Lab Tech PDF

1.
Introduction
1.1. The scientific methods
Objectives:- At the end of this session you should be able to:
Recognize the stages of scientific inquiry as it applies to everyday experiences.
Make observations, formulate hypotheses, make predictions, and design experiments to
test hypotheses.
Biologists use a particular method to find answers to questions about life and living organisms.
This method is called the scientific method, an orderly process that provides scientific
evidence for explanations of natural phenomena. Scientific evidence consists of measurements
or observations that are widely shared with other scientists and that are repeatable by anyone
with the proper tools. Therefore, biologists can repeat the measurements or observations to
either verify or reject the conclusions of other biologists. In this way, the scientific method
makes biology, like other branches of science, an ongoing, self-correcting process of inquiry.
You will participate in the scientific method as you perform the activities in this laboratory
manual. The key steps in the scientific method are shown in Figure 1.1 and described below.
The scientific method of inquiry is an important part of everything we do in our daily lives.
Scientific inquiry involves the steps outlined below.
[Ambo University] Page 1

Figure 1.1 Major steps in scientific method
Steps in scientific methods
A. Observation:
Direct observation: - through the sensory systems (vision, hearing, taste, smell and touch)
Indirect observation: - through the use of special equipment (microscope).
o Observation can be carried out in different stages of scientific methods.
In identifying the problem.
In formulating the hypothesis.
In conducting experiment (to prove or disprove the hypothesis).
B. Identifying the problem or question
Based on accurate observation define the problem by asking question about it (How and why?)

C. Gathering information: - To answer the questions the scientist gathers as many
information (relevant fact) as possible.
D. Forming a hypothesis: - Hypothesis is logical reasoning, an intelligent guess or a tentative
theory formulated based on the information.
o Hypothesis is a proposition that might be true (predication).
o For a single problem there could be alternative hypothesis.
E. Inductive and deductive reasoning: - There are two ways of reasoning to create a
hypothesis.
Inductive reasoning: - is the method of discovering general principles by careful
examination of specific cases i.e. reaching to a general conclusion from specific cases.
Deductive reasoning: - It is the sort of analysis of specific cases using general principles
i.e. reaching to a specific conclusion from general cases.
F. Testing hypothesis: - To test the hypothesis generally involves performing one or more
crucial experiments.
o Not all hypotheses are tested by laboratory experiments.
G. Observing and recording results: - experiment results or observation must be recorded
carefully and systematically and organized in the form of data, tables, charts, and graphs in
addition to verbal explanation.
H. Analysis and interpretation of data: - studying the organized results or observation to
discover the essential facts.
o Treating data using various statistical calculations and finding relationships, trend,
similarities and difference.
I. Drawing conclusion: - on the bases of the result the hypothesis being tested is accepted or
rejected.

J. Evaluation: - Doing experiments over and over again until consistent results are obtained or
comparing your experiment result with others.
K. Reporting and Publishing of results: - research findings are usually published in scientific
Journals. Scientific research report and journals (article) include:-
a. Title of the research
b. Introduction- what did you do? Why?
c. Material and methods (How did y do it?)
d. Results- (what did you find?)
e. Discussion- your interpretation of results
f. Summary- statement of main findings
g. Acknowledgments- who helped you?
h. Reference- Details of references cited.
1.2 Laboratory Safety Rules
Objective:-At the end of this lesson students should be able to describe laboratory safety
procedures and apply them.
A number of chemicals used in biology laboratory are hazardous. For example Phenol and
acids can cause severe burns, some chemicals are toxic if they are swallowed or inhaled and a
few are carcinogens. These chemicals are not harmful if used properly. There fore you should
follow the procedures of laboratory safety rules in the laboratory.
1. Safety goggles must be worn at all times during laboratory work (or upon instructor
request)
2. Long hair must be secured; remove (or secure) hats, pocketbooks, neckties, necklaces and
scarves.

3. It is advisable to wear laboratory aprons or coats to protect clothing.
4. Always wash hands, arms, (and face) before leaving the laboratory. Toxic, pathogens or
otherwise dangerous chemicals may inadvertently be transferred to eyes or mouth.
5. Whenever your skin (hands, arms, face, etc.) comes into contact with chemicals: Call the
instructor immediately; wash it quickly and thoroughly with water. Do not rub the affected
area (especially the face or eyes) before washing.
6. In case of chemicals spilled over a large part of the body: Call the instructor immediately.
(If necessary, use the safety shower to flood the affected area).
7. For fire burns: Call the instructor immediately; place the affected area under running water
for several minutes.
8. In case of chemical spill: Call the instructor immediately; do not attempt to clean the spill.
9. Never taste, smell, or touch a chemical or solutions.
10. In case of accident or injury, even if it is minor, notify your instructor at once.
11. Avoid cuts and burns. All cuts burns etc. reported and treated, no exceptions
12. Do not use any needle, syringe or blade unless you are given by your instructor
II. GENERAL LABORATORY REGULATIONS
1. Come to the laboratory on time. Do not forget to bring your laboratory manual, pencil,
eraser and laboratory note book.
2. Record all your observations carefully in your note book.
3. Drawing should be always done with pencil
4. Work with an open and inquiring mind
5. Unauthorized experiments are strictly forbidden.
6. Maintain an orderly and clean work space.

7. Keep cabinets closed and the aisles free of any obstructions. (i.e. stools, bags, books, etc.)
8. Push all lab stools under the lab benches during experimentation.
9. At the end of the laboratory period:
-clean all equipment used in the experiment
-clean the sink of all debris
-clean the lab bench with a damp paper towel (and properly discard)
10. No drinking, eating, or gum chewing is permitted in the laboratory
11. You are responsible for all laboratory equipments provided to you for use. If you observe
any damage on your equipment, report to your instructor at the beginning of the lab
session, if not you will be held responsible.
12. When you finish your work, do not forget to return the microscope and other materials you
borrowed from the laboratory to their appropriate places and place them properly.
13. Your experiment will be graded on the quality and quantity of your work, your technique
while performing the experiment, your neatness in the laboratory and your report.
14. Be courteous, use your head, and try to enjoy yourself!

1.3 Introduction to Basic Biological Laboratory Equipments
Objective: At the end of this section you should be able to:
Identify the most common biology laboratory equipment.
Handle the equipment properly.
Describe the function of each of the basic equipments.
Probably the most often used tools in biology are the dissection kit and the microscope. During
this laboratory session you will be introduced to these basic tools of the biologist.
I. Components of the dissection kit
The basic components of a dissection kit include scissors, scalpels, forceps, probes
(or needles), droppers, and often camel-hair brush. These basic components of a
dissection kit and some other tools (instruments) often used by biologists are placed
on your table or on the demonstration table in the laboratory.
Table 1.1 . List of commonly used materials and sterilization equipment in biology laboratory
Name Picture Function
S.No
Aquarium
1 Autoclave Used to sterilize
materials
2 Balance Measures mass of

material

3 Beaker A liquid-measuring
container
4 Bunsen burner Gas burner used to

heat things
5 Burette Measures volume

of solution
6 Centrifuge Separates materials

of varying density
7 Clay triangle A wire frame with

porcelain used to
support a crucible

8 Condenser Used in distillation
9 Cover slip Hold specimen

together with the
slide that can be
seen under the
microscope
10 Crucible Used to heat a
small amount of a
solid substance at a
very high
temperature
11 Culture (test) tube Test tubes used for

growing bacteria
12 Test tube Used as holder of

small amount of
solution

13 Dissection kit Used to dissect
small animals
14 Dropper For addition of

liquids, drop by
drop
15 Erlenmeyer flask Used to mix

reagents, heating
and preparation of
microbial culture.
16 Forceps Holds or pick up

small objects
17 Glass funnels For funneling

liquids from one
container to
another, or for
filtering when
equipped with filter
paper.
18 Graduated cylinder For measurement
of an amount of
liquid. The volume
of liquid can be
estimated to the
nearest 0.1 mL with
practice.

18 Graduated pipette Measures solution 1.
volumes
19 Hand lens Magnifies small

objects
20 Microscopes To see microscopic

specimen
21 Mortar and pestle For grinding

chemicals, plants,
etc
22 Oven Used for heating,

drying and
sterilizing metallic
instruments.
23 Petri dish (Petri Plates Used to grow

micro-organisms
24 pH meter Measures acidity of

solutions
25 Pipette Used to transfer
measured
substances into
another vessel
26 Ring stand (with rings or For holding pieces

clamps) of glassware in
place.
27 Round bottom flask Used for distillation

and heating
substances
uniformly
28 Slide( plane and cavity Used to hold

slides) objects for
examination under
a microscope
29 Sprit lamp A lamp that burns a

volatile liquid fuel
such as alcohol,
ethanol or denatured
alcohol.

30 Test-tube holders - For holding test
tubes when tubes
should not be
touched
31 Thermometer Measures
temperature
32 Tong Similar function to

forceps, but are
useful for larger
items
33 Volumetric flasks To measure precise

volumes of liquid
or to make precise
dilutions.
34 Wash bottles For dispensing

small quantities of
distilled water.

