Woldia University

Faculty of Natural and Computational Sciences

Department of Biology

A Module on:
Introduction to Biological Laboratory Techniques
(Biol.1011)

PREPARED By:
Chala Adugna (M.Sc.)

JULY, 2016
Woldia, Ethiopia
Module Introduction

As a beginner in her/his career, a biologist should have basic knowledge in laboratory safety
rules as well as skills in handling biological tools. This enables her/him to gain available
information in life for better understanding of major biological processes and phenomena. To
this end, this module is designed to equip students with the intended primary skills in the
laboratory and help them to perform practical activities efficiently.
The course deals with laboratory safety rules, introduces basic biological tools with more
emphasis on light microscope; parts and function of the light microscope: basic methods of
mounting, focusing, magnification, observation, microscopic investigation of animal cells, plant
cells and microorganisms; Diversity of life, cellular structures, diffusion and osmosis; collection,
sampling and preservation of plant and animal specimen; testing biologically important
molecules; chromatography.
Module Objectives
After completing the course, the student should be able to:
 Recognize biological safety rules and handles biological tools safely.
 develop skills and techniques of using basic biological equipment, chemicals,
reagents and media in the laboratory.
 Use the microscope properly.
 Prepare temporary and permanent microscopic slides for observation.
 Explain cell division processes (mitosis and meiosis).
 Conduct simple biological experiments.
 Discuss mechanisms of material transportation across biological membranes.
 Collect and preserve plant and animal specimens.
 General Laboratory Instruction
Welcome to the exciting field of biological laboratory techniques! The intent of this laboratory
manual is to provide you with basic skills and tools that will enable you to explore a vast biota of
the world. It is hoped that these laboratory studies strengthen the theoretical part of the course.
You have to prepare yourself ahead of each laboratory session by reading this module.
It is of paramount importance that you know all the regulations that are laid down here as
Laboratory Protocol.
Scheduling. During the first week of this course your instructor will provide you with a schedule
of laboratory exercises arranged in the order of their performance.
Before attending laboratory each day, check the schedule to see what experiment or experiments
will be performed and prepare yourself so that you understand what will be done.
Each laboratory session will begin with a short discussion to brief you on the availability of
materials and procedures. Since the preliminary instructions start promptly at the beginning of
the period, it is extremely important that you are not late to class.
Personal Items. When you first enter the lab, place all personal items such as jackets, bags, and
books in some out of the way place for storage. Don’t stack them on your desktop. Desk space is
minimal and must be reserved for essential equipment and your laboratory manual. Your
instructor will indicate where they should be placed.
Attire. A lab coat or apron must be worn at all times in the laboratory. It will protect your
clothing from accidental contamination and stains in the lab. When leaving the laboratory,
remove the coat or apron. In addition, long hair must be secured in a ponytail to prevent injury
from Bunsen burners and contamination of culture material.
The following are the general laboratory rules that all students are expected to follow (respect):
1) Punctuality. Come to laboratory in time and bring your lab manual, pencil, eraser and
ruler.
2) If you miss a lab for reasons beyond your control, report the case to the lab coordinator in
time. Only reasonable excuses are accepted to arrange a make-up lab; or else you will
receive a zero mark for the lab you missed.
3) Unless you have made an arrangement with your lab instructor you are not allowed to do
labs during lab periods that are not with your groups.
4) You shall not change your group and seat without the permission of your instructor
 5) Drawing should be done with soft pencil and labeled with the same
6) If any accidents happen to you in the laboratory, report it to the lab instructor
immediately.
7) You are responsible for all lab equipment provided for your use.
8) At the end of the laboratory periods place your equipment away in proper order and
places
9) When you have finished your work for the day, clean the working area and leave
everything in proper order.

Introduction to biological laboratory techniques

1. Introduction

This laboratory text is designed to guide the student through introduction to biological lab
techniques, procedures and experiments. Throughout this manual, the student will learn the
scientific method and its application. This will allow the student to apply all knowledge gained
throughout this course which will lead to the correct identification of an unknown sample of
different food tests.
1.1. The scientific method

Scientific experiments are designed around a question that requires an explanation. The scientific
method is a guideline by which the explanation may be found and is a general procedure that is
followed by all sciences. The method is modified slightly to fit each individual discipline.

General procedure for biological research:

Definition of the problemReview of the literature pertinent to the problemFormulation of

hypothesisStatement of objectivesAnalysis of variablesDesign of
experimentExperimental procedureAnalysis of data (results)Interpretation of data and
conclusions from interpretations.

Some explanations and definitions of each step of the procedure are important

PROBLEM - two types:

 1. Normally a fact that is assumed to be caused by some factor or factors the researcher is
looking for cause and effect. e.g. why some peoples become tin/effect/? Because 1, they
do not eat meat 2, by nature 3, due to high intention 4, due to work in a load.

2. Involves the quantification of cause and effect. The association of the magnitude of
cause related to the magnitude of effect. Let take cause 1, they do not eat meat. When we
quantify this it requires further investigation like, how many Kkal of protein found in 1
kg of meat?

HYPOTHESIS – an educated guess, a tentative theory. Anything that needs explanation is an

effect. A hypothesis attempts to explain those effects by putting forth causes. A good hypothesis
should:

1. Explain facts not previously explained.

2. Be consistent with all known facts.

3. Account only for phenomena in question.

4. Aid in producing new facts and relations.

5. Be susceptible to verification or refutation.

OBJECTIVE – This should be a carefully thought out and worded statement that Covers the
cause and effect variables whose relationship is being determined. The objective should be
highly specific. The attainment of the objective will support or refute the hypothesis and
therefore should solve the problem under study.

VARIABLES – A thing that may vary or is able to vary.

1. Primary – variables on which the experiment will focus. E.g. temperature

2. Secondary – variables which may influence or be influenced by the primary. E.g. sweeting

3. Independent – variables that are not dependent upon other variables.

4. Dependent – variables that depend upon other variables for magnitude.

These may be grouped together as (1) primary independent variable (cause); (2) primary
dependent variable (effect); (3) secondary independent variable (extraneous variable of
influence); and (4) secondary dependent variable (side effect).

The research should identify ALL variables that may concern or influence the experiment.

The general procedure for handling variables is:

1. Control the secondary independent variables.

2. Manipulate and evaluate or select and evaluate the primary independent variable(s).

3. Evaluate the resulting variations in the primary dependent variable(s).

4. Ignore the secondary dependent variable(s).

EXPERIMENT – A trial or test. The process of learning through observation. Most scientific
experiments are controlled or “pure” experiments which are defined by (1) a predetermined plan;
(2) objective of confirmation or refutation of a cause and effect relationship or degree of effect
between two variables; (3) elimination, equalization, or evaluation of influencing, extraneous
variables; and (4) assumption that the independent variable (cause) will be actively manipulated.

ANALYSIS – May or may not be statistical in nature. Often involves averages of observations.

INTERPRETATION/CONCLUSIONS – Meaning of facts, underlying causes, their affects and

any other implications should be discussed. Researchers should avoid the most common errors in
logic (illusions, generalizations, prejudices, biases, etc.)

1.2. Laboratory safety rules

To insure safety of those working in the lab, as well as the integrity of each experiment, each of
the following rules must be followed:

1. Clothing should be protected by a lab coat or apron. No shorts are allowed - you will
be asked to return home and change if worn to lab class.

2. Hair that is long should always be tied back to avoid contamination as well as safety
when working near the Bunsen burners.
 3. Lab stations must be wiped down at the beginning of lab to lower contamination rates
of cultures by organisms already on the stations as well as safety for the student. Stations
must also be wiped down at the end of every lab session. Station cleaning is best
accomplished with fresh 10% bleach. If there is visible contamination on the bench, wash
with soap and water before the bleach.

4. Avoid direct contact with any microbes being tested by keeping all cultures well below
mouth, nose, and eye regions. Microbial agents normally travel with gravity, so
downward is the basic direction. Because of this movement, to insure integrity of
cultures, avoid coughing, excessive talking, laughing, etc, while working with cultures
while open. Keep cultures at a minimum of exposure to the air for best results.

5. Bunsen burners should be lit from the beginning of each session to the end as this
decreases the risk of contamination of cultures and helps the safety of the lab worker.

6. Gloves must ALWAYS be worn when handling any microbial agent. Gloves are
provided.

7. Lab stations should be kept clear of any extra materials (non-lab books, book bags,
purses, keys, etc.) to avoid contamination as well as accidents.

8. All lab materials must be stored in the appropriate locations at the end of each session
and gloves and other disposables placed in biohazard bags.

9. Tubes and racks should be placed in the appropriate location for autoclaving.

10. All spills should be reported IMMEDIATELY to the lab instructor for proper
cleanup. Unreported spills can result in biohazardous conditions.

