascariasis is the most common human worm infection worldwide. Ascariasis is caused
by infection Ascaris, which is one of the soil-transmitted helminthes and the largest
nematode (round worm) that inhabit the human intestine. The average female length is 2035 cm while the average male length is 15-30 cm.
Ascariasis is spread by Ascaris eggs through contaminated food or dirt. It is found in
areas with poor sanitation and areas using human faeces as fertilizers. People infected with
Ascaris show no symptoms or abdominal discomfort, however, heavy infection can lead to
intestinal blockage and growth problem to children.
Cross-section of an adult female A. lumbricoides,

stained with hematoxylin and eosin (H&E).
Note the presence of the prominent muscle cells
(MU), gravid uterus (UT), intestine (IN) and
coiled ovary (OV). (Source CDC)
93
Cross-section of the cuticle of an adult A.

lumbricoides, stained with H&E. Shown here
are the cuticle (CU), and immediately below the
cuticle, the thin hypodermis (HY). Also shown
are the prominent muscle cells (MU) and one of
the lateral chords (LC). (Source: CDC)
Life cycle:
Life
cycle
of
Ascaris lumbricoides:
A female Ascaris
can lay around 200,000
eggs per day. Eggs
transfer to soil through
faeces, which contain
unfertilized
and
fertilized eggs. Both

can be ingested but
only fertilized eggs can
be embryonated and
becomes infective after
18 days to several
weeks.
Upon
swallowing the infective eggs they hatch and penetrate the intestinal mucosa and reach the
lung through the systemic circulation. The larvae mature in the lung for around two weeks
then penetrate the alveolar walls and reach the throat before swallowed back to the small
intestine where they develop to adults and live for 1 to 2 years. The life cycle of Ascaris
from swallowing the infective eggs to oviposition by female worms takes from 2 to 3
months.
94
3- Ancylostoma duodenale
Ancylostoma duodenale, also called hookworm, is one of the soil-transmitted
helminthes that populate human intestine. Hookworm eggs are deposited on soil through
the faeces of an infected person. The eggs hatch and mature to larvae that can penetrate the
skin of a barefooted person standing on any contaminated area, or if ingested. Risk of
infection of hookworm infection is common in poor sanitation and poor hygiene. Initially,
the infected person may experience no symptoms or may just experience itching and a
localized rash when larvae penetrate the skin. During heavy infection, the infected person
may experience abdominal pain, weight loss, diarrhea, fatigue, loss of appetite and anemia.
Children may experience growth problems due to the consequence malnutrition resulting
from blood loss.
Longitudinal section of an adult hookworm

worm in a bowel biopsy, stained with H&E.
Note the oral cavity (OC) and strong, muscled
esophagus(ES).
(Source:
CDC)
Cross-section of an adult hookworm from the

same
specimen.
Shown
here
are
the
platymyarian musculatures (MU), intestine

with brush border (IN), excretory ducts (ED),
and coiled ovaries (OV). (Source: CDC)
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Life cycle:
Life Cycle (intestinal hookworm infection)
Ankylostoma eggs pass onto soil via faeces of infected persons defecates outside. Eggs
hatch in 1 to 2 days based on the environmental conditions (moisture, warmth, shade). The
emerged rhabditiform larvae grow in faeces and/or soil for 5-10 days before they become
filariform larvae, which are infective form of the worm. The filariform larva can survive in
soil for up to 4 weeks. The filariform larvae penetrate the skin and carried by the blood to
the heart and then to the lungs. They can penetrate the alveoli and reach the pharynx then
swallowed to reach to the small intestine. Filariform larvae mature to adult worms and
attach to the small intestinal wall, which causes blood loss by the infected person.
Occasionally, A. duodenale larvae can become dormant in intestines or muscles.
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4- Tapeworm (Taenia saginata)