35 Watch glasses For holding small
samples or for
covering beakers or
evaporating dishes.
36 Wire gauze Used to spread heat
of a burner flame
1.4 Laboratory report writing methods
Lab Report Format
Each section must be included in each report, unless told differently by the lab instructor.
I. Title page
A. Experiment name
B. Experimenter (author) and partners (co-authors)
C. Experiment date(s)
D. Evaluator
E. Submission date
II. Introduction
A. Provides a brief summary of the background and theory pertaining to the experiment
done.
B. Material for the introduction can be found in books, articles, your lab manual or the
internet (but only from reliable sites be careful!). Any information that is not general
knowledge must be referenced appropriately. Plagiarism is easily recognized.

C. Must answer the questions of:
What was known before the experiment was done?
Why was the experiment carried out?
Was a hypothesis being tested? If so, the hypothesis must be specifically stated.
III. Purpose
Few sentences in order to answer; Why was this experiment performed?
IV. Materials and Methods
What materials and reagents were used?
V. Procedure
A. What was done step by step in reported forms ( for example the instructional
procedures in your lab manual are
- Add two ml of water in a test tube. The reported form should be Two ml
of water was added in to a test tube
Diagrams and flow charts are welcome.
B. Alterations, mistakes or corrections must be included.
V. Results
A. Informing your audience of the purpose of the experiment
B. State the direct outcome of the experiment or procedure.
C. Each experiment should answer a simple question _ i.e. what do the cells of an algae
look like? or what is the concentration of cells in an unknown sample?). Each
answer should be represented by the results of your experiment both in table or figure
AND in words. When writing a description, your audience should be able to use your
words to reconstruct what you observed.
D. The following questions should be answered in the result section of any piece of
scientific writing
Why did you conduct the experiment?
What did you do?
What did you see?
What does it mean? This point is stated in more detail in the discussion, but it
can be simply stated (one sentence) in the results.
E. Figures and tables must be included that show the data generated by your experiment.
Hand drawings and hand written calculations are acceptable, but they must be neat and
labeled.
Tables and figures must be numbered and titled.
Table titles appear at the top, figure titles at the bottom.
Tables: rows and columns are labeled.
Figures: axes are labeled and contain units.
VI. Discussion and conculusion
A. Restate in the first sentence or two the purpose and findings of the experiment.
B. This section is the explanation of the results section.
C. Include explanations of unpredicted or inconsistent results.
D. Place the results into a setting.
E. Compare and contrast results with existing knowledge.
F. Explain why you think the results mean.
G. Referencing other studies is appropriate.
H. Discussion should give the audience a general conclusion about the results and answers
to the questions posed in the introduction.
I. May refer to future experiments that can answer questions raised by this study. This is
not always appropriate.
What would you do next if you were to continue with this study?
What would you change if you were to do the experiment again?
VII. References
A. All thoughts, data or ideas that are not your own must be referenced.
B. If something is a generally known fact, it does not need to be referenced. For example
chemical molecular weights, Species names, molecular composition of known
compounds
C. Be very careful when using websites. Information from the internet can be misleading
or wrong, so you must be critical. Personal websites are not valid references. Any
website used will be highly scrutinized, and if untrustworthy or even questionable, this
will be detracted from your grade.
D. The reference must be given in the text with the name of the author and the year of
publication, and the full reference must be provided in the references section.
E. References must follow standard format. Examples given below.
o Journal: Author, (year) title. Journal volume (issue), pages.
Example: Morehouse, S.I., Tung, R.S., Rodriguez, J.-C., Whiting, J.R. and
Jones, V.R.(1993) Statistical evidence for early extinction of reptiles due to the
K/T event.Journal of Paleontology 17(4), 198-209.
o Book: Author (year) title, number of pages. Edition number. Edition series,
editor. Issue. Number of volumes. Publisher, city.
Example: Billoski, T.V. (1992) Introduction to Paleontology, 212 pp. 2nd ed.
Trans. A. Translator. Series on Paleontology, edited by B.T. Jones, 6. 12 vols.
Institutional Press, New York.
o Book with referred Chapter: Author (date) title. In: book editors (Eds), book
title, edition pages. Volume. Number of volumes. Publisher, City.

o Example: Grosjean, F.O. and chneider, G.A. (1990) Greenhouse hypothesis:
Effect on dinosaur extinction. Trans. M.A. Caterino. In: N.R. Smith and E.D.
Perrault (Eds), Extinction, 3rd ed., pp. 175-189. Vol. 2. 5 vols. Barnes and Ellis,
New York.
o Website: Author (date) Title of page or article (web site).
Example: Gutkind, J. S. (2000). Regulation of mitogen-activated protein kinase
signaling networks by G protein-coupled receptors (http://www.stke.org).
VIII. Appendices
A. Calculations
B. Detailed information about equipment used if required.
C. Answers to questions posed in the lab manual

2. Microscope
Objectives:
At the end of this unit you will be able to:-
Practice proper handling of the light microscope.
Identify the parts of a compound microscope.
Prepare specimen for microscopic study
Discuss the function of each part of the compound microscope
Compare the working principle of compound microscope with that of an electron
microscope.
Explain the basic functioning of other microscopes like the phase contrast microscope,
scanning microscope.
Learn what the position of an object is when viewed through the microscope in relation
to its position on the microscope stage
Introduction
Possibly the most important instrument that aids biologists is the microscope. A
microscope is an instrument used to observe very small organisms that cannot be seen by
naked eyes. An opportunity to learn about and use this valuable instrument is now yours.
2.1. Types of microscope
Objectives: At the end of this section you would be able to identify the different types of
microscopes and tell their uses in to study biological components.
There are three major types of microscope.

1. Simple microscope - It consists only one biconvex lens.
- It enables to observe the external forms of objects
- Its magnification power is between X10 and X20 e.g. hand lens
2. Compound microscope:- consist of two biconvex lenses namely, the eye piece
or ocular and the objective lenses.
3. Electron microscope (EM)
It uses beans of electrons (0.005nm)
o Come into use in the 1950s.
o Magnifies up x 250,000 or more.
o The image formed by electron microscope cannot be seen directly.
There are two kinds of electron microscope.
a. Transmission electron microscope (TEM):- It does not give three-dimensional
picture of the specimens.
b. Scanning electron microscope (SEM):- give three-dimensional picture of specimen.
Electron microscope enables us to see the detail sub cellular structures.
2.2. Parts and Function of light microscope
Objective:-Identify the various parts of your microscope
Introduction
Compound light microscopes contain two lens systems, an objective and an ocular. The total
magnification of an image is calculated by multiplying the magnification of the ocular by the
magnification of the objective. The net effect of the two lenses gives it better magnification
capacity.

Compound microscope uses visible light (400-700nm) and is known as light microscope,
magnifies up to X1500 and used to investigate internal structure of objects.
Fig. 2.1. A monocular compound microscope

Figure 2.2 A binocular compound microscope
1. The Eye piece (Ocular: The eye piece of your microscope has one set of lenses and it may
rest loosely on the top of the Body Tube. The eye piece on your microscope probably has
magnifications of ten times and is labeled as 10X. Some microscopes may have eye pieces
with magnifications power of 8X, 15X and 20X.
2. The objectives: The objectives also have one set of lenses. The objectives are screwed into
a revolving nosepiece or Tricone at the lower end of the body tube. Your microscope may

have three or four objectives. Note that by revolving the nosepiece you can change the
positions of the objectives
The Low Power objective (the shortest with the largest lens opening). Usually has a
magnification of four times and is labeled as 4X. Your microscope may lack this
objective.
The middle power objective (the one with the medium length ) often has a
magnification of 10X
The High power objective (the longer one) has a magnification of 40X or 44X.
An oil Immersion Objective has a magnification of 100X
The microscopes we will use each have a 10X ocular lens and four different objective
lenses listed in the table below.
Objective Magnification Total Magnification
Low power 4X 40 X
Medium Power 10 X 100 X
High Power 40 X or 43 X 400 X or 430 X
Oil Immersion 100 X 1000 X
Light bends when it passes from glass to air or from air to glass because air and glass have
different refractive indices. The bending of light as it passes through the glass slide to the air
and then to the glass lens decreases the resolving power. At high magnification (1000X) it can
prevent a clear image from being viewed. The decrease in resolution can be prevented by
putting oil immersion between the slide and the lens because immersion has the same
refractive index as glass.

The condenser also increases the resolving power of the microscope. When using the oil-
immersion lens, the condenser (located beneath the stage) should be raised to a position very
close to the stage for maximum resolution.
3. The Stage: This is the flat platform upon which an object (Slide bearing the specimen) to be
viewed is placed. There is an opening in the center of the stage (Stage Opening) through which
light passes. On the stage there are two metal Clips serve to hold the slide securely in place.
Some microscopes however, are equipped with a mechanical device that holds the slide in
place and also used to move the sidewise as well as back and forth.
4. The Condenser: The condenser is found immediately beneath the stage. This component of
the microscope causes the light rays to converge at the stage opening. At its lower end the
condenser is equipped with an Iris Diaphragm which can be opened and closed as needed to
control the amount of light entering the body tube. The condenser may be fixed or it may be
raised or lowered by a sub stage Adjustment. Lowering the condenser decreases the amount of
light entering the body tube and ultimately reaching your eye.
5. The mirror: Near the base of some microscopes is a plane- Concave (one side flat and the
other curved- in) mirror. It can be tilted to any desired angle. Its function is to direct light rays
through the condenser into the stage opening. Most of the microscopes in our laboratory have
built- in lamps instead of a mirror. Such microscopes do not need external source of light.
6. The Adjustment Knobs: There are two focusing knobs. The coarse-focusing knob has the
larger diameter and is used to bring objects into rough focus when using the low and medium
power objectives. The fine-focusing knob has a smaller diameter and is used to bring objects
into sharp focus. It is the only focusing knob used with the high-power and oil-immersion
objectives.