Laboratory work should be fun and rewarding. To keep spills, burns, contamination and other
accidents to a minimum, it is wise to stay alert and pay attention to your surroundings at all
times. By following appropriate procedures, an enjoyable and safe lab can be assured.
1.2.1. Protective equipment

1.2.2. Handling common laboratory equipment, chemicals, reagents, media, cultures and other
materials
1.2.3. Laboratory organization

1.3. Introduction to basic biological laboratory equipment

1.3.1. Dissecting kits

Probably the most often used tools in biology are the dissecting kit and the microscope. During
this laboratory session you will be introduced to these basic tools of the biologist. The basic
components of a dissecting kit include scissors, scalpels, forceps, probes (needles), droppers and
often camel-hair brush. These basic components of a dissecting kit and some other tools
(instruments) often used by biologist are placed on your bench or on the demonstration bench in
the lab with the help of your instructors, identify these tools as well as their use.

Figure 1. Dissecting instrument

1.3.2. Sterilization equipment and other materials

Sterilization is the process by which all living cells, spores and acellular entities (e.g.) viruses are
either destroyed or removed from an object or habitat. It can also be defined as the killing or
removal of all viable organisms within a growth medium. A sterile object is totally free of viable
microorganisms, spores and other infectious agents.

A basic knowledge of disinfection and sterilization is crucial for biosafety in the laboratory.
Since heavily soiled items cannot promptly be disinfected or sterilized, it is equally important to
understand the fundamentals of cleaning prior to disinfection (precleaning). In this regard, the
following general principles apply to all known classes of microbial pathogens.
Specific decontamination requirements will depend on the type of experimental work and the
nature of the infectious agent(s) being handled. The generic information given here can be used
to develop both standardized and more specific procedures to deal with biohazard(s) involved in
a particular laboratory.

Contact times for disinfectants are specific for each material and manufacturer. Therefore, all
recommendations for use of disinfectants should follow manufacturers’ specifications.

Various terms such as sterilization, disinfection, germicides, sepsis, and aseptic techniques will
be used here. To be sure that you understand exactly what they mean, the following definitions
are provided.

Cleaning is general removal of debris (dirt, food, feces, blood, saliva and other body secretions).
it reduces amount of organic matter that contributes to proliferation of bacteria and viruses

Sterilization is a process in which all living microorganisms, including viruses, are destroyed.
The organisms may be killed with steam, dry heat, or incineration. If we say an article is sterile,
we understand that it is completely free of all living microorganisms.
Generally speaking, when we refer to sterilization as it pertains here to laboratory safety, we
think, primarily, in terms of steam sterilization with the autoclave. The ultimate method of
sterilization is to burn up the infectious agents or incinerate them. All biological wastes must
ultimately be incinerated for disposal.
Disinfection is a process in which vegetative, non sporing microorganisms are destroyed. Agents
that cause disinfection are called disinfectants or germicides. Such agents are used only on
inanimate objects because they are toxic to human and animal tissues. Removes most organisms
present on surfaces that can cause infection or disease. A physical or chemical means of killing
microorganisms, but not necessarily spores.

Sepsis is defined as the growth (multiplication) of microorganisms in tissues of the body. The
term asepsis refers to any procedure that prevents the entrance of infectious agents into sterile
tissues, thus preventing infection.
Aseptic techniques refer to those practices that are used by microbiologists to exclude all
organisms from contaminating media or contacting living tissues.
Antiseptics are chemical agents (often dilute disinfectants) that can be safely applied externally
to human tissues to destroy or inhibit vegetative bacteria. A substance that inhibits the growth
and development of microorganisms without necessarily killing them. Antiseptics are usually
applied to body surfaces.
Antimicrobial – An agent that kills microorganisms or suppresses their growth and
multiplication.

Biocide – A general term for any agent that kills organisms.

Chemical germicide – A chemical or a mixture of chemicals used to kill microorganisms.

Decontamination – Any process for removing and/or killing microorganisms. The same term is
also used for removing or neutralizing hazardous chemicals and radioactive materials.

Disinfectant – A chemical or mixture of chemicals used to kill microorganisms, but not

necessarily spores. Disinfectants are usually applied to inanimate surfaces or objects.

Microbicide – A chemical or mixture of chemicals that kills microorganisms. The term is often
used in place of “biocide”, “chemical germicide” or “antimicrobial”.

Sporocide – A chemical or mixture of chemicals used to kill microorganisms and spores.

What are the equipment’s used for sterilization?

1.4. Laboratory report writing methods

The main purpose of a lab report is to describe an experiment you have carried out in the lab and
to communicate the results. Writing lab reports is part of learning to be a scientist, and provides
you with experience in writing in a scientific style similar to that used in articles published in
scientific journals.

Because different departments might have different requirements, always check with your
lecturer or demonstrator regarding any particular style and structure requirements they might
have. However, reports that communicate the results of an experiment generally follow the
format known as IMRAD: Introduction, Method, Results, (And) Discussion. Each section has a
specific purpose and contains different information.
Introduction

The Introduction provides an overview of the experiment and informs the reader about what to
expect in the report. It should include the following information:

Define the topic. The first part of a lab report should set up the topic being investigated
by providing background information on it and clearly explaining exactly what you are
testing.
Explain the significance of the topic. How does this topic relate to the world of
biology? Why is this experiment topic important? Why is this experiment important?
Essentially, you should describe why your audience should pay attention to this
experiment.
Present the hypothesis and research question. Your hypothesis when performing the
experiment should be identified in this section along with the research question(s) you are
addressing. If you have more than one hypothesis, they should all be identified.
Define the overall goal. Whether you have one hypothesis or many hypotheses, they
should all be pointing to one overall goal. This goal should be clearly defined and
explained in conjunction with your hypothesis.
Give an overview of the lab methods. Explain what experimental system you will be
using to test your hypothesis and why you chose that particular system. What advantages
does it provide? If the system you used was provided for you in another text, explain why
they chose that particular system. Then you should identify what you are measuring in
the lab.
It should be 1 page in length. The introduction should only be about one page total. If it
exceeds this by much, you can probably eliminate some of the length by making your
language more concise.

Method

In this section you explain to the reader how you carried out the experiment. It explains what you
did and how. It should be written in the past tense and include enough detail to enable a reader to
replicate the experiment.
This means that you need to be specific (e.g. detail the concentration of solution used, the time
that the experiment sat in the water bath, the temperature of the oven). If necessary the Method
section can be divided into subheadings, such as subjects, apparatus (or materials), and
procedure.

It should include the following information:

The materials, subjects, and equipment (e.g. chemicals, experimental animals, and
apparatus) you used.
The steps you took (in chronological order) in carrying out the experiment.
Explain what steps were taken in performing the experiment. This is the section where you
describe the procedures you used in conducting the experiment.
Use the narrative format. This section should not be in a list format or read like a recipe. The
information should be relayed in a story type of writing.
Only include the important details. Not every detail needs to be included. Only the relevant
elements should be mentioned. If a scientist would need to know a certain aspect to repeat the
experiment, then it should be included. Otherwise, leave it out.

Results

This is a very important section of your lab report because it is where you present your findings.
You need to present all relevant findings in a clear, concise, and accurate way that is easy for the
reader to follow and understand. Never falsify your findings. Even if your findings do not
support the hypothesis, they are your findings and should be reported.

You might find it useful to present some of your findings using tables or figures. A figure is
anything other than a table (e.g. graph, diagram, drawing, photo, map).

Sometimes your lecturer will advise you which to use, but if you have to make this decision
yourself, think about the most effective way to present your findings in a clear visual form. For
example:

• Tables are useful for presenting summarised data in rows and columns

• Pie charts are useful for communicating findings expressed as different percentages of a whole
• Graphs are useful for demonstrating numerical difference or trends

It is important that the tables and figures you use summaries or enhance the information you
present in the text of the report rather than merely repeating it.

Tables and figures should be labeled and numbered separately as they appear in the report, so it
is possible to have both Table 1 and Figure 1 in the same report.

The number and label should be provided at the top of a table, and at the bottom of a figure. You
must always refer to a table or figure in the text of your report, and when doing so, try to point
out to the reader what it is you want them to notice. For example,

‘As shown in Figure 1, almost all of the energy consumed (95%) was produced using
non-renewable sources’.

There are some things you should not include in the Results section. You do not discuss your
results here; this occurs in the Discussion section of the report. Raw data (i.e. data that hasn’t
been analyzed or evaluated) should also not be included here but should appear in the
Appendices section.

Distinguish between multiple experiments. If this report includes multiple experiments working
towards the same overall goal, then each experiment should receive its own paragraph in this
section. In each paragraph, explain what you did, why you did it, and what the results were.