Taeniasis is caused by parasitic infection with a species of tapeworm called Taenia.
There are different common Taenia species worldwide, which are Taenia saginata (beef
tapeworm), Taenia solium (pork tapeworm), and Taenia asiatica (Asian tapeworm). People
get infected when eating raw meet contaminated with these worms. Taenia saginata is the
most common in Egypt amongst the three and is the largest as well (reaches up to 10 m).
Tapeworm infection can cause digestive problems such as loss of appetite, abdominal pain,
weight loss, and upset stomach, tapeworm segments maybe discharged in the faeces.
Mature proglottids of T. saginata (Source: CDC)
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Life cycle:
Life cycle of tapeworms
Tapeworm eggs are passed to soil via faeces. Cattle are infected with Taenia saginata
when they ingest contaminated vegetation with eggs or gravid proglottids. The oncospheres
eggs hatch and penetrate the intestinal wall and reach the muscle where oncospheres
develop into cysticerci. Humans get infected when they eat infected raw or undercooked
meat and cycticercus develops into adult tapeworm. The adult tapeworms reside in the
small intestine and attach to the intestinal wall by their scolex.
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5- Fasciola hepatica/gigantica:
Fascioliasis is caused by parasitic infection of Fasciola, which is a flat worm known as
liver fluke. Two species (Fasciola hepatica and F. gigantica) are known to infect sheep
and human, respectively. However, fascioliasis is more common in animals. Fascioliasis
spreads worldwide especially in places where sheep or cattle are reared. Infection usually
starts when people eat contaminated watercress or other water plants with fasciola
immature larval flukes. It is possible to get infection after eating vegetables washed or
irrigated with contaminated water.
The immature larvae migrate through several tissues such as intestinal walls,
abdominal cavities, and livers before they occupy the bile duct where they develop into
mature adult flukes and produce eggs. Most of the time, Fascioliasis symptoms
not
detectable during the acute phase when the immature flukes are moving through the
abdominal cavity and liver. However, during the chronic phase of infection, when the adult
flukes are in the bill ducts, inflammation and blockage of bile ducts occur. Infected person
may experience symptoms such as fever, malaise, abdominal pain, eosinophilia, and
hepatomegaly.
CDC source: http://www.cdc.gov/parasites/fasciola/
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Life cycle:
Life cycle of Fasciola
Unembryonated Fasciola eggs pass to soil through the stool of infected animals or
people. In water, eggs embryonated and release miracidia, which penetrate specific snails
from genera Galba, Fossaria, and Pseudosuccinea. Miracidia develop to sporocysts then
rediae and finally to cercariae, which released from the snail and encyst on water plants as
metacercariae. Animals and humans eat contaminated freshwater plants get infected with
Fascioliasis. When the Metacercariae approach the duodenum they migrate through
intestinal wall, the peritoneal cavity, and the liver parenchyma until approaching the bile
duct where they develop into adult flukes and lay eggs.
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Protists
1- Plasmodium falciparum (Malaria)
Malaria is caused by several parasites, which are Plasmodium falciparum, P. vivax, P.
ovale, and P. malariae. Plasmodium falciparum is the most serious parasite. If not treated,
it may lead to serious infection or even death. Malaria parasites are transferred to human by
female Anopheles mosquito and because parasites infects the red blood cells. Malaria can
also be transferred by blood transfusion, organ transplant, or pregnancy. Symptoms of
infection include fever, nausea, vomiting, fatigue, headache, chills, sweats, and diarrhea.
During late infection, untreated persons may develop kidney failure, mental confusion,
seizures, coma and death.
Life Cycle:
Life cycle of Malaria