There are two types of sophisticated compound microscopes.
a. Phase contrast microscope, which is used to study phenomena in living things.
b. Interference microscope that enables distinction of smaller contracts and allows color
vision.
QUESTIONS
1. Mention differences between a compound microscope and an electron
microscope.
2. Name the microscopes under which we study a living cell and a dead cell.

2.3 Handling the Microscope
Objectives: At the end of this session students will be able
To handle a microscope perfectly.
To use microscope to see small objects that cannot be seen with the naked eyes.
The microscope is an extremely valuable instrument of the biologist and careless handling may
seriously damage it. You should observe the following rules in handling a microscope.
1. Always carry your microscope using your two hands. When carrying your microscope,
grasp the arm (limp) of the microscope firmly with one hand and support the base (foot)
with the other hand. Carrying other object with the other hand is strictly forbidden.
2. Never turn the microscope sideways or upside down or remove a lens for any reason.
3. Place the microscope near, but not on the edge of your laboratory table with its arm
facing you.
4. The magnifying lenses of the microscope can easily be scratched. Therefore, they
should be cleaned only with special lens paper. In the absence of lens paper very soft
toilet paper may serve as a substitute.
5. Before you return your microscope to its place see that no slide is left on the stage and
also make sure that the low power objective (the shortest objective) is in exact position
i.e. right above the stage opening.
6. Do not forget to cover the microscopes with the plastic coats provided..

2.4. Setting the Microscope for Use
In order to study your mounted dot or any other objects under the microscope first you have to
set up your microscope for use. If you follow the following steps, they will enable you to set up
the microscope for use.
1. Place the microscope near the edge, but not on the edge, of your laboratory table with
the arm facing you.
2. Arrange your laboratory stool such that it allows you an easy look through the ocular of
your microscope with out inclining it.
3. The condenser in your microscope can be a type that can be lowered or raised. If the
condenser is in lowered position raise it as near the stage as it will go.
4. Switch on your microscope bulb. If your microscope is not the type with in-built light
source, turn on your microscope table vision, the circular area that you see in the
microscope is uniformly illuminated. Note that once you have adjusted the light you
should not over the microscope or the lamp.
5. Turn and click the primary objective so that it is directly over the stage opening. Make
sure the objective clicks into place.
6. Look through the eyepiece of the microscope. A circle of bright light should now be
visible. This is called the field of view. Adjust the diaphragm to make the field of view
as bright as possible.
7. Look to the side of the microscope. Slowly turn the coarse adjustment wheel back and
forth. Note the movement of the stage in relation to the primary objective.

2.5 Practice in Mounting
Objectives: At the end of this session students will be able:
To mount different specimen on microscope slide.
To observe small dots using low power, medium power and high power of a
microscope.
I. Materials Required:
1. Beakers
2. Cover slip
3. Droppers
4. Microscope
5. Microscope slides
6. Piece of paper ( 1cm by 1cm) with a dote on the center
II. Mounting
Mounting is the process of the preparation of an object for study under a microscope.
There are three types of mounting, namely wet mounting, dry mounting and permanent
mounting.
Wet mounting
Wet mount has the following advantages. The liquid refraction makes it much easier to see
intricate structures. If the specimen is alive, the liquid will make it possible to view both the
natural color and motility patterns.

Wet mounts have disadvantages as well. Finding a moving specimen can be a problem. The
slides also tend to dry out under the light of the microscope. If your wet mounts are drying out
before you are ready, apply an additional drop of liquid under the cover slip.
The procedure of wet mounting is
1. Hold a glass slide by the edge between your forefingers and thumb and clean it
with a piece of soft paper.
1. Place a drop of water in the center of a clean microscope slide.
2. To the drop, add a piece of paper bearing a dot on the center of the slide with
the dot facing upwards.
3. Place one edge of a cover slip at the edge of the water drop and gently lower it
to about 450 so that the water containing the specimen completely spreads out
under the cover slip.

4. Supporting with a needle or a pencil lower the cover slip gently until it covers
the paper. If the above step is carefully done no air bubbles get entrapped.
5. If there is too much water, draw off the excess by touching the corner of a paper
towel to the edge of the cover slip.
6. Examine your wet mount under low power and then under high power.
Remember, care must be taken to avoid touching the cover slip with the
objective lens because it might break the cover slip or scratch the objective lens
Dry mounting
Dry mounts are the easiest microscope slides to make. You will need a glass slide and a cover
slip. Dry mounts work best for samples like blood cells, bacteria, pollen, hair, feathers, or even
dust particles caught in a microfilm filter.
Procedure
1. Place the sample on the slide
2. Allow the sample to dry by air or by moving the slide on a flame.

3. Stain the sample using appropriate staining dye
4. Observe the specimen under your microscope.
If you put a cover slip and then seal the cover slip using Canada balsam, it would become
a prepared slide. Once you are done you can keep them for long time.
2.6 Focusing practice
Objective: At the end of this exercise you should be able to see any specimen under different
magnification power of your microscope.
The objective lenses may be seriously ruined and slides may be broken on the stage if the
correct procedure of focusing is not followed. Therefore, when you are focusing with your
microscope you are expected to follow the focusing steps given below strictly.
1. Put the slide on the stage with the object (dot, in this case) facing up and clip it in place.
2. Move the slide with your thumb and finger until the object (dot) comes in the center of
the stage opening i.e, on the path of the light rays coming from below.
a. Focusing with Low power
3. If the low power objective is not in position (i.e. right above the stage opening) hold
any two objectives between your thumb and the forefinger and turn the nosepiece until
the objectives clicks into position.
4. Looking from the side, raise the stage with the coarse adjustment to 1mm of the slide.
The stages of some types of microscopes cannot be raised to the extent that the low
power objective touches the slide. Thus, it you cannot raise it further, do not apply
force. In some types of microscopes it is the body tube rather that the stage that is
lowered to wards the stage.

5. Looking into the microscope about 2cm away from the eyepiece, slowly lower the stage
with the coarse adjustment until the image comes in to view, Do not plant your eye
right on the ocular, for if you see anything you will only see a reflection of your eye
and eyelashes. Keep both eyes open while looking in to the microscope.
6. If the image viewed is blurred focus it with the fine adjustment until it becomes clear.
7. If the image is off the center of the field of vision, move the slide very gently, while
looking into the microscope, until the image is in the center of the field.
8. Adjust (increase or decrease) the diaphragm opening as necessary. In order to focus the
objects (dot) under the medium power go to step 9 with out changing the focus under
the low power.
b. Focusing with the Middle power
9. Your object is in focus under the low power. Now holding any to of the objectives
between the thumb and the foreigners rotate the nosepieces until the middle power
objective clicks (i.e. you hear a clicking sound ) in to position .
10. Focus with the fine adjustment until you get a clear image.
11. Increase the diaphragm opening if more light is needed. Now you can directly ( i.e.
without changing the focus under the medium power ) go to step 12 in order to focus
the object under the high power.
C. Focusing with the High power
12. Your objective lens is already in focus under the middle power. Taking hold of any two
objectives, rotate the nosepiece until the high power objective clicks into position. Care
should be taken so that the objectives do not hit against the stage clips.

13. Focus with the fine adjustment until you get a clear image. Never use the coarse
adjustment when focusing with the high power objective.
2.7 Magnification, Resolution, and type of image formed
Objectives:- at the end of the lesson students should be able to:-
Determine the diameter of the field of view for microscope objectives.
Estimate the size of an object from the diameter of the field of view.
Accurately determine the size of an object using an ocular micrometer.
2.7.1 The Resolving power of a Microscope
Introduction
A microscope has two features. These are:-
Magnification: -the ratio of the size of the image seen with a microscope to the actual size of
the object.
Resolving power: - the power of the microscope to scatter the image and show fine details
rather than being seen as single blurred point. For Example, if the distance between two dots is
less than 0.1mm, the two dots would appear as a single dot to the unaided human eye. The
resolving power of the human eye is about 0.1 mm.
Resolving power depends on the quality of the lens and wavelength of light. The shorter the
wave length, the greater the resolution power it has. The light microscopes have about 500
times more resolving power than the human eye. The electron microscope has a resolving
power 10,000 greater than the human eye.
The resolving power of a microscope depends up on the kind of illumination used because the
resolving power is X the wave length of the illumination used. Therefore, even with 5500

Angstroms, the objective cannot resolve anything less than 2750 Angstroms (i.e. X 5500) or
0.275 microns.
1millimeter = 1000microns ()
1 micron = 10,000 angstrons( Ao)
Materials Required:
1. Breakers
2. Cover slips
3. Droppers
4. Graph paper
5. Microscope
6. Microscope Slides
7. Typed b
8. Typed dot
Procedure
1 You are provided with a piece of paper about 1cm by 1cm.
2 Using a ball points pen (never use a pencil if you like to get better result) make a
small dot on the small piece of paper.
3 Wet mount the dot following the mounting procedures given in laboratory study
No. 2.
4 Study the dot under the low power and under the medium power following the
steps given in laboratory study No, 2.