 Use visuals where appropriate. A well-executed graph or table representing your results can be
very effective in communicating data from your experiment. However, a figure cannot stand
alone in communicating the results of an experiment. Any figures should be explained in the text
of the results section. For more detailed help on constructing a figure, see
http://faculty.uca.edu/skarafit/bio1/Lab 1/figures.htm.

Discussion

This section of the report requires the most thought. It is where you analyze your findings within
the context of the information you have presented in your Introduction.

Your discussion should be clearly linked to the introduction and might include:
 Whether or not your findings support the hypothesis. If not, can you suggest any reasons
why?
The extent to which your findings agree with previous studies in the area. If not, can you
suggest any reasons why?
Whether there was anything you could have done differently in terms of methodology
and procedures.
This section carries the most weight. Most professors pay the closest attention to this
section because this is where students interpret what they have discovered.
Reiterate the results. Remind the reader of what you found in the results section of the lab
report. If more than one experiment was conducted, separate the individual experiments
into individual paragraphs just as they were in the results portion. Rather than saying the
same information over again though, discuss the results in the context of the overall goal
of the experiment, which was identified in the introduction of the report.
Interpret the results. The reader needs to know what the results of the experiments mean.
Why is the data like it is? Why is this data important to know? Answering these questions
will take raw data and make it meaningful for the reader.
Discuss potential modifications for the experiment. If a person wanted to replicate this
experiment, what might they change about it to create further progress in this topic area?
This is not about correcting mistakes made in conducting the experiment. What should be
discussed here is how to modify the methods of the experiment to push scientific
knowledge in this topic forward

The Discussion section should end with a concluding paragraph that states the significance of
your findings. You should indicate the extent to which your findings are useful or conclusive,
indicate the possible direction of further research, and discuss what improvements could be made
to the methodology for future studies.

Other sections of lab reports

Reference

You will need to include a Reference List at the end of your report: a list of all the references you
have cited in the report (e.g. textbooks, journals, lab manuals, web sites). If you have read a
reference but haven’t cited it in the report, you do not include it here. However, if you are asked
to provide a Bibliography, you include everything you read, whether you have cited it or not.

You need to check with your lecturer or demonstrator regarding the referencing style
requirements in your department.

Appendices

You won’t always have to have an Appendix in your report, but if you have some detailed
information or raw data that you want to include, you can include this at the very end of the
report (following the references). Appendices should be numbered (e.g., Appendix 1, Appendix
2) and have a clear heading.

When you want to refer to information in an Appendix you need to indicate where this can be
found. For example, ‘Detailed figures can be found in Appendix 1’.

Abstract

Some departments may require you to write an Abstract as part of your lab report. The Abstract
is positioned at the very beginning of the report (before the Introduction) and summarizes the
whole report. It is usually brief (around 5% of the total word count) and written in a single
paragraph.

The information in the Abstract should be presented in the same order as it appears in the report,
and should include: the aims of the experiment; an overview of the method; a summary of the
main findings; and your conclusions. The abstract can be difficult to write and is best left until
last when you know exactly what your findings and conclusions are.

Writing style in lab reports

When writing a lab report it is important you use a scientific writing style. This means you
should aim for writing that is clear, objective, accurate and brief.
Format of a laboratory report

As we look at it above there are several sections to a lab report.

Title o Participants (optional)

Introduction o Design
o Theory o Procedures
o Significance Results
o Objective of the experiment Discussion
Method Reffernce
o Equipment (materials) Appendixes (optional)
 2. The Microscope
2.1. Introduction
Microorganisms are much too small to be seen with the unaided eye; they must be observed with
a microscope. The word microscope is derived from the Latin word micro, which means small,
and the Greek word skopos, to look at. It is an instrument used to see objects that are too small
for the naked eye. The science of investigating small objects using such an instrument is called
microscopy. Microscopic means invisible to the eye unless aided by a microscope.
LEARNING OBJECTIVES
 Diagram the path of light through a compound microscope.
 Define total magnification and resolution
 Identify a use for darkfield, phase-contrast, and fluorescence and scanning
acoustic microscopy, and compare each with brightfield illumination.
 Explain how electron microscopy differs from light microscopy.
 Identify one use for the TEM and SEM.
The simple microscope used by van Leeuwenhoek in the seventeenth century had only one lens
and was similar to a magnifying glass. His lenses were ground with such precision that a single
lens could magnify a microbe 300X. His simple microscopes enabled him to be the first person
to see bacteria
Contemporaries of van Leeuwenhoek, such as Robert Hooke, built compound microscopes,
which have multiple lenses. In fact, a Dutch spectacle maker, Zaccharias Janssen, is credited
with making the first compound microscope around 1600. However, these early compound
microscopes were of poor quality and could not be used to see bacteria. It was not until about
1830 that a significantly better microscope was developed by Joseph Jackson Lister. Various
improvements to Lister's microscope resulted in the development of the modern compound
microscope, the kind used in biological laboratories today. Microscopic studies of live specimens
have revealed dramatic interactions between microbes.
 2.2. Types of microscopes

Activity1. Dear students what are the types of microscope?

Microscopes are the important scientific instruments used mainly by the biologists and
researchers. A microscope is a high precision optical instrument that uses a lens or a combination
of lenses to produce highly magnified images of small specimens or objects especially when they
are too small to be seen by the naked (unaided) eye. Depending on the specifications and
methods of use, the microscopes are classified into different categories. These different types of
microscopes are:
1. Light Microscopy
Light microscopy refers to the use of any kind of microscope that uses visible light to observe
specimens. Here we examine several types of light microscopy.
Compound light Microscopes: These microscopes are mainly light illuminated and the image
produced by them is two dimensional. The compound microscope is the most commonly used
microscope and it is powerful enough so that the user can view cells, even the living ones. The
magnification of this microscope is high but the resolution is quite low. The term light refers to
the method by which light transmits the image to your eye. Compound deals with the microscope
having more than one lens. A light microscope uses a beam of visible light to illuminate and
contrast the object being viewed through the scope. The light source is typically a light bulb
which is turned on by a toggle switch at the side of the base. The beam of light shines up from a
lamp in the microscope's base, through a hole or ‘window’ in the stage (area of the scope where
the specimen sits), ultimately up through the specimen.
A modern compound light microscope has a series of lenses and uses visible light as its source of
illumination. With a compound light microscope, we can examine very small specimens as well
as some of their fine detail. A series of finely ground lenses forms a clearly focused image that is
many times larger than the specimen itself. This magnification is achieved when light rays from
an illuminator, the light source, pass through a condenser, which has lenses that direct the light
rays through the specimen. From here, light rays pass into the objective lenses, the lenses closest
to the specimen. The image of the specimen is magnified again by the ocular lens, or eyepiece.
We can calculate the total magnification of a specimen by multiplying the objective lens
magnification (power) by the ocular lens magnification (power). Most microscopes used in
biology laboratories have several objective lenses, including 4x (low power), lOX (middle
power), 40X (high power), and l00X (oil immersion,). Most ocular lenses magnify specimens by
a factor of 10. Multiplying the magnification of a specific objective lens with that of the ocular,
we see that the total magnifications would be l00X for middle power, 400X for high power, and
l000X for oil immersion. Some compound light microscopes can achieve a magnification of
2000X with the oil immersion lens.
Resolution (also called resolving power) is the ability of the lenses to distinguish fine detail and
structure. Specifically, it refers to the ability of the lenses to distinguish two points a specified
distance apart. For example, if a microscope has a resolving power of 0.4 nm, it can distinguish
two points if they are at least 0.4 nm apart. A general principle of microscopy is that the shorter
the wavelength of light used in the instrument, the greater the resolution. The white light used in
a compound light microscope has a relatively long wavelength and cannot resolve structures
smaller than about 0.2 nm. This fact and other practical considerations limit the magnification
achieved by even the best compound light microscopes to about 2000X.
To obtain a clear, finely detailed image under a compound light microscope, specimens must be
made to contrast sharply with their medium (substance in which they are suspended). To attain
such contrast, we must change the refractive index of specimens from that of their medium. The
refractive index is a measure of the light-bending ability of a medium. We change the refractive
index of specimens by staining them. Light rays move in a straight line through a single medium.
After the specimen is stained, when light rays pass through the two materials (the specimen and
its medium) with different refractive indexes, the rays change direction (refract) from a straight
path by bending or changing angle at the boundary between the materials and increase the
image's contrast between the specimen and the medium. As the light rays travel away from the
specimen, they spread out and enter the objective lens, and the image is thereby magnified.
To achieve high magnification (1000X) with good resolution, the objective lens must be small.
Although we want light traveling through the specimen and medium refract differently, we do
not want to lose light rays after they have passed through the stained specimen. To preserve the
direction of light rays at the highest magnification, immersion oil is placed between the glass
slide and the oil immersion objective lens. The immersion oil has the same refractive index as
glass, so the oil becomes part of the optics of the glass of the microscope. Unless immersion oil
is used, light rays are refracted as they enter the air from the slide, and the objective lens would
have to be increased in diameter to capture most of them. The oil has the same effect as
increasing the objective lens diameter; therefore, it improves the resolving power of the lenses. If
oil is not used with an oil immersion objective lens, the image becomes fuzzy, with poor
resolution.
Under usual operating conditions, the field of vision in a compound light microscope is brightly
illuminated. By focusing the light, the condenser produces a bright field illumination. It is not
always desirable to stain a specimen. However, an unstained cell has little contrast with its
surroundings and is therefore difficult to see. Unstained cells are more easily observed with the
modified compound microscopes described in the next section.