101
The life cycle of malaria parasite includes two hosts (human and mosquito). The cycle
starts when an infected female Anopheles mosquito transfers sporozoites into human via a
blood meal. Sporozoites migrate to liver and mature into schizonts. Schizonts rupture and
release merozoites, which infect red blood cells and multiply. The immature trophozoites
(ring stage) mature into trophozoite and form schizonts that rupture to release merozoites.
Some immature trophozoites mature into gametocytes. The microgametocytes (male) and
macrogametocytes (female) are transferred into mosquitos during blood meals. Sporogonic
cycle is referred to as parasite multiplication in mosquitos. Microgametes penetrate
macrogametes and form zygotes in mosquito's stomach. The zygotes become motile and
elongated to form ookinetes, which develop into oocysts after penetrating the mosquito's
midgut wall. Oocysts develop until they are full of sporozites, which are released upon the
rupture of the oocysts.
2- Entamoeba histolytica (Amoebic Dysentery)

Embiasis is a disease caused by infection with a parasite called Entamoeba histolytica. It is
more common in tropical areas with poor sanitation. People can get infected when they
drink, eat or touch anything contaminated with the parasite. Only 10-20% infected people
develop symptoms or get sick. The symptoms are usually mild such as stomach ache,
stomach cramping, and loose faeces. Infected people can get bloody stools and fever during
sever infection that is called Amoebic dysentery.
Trophozoites of E. Histolytica
102
Life Cycle
103
Infection starts when people handle food contaminated with faeces or water with
Entamoeba histolytica. Infected people defecate formed stool or diarrheal stool
contaminated with cysts or trophozoites, respectively. In small intestine, trophozoites
released via excystation and migrate to the large intestine where trophozoites multiply by
binary fission to produce cysts. Because of their sturdy walls, cysts can survive outside the
body under harsh environmental conditions while trophozoites cannot. Therefore, cysts are
the transmission form of the infection.
3- Trypanosoma brucei (African Sleeping Sickness)

African sleeping sickness is caused by microscopic parasites called Trypanosoma
brucei transmitted by the tsetse fly (Glossina species). This disease is a serious problem in
some countries of sub-Saharan Africa.
Trypanosoma brucei ssp. in thin blood smears stained

with Wright-Giemsa
104
Life Cycle:
Infection starts after a blood meal of a tsetse fly on a host. The fly transmits
metacyclic trypomastigotes into the bloodstream of the host. Trypomastigotes multiply by
binary fission in various blood fluids such as blood lymph and spinal fluid. The parasites
transmitted to tsetse flies via blood meals on infected hosts. In the midgut of the infected
fly the parasites transform into procyclic trypomastigotes and multiply by binary fission
then they leave the midgut and transform into epimastigotes. The epimastigotes infect the
salivary gland and multiply inside it by binary fission before the fly can transmit the
parasite again via a blood meal.
105
4- Leshmania (Leishmaniasis)
Leshmaniasis is a disease cause by one of 25 parasites of leshmania species. The
disease found in the tropic, subtropic, and southern Europe countries and transmits by sand
flies. There are different forms of Leshmaniasis; the most common forms are the form that
cause skin sores (cutaneous Leshmaniasis) and the form that affects spleen, liver, and bone
marrow (visceral Leshmaniasis).
Leishmania mexicana in a biopsy specimen from a skin

lesion stained with hematoxylin and eosin
Life Cycle:
106
The sand flies transmit the infective stage of Leshmaniasis (promastigotes) during the
blood meal on the host. Macrophages and other mononuclear phagocytic cells phagocytize
the promastigotes. Inside these cells the phagocytized promastigotes develop and transform
into amastigotes by simple division and released to infect more mononuclear phagocytic
cells. Type of Leshmaniasis (cutaneous or visceral) depends on several factors including
the parasite and the host types. Sandflies get infected then they feed on contaminated blood
meals. Amastigotes develop into promastigotes in the gut of the sandflies before they
migrate to the proboscis.
107
Results and Observation:

Worms
Specimen 1:
Name:
Draw:
Draw life cycle:
108
Specimen 2:
Name:
Draw:
Draw life cycle:
109
Specimen 3:
Name:
Draw:
Draw life cycle:
110
Protists
Specimen 1:
Name:
Draw:
Draw life cycle:
111
Specimen 2:
Name:
Draw:
Draw life cycle:
112
Specimen 3:
Name:
Draw:
Draw life cycle:
113
Questions:
Students name: _________________________