Question
1. What is the resultant total magnification of an object as seen through a microscope with 10
oculars and each of the following objectives? A) 4X B)10X and C) 43X
2. Describe the difference between magnification and resolution.
3. Which part of the microscope is most important in determining its resolving power? Why?
2.7.2 Type of Image formed and Direction of Image Movement
1. Type of Image formed: You are provided with a small piece of paper with the letter
btyped on it to mount and observe under the low power objective,
a. Is the letter inverted from top to bottom?
b. Is it inverted from left to right?
c. If you were to mount the following diagram, draw what the image would look like if seen
under the microscope. Do not attempt to redraw the diagram and observe it under the
microscope as you cannot see the whole diagram at a time.
d. If the above object stands in front of a mirror show with a drawing what its image would
look like.
e. From your above observations how does the image of an object under a microscope differ
from its mirror image?
2. Direction of Image Movement
a. Looking into the microscope, move the slide towards the left. Which way does the image
move?
b. . If you move the slide away from you which way does the image move?

c. How does the direction of movement of an image of an object under the microscope
differ from that of a mirror?
Figure 2.3 Image of letter e seen under a microscope
2.7.3 Microscope Measurements (Micrometry)
There are two methods of micrometry, stage micrometry and ocular micrometry.
Procedure
A. The size of objects viewed with the compound microscope can be estimated by first
determining the diameter of the field of view for a particular microscope objective and then
estimating the size of the specimen by comparing it with the total diameter of the field of
view.
1. Place a 1cm by 1cm graph paper on the center of the stage (diameter). Suppose the
ocular field view under low power is 4 mm, what is the diameter in micrometers?
Since 1 mm is 1000 m then 4x 103m = 4000 m
2. Calculate the diameters of the fields of view using the other objectives on your
microscope.

Objectives Field of view in mm Field of view in m
Low power
Medium power
High power
Oil emulsion
2. Obtain a prepared slide of cells. Estimate the length of one cell in micrometers (Hint: Use
the diameter of the field of view to determine the length of a strand of cells, then divide by
the number of cells):
Example1. . If the diameter of the field under low power objective is 1200 m, and the
objects diameter is about of the low power field, what is the approximate diameter of the
object?
Answer:-1200m x = 300m.
Example 2. If the field of view when using the 10x objective (100x total magnification) is 2
mm and 8 plant cells extend across the field of view (2 mm) as shown in the figure below,
what is the approximate diameter of each cell?
Answer: the length of each cell is 2/8 = 0.25 mm long(250 m)

Example 3. Given that the diameter of the average human red blood cell is 7.5m, how many
such cells can be lined up across the diameter of your microscope if the field view of your
microscope is 1200 m?
Answer:- 1200um /7.5m = 160 cells
B. To determine the diameter of the field of view more accurately, you can use an ocular
micrometer,( a small glass-disc on which uniformly spaced lens) shown as in Figure 2.4
below. Notice there are numbered divisions, but no units per division. The smallest
divisions are 0.01 mm (10m) in length.
Figure 2.4 The occular micrometer

3. Observing cellular Structure
3.1 Observing Plant cell (Onion epidermal cell)
Objective: At the end of this experiment you should be able to observe plant epidermal cell
and identify the cell membrane/cell wall, cytoplasm, nucleus and vacuole.
Materials and apparatus:
Microscope Microscope slide
Cover slip Dropper
Iodine/ Potassium iodide solution Forceps
Onion bulb
Procedure:
1. Peel the inner epidermis from one of the leaves
2 . Place a piece of the epidermis on a glass slide in a drop of water and then cover it with
cover slip.

5. Examine it under low power and medium power.
6. Remove the slide from the stage and place a drop of iodine solution at one end
holding a piece of absorbent paper at the opposite end of the cover slip. Wait for
about 5 minutes tills the cells get stained.
7. Examine under the middle power and draw two or more adjacent cells and
label the parts.
Figure3.1 An onion epidermal cell seen under a microscope (100X)

Discussion
1. Which part( s) of the cell become more deeply stained?
2. What structures observed in onion epidermal cells
3. Indicate the description that best describes the onion epidermal cells:
3.2 Observing Animal cell (cheek cells)
Objectives:- At the end of the this section you will be able to observe your own cheek
cells.
Microscope Microscope slide
Cover slip Dropper
Methylene blue Tooth pick
Procedure:
1. Gently scrap the inside of your cheek or the inside of your lip with the broad end of a
clean tooth pick.
2. Stir the scraping into a drop of water on a clean glass slide.
3. Place a cover slip on it and examine under a middle power
a. Are the parts of the cell clearly visible?
b. What is the color of the cells?
4. Lift the cover slip and put a drop of methylene blue. Cover with the cover slip and wait
for about 5 minutes until the cells get stained.

5. Examine under the middle power or high power and draw one or more cells and label it.
a. Can you now distinguish the nucleus and the cytoplasm?
b. Compare the position of the nucleus in cheek cells with that of onion cells.
Figure3.2 A cheek cells under a compound microscope ( 100X)
Discussion
1. Are human epithelial cells thinner than onion epidermal cells?
2. Diagram a few cells from your slides and label the parts observed.
3. Indicate the description that best describes the human epithelial cells
4. Which of the structures are unique to plant cells?
5. Which structures are common to both plant and animal cells?

3.3 Observing Microorganisms (e.g. Hay infusion)
Objective: In this practical activity you would be able:
To prepare hay infusion
To observe motile protozoan and other small metazoan and identify paramecium.
Introduction
A hay infusion is a preparation to culture and grow microscopic organisms such as protozoa,
microscopic worms and arthropods by sampling them from their natural habitat. Aquatic
protozoa feed on decaying vegetation and bacteria, or are carnivorous and use other species of
protozoa for food. Many species of protozoa and other invertebrates such as rotifers, water
fleas, flatworms and the like may prove detrimental to culture by controlling the populations of
protozoa. The hay infusion contains
Freshwater Ciliate Protozoa
Freshwater Amoeboid Protozoa
Freshwater Euglenoid Protozoa
Symbiotic Protozoa
Place about 1/4 inch of pond water in a finger bowl and add some vegetation. After a week
make several wet mounts from each culture
Microscope
Microscope slide
Cover slip
Dropper

Procedure:
1. Clean a slide and cover slip and transfer a few drops of hay infusion culture material to
the cavity slide.
2. Grasp the cover slip between the thumb and first finger and lower it to the slide so that
it rests at the edge of the drop of the culture media.
3. Finally support the upper edge of the cover slip with the dissecting needle (probe) and
slowly lower the cover slip into place.
4. In the process, air will naturally be expelled and few air bubbles will be included in the
slide.
5. Examine under the low power objective and proceed to the next higher power as
required.
Questions
1. Do you see motile organisms?
2. What are these organisms?
3. Identify and record the types of the organisms in the culture.

4. Dialysis, Diffusion and Osmosis
Objectives: At the end of this section you should be able:
To list factors that determines the rate of diffusion.
To separate different molecules in a solution
To distinguish a turgid and plasmolysed plant cells
Materials Required:
1. Absorbent cotton 17. Microscopes
2. Ammonia solution 18. Microscope slides
3. Beakers 19. Onion bulb
4. Biuret 20. Petri dishes
5. Cover slips 21. Pipettes
6. Dialysing tube 22. Saline solution (10%)
7. Distilled water 23. Potassium permanganate crystals
8. Droppers 24. Sewing thread
9. Forceps 25. Saline solution (0.9%)
10. Glass rods 26. Saline solution (IN)
11. Glass tubes 27. Scalpel
12. HCI (Cone.) 28. Starch suspension (5%)
13. Iodine solution 29. Silver nitrate solution
14. Masking tape 30. Test tubes
15. Meter ruler 31. Solidified agar
16. Methylene blue crystals 32. Sterilized pins

4.1 Diffusion:
The random movement of molecules from a region of higher concentration to a region of lower
concentration is known as diffusion.
Factors that affect the rate of diffusion
1. Size of molecules: - small molecules move faster than large ones do at the same
temperature
2. Temperature:- As temperature increases, the molecules more faster and the rate of
diffusion increases
3. Concentration differences between two regions:- the greater gradient difference, the
faster the rate of diffusion.
4. Surface area of membrane.
5. The distance through which the diffusion substance moves. Rate of diffusion
deceases rapidly as distance increases.
Therefore, Diffusion is effective over a very short distances Ficks law states that
Diffusion rate= surface area of the membrane x difference across them
Thickness of the membrane:
6. Type of Medium: rate of diffusion is fastest in gases, medium in liquids and least or
absent in solids.

4.1.1 Diffusion in Solids:
Procedure
1. You, in a group, are provided with a petri dish containing solidified agar.
2. Place a few crystals of potassium permanganate (molecular weight =158) at a point on
one side of the petri dish and on the other side of the petri dish place a few crystals of
ethylene blue (molecular weight = 374).
3. After about 30 minutes compare the areas of the agar through which the chemicals
diffused.
1. Which chemical diffused faster?
2. How do account for the difference in the diffusion rates of the two
chemicals?
4.1.2. Diffusion of Gases:

This experiment is to be carried out in groups.
A. You, in a group, are provided with glass tubing.
B. Place it in a horizontal position on your table.
C. Take two plugs of absorbent cotton and on one of them place a few drops of ammonia
solution (specific gravity 0.88 to 9.91) and on the other cotton plug place some
concentrated HCI (specific gravity 1.18). Protect your fingers from the HCI.