Dissection Microscopes: like the compound microscope is also light illuminated. However, it
produces a three dimensional image and is commonly used for dissection of large specimen.
Compared to the compound microscope, it has low magnification.
Darkfield Microscopy
It is used to examine live microorganisms that either invisible in the ordinary light microscope,
cannot be stained by standard methods, or are so distorted by staining that their characteristics
then cannot be identified. Instead of the normal condenser, a dark field microscope uses a dark
field condenser that contains an opaque disk. The disk blocks light that would enter the objective
lens directly. Only light that is reflected off (turned away from) the specimen enters the objective
lens. Because there is no direct background light, the specimen appears light against a black
background-the dark field. This technique is frequently used to examine unstained
microorganisms suspended in liquid.
Phase-Contrast Microscopy
Another way to observe microorganisms is with a phase-contrast microscope. It is especially
useful because it permits detailed examination of internal structures in living microorganisms. In
addition, it is not necessary to fix (attach the microbes to the microscope slide) or stain the
specimen procedures that could distort or kill the microorganisms.
The principle of phase-contrast microscopy is based on the wave nature of light rays and the fact
that light rays can be in phase (their peaks and valleys match) or out of phase. If the wave peak
of light rays from one source coincides with the wave peak of light rays from another source, the
rays interact to produce reinforcement (relative brightness). However, if the wave peak from one
light source coincides with the wave trough from another light source, the rays interact to
produce interference (relative darkness). In a phase-contrast microscope, one set of light rays
comes directly from the light source. The other set comes from light that is reflected or diffracted
from a particular structure in the specimen. (Diffraction is the scattering of light rays as they "
touch" a specimen's edge. The diffracted rays are bent away from the parallel light rays that pass
farther from the specimen.) When the two sets of light rays- direct rays and reflected or
diffracted rays- are brought together, they form an image of the specimen on the ocular lens,
containing areas that are relatively light ( in phase), through shades of gray, to black (out of
phase), in phase-contrast microscopy, the internal structures of a cell become more sharply
defined.
Fluorescence Microscopy
Fluorescence microscopy takes advantage of fluorescence, the ability of substances to absorb
short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible). Some
organisms fluoresce naturally under ultraviolet light; if the specimen to be viewed does not
naturally fluoresce, it is stained with one of a group of fluorescent dyes called fluorochrome.
When microorganisms stained with a fluorochrome are examined under a fluorescence
microscope with an ultraviolet or near ultraviolet light source, they appear as luminescent, bright
objects against a dark background.
The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent -
antibody (FA) technique, or immunofluorescence. Antibodies are natural defense molecules that
are produced by humans and many animals in reaction to a foreign substance, or antigen.
Fluorescent antibodies for a particular antigen are obtained as follows: an animal is injected with
a specific antigen, such as a bacterium, and the animal then begins to produce antibodies against
that antigen. After a sufficient time, the antibodies are removed from the serum of the animal.
Next, a fluorochrome is chemically combined with the antibodies. These fluorescent antibodies
are then added to a microscope slide containing an unknown bacterium. If this unknown
bacterium is the same bacterium that was injected into the animal, the fluorescent antibodies bind
to antigens on the surface of the bacterium, causing it to fluoresce.
This technique can detect bacteria or other pathogenic microorganisms, even within cells,
tissues, or other clinical specimens. Of paramount importance, it can be used to identify a
microbe in minutes. Immunofluorescence is especially useful in diagnosing syphilis and rabies.
2. Electron Microscopy
Objects smaller than about 0.2 micro meters, such as viruses or the internal structures of cells
must be examined with an electron microscope. In electron microscopy, a beam of electrons is
used instead of light. Like light, free electrons travel in waves. The resolving power of the
electron microscope is far greater than that of the other microscopes described here so far. The
better resolution of electron microscopes is due to the shorter wavelengths of electrons; the
wavelengths of electrons are about 100,000 times smaller than the wavelengths of visible light.
Thus, electron microscopes are used to examine structures too small to be resolved with light
microscopes. Images produced by electron microscopes are always black and white, but they
may be colored artificially to highlight certain details.
Instead of using glass lenses, an electron microscope uses electromagnetic lenses to focus a beam
of electrons onto a specimen. There are two types of electron microscopes: the transmission
electron microscope and the scanning electron microscope.
Transmission Electron Microscopy (TEM)
It is also electron illuminated but provides the user with a two dimensional view. This
microscope comes with high magnification and resolution. In the transmission electron
microscope (TEM), a finely focused beam of electrons from an electron gun passes through a
specially prepared, ultrathin section of the specimen. The beam is focused on a small area of the
specimen by an electromagnetic condenser lens that performs roughly the same function as the
condenser of a light microscope directing the beam of electrons in a straight line to illuminate the
specimen.
Electron microscopes use electromagnetic lenses to control illumination, focus, and
magnification. Instead of being placed on a glass slide, as in light microscopes, the specimen is
usually placed on a copper mesh grid. The beam of electrons passes through the specimen and
then through an electromagnetic objective lens, which magnifies the image. Finally, the electrons
are focused by an electromagnetic projector lens (rather than by an ocular lens as in a light
microscope) onto a fluorescent screen or photographic plate. The final image, called a
transmission electron micrograph, appears as many light and dark areas, depending on the
number of electrons absorbed by different areas of the specimen.
In practice, the transmission electron microscope can resolve objects as dose together as 2.5 nm,
and objects are generally magnified 10,000 to I00,000X. Because most microscopicmens are so
thin, the contrast between their ultrastructure and the background is weak. Contrast can be
greatly enhanced by using a "stain" that absorbs electrons and produces a darker image in the
stained region. Salts of various heavy metals, such as lead, osmium, tungsten, and uranium, are
commonly used as stains. These metals can be fixed onto the specimen (positive staining) or
used to increase the electron opacity of the surrounding field (negative staining). Negative
staining is useful for the study of the very smallest specimens, such as virus particles, bacterial
flagella, and protein molecules.
Transmission electron microscopy has high resolution and is extremely valuable for examining
different layers of specimens. However, it does have certain disadvantages. Because electrons
have limited penetrating power, only a very thin section of a specimen (about 100 nm) can be
studied effectively. Thus, the specimen has no three-dimensional aspect. In addition, specimens
must be fixed, dehydrated, and viewed under a high vacuum to prevent electron scattering. These
treatments not only kill the specimen, but also cause some shrinkage and distortion, sometimes to
the extent that there may appear to be additional structures in a prepared cell. Structures that
appear as a result of the method of preparation are called artifacts.

Scanning Electron Microscopy

The scanning electron microscope (SEM) overcomes the problem of sectioning associated with a
transmission electron microscope. It provides striking three -dimensional views of specimens and
an electron gun produces a finely focused beam of electrons called the primary electron beam.
These electrons pass through electromagnetic lenses and are directed over the surface of the
specimen. The primary electron beam knocks electrons out of the surface of the specimen, and
the secondary electrons thus produced are transmitted to an electron collector, amplified, and
used to produce an image on a viewing screen or photographic plate. The image is called a
scanning electron micrograph. This microscope is especially useful in studying the surface
structures of intact cells and viruses.
In practice, it can resolve objects as dose together as 10 nm, and objects are generally magnified
1000 to 10,000X. It uses an electron illumination and produces a three dimensional image. This
is an expensive microscope which has high magnification and resolution. It can take black and
white pictures of the specimen.

a) Transmission electron microscope B) scanning electron microscope

Activity 2.1 Distinguishing the Different Types of Microscope· Identify the main differences
between the different types microscope?

2.3. Parts and functions of the compound light microscope

Before you buy or use a microscope, it is important to know the parts and the functions of each
one. The parts of a typical modern compound light microscope are described below

EYEPIECE or OCULAR LENS- the lens at the top that you look through. The
eyepieces standard lens powers are 5X or 10X of 15X or 16X power.
Revolving nosepiece (RN): This moveable part house two or more objectives lenses.
Nosepiece (NP): You often find three or four objective lenses on a nosepiece with 4X,
5X, 10X, 40X, 100X powers. The low powers (4X, 5X, 10X, 40X) are called “dry” scan
objectives. Power 100X forms the oil immersion. You should note that the shortest lens
has the lowest power; the longest one has the highest power.
Condenser Lens (CL): You may use CL to focus light onto the specimen on your
microscope stage. You need to use it most at a higher powers (40X and 100X) to give
you a sharper image than those microscopes without CL. Your CL adjusting knob is
located on the other side of your microscope to move the CL up and down to focus a
good quality images. The CL rated at 0.65 – 1.25 will give you maximum benefit if you
operate your microscope at 40X and 100X objective powers. You should set very close to
the slide at 100X and further down away at the lower powers.
Stage (ST): The flat plate where you place your slide for viewing.
Diaphragm Knob (DN): This knob controls the disc directly above the CL. You may
use the DN to vary the amount and size of cone light reaching the slide from below. No
rule of thumb as to setting you should use for a particular power. The setting is a function
of transparency of specimen, contrast, and objective lens in use.
Illuminator (IL): A lighting source used in place of a mirror
Base (BA): The bottom of the microscope use for support and house the power source,
fuse and illuminator.
Power Switch (PS): This switch controls light supply to the microscope
Illumination Control: This bar control the amount of light passing through the
illuminator condenser.
Power Cable (PC): This wire connects to the power source to provide light to the
microscope through the illuminator (IL)
Condenser Mover (CM): A knob you use to move the condenser up or down, to attain a
good quality of light.
Focus Knobs (FN): Knobs that make rough and fine adjustments to be focused. You
have two knobs on a m microscope, large knob is for coarse focus, and the small one is
for fine focus. The latter is used at a higher power objective. FN moves the stage up and
down when you turn the knobs.
Rack Stop (RS): You may use this knob to lock the stage level after adjusting how close
the objective lens can get to the slide. You do this to avoid making the high power
objective lens down into the slide and break it! It is often set at the factory.
Stage Holder (SH): Seat on which the stage is resting, connect it with me FK knob,
which in turn move the stage up or down when you turn the FK knob.
 Stage Clip (SC): The stage clip holds the slide in place.
Arm (AR): It attaches the eyepieces and the objectives to the base.
Stage and Slide Mover: Stage mover knob (big upper knob) is used to move the stage
forward or backward to position the best quality specimen. While, the small lower knob
moves the slide to either left or right to position the best specimen for view (Look at the
other side of your microscope for the combined knobs).
2.3. Handling of the microscope

Care and Handling

1. Transporting- When you pick up the microscope and walk with it, grab the arm with one
hand and place your other hand on the bottom of the base. DON'T SWING THE
MICROSCOPE!
2. Handling & Cleaning- Never touch the lenses with your fingers. Your body produces oil
that smudges the glass. This oil can even etch the glass if left on too long. Use only
Lenses Paper to clean the glass. TOILET PAPER, KLEENEX, AND PAPER TOWELS
HAVE FIBERS THAT CAN SCRATCH THE LENSES.
3. Storage- When you are finished with your "scope" assignment, rotate the nosepiece so
that it's on the low power objective, roll the nosepiece so that it's all the way down to the
stage, then replace the dust cover. DON'T FORGET TO USE PROPER
TRANSPORTING TECHNIQUES!
4. Clean up- Clean all slides, materials, and work area when you're done. Please, be careful
with the slides and cover slips. They are made of glass and if broken, you will get cut and
you will bleed. DON'T CUT YOURSELF, THERE ARE NO BAND AIDS IN THIS
ROOM.
5. When carry the microscope in an upright position. If tilted, the ocular lens may fall and
be damaged.
6. Place the microscope near, but not on the edge of your lab bench with its arm facing you
7. Place the microscope in its box with its arm facing you

2.4. Setting the microscope for use

Directions When Using the Microscope

 1. To carry the microscope grasp the microscopes arm with one hand. Place your other hand
under the base.
2. Place the microscope on a table with the arm toward you.
3. Turn the coarse adjustment knob to raise the body tube.
4. Revolve the nosepiece until the low-power objective clicks into place.
5. Adjust the diaphragm. While looking through the eyepiece, also adjust the mirror until
you see a bright white circle of light.
6. Place a slide on the stage. Center the specimen over the opening on the stage. Use the
stage clips to hold the slide in place.
7. Look at the stage from the side. Carefully turn the coarse adjustment to lower the body
tube until the low-power objective almost touches the slide.
8. Looking through the eyepiece, VERY slowly the coarse adjustment knob until the
specimen comes into focus.
9. To switch to the high-power objective lens, look at the microscope from the side.
CAREFULLY revolve the nosepiece until the high-power objective lens clicks into place.
Make sure the lens does not hit the slide.
10. Looking through the eyepiece, turn the fine a adjustment knob until the specimen comes
into focus.
3. Practice in Mounting and focusing.
An object that is to be studied under microscope is placed on a glass slide and is covered
with a coverslip. You will practice this using a dot provided.
1) Put a dot on a piece of paper and lay it on a microscope slide. Using a dropper put a
small drop of water on the dot (wet mounting).
2) Cover the dot with a clean coverslip. To do this, first make the coverslip stand at right
angle to the glass slide and slowly lower it until it covers the object. If this done
carefully, there should be no air bubbles imprisoned in the water between the slide
and the coverslip. You may practice this procedure of mounting until you have
perfected it.
3) Put the slide on the stage of microscope, and move the slide until the object (dot)
comes in the center of the stage opening.

Focusing with lower power

 a) If the low power objective is not in position hold any of the two objectives between your
thumb and the forefinger and turn the nosepiece until the objective clicks into position.
b) Looking from the side, lower the body tube with the coarse adjustment to within 5mm of
the slide. If the body tube is not lowered, it is the stage that is raised up towards the
objective instead.
c) Looking into the microscope about 2 cm away from the eyepiece, slowly raise the eye
body tube with the coarse adjustment until the image comes into view. Keep both eyes
open while looking into the microscope.
4) If the image is blurred, focus it with the fine adjustment until it becomes clear.
5) If the image is off the center of the field of vision, move the slides very gently, while
looking into microscope, until the image is in the center of the field.
6) Adjust (increase or decrease) the diaphragm opening as necessary

Focusing with the high-power

Now you can directly go to step 7 in order to focus the object under high power objective

7) Your objective is already in focus under lower power. Taking hold of any two
objectives rotate the nosepiece until the high power objectives clicks in position. Care
should be taken so that the objectives do not hit against the clips
8) Focus with the fine adjustment until you get clear image. Never use the coarse
adjustment when focusing with the high power objectives.
4. Magnification and resolution

A compound microscope is composed of two elements; a primary magnifying lens and a

secondary lens system, similar to a telescope. Light is caused to pass through an object and is
then focused by the primary and secondary lens. If the beam of light is replaced by an electron
beam, the microscope becomes a transmission electron microscope. If light is bounced off of the
object instead of passing through, the light microscope becomes a dissecting scope. If electrons
are bounced off of the object in a scanned pattern, the instrument becomes a scanning electron
microscope.

The function of any microscope is to enhance resolution. The microscope is used to create an
enlarged view of an object such that we can observe details not otherwise possible with the
human eye. Because of the enlargement, resolution is often confused with magnification, which
refers to the size of an image.

In general, the greater the magnification, the greater the resolution, but this is not always true.
There are several practical limitations of lens design which can result in increased magnification
without increased resolution.

The eyepiece the Microscope magnifies objects 10 times. Each lens then further magnifies the
objects: 4X (or 4 times magnification), 10X, and 40X, as marked on the lens used. These
magnification numbers combine to give the actual magnification of the object. For example,
using the eyepiece plus the 4X lens, the magnification is 10 x 4 or 40 times or 40X. Any object
viewed this way will appear 40 times its actual size. Can you see the relationship between
magnification and resolution? How it is calculated?

Also, as the magnification increases, the field of view, or total area that can be seen, decreases.
At 40X, the field of view is a circle about 4.5 mm in diameter. The change in the field of view is
inversely proportional to the magnification increase. It’s like zooming in: the closer you go, the
less area you see.
Activity 2.3: Learning to use a Microscope
You will need
· a microscope, a lamp, a piece of graph paper and a prepared slide of stained human cheek cells
Method
Remember, microscopes are delicate pieces of equipment so always take care of them and handle
them safely.
1. Set up your microscope with the lowest power lens (the smallest lens) in place.
2. Clip the prepared slide clips. Position on the stage using the stage clips. Position the piece of
graph paper over the hole in the stage.
3. If your microscope has built-in lamps switch it on. If it has a mirror, adjust the angle of the
mirror until the specimen is illuminated.
4. Now look through the eyepiece lens and adjust the iris diaphragm until the light is bright but
doesn’t dazzle you. The illuminated area you can see is known as the field of view.
5. Looking at your microscope from the side (not through the eyepiece lens) and using the coarse
focusing knob, move the objective lens down slowly so it is as close as possible to the paper
without touching it.
6. Now look through the eyepiece lens again. Turn the coarse focusing knob very gently in the
opposite direction to move the objective lens away from the slide. Do this while you are looking
through the eyepiece lens and the lines on the graph paper will gradually appear in focus. Once
you can see the specimen clearly, use the fine focusing knob to get the focus as sharp as you can.
7. You may find that if you now shut the iris diaphragm down future, so that the hole for the light
to pass through gets smaller you will see the specimen better (the contrast is grater).
8. To use the higher magnification, rotate the nosepiece so that next lens clicks into place. Do not
adjust the focusing knobs at this point as the specimen should still be in focus and, with the
coarse focusing knob in particular, it is very easy to break a slide. It is good to practice this using
graph paper, which will not break!
If you do need to adjust the focus, use the fine focusing knob only with higher magnifications.
Take great care to avoid letting the lens touch the slid/paper. You may want to adjust the iris
diaphragm as well.
9. Make simple drawing to show how much of the graph paper you can see at each
magnification. This will help you to get an idea of how much microscope is magnifying what
you are seeing. Notice how the appearance of the smooth lines changes as you see them at grater
magnification.
10. Return the microscope lenses to their original positions.
5. Observing cellular structures

Dear students, our aim is to prepare stained temporary mounts of onion peel and human cheek
cells and to record the observations. Before exploring the details of cell structure, let's understand
the differences in the structure of an onion cell and a human cheek cell.

5.1 Plant cell (onion epidermal cell)

An onion is a multicellular (consisting of many cells) plant organism. As in all plant cells, the
cell of an onion peel consists of a cell wall, cell membrane, cytoplasm, nucleus and a large
vacuole. The nucleus is present at the periphery of the cytoplasm. The vacuole is prominent and
present at the center of the cell. It is surrounded by cytoplasm. The presence of a cell wall and a
large vacuole are indicators that help identify plant cells, such as seen in the onion peel.
5.2 Animal cell (human cheek cells)

As in all animal cells, the cells of the human cheek do not possess a cell wall.   A cell
membrane that is semi-permeable surrounds the cytoplasm. Unlike plant cells, the cytoplasm in
an animal cell is denser, granular and occupies a larger space. The vacuole in an animal cell is
smaller in size, or absent. The nucleus is present at the center of the cytoplasm. The absence of a
cell wall and a prominent vacuole are indicators that help identify animal cells, such as cells
seen in the human cheek.

 
Cell Organelles
Have you ever wondered what the inside of a cell looks like? If you think about the rooms in our
homes, the inside of any animal or plant cell has many similar room-like structures called
organelles. Here are some names and descriptions of organelles and other parts commonly found
in cells.

Cell Wall: Protective Coat in Plant Cells

The presence of a cell wall is what provides the most significant difference between plant
and animal cells, as it is present only in plant cells and covers the cell membrane. The cell
wall is rigid and is composed of cellulose fibre, polysaccharides, and proteins. Despite the
rigidity of the cell wall, chemical signals and cellular excretions are allowed to pass between
cells.
Cell Membrane: Protective Coat in Animal Cells
The cell membrane is found in both plants and animals, and it is the outer most layer in the
animal cell, that separates the contents of the cell from the outside world. It consists of both
lipids and proteins and is selectively permeable, which means it permits only some molecules
to pass through it.
Cytoplasm: Cell’s Inner Space
Cytoplasm is a jelly-like material that is eighty percent water and is usually clear in colour. It is
also called cytosol. Cytoplasm contains all the organelles inside the cell membrane. The cytosol
contains dissolved nutrients, helps break down waste products, and moves material around the
cell through a process called cytoplasmic streaming.
Nucleus: The Control Centre
The nucleus is known as the control center of the cell. It contains the regulatory machinery
responsible for all the activities inside the cell. It is the largest organelle in the cell and it
contains the DNA of the cell. DNA contains all the information that helps cells live, perform
their functions and reproduce. The nucleus has a double layered covering called nuclear
membrane.
Vacuoles: Cell’s Storage Space
A vacuole is a membrane-bound organelle that stores solid and liquid contents. Vacuoles
are found in both animal and plant cells, but are much larger in plant cells. Vacuoles are formed
by the fusion of multiple membrane vesicles and are effectively just larger forms of these. The
organelle has no basic shape or size; its structure varies according to the needs of the cell.
Similarities and Differences between Plant and Animal Cells
Animal and plant cells have a number of key similarities, but also some noted differences. Here
are some of the common similarities and differences between plant and animal cells.

Features Animal Cell Plant Cell

Cell Shape Round (irregular shape) Rectangular (fixed shape)
Present and is formed of
Cell Wall Absent
cellulose
Present and is covered by the
Cell Membrane Present
cell wall
Nucleus Present Present
A large central vacuole taking
Vacuole One or more small vacuoles
up 90% of the cell volume
Plastids Present Present
Present and make their own
Chloroplast Absent
food
Endoplasmic Reticulum Present Present
Ribosomes Present Present
Mitocondria Present Present

To Prepare Stained Temporary Mount of Onion Peel

Materials Required
Real Lab Procedure
 Pour some distilled water into a watch glass.
 Peel off a leaf from half a piece of onion and using the forceps, pull out a piece of
transparent onion peel (epidermis) from the leaf.
 Put the epidermis in the watch glass containing distilled water.
 Take a few drops of safranin solution in a dropper and transfer this into another watch
glass.
 Using a brush, transfer the peel into the watch glass containing the safranin solution.
 Let this remain in the Safranin solution for 30 seconds, so that the peel is stained.
 Take the peel from the Safranin solution using the brush and place it in the watch glass
containing the distilled water.
 Take a few drops of glycerine in a dropper and pour 2-3 drops at the center of a dry glass
slide.
 Using the brush, place the peel onto the slide containing glycerine.
 Take a cover slip and place it gently on the peel with the aid of a needle.
 Remove the extra glycerine using a piece of blotting paper.
 Place this glass side on the stage of the compound microscope and view it.
Observations
  There are a large number of regularly shaped cells lying side by side and each cell has a
distinct cell wall.
 A distinct nucleus is present on the periphery of each cell.
 Lightly stained cytoplasm is observed in each cell.
 A large vacuole is present at the center of each cell, and is surrounded by the cytoplasm.
Conclusion
As cell walls and large vacuoles are clearly observed in all the cells, the cells placed for
observation are plant cells.
Precautions
 Use a brush to transfer the peel from one apparatus to another.
 Staining of peel should neither be too dark, nor too light.
 Extra glycerine stain should be removed using blotting paper.

Activity 2.3: Using the Microscope to Look at a Plant Cells

In this activity you are going to learn how to make a slide of plant tissue and stain it so that the
cells are easier to see.
You will need:
a microscope, microscope slides, cover, slips forceps
a mounted needle, a pipette and A lamp
apiece of onion skin
Iodine solution
Method
Remember, microscopes are expensive and delicate pieces of equipment so always take care of
them and handle them safely.
Onion cells (the sample taken) do not contain any chlorophyll so that are not colored. You can look
at them as they are, or stain them using iodine, which reacts with the starch in the cells and turns
blue black.
1. Take your piece of onion and remove a small piece of the thin skin (inner epidermis) on the
inside of the fleshy part using your forceps. It is very thin indeed and quite difficult to handle.
2. Place the epidermis onto a microscope slide and add a drop of water. Make another identical
slide and add a drop of iodine very gently from a pipette.
3. Using the mounted needle (or a sharp pencil), lower the cover slip very gently over the first
specimen. Take great care not to trap any air bubbles – these will show up as black ringed circles
What is magnification means? Can you see any differences between the terms magnification and
resolution?
What plant organelles did you observe from stained slides?
Which organelles are only seen in plant cells as compared to animals?
To Prepare Stained Temporary Mount of Human Cheek Cells
Materials Required

Real Lab Procedure

 Gently scrape the inner side of the cheek using a toothpick, which will collect some
cheek cells.
 Place the cells on a glass slide that has water on it.
 Mix the water and the cheek cells using a needle and spread them.
 Take a few drops of Methylene blue solution using a dropper and add this to the mixture
on the slide.
 After 2-3 minutes remove any excess water and stain from the slide using  a blotting
paper.
 Take a few drops of glycerine using a dropper and add this to the test mixture.
  Take a clean cover slip and lower it carefully on the mixture with the aid of a needle.
 Using a brush and needle, press the cover slip gently to spread the epithelial cells.
 Remove any extra liquid around the cover slip using a blotting paper.
 Place this glass side on the stage of the compound microscope and view it.
Observation
 A large number of flat and irregular-shaped cells are observed.
 The cells do not have a cell wall.
 However, each cell has a thin cell membrane.
 A deeply stained nucleus is observed at the centre of each cell.
 No prominent vacuoles are observed in the cells.
6. The Cell and Its Environment

Your cells need to take in substances, such as oxygen and glucose, and to get rid of waste
products and chemicals that are needed elsewhere in your body. In human beings, just like all
other living organisms, dissolved substances can move into and out of your cells across the cell
membrane in three different ways-by diffusion by osmosis and by active transport. In this section
you will look at each of these processes by doing different activities in the laboratory or the
teacher will demonstrate each of the methods.

WHAT IS DIFFUSION?

Diffusion is the flow of matter from a higher concentration to a lower concentration, resulting in
a homogeneous distribution. Diffusion of matter occurs most rapidly in gases, more slowly in
liquids, and most slowly in solids. The spreading of odoriferous molecules (a smell) throughout a
room is a common example of gaseous diffusion. A solid may dissolve and diffuse through a
liquid, as when a lump of sugar is placed in a cup of water. This process is much slower than the
diffusion of a gas; if the water is not stirred, it may take weeks for the solution to become
homogeneous. An example of the slowest diffusion process, a solid diffusing into a solid, occurs
when gold is plated on copper. The gold will diffuse slowly into the surface of the copper;
however, diffusion of an appreciable amount of gold more than a microscopic distance normally
requires thousands of years. Look Figure 5 to have an understanding about the process of
diffusion.
Figure 5: The Process of Diffusion

Activity 2.5: Detecting the Process of Diffusion

You will need:

· Stopwatch or timer

Method

1. Your teacher will open a bottle of a strongly scented chemical such as ammonia or spray some
perfume at the front of your class.

2. Start timing as the spray is released, and then put your hand up when you can smell the scent.
You’ll see a wave of hands moving from the front to the back and sides of the class as the
molecules spread out by diffusion.

3. Time how long it takes to reach the last person.

Activity 2.6: The Effect of Temperature on the Rate of Diffusion

Potassium Manganate (VII) (potassium permanganate) forms purple crystals that dissolve in
water. This activity demonstrates both simple diffusion and the impact of temperature on the rate
of diffusion.

You will need

3. Two identical beakers (100,200 or 250 cm³)

4. Cold water
5. Warm/hot water
6. Two crystals of potassium manganate (VII)
7. A stopwatch
Method

1. Half fill one beaker with cold water

2. Put exactly the same amount of warm or hot water in the second beaker.(N.B. if the water is
hot, be careful how you handle it.)

3. Drop a crystal of potassium manganate (VII) in each beaker at the same time. Simultaneously
start the stop- watch.

4. Time how long it takes the purple color to reach different points in your beaker, and (if
possible) the total time it takes for the liquid to become purple.

5. Write up your investigation and explain your results in terms diffusion and the

Q. In which beaker diffusion is faster?

Q. What other factors do you think affect the rate of diffusion?

Q. Can you mention areas (biological processes) where diffusion is important in the body
of human beings?

6.3. Osmosis

What is osmosis?

Osmosis is a special type of diffusion where only water moves across a partially permeable
membrane from an area of high concentration of water to an area lower concentration of water.
A cell is basically a solution inside partially permeable bag (the cell membrane) the cell contents
contain a fairly concentrated solution of salts and sugars. Water moves from a high concentration
of water particles (a dilute solution) to a less concentrated solution of water particles ( a
concentrated solution) across the membrane of the cell. The sugars and salts cannot cross the
membrane. In the next activity the teacher will demonstrate you the process of osmosis by using
model cells. In addition you will also able to investigate the factors affecting the process of
osmosis and the effect of osmosis on plant and animal cells.
Q. Can you mention areas (biological processes) where osmosis is important the body of
human being?
Q. Explain the differences between diffusion and osmosis.

Activity 2.8: Demonstrating Osmosis Using Potato cubes as Osmometers

You can use simple potatoes to demonstrate osmosis. A system that shows or measures osmosis
is an osmometer.
Three raw potatoes and one cooked potato (or three raw half potatoes and one cooked
half potato)
Four containers e.g. beakers, bowls
Strong sugar solutions/sugar
Water
Method
1. Take each potato or half potato and cut one end to make it flat. Pell the layer directly above
the flat end.
2. Hallow out the other end of the potato to make a cup
3. Set up the experiment
4. In B place sugar or strong sugar solution in the cup, mark the level before you start the
experiment.
5. In C place water in the potato cup and mark the level. Place strong sugar solution in the
container.
6. In D you will be given a cooked potato, or you must cook it yourself before starting the
experiment. Place sugar or strong sugar solution in the cup. Mark the level. Place water in the
container.
7. Leave the investigations for several hours or overnight.
8. Record your results carefully.
· Write up the investigation and make some conclusions.
· Explain your results in terms of osmosis.
· Do you think varying the strength of the sugar solutions can affect the rate and amount of
osmosis? How?

7. Testing for biological important molecules

All living things contain organic macromolecules: Lipids, proteins, carbohydrates and nucleic
acids. Characteristic for these organic molecules is that they are made up of only a small number
of elements: carbon, hydrogen, oxygen, and to smaller amounts nitrogen, phosphorus and sulfur.
They are called "macromolecules" because they are very large, containing long chains of carbon
and hydrogen atoms and often consist of repeating smaller molecules bonded together in a
repeating pattern (polymers).

Macromolecule    building block

protein                   amino acids
carbohydrates         monosaccharides
lipids                      glycerol + fatty acids
nucleic acids           nucleotides

7.1. Test for carbohydrates

Carbohydrate, any of a large group of compounds in which hydrogen and oxygen, in the
proportions in which they exist in water, are combined with carbon; the formula of most of these
compounds may be expressed as Cm(H2O)n. Structurally, however, these compound are not
hydrates of carbon, as the formula would seem to indicate.

Carbohydrates, as a class, are the most abundant organic compounds found in nature. They are
produced by green plants and by bacteria using the process known as photosynthesis, in which
carbon dioxide is taken from the air by means of solar energy to yield the carbohydrates as well
as all the other chemicals needed by the organisms to survive and grow.

The carbohydrate group consists principally of sugar, starch, dextrin, cellulose, and glycogen,
substances that constitute an important part of the human diet and that of many animals. The
simplest of them are the simple sugars, or monosaccharides, which contain either an aldehyde or
a ketone group. The most important is glucose. Two monosaccharide molecules joined together
by an oxygen atom, with the elimination of a molecule of water, yield a disaccharide, of which
the most important are sucrose (ordinary cane sugar), lactose, and Maltose. Polysaccharides have
enormous molecules made up of one type or several types of monosaccharide units—about 10 in
glycogen, for example; 25 in starch; and 100 to 200 in cellulose.

Molisch test
Molisch test is Specific for all carbohydrates – To differentiate between Proteins & Amino Acids
(-ve), and Carbohydrates (+ve). The Molisch reagent reacts with all carbohydrates larger than
tetroses. This test is based on the fact that pentoses and hexoses are dehydrated by conc. H2SO4
acid to form furfural or hydroxymethylfurfural, respectively. Oligo-and polysaccharides first
undergo hydrolysis with H2SO4 acid to give pentoses /or hexoses, which on subsequent
dehydration yield furfurals. These furfurals condense with α-naphthol to give colored complexes.

Procedure & observation:

To 2 mL of carbohydrate solution in a test tube, add 2 drops of Molisch’s reagent [1% α-

Naphthol in absolute alcohol].
Mix well; add carefully at the side wall of the test tube 2 mL conc. H2SO4 without
mixing.
o Reddish violet ring will be formed at the interface of the two solutions

Activity 3.1: Testing the presence of Starch

You will need:
· A 1% starch solution made by boiling a mixture of starch powder and cold water
· Two clean test tubes • Iodine solution
Method
1. Pour about 1 cm³ of starch solution into a clean test tube and the same volume of water into
the other test tube.
2. Add tow drops of iodine solution to each tube.
3. Record your observations and conclusion In tabular form. What did you find?

Activity 3.2: Benedict’s for Simple (Reducing) Sugars)

Benedict's solution is used to test for simple carbohydrates. Benedict's solution is a blue colored
liquid that contains copper ions. When Benedict's solution and simple carbohydrates are heated,
the solution changes to orange red/ brick red. This reaction is caused by the reducing property of
simple carbohydrates. The copper (II) ions in the Benedict's solution are reduced to Copper (I)
ions, which causes the color change. Sometimes a brick red solid, copper oxide, precipitates out
of the solution and collects at the bottom of the test tube. Complex carbohydrates such as
starches DO NOT react positive with the Benedict's test unless they are broken down through
heating or digestion (try chewing crackers and then doing the test). Table sugar (disaccharide) is
a non-reducing sugar and does also not react with the iodine or with the Benedict Reagent. Sugar
needs to be decomposed into its components glucose and fructose then the glucose test would be
positive but the starch test would still be negative. Some sugars react really with Benedict’s
solution. They reduce copper (II) ions to copper (I) ions and for this reason they are known as
reducing sugars. So there is a straightforward chemical test for the reducing sugars. Include all of
the single sugars and some double sugar.
Benedict test is Specific to reducing sugars, sugars with a free aldehyde or ketone group) such as
Fructose, Glucose, Galactose, Lactose & Maltose. It is used to differentiate between non-
reducing (e.g. Sucrose)& reducing sugars. Benedict is formed from sodium carbonate + sodium
citrate dihydrate + copper sulfate pentahydrate. The copper sulfate (CuSO4) present in Benedict's
solution reacts with electrons from the aldehyde or ketone group of the reducing sugar to form
cuprous oxide (Cu2O), of yellowish orange or red ppt.
CuSO4 Cu++ + SO4--
2 Cu++ + Reducing Sugar (electron donor) Cu+ (CuOH)
Cu+ Cu2O (precipitate)
You will need:
A Bunsen burner
Tripod, gauze and heat- proof mat
A large beaker half filled with water
Some glucose powder or food to be bested
Benedict’s solution
Different food samples to analyse (e.g. bread, fruit)
Method
1. Bring the water in the beaker to the boil, using the Bunsen burner.
2. In one boiling tube add water to a depth of about 2cm – this will act as you control.
3. In another tube add a sample of glucose powder and water to a depth of about 2 cm.
4. Place any food samples to be tested in other boiling tubes in the same way.
5. Add a few drops of Benedict’s solution to each boiling tube. Add enough to colour the mixture
blue.
6. Place the tubes in the boiling water and leave for several minutes. TAKE CARE with the
boiling water.
4. Record your observations and conclusion In tabular form. What did you find?

7.1.4. Acid hydrolysis of disaccharides and polysaccharides

8. Testing for fats and oils

A lot of lipids function as long-term energy storage. One gram of fat stores more than twice as
much energy as one gram of carbohydrates. Lipids are also an important component of the cell
membrane. Lipids consist of glycerol and fatty acids "tails". The fatty acid "tails" are long chains
of carbon and hydrogen that contribute to the non-polar behavior of fats - they don't mix with
(polar) water. The fatty acid chains can be saturated, with all carbons saturated with hydrogen
atoms forming a straight chain without double bonds. Unsaturated fatty acids contain double
bonds within the carbon chain, which results in a bend of the chain.
Vegetable fats and oils are lipid materials derived from plants. Physically, oils are liquid at room
temperature and fats are solid. Chemically, both fats and oils are composed of triglycerides, as
contrasted with waxes which lack glycerin in their structure. Although many plant parts may
yield oil, in commercial practice, oil is extracted primarily from seeds. The melting temperature
distinction between oils and fats is imprecise, since definitions of room temperature vary, and
typically natural oils have a melting range instead of a single melting point since natural oils are
not chemically homogeneous. Although thought of as esters of glycerin and a varying blend of
fatty acids, fats and oils also typically contain free fatty acids, monoglycerides and diglycerides,
and unsapoifiable lipids.

Vegetable fats and oils may or may not be edible. Examples of inedible vegetable fats and oils
include processed linseed oil, tung oil, and castor oil used in lubricants, paints, cosmetics,
pharmaceuticals, and other industrial applications. Fat consists of fatty acids attached to a
substance called glycerol. Dietary fats are classified as saturated, monounsaturated, and
polyunsaturated according to the structure of their fatty acids. Animal fats—from eggs, dairy
products, and meats—are high in saturated fats and cholesterol, a chemical substance found in all
animal fat. Vegetable fats—found, for example, in avocados, olives, some nuts, and certain
vegetable oils—are rich in monounsaturated and polyunsaturated fat. As we will see, high intake
of saturated fats can be unhealthy.

Activity 3.4: Test for Lipids

a) Filter paper test

The filter paper test is also known as the grease spot or translucent mark test.

You will need:

 Cooking oil or cooking fat
A clean filter paper or sheet of paper
dropper

Method

1. Put a drop of cooking fat (or smear alitle cooking fat) on a clean white sheet of paper.

2. Leave the paper for a few the cooking oil was dropped while holding the paper against light
(not a flame!). Use light coming in through the window or from the electric bulb or tube.

A permanent translucent mark is formed by lipids on paper. A translucent mark is one that does
not allow all the light to pass through. If you squeeze a food sample between two bits of filter
paper any water that has been squeeze out will evaporate from the paper. Any lipids will leave a
translucent mark that does not dry out and disappear. However, this test, although effective, is
not very scientific because it does not depend on a chemical reaction.

b) Emulsion test

You will need:

 Clean, dry test tubes – they MUST be dry

 Ethanol
 Cooking oil or cooking fat

Method

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

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

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

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

5. Carefully pour the contents of the tube containing the oil fat or food sample into one of the
tubes containing water.
6. Pour about 2 cm³ pure ethanol into the other tube containing water and compare the two.

If lipid is present, a white, cloudy layer forms on top of the layer of water.

· Use this test to investigate a number of common foods and find out if they contain lipids.

· Write up your method and the results of any foods you have tested.

9. Testing for proteins

Proteins are complex, specialized molecules composed of carbon, oxygen, hydrogen, nitrogen
and sometimes sulfur. The building blocks of proteins are amino acids. There are 20 different
amino acids that combine to form polypeptides (proteins). The different amino acids are similar
in structure: at the center of the molecule is the alpha carbon that is connected to an amino group,
a carboxyl group, a hydrogen atom and the R group (the side chain). The different amino acids
have different side chain, but are otherwise identical. Proteins have many important roles in
organisms. Structural proteins such as collagen or elastin, provide support. Regulatory proteins
such as enzymes control cell processes. Proteins also play an important part in the immune
system (antibodies), oxygen transport (hemoglobin), movement (muscles) etc.

Proteins are made of smaller units called amino acids. Of the more than 20 amino acids our
bodies require, eight (nine in some older adults and young children) cannot be made by the body
in sufficient quantities to maintain health. These amino acids are considered essential and must
be obtained from food. When we eat food high in proteins, the digestive tract breaks this dietary
protein into amino acids. Absorbed into the bloodstream and sent to the cells that need them,
amino acids then recombine into the functional proteins our bodies need. Animal proteins, found
in such food as eggs, milk, meat, fish, and poultry, are considered complete proteins because
they contain all of the essential amino acids our bodies need. Plant proteins, found in vegetables,
grains, and beans, lack one or more of the essential amino acids. However, plant proteins can be
combined in the diet to provide all of the essential amino acids. A good example is rice and
beans. Each of these foods lacks one or more essential amino acids, but the amino acids missing
in rice are found in the beans, and vice versa. So when eaten together, these foods provide a
complete source of protein. Thus, people who do not eat animal products can meet their protein
needs with diets rich in grains, dried peas and beans, rice, nuts, and tofu, a soybean product.
Experts recommend that protein intake make up only 10 percent of our daily calorie intake.
Some people, especially in the United States and other developed countries, consume more
protein than the body needs. Because extra amino acids cannot be stored for later use, the body
destroys these amino acids and excretes their by-products. Alternatively, deficiencies in protein
consumption, seen in the diets of people in some developing nations, may result in health
problems. Marasmus and kwashiorkor, both life-threatening conditions, are the two most
common forms of protein malnutrition.

Activity 3.3: Biuret Test for Proteins

When we test foe proteins sometimes we add two separate chemicals (5% potassium or sodium
hydroxide solution and 1% copper sulphate solution) to our test food
You will need:
· Test tubes
· 5% potassium or sodium hydroxide solution and 1% copper sulphate solution OR
· Biuret reagent • Different food samples
Method
1. In one test tube add water to a depth of about 2 cm – this will act as your control.
2. In another tube add a sample to be tested in other test tubes in the same way.
3. Place any other food samples to be tested in other test tubes in the same way.
4. Add an equal volume of dilute potassium or sodium hydroxide solution in all the test tubes and
mix.
5. Add a few drops of dilute copper ion. (If you are using Biuret reagent steps 4 and 5 are
combined in one.)
Write up your method and the results for the different foods you have tested.

9.2 Xanthoproteic test

10. Cell Division –Mitosis

11.Cell Division –Meiosis

12. Photosynthesis

12.1 Test for starch in leaves

12.2 Chromatographic separation of chlorophyllous

Pigments

13. Collection and preservation of plant and animal specimens