ID: ________
1-
Mention the difference in shape between Schistosoma and other flakes.
2-
How do Tapeworms obtain their food without having a digestive system?
3-
Mention the difference between mature and gravid proglottids.
4-
How do you differentiate between male and female Ascaris?
5-
Compare the body cavity of nematodes with flatworms?
6-
What is Trophozoites of Plasmodium Falciparum?
7-
Compare cysts and trophozoites of Entamoeba histolytica.
8-
What is the infective stage of Trypanosoma brucei?
9-
Mention five different leshmania species that can cause Leshmaniasis.
10-
What is amebiasis? Describe its symptoms.
114
The energy content of food
115
13- The Energy Content of Food
Objectives
1. Calculate and compare the caloric content of a specific amount of different types
of food.
2. Know how to read a nutrition label.
3. Lean the general impact of t obesityon health.
Introduction
Food is the source of energy for all human activities. The three main sources of
calories in food are carbohydrates (1gm=4 cal.), proteins (1gm=4 cal.) and fats (1gm=9
cal.). The average adult at rest metabolizes about 1kcal/h for every kilogram of body
weight. The daily expenditure of energy for a typical student is 2300 to 3100 Kcal,
depending on the sex, height, body frame, and exercise . The caloric expenditures of
different individuals vary considerably depending on the type of physical activity they
perform. Examples are shown in the following table:
Activity
Energy Expenditure (Kcal/hr)
Sleeping
60
Sitting
100
Walking
250
Running
600
A healthy eating plan will show you how much you need from each food group to stay
within your caloric needs and promote good health. A healthy eating plan can also help
you learn:
How many calories you need each day and how to balance your calorie needs.
116
How much of each food group you should consume.
How to make healthy choices in each food group.
An eating plan that is unbalanced or contains high caloric intake without adequate
activity may lead to overweight, obesity and other health problems.
1. Body Mass Index (BMI)
Obesity is defined as abnormal or excessive fat accumulation that presents a diverse
group of health risks. . The most common measure of obesity is the Body Mass
Index or BMI. Body mass index (BMI) is a number calculated based on the persons
weight and height, and it is used to generally measure the relative weight of a person
compared to the ideal weight, taking into consideration the persons hight. The BMI
is given by the formula:
The BMI number that results from this formula is used to assess the persons body
weight as follows:
BMI< 19 kg/m2: underweight.
BMI= 19-24.9 kg/m2: Normal
BMI= 25-29.9 kg/m2: Overweight.
BMI> 30 kg/m2: Obesity.
117
Fig.1: A range of estimate normal body weights based on measures of weight

and height.
(Image source: http://www.news-medical.net).
"Morbid obesity" means that a person is 50%-100% over normal weight, more than
100 pounds over normal weight, has a BMI of 40 or higher, or is sufficiently
overweight to severely interfere with health or normal function.
Overweight and obesity are major risk factors for a number of chronic diseases,
including diabetes, cardiovascular diseases and cancer. Once considered a problem
only in high income countries, overweight and obesity are now dramatically on the rise
in low- and middle-income countries, particularly in urban settings.
118
2. Nutrition Labels
Knowing how to read a nutrition facts label is crucial for you to balance your food,
control your caloric intake and, accordingly, maintain a healthy eating plan.
Start here: Note the size of a single serving and how many servings are in the
package.
Check total calories per

serving: Look at the serving
size and how many servings
you are really consuming. If
you double the servings you
eat, you will double the
calories and nutrients,
including the Percent Daily
Value (% DV).
Limit these
nutrients: Remember, you
need to limit your total fat to
no more than 5678 grams a
day including no more than
16 grams of saturated fat, less
than two grams of trans fat, and
less than 300 mg cholesterol
(for a 2,000 calorie diet).
Fig.2: A general guide to reading a sample nutrition label. (Image source:

www.heart.org)
119
Get enough of these nutrients: Make sure you get 100 percent of the fiber,
vitamins and other nutrients you need every day.
Quick guide to The Percent Daily Value (% DV):