D. Insert the two plugs into the opposite ends of the tube at the same time and note the
time. Watch until a white ring of ammonium chloride (NH.CI) is formed.
E. When the ring is formed, again not the time, measure the distance of the ring from the
inner ends of the two plugs of cotton.
a. How long did it take for the ring to form?
b. What is the distance of the ring from the HCI end?
c. What is the rate of diffusion of the HCI in cm/sec.?
d. What is the distance of the ring from the ammonia solution end?
e. What is the rate of diffusion of ammonia in cm/sec.?
f. If the ratios of the diffusion of the two gases are different, how do you explain
the difference?
4.2 Dialysis:
A mixture of particles of different sizes can be separated by means of dialysis if a membrane
having the appropriate pore size is used.
1. You are, in a group, provided with a dialysing tube about 20 cm long. Tie one end of the
tube tightly with a sewing thread. It should be water tight.
2. Fill the tube about 2/3 full with a mixture of 1N NaCl and 5% starch suspension.
3. Bend the tube into a loop and tie both ends together. Note the volume.

4. Using a glass rod suspend the loop in about 100 ml of distilled water in a beaker.
5. Allow the dialysis to take place for about 1 hr.
In the mean time do the next
6. After about an hour place about 5 ml of the dialysate (the liquid in the beaker)in test
tubes A and B.
7. To test tube A add one or two drops of silver nitrate solution (test for NaCI ) and to
test tube B add one or two drops of 1% iodine solution (test for starch) and shake.
a. Is the test in test tube A negative or positive for NaCI?
b. . Is the test in test tube B negative for starch?
c. . From the result of your experiment what can you say about the nature of the
membrane?
8. Note the volume of the solution in the dialysing tube.
a. Has it increased or decreased compared to the initial volume?
b. How do you account for the change in volume if you have observed any change?
4.3 Osmosis:
Osmosis is the diffusion (net movement) of solvent particles (molecules) across a semi
permeable membrane from a region of their higher concentration (i.e. from weak solution) to a
region of their lower concentration (i.e. to strong solution).

In biological systems since the solvent is water and the membrane is the cell .membrane,
osmosis can be defined as the diffusion of water molecules across the cell membrane from a
region of their higher concentration to a region of their lower concentration.
Whether the net movement of water is into or out of the cell depends, upon the relative
concentration of the protoplasm and the solution surroundings the cell.
When osmosis results in the net movement of water out of a cell, the protoplasm or the cell
shrinks. This shrinkage of the protoplasm as a result of net loss of water is called Plasmolysis.
The shrinkage of animal cells is called Crenation.
When osmosis results in a net movement of water into a cell, the protoplasm swells. If it is a
plant cell, the swelling protoplasm exerts a pressure (Turgor Pressure) against the cell wall.
Eventually the net movement of the water into the cell is stopped when the pressure exerted
inwards by the cell wall and the pressure exerted outwards by the protoplasm is equalized. If it
is an animal cell (e.g. blood cell) since it does not have a rigid cell wall, it may burst if it takes
in excess water.

1. Osmosis in Onion Epidermal Cells
1. Remove a small piece of onion epidermis and mount it in water. The outer pinkish
epidermis may be preferred since the cytoplasm can easily be seen under the microscope.
2. Using a clean slide mount a fresh onion epidermis in a drop of distilled water.
3. Observe your preparation under the middle power and draw 2 or 3 adjacent cells and
label the parts.
4. Take another clean slide and mount a fresh epidermis in a drop of 1N NaCl.
5. Observe it under the middle power and draw 2 or 3 adjacent cells and label the parts.
6. After about 20 minutes observe them again and draw 2 or 3 adjacent cells and label the
parts.
a. What major differences do you observe between the cells mounted in water and
those mounted in NaCl solution?
2. Explain how this difference could come about.

5. Testing for Biologically Important Molecules
Objective: At the end of this section you would be able to :
Identify biological molecules using qualitative tests
Identify the test reagents and the positive test results for the various organic molecules.
1.1. Tests for carbohydrates
Introduction
Carbohydrates, fats, proteins and nucleic acids (DNA and RNA) are among the large
biologically important molecules. In this practical you will apply chemical tests on the first
three classes of molecules.
Carbohydrates: Carbohydrates are compounds of carbon, hydrogen and oxygen occurring in a
variety of forms in both plants and animals. They include such substances as sugar, starch,
glycogen and cellulose. Carbohydrates serve as structural substances (e.g. cellulose in cell
wall) as immediate source of potential energy reserve.
On the basis of size (i.e. number of sugar units they are composed of) carbohydrates are known
as monosaccharides, disaccharides, oligosaccharides and polysaccharides.
1. Monosaccharides: These, otherwise known as simple sugars, have the general formula of
CaH2nOna or (CH2O)n. The value of "n" ranges from 3 to 8.
2. Disaccharides: Disaccharides are composed of two identical of different monosaccharide
units compounded together. Upon hydrolysis, a monosaccharide, for example, maltose gives
two molecules of glucose while sucrose gives one molecules of glucose and another of
fructose.

No. of carbon atoms Name of sugar
3 Triose
4 Tetrose
5 Pentose
6 Hexose
7 Heptose
8 Octose
3. Oligosaccharides: Carbohydrates containing more than two, but less than ten, sugar units
are usually known as Oligosaccharides.
4. Polysaccharides: The general formula of polysaccharides is (C6 H12O6)n. Upon complete
hydrolysis a polysaccharide splits into simple sugars. Cellulose and chitin are structural
polysaccharides of plants and arthropods, respectively. Plants store their reserve carbohydrates
in the form of starch and animals in the form of glycogen.
Materials Required:
1. Alpha-naphthol
2. Benedict's solution 10. Iodine solution (0.05N)
3. Burettes 11. Masking tape
4. Bunsen burner 12. Pipettes
5. Dextrin (1%) 13. Sodium hydroxide (6 N)
6. Droppers 14. Starch Solution (1%)
7. Funnel 15. Sucrose (O.IM)
8. Glucose (O.IM) 16. Sulfuric acid (Conc.)
9. Hydrochloride acid (6 N) 17. Test tubes

5.1.1 Molisch Test: This is a general test for carbohydrates. Carry out the test on O.1 M
solution of glucose and sucrose, and on 1% starch solution.
a. Place 5 ml of the solution (glucose, sucrose and starch) in clean test tubes.
b. Add 2 drops of alpha-naphthol. Mix the contents well.
c. Incline the test tube. Slowly add 2-3 ml of concentrated sulphuric acid down the side.
A purple or violet ring at the junction of the acid and the solution indicates the presence of
carbohydrates.
5.1.2 Benedict's Test: Some sugars have the capacity of reducing certain compounds.
Such sugars are called reducing sugars. These are mostly monosaccharides. Those
disaccharides with free aldehyde or ketone groups such as maltose, lactose, cellobiose, are also
reducing sugars.
Benedict's solution contains cupric sulphate, sodium carbonate, and sodium citrate.
When the cupric sulphate is reduced a red precipitate of cuprous oxide is formed.
a. Place 2 ml of Benedict's solution in a test tube.
b. Add 2 ml of 0.1M glucose solution and mix well.
c. Heat the mixture over a spirit lamp or in a hot-water bath. Shake the tube constantly while
heating over a flame: Otherwise the contents may shoot out.
A brick red, sometimes orange precipitate indicates the presence of a reducing sugar.
5.1.3 Iodine Test: This is a test for the polysaccharides starch and dextrin.
1. Place 2 ml of 1% starch solution in a test tube.
2. Add a drop or two of iodine solution.
3. Mix the contents by shaking the tube.
A blue or a blue-black coloration is the positive test for starch.

4. To 2 ml of 1% dextrin solution add a drop or two of 0.05 iodine solution. Note a brownish
red color.
5.1.4 Acid Hydrolysis of Disaccharides and Polysaccharides: An acid can
split disaccharides and polysaccharides into simple sugars. The presence or the production of
the latter can be demonstrated by applying Benedict's test.
a. Take two test tubes and place 2 ml of 0.lM sucrose solution into each.
b. Apply Benedict's test to one of the tubes and see if a red precipitate is produced.
c. To the second test tube add 2 ml of 6N HCl. Mix and heat the contents for
15minutes.
d. Then cool the test tube under running tap water and add 2 ml of 6N NaOH to
neutralize the acid. Apply Benedict's test (see test procedure (b) above).
A red precipitate shows that sucrose has been reduced into simple reducing' sugars by the
acid.
5.1.5 Acid Hydrolysis of a Polysaccharide:
1. Take two tubes and add 2 ml of 1% starch solution into each.

2. Apply Benedict's test to one of the tubes. Is a red precipitate produced?
3. To the second test tube add 2 ml of 6N HCl. Heat the contents for about half an h
our. Cool the tube; add 2ml of 6N NaOH to neutralize the acid. Apply Benedicts test.
Do you get a red precipitate?
5.2 Tests for Fats and Oils
Introduction
Lipids (fats and oils) are made up of two different groups of compounds namely fatty acids
and glycerol. Three molecules of identical or different fatty acids combine with a molecule of
glycerol to make one molecule of a fat or oil. There is not much difference between oils and
fats except that oils are composed of unsaturated fatty acids and are therefore liquid at room
temperature. In other words oil is a liquid with a low melting point and fat is a liquid with a
relatively high melting point.
Materials Required:
1. Absolute alcohol 7. Microscopes
2. Blank paper 8. Oil
3. Bunsen burner 9. Pipettes
4. Droppers 10. Sudan III solution
5. Match box 11. Test tubes
6. Microscope slides
5.2.1 Grease Spot Test:

Procedures
1. Place a drop of oil 'on ordinary paper.

2. For the sake of comparison, place a drop of water near the drop of oil.
3. Warm, the paper gently for a few minutes.
4. Then, hold the paper up to the light and examine.
The oil leaves a translucent spot. d
5.2.2 The Emulsion Test:

1. Add 2 ml of absolute alcohol and drops of oil (e.g. Noug oil) in a test tube.
2. Shake the contents vigorously.
3. Then allow the solid matter to settle down.
4. Pour the "ethanol" into a second test tube, and add 2 ml of cold tap water.
A milky emulsion is the characteristic appearance.
Keep the emulsion for test 5.2.3
5.2.3 Test with Dyes:

1. Place a drop of the emulsion on a clean slide and add a drop of Sudan III.
2. Examine the slide under the low power of your microscope.
3. Do not cover your preparation with cover slip.
Some dyes such as SUDAN III are specifically soluble in fats and oils.
The characteristic result is the formation or red droplets.

5.3 Test for Proteins
Introduction
Proteins are compounds of carbon, hydrogen, oxygen, nitrogen and often sulfur.
They are important constituents of the protoplasm. Besides, 'all known enzymes and many
hormones are proteins.
Complete hydrolysis of proteins gives the building blocks known as amino acids. About
twenty-two different amino acids are known to occur in common proteins. The type and the
sequence of amino acids contribute to a great extent to cause the uniqueness of protein.
Several of the amino acids cannot be synthesized in the animal body. Such amino acids are
known as Essential Amino Acids. The amino acid essential for the human body are
valine, leucine, isoleucine, lysine, methionine, threonine, phenylalanine (or tyrosine) and
tryptophan. The remaining amino acids required for protein synthesis can be synthesized by
human himself.
Materials Required:
1. Ammonia solution (15N) 7. Match box
2. Biuret 8. Nitric acid (Conc.)
3. Copper sulfate (0.1%) 9. Pipettes
4. Droppers 10. Sodium hydroxide (6N)
5. Egg albumin (2%) 11. Test tubes
6. Masking tape
Procedures
5.3.1 The Biuret Test:
Although not very specific, it is a good general test for proteins.

1. Place 2 ml of 2% egg albumin solution in a test tube.
2. Add 2 ml of 6N NaOH solution.
3. Add drop by (maximum of 10 drops) 0.1% CUS04, Solution.
The production of pink or violet coloration indicates the presence of proteins.
5.3.2 . The Xanthoproteic:
The amino acids tyrosine, phenylalanine and to some extent tryptophan are responsible for the
xanthoproleic reaction.
1. Place 2ml of 2% egg albumin solution in a test tube.
2. Add about 2 ml of concentrated nitric acid.
3. Mix carefully and note the formation of white precipitate (which may eventually become
yellow).
4. Heat the contents carefully. Note the formation of a yellow colored solution.
5. Cool the test tube under running tap water and then very carefully add 2 to 5 ml of 15N
ammonia solution The yellow color deepens to orange.

6. Cell Division
All cells undergo a process of growth and division called the cell cycle. During the cell cycle, a
cell grows, prepares for division, and divides to form two daughter cells, each of which then
begins the cycle again.
The cell cycle consists of four (4) phases. Mitosis and cytokinesis take place during the M
phase; G1 phase is the time when cells grow; chromosome replication takes place during the S
phase; G2 phase is when the cell produces the structures needed for replication.
Objectives
Upon completion of this activity, you should be able to:
Identify cells undergoing the process of mitosis
Identify the stages of mitosis in cells
Make drawings of structures seen with a microscope
Materials:
-Pins, - Cover slip,
- Test tubes, -onion, scalpel,
-distilled water, -fine needles,
-acetic alcohol, -blotting paper (soft paper),
-hydrochloric acid, - Small corked tube,
- feulgen stain, -forceps,
- Microscopic slide, - Petri dishes,
-water bath,
-microscope,

Procedure:
2. Place a pin through a bulb of onion and suspend in a test-tube full of water so that
the base of the bulb is covered with water. Leave the contents for 3-4 days without
any disturbance.
3. When several roots have grown to 1-2 cm long, remove the bulb and cut 1 cm of
the roots from the terminal part.
4. Transfer the roots to a small corked tube containing acetic alcohol and leave the
contents overnight at room temperature. The process will fix the material.
5. Remove the root tips with forceps by grasping the cut end of the root and transfer
them to a petri dish containing distilled water. Finally wash the roots for a few
minutes to remove the fixative.
6. Transfer the root tips to a test tube containing 1N hydrochloric acid (HCI), which
is maintained at 60C for 6-10 minutes.
7. Pour the root tips and acid into a petri dish. Transfer the roots into another petri
dish containing distilled water and wash them thoroughly to remove the acid.
Leave the contents for about 5 minutes:
8. Transfer the roots to a small corked tube containing Feulgen stain. Leave in a cool
place (preferably a refrigerator) for a minimum of 2 hours.
9. Remove a root tip and place in a drop of acetic alcohol on a clean microscopic
slide. Cut off the terminal 1-2mm of the root tip and discard the rest of the root.
10. Cover the slide with a cover slip and .using blotting paper press down firmly over
the cover slip with the ball of the thumb. Do not allow the cover slip to move
sideways.
11. Examine the slide under the low, middle and high power objectives of the
microscope.
Questions
a. Identify cells showing different phases of mitosis.
b. Draw and label nuclei showing the various phases.
c. Why do we use HCI (hydrochloric acid) in the above experiment?
d. Why do we use root tips in this experiment? 0opper.

7. Photosynthesis
Photosynthesis is the process by which green plants manufacture organic molecules from
inorganic molecules (CO2 and H2O) using light energy. The purpose of photosynthesis is to
convert radiant energy into stored chemical energy
6CO2 + 6H2O + light energy absorbed by pigment C6H12 O6 + 6O2
The products of photosynthesis (glucose and oxygen) are the most essential for survival of life
on earth. In order to carry out photosynthesis, a plant must have light, but how much light does
a plant need? Some plants need a lot of light; others seem to thrive in the shade. Does more
light lead to more photosynthesis?
7.1 Test for starch in leaves
Objective: At the end of the experiment you should be able to
Material Required
Potted plant
Aluminum foil or black paper
Iodine solution
Ethanol
Beaker for boiling water, 250 cm3

Forceps
Boiling tube
White tile

Procedure
1. Take aluminum foil or black paper a little larger than the leaf and cut a simple shape
such as X from the piece of aluminum foil or black paper. The cut shape should allow
light to pass through.
2. Fasten the foil or black paper firmly over both sides of a leaf of a potted plant .
3. Put the plant in sunlight for three hours.
4. Detach the leaf from the plant.
5. Remove the cover and put the leaf in boiling water for one minute.
6. Put in to alcohol and warm the alcohol and leaf in hot water bath until all the color
disappears from the leaf. The alcohol dissolves the chlorophyll. Do not heat the ethanol
in a Bunsen burner flame. This is not safe because ethanol is highly flammable.
7. Using forceps remove the leaf from the boiling tube and rinse the leaf in cold water.

8. Spread the leaf out on a white tile and use the iodine solution to test for starch. A
blue-black color indicates starch is present.
7.2. Light intensity and Rate of photosynthesis

Objective:- You should be able to examine how the intensity of light affects photosynthesis
Materials Required:
Test tube
Freshly cut sprig of an evergreen or Elodea
400-600 ml beaker

Sodium bicarbonate solution (baking soda)
Forceps
Watch or clock with second indicator
Source of bright light
Hand lens
Procedure
1. Working with a partner, completely fill a test tube and a beaker with a sodium
bicarbonate solution. Sodium bicarbonate will provide a source of carbon dioxide.
2. Using forceps, place a plant sprig about halfway down in the test tube. Be sure that the
cut end of the sprig points downward in the test tube.
3. Cover the mouth of the test tube with your thumb and turn the test tube upside down.
Try not to trap any air bubbles in the test tube.
4. Place the mouth of the test tube under the surface of the sodium bicarbonate solution in
the beaker. Remove your thumb from the mouth of the test tube.
5. Gently lower the test tube inside the beaker so that the test tube leans against the side
of the beaker.
6. Put the beaker in a place where it will receive normal room light. As soon as see small
bubbles coming from the cut end of the stem, time the reaction for 10 minutes. If you
do not see bubbles, cut the stem again and recrush.
7. Calculate the net photosynthesis in bubbles/min. (Divide the number of bubbles by 10
minutes.)
8. Remove your test tube from the bright light. Observe and record the rate of bubbles
without direct light.
9. Record the number of bubbles in the Data Table.
10. Turn the lights back on in the room and shine a bright light on the sprig for 5 minutes.
Again, count the number of bubbles produced in 5 minutes. Record the number in the
Data Table.