This section tells you the percent of each nutrient in a single serving, in terms of
the daily recommended amount. If you want to consume less of a nutrient (such
as saturated fat, cholesterol or sodium), choose foods with a lower % DV 5
percent or less is low. If you want to consume more of a nutrient (such as fiber),
seek foods with a higher % DV 20 percent or more is high. (Read more:
www.heart.org)
In this experiment, we will determine the energy content (in calories) of various
food materials, and compare them to each other.
Materials
1. One Test tube.
2. Clamp scissors or blunt tweezers.
3. Clamp stand.
4. Bunsen burner.
5. Balance.
6. Thermometer.
7. Food samples (Cheetos, sugar cubes, cheese, biscuits, marshmallows, and
chocolate.)
Experimental Procedure
1. Measure 10 ml water into a test tube.
2. Clamp the test tube to the clamp stand.
3. Record the temperature of the water with the thermometer.
4. Record the mass of the piece of food.
120
5. Remove the thermometer from the test tube. Light the Bunsen burner and hold
the food in the flame until it catches alight.
6. Burn the food below test tube.
7. Make sure that the heat from the burning food is transferred to the water by
keeping the flame directly under the tube.
8. After the complete combustion of the food, record the temperature of the water
again.
9. Calculate the energy released from the food using this equation:
Q= M X C X T
Where,
Q: Heat absorbed by water (calories)
M: Mass of water (1ml=1g)
C: Specific heat of water (1cal. / (g.C))
T: Final Temperature - Initial Temperature
Fig.3: Burning a food sample under a test tube. (Image Source:

http://scienceteacher.org.uk/?page_id=239).
121
Observation
Food
sample
Initial
Initial
mass of
temp of
sample
water
Final
Final
mass
temp
of
of
sample
water
122
Questions
Students name: _________________________

ID: ________
1- For one of the food samples you used in the experiment, compare the caloric
content you calculated with the caloric content obtained by your colleague.
2- Compare your answer for the energy in any food to the official value given for
the food on its packet. Is your result close to the official numbers?
3-
What is the name of the process in living

organisms that releases energy from food
4- Using the nutrition label on the right, answer

the following questions:
a) What is the serving size for this food?
b) How many grams of proteins you will
get from 5 servings of this food
Take home questions:

1-
Record your food intake for the following 3 days.

Knowing that 1gm of carbohydrates is worth 4
calories, 1gm of protein is worth 9 calories and
1gm of fat is worth 9 calories; calculate your
calories intake for each day.
123
Permeability of living and dead cells
124
14- Permeability of Living and Dead Cells

Objectives
1. Describe the composition of the biological cell membrane
2. Learn the effect of disruption of the cell membrane composition by heat and
chemical treatments
3. Observe the results of heat and chemical treatments compared with control.
4. Interpret the type of cell membrane damage caused by different chemicals
Introduction
Both eukaryotic and prokaryotic cells are enclosed by cell membrane. The cell membrane
(plasma membrane) of the living cells is a biological membrane that protects the interior of
the cell from the surrounding environment (Figure 1). In plant cell, the cell membrane is
surrounded by cell wall which is absent in the animal cell. The cell membrane consists of a
phospholipid bilayer with embedded proteins. The phospholipid bilayer is composed of
phosphatidylcholine and sphingomyelin on the outer leaflet of the plasma membrane,
whereas phosphatidylethanolamine and phosphatidylserine are of the inner leaflet.
Embdded proteins constitute approximately 50% of the plasma membrane and they are
formed of integral and peripheral plasma membrane proteins. The intact cell membrane has
selective permeability that allows certain substances to pass in and out of the cell
Exposing the cell to various kinds of stress (physical, chemical, heatetc.) may
destabilize the phospholipid bilayer of the plasma membrane and denature its proteins
which consequently affect its selective permeability.
125
Outer Face
Glycoprotein
Phosphatidylcholine &
Sphingomyelin
Phosphatidylserine&
Phosphatidylethanolamine
Integral
proteins
Peripheral
proteins
Inner face
Hydrophilic head
Hydrophobic tails
Hydrophobic tails
Hydrophilic head
Phospholipid molecule
Figure 1. Cell membrane composition
Living cells of beet root (Beta vulgaris) contain a water soluble red pigment called
betacyanin, localized inside central vacuoles and surrounded by a vacuolar membrane
called a tonoplast. The betacyanin remains inside the vacuoles as long as the cells and their
membranes are intact. Heat or acetic acid treatments of beet root cells will denature the
plasma membrane proteins, while chloroform will dissolve the phospholipid bilayer. This
126
permits red pigment to leak out the stressed cells and the amount of leaked pigment
correlates with the severity of membrane damage.
Materials
a. Knife
b. Four Beakers (400ml)
c. Two Pipettes
d. Tripod
e. Wire gauze
f. Bunsen burner
g. Tubing
h. Acetic acid
i. Chloroform
Procedures:
1. Cut a transverse section (about 2 cm thickness) out from fresh beet root and then
divide it into quarters of the same size.
2. Wash each quarter under running water until getting no colored water
3. Put each quarter into a beaker filled with 300 ml water
4. Add 5 mL acetic acid and 5 mL chloroform to the first and second beaker
respectively. Boil the third beaker for 1 minute and leave the fourth beaker
unchanged.
5. Leave the four beakers at room temperature for one day
127
Observation
After one day, the water in the first, second and third beaker is turned red whereas
the water in the fourth beaker is still clear and uncoloured.
The addition of acetic acid to the first beaker as well as boiling of the beaker causes
denaturing of cell membrane proteins while addition of chloroform increases the fluidity of
membrane by dissolving lipids. All previous treatments alter the cell membrane integrity
and increase its permeability which results in betacyanin diffusion into the water
surrounding the beet roots, producing the red colour
128
Questions:
Students name: _________________________

ID: ________
1. What is the composition of the cell membrane?

2. What the source of the red pigment coming out from the beet root quarters during
the first wash? (1 pts.)
3. Why does the cell membrane leak betacyanin after chemical or heat treatment? (1
pts.)
4. What is the difference between damage caused by heat and chloroform treatment?
(2 pts.)
5. Does different temperature treatments of the beet root quarters result in different red
colour intensity? Explain (2 pts.)
6. Why do we use beet roots, ad not potatoes for example to study the damage of
cellular membranes? (2pts.)
129
Antibiotic Resistance
130
11. Antibiotics Test and Detection of Resistant Mutants

Objectives
1. Understand the antibacterial effect of antibiotics by measuring the bacteriafree zones.
2. Explain how antibiotics affect the growth of microorganisms.
3. Understand how misuse of antibiotics can lead to the evolution of antibiotic
resistant bacteria
Introduction:
Chemical antimicrobial agents are chemical compounds capable of either inhibiting
the growth of microorganisms or killing them outright. Bacteria demonstrate two kinds of
resistance to antibiotics: intrinsic resistance and acquired resistance. Intrinsic resistance
means that the species was resistant to an antibiotic even before its introduction. Acquired
resistance means that the species was originally susceptible to an antibiotic, but later
became resistant. Bacteria can acquire antibiotic resistance either by mutation or through
exchange of genetic material among same or closely related species.
The sudden acquisition of resistance to antibiotics poses difficulties in treating
infections. Resistance to several different antibiotics at the same time is even a more
significant problem. It is because of the acquired resistance that bacterial isolates must be
subjected to antibiotic susceptibility testing. Bacteria showing reduced susceptibility or
resistance to an antibiotic implies that it should not be used on the patient.
Materials:
1. Antibiotic disks are placed in 12 cartridge dispenser, kept in fridge (2-8C), until
use:
Vancomycin
Penicillin G
Gentamicin
131
Amoxicillin
Streptomycin
Tetracycline
Chloramphenicol
2. Mueller Hinton (MH) agar plate, 150 mm , kept in fridge (2-8C), until use
3. Bacterial strains.
4. Sterile saline or tryptic soy broth (TSB)
5. Sterile swabs
6. Bunsen burners.
7. 0.5 McFarland barium turbidity standard / photometer (colorimeter)
8. Kirby Bauer chart
Experimental procedure (Disk diffusion test):

1- Grew standardized bacterial isolate in broth and incubate the tube 24 hours at 37 C.
2- The inoculum density is standardized using 0.5 McFarland standard (barium sulfate
standards). The reasons are because if the size of the inoculum is too small, the zone
of inhibition will be larger than what it is supposed to be and if the inoculum is too
large, the zone of inhibition will be smaller.
3- Transfer bacteria from the liquid culture to an agar plate using a sterile swab to give
a lawn culture.
4- Place the paper disc containing specific concentration of antibiotics on the surface
of inoculated agar and incubate at 37oC overnight.
5- Incubate the plate 24 hours at 37 C.
6- Check if bacteria-free zones have formed, measure the diameter of the zone of
inhibition (see diagram below) and note the results down.
7- Results read from the Kirby Bauer chart as sensitive, intermediate or resistant.
132
Zone of Inhibition
Bacterial Lawn
Paper Disk
133
Observations:
Record the measurements of inhibition zones and the culture response in the
following table:
Bacterium: Streptococcus faecalis
Antibiotic used
Inhibition zone size

(mm)
134
Culture response
Questions:
Students name: _________________________

ID: ________
1. What is antibiotic resistance?

2. How did bacteria respond to the different treatments? What does that tell us about
resistance?
3. Does the degree of inhibition of Bacitracin, Penicillin and Streptomycin on E.Coli
growing is equal? (2pts)
4. How does antibiotic resistance affect humans?
5. What are the benefits and drawbacks of the use of antibiotics?
135
136
Origin of acid Rain
15. Origin of Acid Rain:

Observing the effects of acid rain
Objectives
1. Generate components of acid rain (gases).
2. Observe the effects of different gases on the pH of water.
3. Observe the effects of low pH on lime stone or flowers.
Introduction
Acid rain is caused by emissions from power plants, households and traffic. Gases
such as sulfur dioxide, nitrogen dioxide and carbon dioxide dissolve in rainwater, the
products of which form the acids (acids containing sulfur, nitrous acid, nitric acid, carbonic
acid). Acid rain reduces the pH of soils and waters. Environmental damage such as
corrosion of historical buildings, agriculture dieback, or health problems are consequent
results.
Gas
Source
Product in water
CO2
common air pollutant
Carbonic acid
NO2
cars exhaust
Nitrous acid and nitric acid
SO2
burning of coal containing sulfur

impurities
Sulfurous acid
Experimental procedures
Observing the effects of different gases on the pH of water:
o List of needed material
Two test tubes
Distilled water
pH meter
Clamp stand
137
Origin of acid Rain
One-hole stopper
One beaker
Rubber pipe
Clamp stand
Pipettes
Spatula
Pipette aid
Chemicals: CaCO3, HCL, HNO3, Cu
Procedures:
1. Connect a piece of rubber pipe to the one-hole stopper.
2. Place around 20 ml distilled water in a beaker.
3. Measure the pH of water.
4. Fix one test tube using the clamp stand.
5. Add small amount of CaCO3 into the test tube.
6. Cautiously add few drops of HCL to the walls of the test tube.
7. Plug the tube with the one-hole stopper, and insert the tube ending into the beaker.
8. Wait for few minutes, then measure the pH of water inside the beaker.
9. In a new test tube, repeat the experiment using Cu with the cautious addition of
few drops of HNO3 to the walls of the test tube.
Experimental Chemical reaction:
CaCO3 + HCL -> CO2 + CaCl2 + H2O
4HNO3 + Cu -> Cu(NO3)2 + 2NO2 + 2H2O
Formation of Acid rain:
CO2 + H2O<==> H2CO3
2NO2 + H2O -> HNO3 + HNO2
o CAUTION:
You are advised to wear goggles and avoid direct inhalation of fumes generated during
the chemical reaction.
HCl is a strong acid. Gently hold the pipet with the stem pointing up, so that the HCl
drops do not escape.
Observing the effects of low pH on lime stone or flowers
138
Origin of acid Rain
List of needed material

Distilled water
pH meter
Two beakers
Vinegar
Chalk
Producers
1. Place equal amounts of distilled water or vinegar into each beaker.
2. Measure the pH.
3. Scratch the endings of 2 pieces of chalk then place them into each beaker.
4. After one hour, monitor the effects of acidity on the dissociation of chalk.
Observation
Gas
Initial pH
Final pH
CO2
NO2
Conclusion
Water
Vinegar
Effect on limestone
Conclusion
139
Origin of acid Rain
Questions:
Students name: _________________________

ID: ________
1. For each of the two gases, calculate the change in pH (pH).

2. Which gas caused the smallest drop in pH?
3. How would energy production be a source for acid rain?
4. What is the source of acid rain from unpolluted air?
5. What happens to flower petals if we place it in acidic water?
6. High temperatures in the automobile engine cause nitrogen and oxygen gases
from the air to combine to form nitrogen oxides. What two acids in acid rain
result from the nitrogen oxides in automobile exhaust?
7. Why is acid rain more of a problem in Europe than in Egypt?
8. How many grams of proteins you will get from 5 servings of this food?
9. How would acid rain affect metal?
10. How many grams of proteins you will get from 5 servings of this food?
11. What happens to flower petals if placed in acidic water?
140

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Laboratory safety practices

Biology Laboratory Manual

Laboratory safety practices

Biology Laboratory Manual

1. LABORATORY SAFETY PRACTICES

Important notes in biology laboratory session:

Laboratory safety practices

Biology Laboratory Manual

Laboratory safety practices

Biology Laboratory Manual

Fig. 1: important signs

Biology Lab Rules:

Laboratory safety practices

Biology Laboratory Manual

Laboratory safety practices

Biology Laboratory Manual

Fig. 2: Types of hazards.

Laboratory safety practices

Biology Laboratory Manual

In the laboratory, be familiar with:

Laboratory safety practices

Biology Laboratory Manual

How to handle chemicals:

color-coded diamond to represent four different hazards.

Fig. 3: color-coded diamond

Laboratory safety practices

Biology Laboratory Manual

Students name: _________________________

1- What is the difference between fume hood and safety cabinet?

2- In your Biology lab, you found the following signs on chemical

Laboratory safety practices

Biology Laboratory Manual

3- What does each of the following hazard sign refer to?

Laboratory safety practices

Biology Laboratory Manual

4- What does each of the following signs indicates?

The Scientific Method

Biology Laboratory Manual

The Scientific Method

Biology Laboratory Manual

The Scientific Method

The Scientific Method

Biology Laboratory Manual

3. Formulate a hypothesis and make a prediction

The Scientific Method

Biology Laboratory Manual

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

The Scientific Method

Biology Laboratory Manual

Students name: _________________________

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

Biologically Important Molecules

Biology Laboratory Manual

Biologically Important Molecules

Biology Laboratory Manual

3- Biologically Important Molecules

Figure 1. Dehydration synthesis and hydrolysis of a polymer

Biologically Important Molecules

Biology Laboratory Manual

Identification the major organic compounds

Polymers of monosaccharides (Polysaccharides)

Figure 2. Examples of monosaccharides (glucose),

Biologically Important Molecules

Biology Laboratory Manual