Bright Light Dim Light
Bubbles/min __________ Bubbles/min ____________
Discussion
1. What are the bubbles? Explain why bubbles happen.
2. What are the products of photosynthesis? Which of these products is released from
leaves as a gas?
3. What can you tell about photosynthesis if a leaf begins to produce more gas bubbles?
Fewer gas bubbles?
4. What are the reactants of photosynthesis? Which of these reactants is taken into the
leaves as a gas?
5. Write out the chemical formula for photosynthesis
6. Did the number of bubbles change when the light intensity was reduced? Explain why
this would occur.
7. What was the purpose of adding sodium bicarbonate (baking soda) to the plant? Hint:
look at the formula for photosynthesis
8. Explain why plants cannot grow in the depths of the ocean.

7.2 Chromatographic separation of chlorophyllous pigments
Introduction
Paper chromatography is a process that uses special filter paper to separate and identify the
different substances in a mixture. Chromatography means to write with color. The
substances in the mixture dissolve in the solvent and move up the paper. The heavier
substances move up the paper more slowly. The lighter substances move up the paper more
quickly. So, heavy and light substances get separated from one another on the paper.
As the solvent slowly travels up the paper, the different components of the mixtures travel at
different rates and the mixtures are separated into different colored spots. The distance
travelled relative to the solvent is a constant for a particular compound as long as you keep
everything else constant - the type of paper and the exact composition of the solvent.

The distance travelled relative to the solvent is called the Rf value. For each compound it can
be worked out using the formula:
For example, if one component of a mixture travelled 9.6 cm from the base line while the
solvent had travelled 12.0 cm, then the Rf value for that component is:
There are two major types of pigments in higher plants. These are:-
A. Chlorophylls:- There are several kinds of chlorophyll molecules. The most important are
chlorophyll a and chlorophyll b.
i) Chlorophyll "a" is bright - green pigment
ii) Chlorophyll "b" is yellow - green pigment
B. Carotinoids: - are yellow, orange, red and brown pigments which are two types:-
i) Xanthophylls - a yellow or brown pigment
ii) Carotene - a yellow or orange pigment
These different kinds of pigments can be separated by paper chromatography technique.

Materials
Chromatography tank Filter paper
Whatman No. 1paper Pencil
Acetone Mortar and pestle
Funnel Reagent bottle
Dropper Ruler
A. Separation of photosynthetic pigment from leaves
Procedure:
1. Take about four green leaves and grind with mortar and pestle in the chosen solvent (
Acetone)
2. Filer the extracts using filter paper
3. Pour the solvent system in to the chromatography tank so that it is about 2cm deep
and cover the lid
4. Prepare strip of Whatman No. 1 chromatographic paper to fit in to your developing
chamber.
5. With pencil draw a straight line about 3 cm away from the base of the paper.
6. Add a drop of chlorophyll extract on the center of the line and allow drying.
7. Place the chromatography paper in the tank containing the solvent so that its tip is
submerged in the solvent taking care that the spot is above the surface of the solvent
8. Watch as the solvent moves up the paper.

9. Remove the chromatography paper before the solvent front reaches the top of the
paper.
10. Mark with pencil the distance that the solvent front traveled and set the paper aside to
dry.
Discussion
1. Did the leaf you tested contain different pigments? Use your results to support your
answer.
2. Observe the color bands and calculate the Rf value of each pigment.

8. Collection and preservation of plant and animal
specimens
8.1 A herbarium specimen

.
Introduction
A herbarium specimen is a pressed plant sample deposited for future reference. It supports
research work and may be examined to verify the identity of the specific plant used in a study.
Why preserving specimens is needed? Plant classification is constantly changing. Shifts in
species alignments and groupings are made as new evidence comes to light. Identifications are
subject to change. Preserved specimens help cross-reference these changes to previous
research.
Preplanning for the preparation of herbarium specimens is crucial. Arrangements should
include:
targeting collection locations and date periods to obtain useful specimens;
obtaining collection permits from appropriate agencies (this can take months); and
Establishing official contact with government, herbarium, and research personnel in
the area you will be working. This is required by law in most countries.

PRESSING AND DRYING PLANT SPECIMENS
Specimens are pressed in a
plant press, which consists of
a wooden frame (for rigidity),
corrugated cardboard
ventilators (to allow air to
flow through the press),
blotter paper (to absorb
moisture), and folded newspaper (to contain the plant material). The plant press is tightened
using straps with buckles or bolts with wing nuts. The objective of pressing plants is to extract
moisture in the shortest period of time, while preserving the morphological integrity of the
plant, and to yield material that can be readily mounted on herbarium paper (an acid-free
cardstock) for long-term storage.
In order to fit on a standard herbarium sheet, a plant specimen should be pressed flat to no
more than 11 X 16 inches. If the specimen will not fit those dimensions, it may be folded or cut
into sections. Multiples of smaller plants may be pressed together in order to provide ample
material for mounting and study. Small loose pieces, such as seeds, may need to be placed in a
small paper packet inside of the newspaper. Large fruits or bulbs are often cut in half
lengthwise or in slices prior to pressing. In order to insure rapid and thorough drying,
extremely succulent materials such as cactus stems may need to be sliced open and some of the
fleshy interior scraped out. Each specimen should consist of a stem with attached leaves and, if
at all possible, flowers and/or fruits. The roots of herbaceous plants should also be included. In
the case of very large trees, shrubs, or vines, pieces should be selected to illustrate to the

greatest extent possible the overall characteristics of the plant and the range of variation in
flowers, leaves, and other structures. Each collection, i.e. gathering of a plant specimen, should
be assigned a collection number. Data for each collection should be entered in a field
notebook (see discussion of label data below). If ample material is available, a minimum of
three specimens should be pressed for each collection, especially if collecting in a region where
the flora is poorly known. This will help facilitate the identification of the plants through the
distribution of specimens to various herbaria and researchers. An ethical collector will insure
that his/her collecting activities do not pose a significant threat to the survival of endangered
species or habitats.
Care should be taken to make good specimens.
Pressing material immediately upon
collection results in the best specimens.
Samples that are allowed to wilt prior to
pressing will generally produce inferior
specimens. Plants should be carefully arranged as they are placed in the press to maximize
preservation of diagnostic features. Leaves, flowers, and fruits should be spread out so that
they do not overlap and can be observed from different perspectives. The collection number
should be clearly written on the outside of the newspaper containing each plant specimen. The
plant press must be kept tight; this prevents shrinkage and wrinkling of the plant material and
yields specimens that are easier to mount securely on herbarium paper. The pressed plants must
also be thoroughly dried prior to storage and mounting. Best results are obtained with the use
of an electric drier that holds the presses and provides steady bottom heat between 95F and
113F. A low ambient humidity and good airflow around and through the presses also
insures rapid and thorough drying of plant material. As the specimens dry, it may be necessary

to further tighten the straps on the press to minimize shrinkage and wrinkling. Rapid drying
promotes the best retention of plant color, but excessively high temperatures or long drying
periods can result in blackened, discolored, and brittle specimens.
Mounting and storage of specimens require a considerable financial commitment in the form of
archival materials, labor, and storage cabinets. Herbaria have the prerogative not to accept
specimens if the cost of labor/materials for processing is excessive or if the quality of
specimens or accompanying data is unsatisfactory. Due to differences in mounting
methodologies and materials, most herbaria prefer not to accept already mounted specimens.
Because plant classification is generally based on the morphology of flowers and fruits, in most
cases sterile (non-flowering or -fruiting) specimens will not be accepted.
HERBARIUM SPECIMEN LABELS
A plant specimen is incomplete without label data. Label data is a form of field data and must
be accurate. The following are important elements:
Scientific name: genus, species, authority, infraspecific information
Determiner of the scientific name: the name of the person who identified the plant
Detailed location; the location is used by researchers on several levels:
o for general mapping to region, county or province;
o for detailed mapping, as in GIS computer applications;
o to physically locate the plant(s) in order to obtain further research material. The
location should consist of: country, state or province, county or municipality
and a description of the location in reference to roads, road junctions, mile
markers and distances from cities and/or towns. Latitude and longitude, section,

township and range, and elevation may also be helpful. A location taken with a
Global Positioning System (GPS) is a desirable complement to the locality
description. GPS coordinates MUST include a datum!
Habitat: the type of plant community where the plant is growing and, if known, other
plants growing in association
Plant habit: describes the form of the plant (tree, shrub, vine, herb) and its height.
Examples: tree, ca. 50 ft. tall. sprawling herb
Frequency: is the plant rare, occasional, frequent or common?
Plant description: describe characteristics of the plant which may be lost upon drying,
such as flower/fruit color and fragrance, leaf orientation and aroma
Collector name: it is recommended that the collector be consistent and use their full
first name, middle initial (or full name) and full last name.
Other collectors (*see label examples note below) present with the collector
Collection number: a sequential straightforward numbering system (1,2, 3, ...) is
preferable.
Date of collection: a format with the month spelled out or abbreviated and 4 digit year
will prevent confusion. E.g., 3 May 2003, not 3/5/03 or 5/3/03.

8.2 Animal preservation
Preservation methods
1. Cooling, refrigeration: Freezing at about - 10C is a good method for preserving
specimens for skeletal preparation, study skins or mounting, certain pathologic
specimens and legal evidence. Laboratory freezers with - 70-80C are best
2. Taxidermy: is the act of mounting or reproducing dead animals for display or for
other sources of study. Taxidermy can be done on all vertebrate species of animals,
including mammals, birds, fish, reptiles, and amphibians. The animal is first skinned in
a process similar to removing the skin from a chicken prior to cooking. This can be
accomplished without opening the body cavity, so the taxidermist usually does not see
internal organs or blood. Depending on the type of skin, preserving chemicals are
applied or the skin is tanned. It is then either mounted on a mannequin made from
wood, wool and wire, or a polyurethane form. Clay is used to install glass eyes. Forms
and eyes are commercially available from a number of suppliers. If not, taxidermists
carve or cast their own forms.
3. Drying: For certain samples, for instance for stomach contents and faeces, air-drying is
recommended
4. Chemical preservation:
A. Formalin preservation can be used for specimens for histological examination and
for fluid-preserved specimens. Small animals such as worms, arthropods, small reptiles
and amphibians can be preserved in formalin.
A. Ethyl alcohol The better solution for long term storage of invertebrate specimens is
in an 80% solution of ethyl alcohol. Ethyl alcohol can be found in the painting

supplies, though it may not be specifically labeled as ethyl alcohol. It may be labeled
denatured alcohol and should list ethyl alcohol, or ethanol, or methanol in the
contents. This product can also be used for thinning shellac, cleaning glass and metal
and as a clean burning fuel for marine stoves. You should also buffer the solution
with the glycerin/antacid tablets.
B. Isopropyl alcohol Isopropyl alcohol is relatively cheap and easy to obtain. It is
rubbing alcohol and can be found in the health and beauty section of your local
retailer. There are different strengths available (70% and 90%), so if you use
isopropyl you want to dilute it to a Isopropyl alcohol is relatively cheap and easy to
obtain. It is rubbing alcohol and can be found in the health and beauty section of
your local retailer. There are different strengths available (70% and 90%), so if you
use isopropyl you want to dilute it to a 40% alcohol solution. Isopropyl alcohol can
be hard on the specimens and tends to make them brittle over time. You can buffer it
with a few drops of glycerin and a pinch of calcium carbonate antacid tablets (crush
the tablets to a powder and add).

References
1. Collee, J.G, Duguid, J.P., Fraser, A. G., and Marmion. B.P. (1989). Practical Medical
Microbiology, Vol, 2.
2. Enger, E.D. and Ross F.C.(2008)) Laboratory Manual Concept s in Biology. 13th ed
http://www.primisonline.com
3. Gunstream,S.E. (2012).Exploration in Basic Biology.12th ed. Benjamin Cummings Boston
Columbus Indianapolis New York
4. Helms,D.R.,Helms,C.W.,Kosniski,R.J. and Cummings,J.R.( ) Biology in the
Laboratory.,2nd ed,
5. http://www.dsbn.edu.on.ca/schools/Westlane/Science/simon/SBI3C1/micro.gif
6. http://www.practicalbiology.org/areas/introductory/energy/photosynthesis/testing-
leaves-for-starch-the-technique,73,EXP.html
7. Jean D. (2003). Laboratory Investigation for Biology. 2nd ed., Benjamin Cumming.
8. Kenneth, M. and h Levine,J. (2006). Biology High School Assessment Student Lab
Manual Prince Georges County Public Schools Pearson Prentice Hall
9. Roberts K. (1987). Biology a Functional Approach: Students Manual.2nd edition,
Thomas Nelson & Sons Ltd.

Contents
1. Introduction ......................................................................................................................... 1
1.1. The scientific methods .................................................................................................... 1
1.2 Laboratory Safety Rules.................................................................................................. 4
1.3 Introduction to Basic Biological Laboratory Equipments ........................................... 7
1.4 Laboratory report writing methods ............................................................................. 14
2. Microscope ........................................................................................................................ 19
2.1. Types of microscope ................................................................................................. 19
2.2. Parts and Function of light microscope ...................................................................... 20
2.3 Handling the Microscope .............................................................................................. 26
2.4. Setting the Microscope for Use ............................................................................... 27
2.5 Practice in Mounting ............................................................................................... 28
2.6 Focusing practice ..................................................................................................... 31
2.7 Magnification, Resolution, and type of image formed................................................ 33
2.7.1 The Resolving power of a Microscope ..................................................................... 33
2.7.2 Type of Image formed and Direction of Image Movement ...................................... 35
2.7.3 Microscope Measurements (Micrometry)................................................................. 36
3. Observing cellular Structure ............................................................................................ 39
3.1 Observing Plant cell (Onion epidermal cell) ............................................................... 39
3.2 Observing Animal cell (cheek cells)........................................................................ 41
3.3 Observing Microorganisms (e.g. Hay infusion) .................................................... 43
4. Dialysis, Diffusion and Osmosis....................................................................................... 45
4.1 Diffusion .......................................................................................................................... 46
4.1.1 Diffusion in Solids: ................................................................................................ 47
4.1.2. Diffusion of Gases: .................................................................................................. 47
4.2 Dialysis: .......................................................................................................................... 48
4.3 Osmosis ........................................................................................................................... 49
5. Testing for Biologically Important Molecules ................................................................. 52
1.1. Tests for carbohydrates ........................................................................................... 52
5.1.1 Molisch Test: ............................................................................................................ 54
5.1.2 Benedict's Test ............................................................................................................ 54
5.1.3 Iodine Test: ............................................................................................................. 54
5.1.4 Acid Hydrolysis of Disaccharides and Polysaccharides ........................................... 55
5.1.5 Acid Hydrolysis of a Polysaccharide: .................................................................... 55

5.2 Tests for Fats and Oils ................................................................................................... 56
5.2.1 Grease Spot Test: ...................................................................................................... 56
5.2.2 The Emulsion Test: ................................................................................................... 57
5.2.3 Test with Dyes: ......................................................................................................... 57
5.3 Test for Proteins ............................................................................................................. 58
5.3.1 The Biuret Test: ........................................................................................................ 58
5.3.2 . The Xanthoproteic .................................................................................................. 59
6. Cell Division ...................................................................................................................... 60
7. Photosynthesis ................................................................................................................... 63
7.1 Test for starch in leaves ................................................................................................. 63
7.2. Light intensity and Rate of photosynthesis ................................................................. 65
7.2 Chromatographic separation of chlorophyllous pigments ......................................... 69
8. Collection and preservation of plant and animal specimens............................................... 73
8.1 A herbarium specimen .................................................................................................. 73
8.2 Animal preservation ...................................................................................................... 78

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1.

Objectives:- At the end of this session you should be able to:

Recognize the stages of scientific inquiry as it applies to everyday experiences.

Make observations, formulate hypotheses, make predictions, and design experiments to

evidence for explanations of natural phenomena. Scientific evidence consists of measurements

Scientific inquiry involves the steps outlined below.

[Ambo University] Page 1

Steps in scientific methods

Indirect observation: - through the use of special equipment (microscope).

o Observation can be carried out in different stages of scientific methods.

In identifying the problem.

In formulating the hypothesis.

In conducting experiment (to prove or disprove the hypothesis).

B. Identifying the problem or question

[Ambo University] Page 2

information (relevant fact) as possible.

D. Forming a hypothesis: - Hypothesis is logical reasoning, an intelligent guess or a tentative

theory formulated based on the information.

o Hypothesis is a proposition that might be true (predication).

o For a single problem there could be alternative hypothesis.

Inductive reasoning: - is the method of discovering general principles by careful

i.e. reaching to a specific conclusion from general cases.

o Not all hypotheses are tested by laboratory experiments.

G. Observing and recording results: - experiment results or observation must be recorded

addition to verbal explanation.

H. Analysis and interpretation of data: - studying the organized results or observation to

discover the essential facts.

similarities and difference.

[Ambo University] Page 3

comparing your experiment result with others.

Journals. Scientific research report and journals (article) include:-

a. Title of the research

b. Introduction- what did you do? Why?

c. Material and methods (How did y do it?)

d. Results- (what did you find?)

e. Discussion- your interpretation of results

f. Summary- statement of main findings

g. Acknowledgments- who helped you?

h. Reference- Details of references cited.

1.2 Laboratory Safety Rules

procedures and apply them.

follow the procedures of laboratory safety rules in the laboratory.

[Ambo University] Page 4

otherwise dangerous chemicals may inadvertently be transferred to eyes or mouth.

area (especially the face or eyes) before washing.

for several minutes.

9. Never taste, smell, or touch a chemical or solutions.

II. GENERAL LABORATORY REGULATIONS

eraser and laboratory note book.

2. Record all your observations carefully in your note book.

3. Drawing should be always done with pencil

4. Work with an open and inquiring mind

5. Unauthorized experiments are strictly forbidden.

6. Maintain an orderly and clean work space.

[Ambo University] Page 5

9. At the end of the laboratory period:

-clean all equipment used in the experiment

-clean the sink of all debris

10. No drinking, eating, or gum chewing is permitted in the laboratory

session, if not you will be held responsible.

14. Be courteous, use your head, and try to enjoy yourself!

[Ambo University] Page 6

Objective: At the end of this section you should be able to: