LABORATORY MANUAL FOR BIOLOGY

(BIO F110 BIOLOGY LABORATORY)

BIOLOGICAL SCIENCES FACULTY

EDUCATIONAL DEVELOPMENT DIVISION

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE

PILANI 333031 (RAJASTHAN)

2018
 Contents

Preface

Laboratory Instructions

Index Table
Experiment – 1: Scientific Process Lab

Experiment – 2: Microscopy Lab: Introduction to microscopy, different cell types,

visualizing different cell types, preparation of temporary mount

Experiment – 3: Protein Lab: Measurement of total protein content in the given

sample by Lowry’s method

Experiment – 4: DNA Lab: Extraction of DNA from cells

Experiment – 5: Electrophoresis Lab: Visualizing the extracted DNA using Gel

Electrophoresis

Experiment – 6: Mitosis Lab: Measurement of Mitotic Index and duration of

mitosis in the given plant tissue and observation of various stages
of Mitosis through readymade slides.

Experiment – 7: Blood Group Determination Lab: Determining blood group and

Rh factor in blood samples

Experiment – 8: Enzymatic Activity Lab: Assessing lactase activity

Experiment – 9: Transport Lab: study the phenomenon of plasmolysis in onion

peel

Experiment –10: Transpiration Lab

Experiment- 11 Photosynthesis Lab

Bibliography
 Preface

The spectacular progress over the past two decades in our understanding of biological processes
at the molecular level has been made possible by the coming together of physical, chemical,
biochemical and genetic approaches and by the availability of a wide range of analytical
techniques.
The need for re-writing this laboratory manual was felt because of the special status of the newly
designed course Biology Laboratory (BIO F110), which is being offered to the students across the
campuses of BITS Pilani with the objective to give them their first Laboratory exposure in Biology.

Description of experimental set-up and working of specialized instruments

 An adequate theoretical explanation of the protocol to help the student prepare and
understand the experiment.
 A built-in format of the manual including tables and graphs for recording the experiments.

The authors gratefully acknowledge the continuous interest and encouragement received from
Director, Prof A.K. Sarkar, Dean ID Prof. A.P. Singh and Associate Dean, Pilani campus, Prof.
Srikanta Routroy. Our sincere thanks are due to Head of Department Biological Sciences Dr.
Prabhat Nath Jha for his overall support in conducting the course. Our thanks are especially due to
Prof. S.K. Verma, Prof. A.K.Das, Prof. V.N.Sharma and Dr. Neeru Sood for their contribution in
developing the first edition of this manual. Thanks are due to Dr. Uma S. Dubey and Dr. B.Vani,
who have been made possible the increase in number of experiments from 6 to 10 to support the
restructured new course and ensure the workability of the same within the required time frame of
1h 50minutes. Thanks are also due to earlier course ICs like Dr. Sandhya Mehrotra, Dr. Lalita
Gupta and Dr. Santosh Padhi and Dr. Shilpi Garg for their subsequent inputs. We are also grateful
to Mr. Casey Hanley, lab co-ordinator, Biology department, University of Dayton for letting us
use the ideas of new labs.
It was the intense discussion and constructive criticism amongst the faculty members, specially
the Ex DCA convener Dr. Uma S. Dubey who along with the other faculty members, Dr. Manoj
Kannan, Dr. Pankaj Sharma, Dr. Sandhya Marathe, Dr. Shilpi Garg, Dr. Sudeshna Mukherjee, Dr.
Syamantak Majumder and, Dr.Vishal Saxena, for providing inputs in planning and developing the
manual. Also this effort would never have been successful without the constructive criticism and
suggestions from our highly dedicated Research scholars & Teaching Assistants who as course
Instructors have time to time continuously contributed towards the betterment of the earlier form
of this course and the present manual.
 General Laboratory Instructions

 The objective of these laboratory exercises is to acquaint you with the techniques
emphasizing the need of measurements in the field of biology. We want you to take serious
interest in conducting these experiments. It will help you in understanding some basic
concepts in modern biology.

 Begin your experiment, without any preconceived notions about the outcome of the
experiment. Follow directions carefully, and see what actually does happen. Be meticulous
in recording the observations accurately.

 You must be on time in laboratory and come well prepared for the experiment.

 You are kindly requested to follow the dress code full pants and closed toed shoes in
accordance to safe laboratory practice.

 Every student has to have his/her individual copy of the manual. The laboratory manual
has to be brought when you come to the laboratory.

 The laboratory work will continue for 1 hour 50 minutes. .

 For accurate measurements, work carefully and co-operate with your team members.

 Presentation of data, tables, graphs and calculations should be neat and carefully done in
the manual itself and should be verified by your instructor after each experiment.

 Bring your marker pen, calculator, scale and pencil etc. and try to complete the record in
the laboratory itself.

 Handle the glassware and apparatus with care while working. Report any sort of breakage
to the instructor.

 Refer to the course handout for the evaluation components. For further reading the related
chapters in the first year General Biology text book should be taken into account.

 Regularity is expected and make-ups will not be encouraged.

 BIOLOGY LABORATORY

Second Semester 2018 to 2019

Name:………………………………………. ID No:. ………………………………….

Section No:. ………..... Day: …………………….. Hr: ………………

S. No. Experiment Date Instructor’s

Signature
1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.
 EXPERIMENT No. 1
Exercise 1

The Scientific Process

Objectives

 Make educational observations that lead to further thought and analyses

 Raise appropriate questions based on observations
 Generate hypotheses that can be tested using science
 Properly setup an experiment to test the hypothesis
 Identify the control, independent and dependent variables of the test
 Discuss results and draw conclusions based on these results

Introduction

What is science? If we look up the definition in a dictionary, we find “a branch of knowledge

or study dealing with a body of facts or truths systematically arranged and showing the operation
of general laws”. Sounds a little daunting, maybe even a little boring… For biology, the ‘branch
of knowledge’ alluded to includes the activities and processes of all living organisms and their
interactions with each other and their environment. So yes, a good amount of factual knowledge
and memorization is required, but scientists don’t just sit around spouting off facts all day. They
are active participants in the revision of old knowledge, application of current knowledge, and the
creation of new knowledge. Science and technology are on the ‘cutting edge’. The old stereotype
of a middle-aged white man in a lab coat mixing chemicals rarely applies. Check out the characters
of mainstream media like the Myth-busters team and the numerous crime shows. Glamorous video
documentaries like Planet Earth and Life are in the zeitgeist. Science is interesting and every one
of us has a little of that in them. We all seek to understand the world and our place in it.

The synonyms of science are “art, technique, method, discipline”. Thus, we find that science
is a bit more dynamic and quite a lot more exciting than it would seem at first glance. It is an ever-
changing body of knowledge that needs constant attention if we are to be sure our answers to life’s
many questions are correct. The art of science involves the challenging of old ideas and the
discovery of new ones. One must be creative in designing new experiments and meticulous in
drawing conclusions from gathered data. Paramount to all is the ability to ask testable questions
of the universe based on your own observation.

The goal of this entire semester’s lab experience is to get you started using the skills and practices
necessary to conduct science: asking questions, designing experiments, collecting data, analyzing
it, then generating conclusions from it; all while adding to your factual knowledge along the way.
Science is a process; a Scientific Process!!

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What is a scientific Process?

The scientific process is a universally agreed upon method used to ensure that the answers to our
questions are discovered in a systematic manner based on empirical evidence. It is a cheat sheet
in the creation of a good experiment. It provides (1) a step-by-step process to guide you through
scientific investigation and (2) a standard rubric to use when presenting new findings to others.
You have probably used pieces of the process in your own life, without even being aware of it. In
this first lab, we will outline the overall purpose of each step in the process.

The most common misconception about the scientific process is that it is linear. All science begins
with an observation that leads to a hypothesis followed by an investigation. But through that
investigation, new observations are made which lead to new questions and thus new hypotheses.
Science is a cyclic process that is constantly questioning and revising itself. Without revision, we
would still believe the earth was flat, the sun revolved around us, disease was caused by immoral
behavior and blood-letting was a good way to cure it.

Another common misnomer regarding science is that experiments are designed to “prove” a
hypothesis. This statement embodies two misconceptions – 1) that science can “prove” something
to be completely true and 2) that experiments are designed to accept a hypothesis rather than reject
one.

USING THE SCIENTIFIC PROCESS

Step 1: Making Observations

Scientists begin investigations because they have noticed something they do not understand or
want to find a solution to a problem. There are several ways a problem may come to your attention.
Someone may assign you the problem (this happens often in school or at work), the problem may
thrust itself upon you (your vehicle won’t start), or you may discover the problem simply by being
curious (there are more peafowl birds on the specific side of this campus). Scientists look for
patterns or differences that might be causing what they are seeing. They compile all of their
observations and then pull testable variables from them, around which they can design an
investigation. For example:

Question: Why are there more peafowl birds on this campus?

Observations: A) Campus fulfills their food, water and shelter requirements better.
B) Campus also runs a peafowl restoration project.

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Testable variables: Type of food, water birds get; type of arrangements made for restoring
populations…

Step 2: Formulating a Hypothesis

Observations and testable variables lead to the formulation of hypotheses. Use the situation
described in Step 1 to do this. Now, think of testable hypotheses regarding the non-uniform plant
growth across the island, write it in the space provided below:

You probably wrote a single sentence for each hypothesis. You may even have used an if/then
statement, having been taught that in the past. Both of these are correct, but incomplete. We are
going to extend your knowledge of hypotheses and expect you to use the following model from
this point on. Now that you know science does not prove, but rather disproves, we have to format
your standard hypothesis to fit that model. For each investigation, you must have two separate
hypotheses: a null and an alternate. There must be two because regardless of the outcome of your
experiment, you must reject one in order to accept the other. For this reason, they must also be
formatted very specifically.

The basic ‘if/then’ statement can usually take the role of the alternate hypothesis (Ha) and is most
often formatted this way:

Ha: If I change (_____________), then I WILL see (_______________).

i.e. If I change the amount of food the birds receive, then I WILL see a difference in the
number of birds that survive.

This is most likely the first statement that comes to mind when you are forming a hypothesis. It
states that the variable you are testing IS causing the outcomes you are seeing.

The null hypothesis states the opposite; that the variable you are testing IS NOT causing the
outcomes you are seeing. Any variation in the outcome is too small and simply due to the
inherent variability of nature or chance. It is formatted this way:

Ho: If I change (______________), then I WILL NOT see (______________).

i.e. If I change the amount of water the plants receive, then I WILL NOT see a difference
in the number of plants that survive.

These hypotheses must be setup as exact opposites and be general enough to ensure that no matter
the outcome of the experiment, one hypothesis can be rejected, thereby forcing the acceptance of
the other. Notice, the alternate does not end by stating that more or less plants will survive, just
that the number that will survive will change. This ensures that regardless of the experimental
outcome, one hypothesis can always be rejected while the other is accepted. The hypothesis pair

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setup is one of the main reasons experiments must be designed to test a single independent variable
at a time.

Step 3: Experimental Design

Obtaining valid conclusions from an experiment relies heavily on designing the experiment
properly. You must consider the variables being tested, the types of treatments being applied, the
method by which treatments are applied, and how outcomes of those treatments will be compared.
The following paragraphs outline three basic factors used in designing experiments.

Variables

There are three variables that must be defined prior to actual experiment implementation. The
dependent variable, independent variable, and the constants.

Dependent variable: the outcome being measured. This can be one or many outcomes, but always
includes the observation the scientist originally made. In the island example, the observation
revolved around the number of green plants on different parts of the island, so in this case the
dependent variable would be the number of green plants. Before performing an experiment,
scientists must determine what variables they will be measuring, how they will measure them, and
how they analyze the data once it is collected.

Independent variable: the condition the investigator varies during the experiment. This can be
any change the investigator chooses; however, scientists usually limit themselves to one
independent variable per experiment. Testing one variable at a time, allows scientists to be certain
it is only the variable they are manipulating that is causing the change in the dependent variable.
Testing multiple variables can leave researchers confused as to which one(s) are actually causing
the change.

Helpful mnemonic: The dependent variable, the one being measured, is “dependent” on the
independent variable, the one being manipulated by the researcher.

Both of these variables should be mentioned in the hypotheses of any experiment. Using the setup
described above, these variables fit into a hypothesis like this:

Ha: If I change the independent variable, I will see a change in the dependent variable.

Constants: all other variables within the experiment, which are held constant. The only two
variables allowed to change in an experiment are the dependent and independent. All other
components which could alter the outcome of the experiment must be held constant. There are
usually multiple constants in a single experiment. This ensures that the only variable responsible
for the change seen in the outcome (dep. variable) is the one intentionally changed by the
investigator (indep. variable).

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Treatments There are two treatment types to consider when designing a proper experiment, the
experimental treatments and the control.

Experimental treatments: these treatments consist of the groups being subjected to the change
in the independent variable, often in varying degrees of strength. There can be as many of these
groups as the researcher feels is needed to adequately answer the question being posed.

The control: this treatment is the sole group not subjected to the changes in the independent
variable. It is a group maintained under “normal” conditions and is used to eliminate the possibility
that other factors not taken into account are affecting the experimental outcome. If the control acts
as predicted, it is used as a baseline against which the outcomes of the experimental groups can be
compared. If the control does not act as predicted, there could be some other unaccounted for
variable in play and it becomes necessary to go back to the drawing board.

Replication

All good experiments are repeated to ensure the results are consistent each time it is performed.
By repeating the experiment, errors due to variability differences in the test subjects,
measurements, and external or unforeseen conditions can be reduced. Replication can often be
built into an experiment, which saves time and energy in the long run.

Step 4: Data Collection

Scientists use lab notebooks to record all parts of their experiments, but most importantly their
data/results. All data must be kept organized and specifically labeled so it can be easily understood
in the future. You will be using this lab manual and will be graded on your content and
organization of all information collected in each lab.

Step 5: Data Analysis

Data comes out of experimentation in what is called ‘raw’ form. Oftentimes, there is so much data
and the inherent variability within it is so chaotic, it can look wholly unmanageable and incoherent.
For this reason, data is never kept and rarely presented in its raw form. Instead , scientists
summarize data into easily readable tables and figures after condensing it through statistical
analysis.

‘Eyeballing it’ does not hold water in the scientific community. If you want other researches to
take your results seriously, you must have some statistical analysis (some math) backing your
conclusions. Statistical analysis shows how well your data supports or rejects your hypotheses
and tells other scientists the level of confidence you have in your results. The simplest form of
statistical analysis, which you will be using most in this course, is the average.

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Step 6: Make Conclusions:
Finally, the end goal of the process, using the results of the data analysis to make conclusions
based on the original hypotheses. In this step, scientists tease out a general conclusion from their
data analysis They do this by determining which of their two hypotheses their data supports and
attempting to explain why. Results do not always show what was predicted, or even what was
hoped for, so researchers must consider all alternate explanations, sources of experimental error,
and the outcomes of similar investigations performed by other scientists. If an experiment is setup
completely and correctly from the start, then even a lack of results can be a result. Life is a fickle
thing and resists being restrained into well-defined categories and laws, but we try our best to stick
to the data and make sense of what it shows.

Step 7: Repeat

Once is never enough. Repetition built into an experiment shows the results of one manipulation
at one point in time. Experiments must be repeated in another place, at another time, by another
person; checked and rechecked to make sure the same results present every time. You would not
take a new medicine because someone told you it worked once for one other person. You would
want proof, to see for yourself, or at least know that rigorous testing was done on multiple subjects
to ensure efficacy. Repetition is key, because you just never know.

Investigation

You will now practice using the scientific process to complete an investigation on temperature of
different people. You will begin by confirming or rejecting observations of temperatures made
by a fellow scientist. You may use this page to keep track of your observations.

Observation of Temperatures:

Obtain a thermometer from lab instructor for your group and note down temperature of at least
three group members. Write it down in the space below:

1.

2.

3.

Now, get ready to design Your Own Experiment:

Based on the observations and discussion, you will now design your own investigation to explain
the observations of temperature of three different people. On the next two pages is a template for
helping you to design, implement, and complete an experiment. (Hint: you can use all different
ways those will result in affecting temperature of an individual’s hands; make them cold/hot)

Experimental Design

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Before you begin, describe your experiment below and show your answers to your instructor.
Do not proceed with your experiment until your instructor has given you the go-ahead.

 What is your hypothesis (state both the null and the alternate)?

 What is your dependent variable?

 What is your independent variable?

-Why/How do you think this independent variable will affect the behavior?

 What is your control treatment?

 How will you include replications?

 What results would support your hypothesis?

 What materials will you need?

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  Describe your methods.

Perform Your Experiment

Before you begin, make sure that everyone at your lab group understands the procedures and
treatments you will be using. As you carry out your experiment you will want to record your
procedures, results (including table/s to collect data and observations), and conclusions in your
notebook. Be thorough and detailed as you record your results. If you have problems, questions,
and/or errors during the experiment, be sure to write these down. Use the following information
to guide you in writing your results and your conclusions:

Results - Describe your results in general. Do not explain why you got these results yet. Decide
how best to present your results – as a table and/or as a graph – and then complete your tables
and/or graphs before you interpret your results.

 Conclusions –
o Look back at your hypothesis and look at your observations. Do your results
support or refute your hypothesis? Explain by using your data.
o If your results are not what you predicted, explain why that may be.
o If you had an opportunity to redo this experiment, how might you do it differently
to make it more convincing?
o Summarize the conclusions you have drawn from your results – what do your
results mean?

MARKS OBTAINED

SIGNATURE OF THE INSTRUCTOR

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 Exercise 2: Microscopes and Cells

Lab Objectives

 Use proper microscope techniques

 Locate and focus organisms in the field of view
 Determine and compare the size of different organisms
 Identify similarities and differences between the plant and animal kingdoms
 Identify similarities and differences between prokaryotic and eukaryotic cells

Introduction to Microscopy

The microscope has been an invaluable tool to biologists ever since Hans Janssen and his son
Zaccharias invented it in the seventeenth century. Since many biological specimens are too small
to be seen by the naked eye, microscopes ranging from a hand lens to an electron microscope can
be used to magnify an object. A simple microscope such as a magnifying glass has only a single
lens; whereas, a compound microscope has two or more lenses. The lens closest to the object
magnifies the image, and the second lens further magnifies the image before the light reaches your
eye. Compound light microscopes (Fig 1.1) use light rays and glass lenses to magnify small objects
or sections of specimens. The dissecting (stereoscopic) microscope is used to observe objects that
are too large or thick for the compound light microscope. A dissecting microscope does not
magnify as greatly as a light microscope, but the object is seen in three dimensions.

Parts of a Compound Light Microscope

Refer to Figure 1.1 as you read the following. You will be held responsible for knowing the
following microscope parts and their functions.

Part Function

stage provides a flat surface on which to lay the

microscope slide
stage clips secure the slide

stage manipulator moves the slide to allow viewing of different areas

objective lenses magnify the specimen's image

4x (scanning power) - used for initial viewing

10x (low power) - provides increased magnification
40x (high power) - provides highest magnification
revolving nosepiece allows viewer to select an objective lens

ocular (eyepiece) the lens the viewer looks through; magnifies image
produced by objective lens

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light source provides light

condenser a series of lenses immediately below the stage that focuses

light on the specimen

iris/diaphragm regulates amount of light reaching the specimen

coarse control moves stage toward or away from objective lens;

used for initial focusing

fine control moves objective lens (or stage) slightly;

used for "fine tuning" focus

base & arm provide mechanical support; microscope

should be held here when carrying

Figure 1.1: Parts of a Compound Light Microscope

Terms Used in Microscopy

magnification - enlargement in one direction.

If an objective lens is labeled 10x, it makes an object appear 10x longer than its actual size.
The object will also appear ten times wider.

total magnification - the product of the magnifications of the ocular lens and the objective lens
being used. For example, if your ocular reads 15x and you are working on scanning power
(4x), the total magnification would be 60x.

resolving power (resolution) - ability to distinguish detail.

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Resolving power may be determined by measuring the distance required for two points to appear
as two distinct points rather than one blurry dot. A short distance indicates high resolution.

working distance - distance between objective lens and specimen.

field of view - area visible when viewing through ocular.

The field of view appears as a circle. The area of the field of view decreases dramatically with
increasing magnification.

field diameter - distance across the field of view.

Field diameter is usually measured in micrometers (10-6m). A micrometer is one-millionth

(1/1,000,000) of a meter or one-thousandth (1/1000) of a millimeter.

parfocalism -the ability of a microscope to remain basically in focus when switching from one
objective lens to another.

image reversal -the apparent reversal of an object (from top to bottom and left to right) seen when
viewing an object through a microscope.

depth of field - the vertical distance in which an object remains in focus.

temporary wet mount - the specimen is placed in a drop of water and covered with a cover slip.

permanent mount - the specimen is embedded in a mounting compound which hardens, allowing
the slide to be kept permanently.

The principle function of a microscope is magnification. In other words, the microscope increases
the apparent angle subtended at the eye by objects within the microscopic field. This property of a
microscope is quantified as the Magnification (M). Resolving power specifies the smallest detail
that a microscope can resolve in imaging an ideal specimen. The distance between two points in
the microscopic field that can just be distinguished from one another is called the minimum
resolvable distance, Dmin. This is defined by the equation

0.5  .
Dmin 
N sin 
where, λ is Wavelength of light source.
α is the Aperture angle of the objective lens
N is the Refractive index of medium between specimen and the objective lens.

 Magnification leads to enlargement of a sample. The angular magnification is given by

 Magnification leads to enlargement of a sample. The angular magnification is given by

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 where is the magnification of the objective and the magnification of the
eyepiece. The magnification of the objective depends on its focal length and on the
distance between objective back focal plane and the focal plane of the eyepiece (called
the tube length):

.
In order to be visible through a microscope an object must possess a certain degree of contrast with
its surrounding medium. This contrast is a result of the fact that less light is transmitted through
the object than through the medium. This decreased light transmission is caused by two factors-
light absorbed by the object and light refracted out of the optical path of the microscope by a
difference in the refractive index between the object and the surrounding medium. Contrast can be
greatly increased by staining procedures: treatment with dyes that bind selectively either to the
whole cell or to certain cell components, thus producing a much greater absorption of light. Thus
we see that magnification, resolution and contrast are three important factors in microscopy.

Introduction to Cells

At the most basic level, a cell is an ordered assemblage of atoms and molecules, just as your tissues
are an ordered assemblage of cells, and your organs are an ordered assemblage of tissues, and so
on. All of these pieces of matter are used to build a living thing, but not all the pieces are
considered to be alive. Although life cannot be defined simply, all living organisms possess certain
common characteristics.

All organisms are composed of cells. Some organisms such as bacteria, Paramecium, Amoeba,
Euglena, and yeast are themselves a single cell. By contrast, the typical human is composed of
approximately one hundred trillion (100,000,000,000,000) cells!

To make some sense of these two forms and everything in between, we try to classify organisms
based on their similarities and differences. The most general of these classifications is based on
the fundamental characteristics of the cells that makeup organisms. We call this category the
Domain and it has three groups: eubacteria, archaebacteria and eukaryotes. Both bacteria
categories can be lumped together in a group called prokaryotes. These cells are extremely small
and represent the oldest forms of life on our planet. Fossil records indicate that these first appeared
on Earth around 3.5 billion years ago. Prokaryotes have no organized nucleus, at times a single,
circular loop of naked DNA, called a plasmid, and no organelles (small, membrane-bound
structures that perform specialized tasks within the cell, much like organs in your body).
Eukaryotic cells, on the other hand, are much younger evolutionarily speaking. The earliest
examples of these show up around 1.5 billion years ago. The typical eukaryotic cell is
approximately one thousand times larger and much more complex than the typical prokaryotic
cell. The DNA of a eukaryotic cell is organized into linear chromosomes which are stored in a
membrane-bound nucleus. Eukaryotic cells also contain many organelles such as the endoplasmic
reticulum, chloroplast, Golgi apparatus and mitochondria. For further clarification, eukaryotes are

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divided into four kingdoms: protista, fungi, plantae, and animalia. Examples of organisms
containing eukaryotic cells are Amoeba, bread molds, cacti, and humans.

Examples of Cellular Structures and Their Functions

Structures Function

Plasma membrane regulates passage of molecules in and out of a cell

Cell wall protects cell; maintains shape

Structures in Eukaryotes Only

Chloroplast site of photosynthesis

Endoplasmic reticulum manufacture of macromolecules

Golgi Apparatus packages proteins for transport

Mitochondria site of cellular respiration, the production of ATP

from energy in organic molecules

Nucleus controls cellular activities; stores genetic

information
INVESTIGATIONS

How to use a Microscope

1. Using coarse control, lower the stage (or raise the nosepiece) as far as possible.
2. Place slide on stage; secure with clips or slide holder.
3. Rotate the lowest power objective lens (4x scanning power) into place.
4. Using coarse control, slowly raise the stage until the specimen appears in view. Look
through the ocular as you do this step.
5. CENTER the specimen.
6. Focus using fine control.
7. Rotate the low power (10x) objective lens into position.
8. Focus using coarse control; center; focus with fine control.
9. Rotate the high power (40x) objective lens into position. THE LENS WILL NOT TOUCH
THE SLIDE.
10. FOCUS using FINE CONTROL ONLY!
11. Draw your specimen and record any observations.
12. Record total magnification.
13. Rotate scanning power objective back into position.
14. Remove your slide.

A. Image Reversal and Field of View

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 1. Using coarse control, lower the stage (or raise the nosepiece) as far as possible.
2. Obtain a prepared slide from the box at your table and place the slide on the stage; secure
with clips or the slide holder.
3. Rotate the lowest power objective lens (4x scanning power) into place.
4. Using coarse control, slowly raise the stage until the specimen appears in view. Look
through the ocular as you do this step.
5. CENTER the specimen.
6. Focus using fine control.
7. Draw what you see.
8. Rotate scanning power objective back into position.

B. Preparing a wet mount

1. Make a wet mount of your cheek. Obtain a fresh toothpick from your instructor and scratch
the inner side of your cheek.
2. Put this scratched material on the slide, try to make a thinner layer on the slide.
3. Add a drop of methylene blue to the smear.
4. Put a cover slip and observe under the microscope. Draw what you see.

Characteristics of Cells

Using the techniques, you just learned about microscopes, you will now look at a variety of
different organism’s cells. You will compare these cells to each other. Some key questions you
should be asking yourself are:

1. What is the difference between cells in different kingdoms?

2. How are single celled organisms different from multicellular organisms?
3. How are plant cells different from animal cells?
Procedure

***Everyone should use microscope, and should be observing specimen. ***

Domain Prokaryote: ______________________

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Kingdom Protista: ______________________

Kingdom Fungi: ______________________

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Kingdom Plantae: ______________________

Kingdom Animalia: ______________________

Marks Obtained

Signature of Instructor

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 EXPERIMENT NO. 3
OBJECTIVE
Measurement of total protein content in the given sample by Lowry’s method.

THEORY

Photometery: Photometry involves measurement of light transmitting power of a solution in order

to determine the concentration of light absorbing material present. Analytical instrument, based on
the direct measurement of color intensity in terms of light absorbed at a specific wavelength is
called a photometer. Colorimetric procedures are limited to the visible portion of the spectrum
whereas photometric procedures however involve the usage of Ultraviolet, visible and Infra red
portions of the spectrum. Photometers use a light filter for the wavelength, at which the
photometric measurement is made. Whereas in case of a spectrophotometer, a diffraction grating
or a prism along with slit is used for getting the monochromatic light. Light filters do not transmit
monochromatic light but they narrow the range of spectral region. Spectrometers have the
advantage of greater flexibility, increased sensitivity, spectral range and narrowness besides the
flexibility and convenience over photometers but are more expensive.
The working of spectrometers and photometers are based on Beer’s and Lambert’s laws. The
simplest expression of these laws is as follows:
Beer’s law: It states that the optical density of a solution is directly proportional to the
concentration of the solution.
Lambert’s law: It states that the optical density of a colored solution is directly proportional to
the path of light i.e. diameter of the cuvette.
Note: If diameter of cuvette is doubled, OD will also be doubled but since cuvettes of the same
diameter are used for most analytical purposes, so automatically Lambert’s law is observed.

Beer and Lambert’s law states that the absorbance of a solution containing light absorbing
material depends on the follow factors:
(1) The nature of the substance
(2) The wavelength of light
(3) The path of light
(4) The amount of colored material in the light path.
If the incident light is I0 , the transmitted light (light emerging from cuvette containing solution) is
I, then the ratio I/I0 is the transmittance (T) of the colored solution. If the material does not absorb
at all then I and I0 are the same, and so the %T = 100. However, if a substance does not transmit
at all, then transmittance is zero and the substance is opaque. By using negative logarithm of T,
the measurements are transformed to the light absorbed (1/T). The equation now becomes A = 2 –
log T and since 2 is the log of 100, the formula becomes A = 2 – log T.
According to Beer- Lambert’s law T= 10-kcl, where.
T = Transmittance
k = Molar extinction coefficient constant (depends on the characteristic of the solution)
c = Concentration of the colored solution
l = Path of light through the colored solution
By converting this expression through logarithmic form
Log T = - kcl
- log T = kcl

18
 O.D. = kcl
O.D. is constant for a particular solution and path length is also constant, as the diameter of cuvette
is fixed. Therefore, the optical density is directly proportional to the concentration. Absorbance is
also called optical density. A = kIc
Molar extinction coefficient is the O.D. or absorbance given by a substance concentration 1
mol/litre in a path length of 1 centimetre. Graphs of absorbance vs. concentration, or log %T vs.
concentration are known as Beer’s Law plots. They are made by measuring the light absorbed by
solution of varying concentration. Cell width and wavelength of light are maintained constant. If
a linear plot is obtained (showing that the Beer-Lambert relationship holds for the solution at that
wavelength), it may be used to determine concentration of unknown solution(s).

Applications: Photometry has a very vast application range such as studies on chemical
compositions of substances, enzyme analysis, soil sample analysis, microflora studies etc. Most
interestingly it can be use in diagnosis of many diseases.

Diagnosis of diseases: Body fluids such as blood (serum and plama), cerebrospinal fluid (CSF)
and urine contain several organic and inorganic substances. For example, blood contains glucose,
urea, uric acid, creatinine, proteins, etc. It also contains inorganic anions cations like sodium,
potassium, calcium, etc. and anions such as phosphorus, chlorides, bicarbonates, etc. Most
chemicals are in equilibrium, i.e. the rate of production and rate of loss (by degradation or
excretion) are equal. The normal concentration of most of these substances varies within narrow
limits. Diseases alter this equilibrium in many ways. Thus the alteration and its extent can be useful
in the diagnosis of a very broad range of disease like diabetes, anaemia liver and kidney
dysfunctions.
(See Table 1 for commonly used colorimetric assays).

Table 1: Common colorimetric assays:

Substance Reagent Wavelength(nm)

Inorganic Ammoniun molybdate; H2SO4; 600

Phosphate 1,2,4-aminonaphthanol; NaHSO3; Na2SO3

Amino acids a. Ninhydrin 570 (Proline 420)

b. Cupric salts 620
Peptide bonds Biuret (alkaline tartarate buffer, cupric salt) 540
Phenols, tyrosine Folin (phosphomolybdate, phosphotungstate, 660 or 750
cupric salt) (750 more
sensitive)
Protein a. Folin 660
b. Biuret 540
c. BCA reagent(Bicinchoninic acid) 562
d. Coomassie Brilliant Blue 595
Carbohydrates a. Phenol, H2SO4 Varies, For e.g.
b. Anthrone, H2SO4 Glucose 490
Xylose 480,
620 or 625

19
 Reducing sugars Dinitrosalicilate, alkaline tartarate buffer 540

Pentoses a. Bial (orcinol, ethanol, FeCl3, HCl) 665

b. Cysteine,H2SO4 380-415

Hexoses a. Carbazol, ethanol, H2SO4 540 or 440

b. Cysteine, H2SO4 380 – 415
c. Arsenomolybdate Usually 500-570
Glucose Glucose oxidase, peroxidase, O-dianisidine, 420
Phosphate buffer
Ketohexose a. Resorcinol, thiourea, ethanoic acid, HCl 520
b. Carbazole, ethanol, cysteine, H2SO4 560
c. Diphenylamine, ethanol, ethanoic acid, 635
HCl

Hexosamines Ehrlich (dimethylaminobenzylaldehyde, ethanol, 530

HCl)
DNA Diphenylamine 665
RNA Bial ( orcinol, ethanol, FeCl3, HCl)

α- Oxo acids Dinitrophenylhydrazine, Na2CO3, ethyl acetate 435

Sterols Liebermann-Burchardt reagent ( acetic anhydride, 625

H2SO4, chloroform)

Description of MINI SPEC SL171: Spectrophotometer mainly comprises of light source, a

monochromator, a photo diode, processing electronics and readout.
Light from the tungsten filament halogen lamp/equivalent source is focused on to the entrance slit
of the monochromator (which also a diffraction grating and an
exit slit) by condensing optics. The light from the slit is
collimated and directed on diffraction grating with 600
lines/mm. This disperses the beam and the resultant spectrum
is focused on to the exit slit. Various wavelengths are scanned
by rotating the grating on its axis by rotating the wavelength
disc which is coupled to the grating mounting. The
monochromatic light, isolated by exit slit, passes through
Blank, Standard or Sample (held in a cuvette) and the
transmitted light falls on a photo-diode. The output signal
from the photo-diode is amplified, processed, read to
Percentage Transmittance (%T), Absorbance (Abs.) and
Concentration (Conc.) as selected.
White light emanating from the tungsten halogen lamp of the
MINI SPEC SL171 passes through the monochromator
containing entrance slit, diffraction gratings ruled 600lines/mm and an exit slit. Of the dispersed
beam a narrow band of similar wavelength light passes through a second slit into a sample solution
being measured. Any of this light which is not absorbed by the sample solution, but which passes

20
through the solution, falls upon the phototube of the instrument, where the intensity of the
transmitted light is measured electronically.

Description of the Spectronic 20: White light emanating from the tungsten lamp of the Spectronic
20 passes through an entrance slit and is dispersed by a diffraction grating. Of the dispersed beam
a narrow band of similar wavelength light passes through a second slit into a sample solution being
measured. Any of this light which is not absorbed by the sample solution, but which passes through
the solution, falls upon the phototube of the instrument, where the intensity of the transmitted light
is measured electronically.
The diffraction grating is a precision replica grating having 600 grooves to the millimetre and
accurately spaced. The white light falling upon the grating is dispersed into a horizontal fan of
beams (violet and ultra-violet) at one end and the long wavelengths (red and infrared) at the other.
The spectrum of light falls on a dark screen with a slit cut in it. Only that portion of the spectrum
which happens to fall on the slit goes through onto the sample, and we can project any part of the
spectrum onto the slit that we wish simply by turning the grating.
The grating is turned by the knob on the top of the instrument (the wavelength control knob).
Attached to this knob is a dial calibrated in wavelengths. This dial may be set to the wavelength
wanted. The wavelengths are given in millimicrons (1mµ = 10 A = 10-7 cm). The slit of the
instrument passes a band of wavelengths of 20mµ. Because of the linear diffraction of the grating,
this bandwidth of 20mµ is constant over the entire wavelength region.
The colorimeter is turned on by rotating the amplifier control (left hand knob) clockwise. This
should be done at least 20 minutes before measurements are made. After the instrument has
warmed up, the amplifier control knob may be adjusted so that the meter needle will read “0” on
the percentage transmission scale when no light is striking the phototube.
The right-hand knob regulates the amount of light passing through the second slit to the phototube.
The need for this control knob arises from the fact that the light source does not emit light of
different wavelengths at equal intensities and the phototube is not equally responsive to light of
varying wavelength. Besides this, the “blank” solution (the medium in which the substance being
measured is located) may itself absorb light of certain wavelengths. In order to measure the
absorbance due to only a particular species in solution, such “side effects” which affect the %T
reading must be compensated for. Therefore, after the colorimeter has been “zeroed” (by means of
the amplifier control knob), a “blank” solution is placed in the light path and the light control knob
is rotated until the dial reads 100 %T, to achieve this desired compensation. If a sample solution
is now placed in the light path, any change in the %T reading is due to the particular light–
absorbing species in the sample and the %T reading is a measure of the quantity of that species
present.
Whenever a wavelength is changed, the 0 %T and 100 %T must then be reset, since the amount of
“compensation” needed varies with wavelength. The meter needle should go to 0 %T whenever
the cuvette is removed from the sample holder, because removing the cuvette releases an occluder,
which drops into the light beam and prevents the beam from reaching the phototube. Adjusting the
zero point o the scale is therefore always done with the sample holder empty. The phototube is a
cesium-antimony surface type photoemissive cell (type S-4). The relative response of the
phototube to a beam of monochromatic light of constant intensity is:

Wavelength Response (Relative)

350 90%

21
375 98
400 100
450 91
475 81
500 68
512 61
525 53
550 37
575 21
600 10
612 7
625 5
Thus it is seen that the phototube is much more sensitive to light of wavelength 400 mµ than to
light of wavelength 600mµ.This means that the phototube will require a greater flux of 600mµ
than of 400mµ monochromatic light, in order for the same %T reading to be registered upon the
colorimeter dial.
Handling of Cuvettes: The handling of cuvettes is extremely important. Often two cuvettes are
used interchangeably; one for the “blank” solution and one for the samples to be measured. Yet
any variation in the cuvette (such as a change in cuvette width or the curvature of the glass, stains,
smudges or scratches) will cause varying results. Thus, it is essential, in dealing with cuvettes, to
follow several invariant rules:

 Do not handle the lower portion of a cuvette (through which the light beam will pass).
 Always rinse the cuvette with several portions of the solution before taking a measurement
cuvette, with a clean Scott Wiper before placing the cuvette in the instrument. NEVER wipe
cuvettes with towels or handkerchiefs.
 Wipe off any liquid drops, or smudges on the lower half of the Standard Solution (200µg/ml)
when inserting a cuvette into the sample holder:
 To avoid any possible scratching of the cuvette in the optical path, insert the cuvette with the
index line facing towards the front of the instrument.
 After the cuvette is seated, line up the index lines exactly.

The cuvette should be removed in the reverse manner.

 When using two cuvettes interchangeably, use one of the cuvettes always for the “blank”
solution and the other cuvette always for the various samples being measured. Under apparatus,
these are called “matched” tubes.

These rules must be observed in all experimental work with the colorimeter. The equipment used
in these experiments is expensive and hence, we expect you to be careful while using the
equipment.
Lowry’s method for measuring Protein concentration: In this method protein samples are
treated with Biuret reagent and Folin-Ciocalteu reagent subsequently. The Biuret reaction involves
the binding of Cu+2 under alkaline conditions to nitrogen in the peptide bonds of the proteins. The
Folin- Ciocalteu reagent, which contains phosphomolybdotugstate acids, is reduced by Tyrosine,
tryptophan and polar aminoacids present in proteins. This process is facilitated by copper (II) ions

22
from the Biuret reaction. In this reaction, heteropolymolybdenum blue is detected at 740nm (red
filter) because at this wavelength the blue complex absorbs maximum light. The intensity of the
blue color of the solution is directly proportional to the amount of these aromatic amino acids
present in the sample It has many applications in disease diagnostics.

REQUIREMENTS

1. Reagent A: Dissolve 20 g Na2CO3 and 4 gm NaOH in 1 litre of distilled water.

2. Reagent B: Dissolve 0.5 g CuSO4.5H2O and 1 g sodium potassium tartarate in 80ml of
distilled water. Make up the volume to 100 ml. (Precaution: This reagent is not to be shaked
before use – only its supernatant should be used).
3. Reagent C: To 50 ml reagent A, add 1 ml reagent B (freshly prepared).
4. Reagent D: Diluted (1N) Folin’s and Ciocalteu reagents.
5. Examining samples:
6. Protein Standard Solution:
a. Stock Solution: Dissolve 50 mg of bovine serum albumin (BSA) in 50 ml of
distilled water and store in refrigerator.
b. Working Standard: Dilute the stock solution 5 times so that final protein
concentration is 200µg/ml.

PROCEDURE
1. Take 6 clean test tubes and mark them as B (blank), S1, S2, S3, S4 (different concentrations
of standard) and T ( test solutions). Fill these tubes in the following way:

B S1 S2 S3 S4 T
Standard Solution -- 0.25 ml 0.50 ml 0.75 ml 1.0 ml --
Water 1.0 ml 0.75 ml 0.50 ml 0.25 ml -- --
Test Solution -- -- -- -- -- 1.0 ml
Final conc.(µg/ml) 0.00 50.0 100.0 150.0 200.0 ?

2. Add 5 ml of alkaline copper solution (reagent C) in each tube. Allow them to stand for 10
min at room temperature.
3. Add 0.5 ml of reagent D (diluted Folin’s reagent) in each tube. Mix immediately and
incubate at room temperature for 20 min, until blue color develops.
4. Read OD at 740 nm (i.e. filter 4 in the given model) using blank solution to set zero.

OBSERVATION

1. Read OD in the following table:

Tube Number Concentration of protein OD at 740 nm

B
S1
S2
S3
S4

23
 T
2. Plot the values of OD 740 nm against different concentrations of standard protein solution
in the graph paper for preparation of standard curve.
3. Match the OD of T (test sample) on the standard curve. The corresponding concentration
on X-axis will be the actual protein concentration (in g/ml) in the given sample.

RESULTS
The protein concentration in the given sample is ------------------- g/ml.

MARKS OBTAINED

SIGNATURE OF INSTRUCTOR

24
 EXPERIMENT No. 4
OBJECTIVE
To study the effect of the enzyme lactase on milk.

THEORY
Lactose, sugar found in milk is a disaccharide compound composed of glucose and galactose.
Ordinary table sugar sucrose is also a disaccharide composed of fructose and glucose. The
enzyme lactase breaks down lactose into glucose and galactose. Although lactose is similar to
sucrose, lactase will breakdown only lactose because of the shape of sugar. The specificity of
enzyme reaction of breaking down of sucrose and lactose is based on shape. When hydrolysed
by the enzyme lactase, lactose yields equi-molar quantities of D-glucose and D-galactose.
We will also observe what happens if the shape of lactose is changed due to heating.

LACTOSE Lactase D- GLUCOSE + D-GALACTOSE

SUCROSE Lactase GLUCOSE + FRUCTOSE (No reaction)

GLUCOSE
Glucose is an aldohexose, belonging to the carbohydrate family, and is found in large quantities
throughout, the living world. It is the primary fuel for living cells. Dietary sources include plant
starch, lactose, maltose and sucrose. In nature, glucose exists as D-glucose (Dextrose).
Glucose is a simple sugar (monosaccharide). In solution, it exists as open chain form (less than
1%), and cyclized ring forms –α- D-glucose (36%) and β-D-glucose (63%).

Glucose is a reducing sugar. It is able to function as a reducing agent, because free or potentially
free, aldehyde group is present in the molecule. This aldehyde group is readily oxidized to
glucoronic acid at neutral pH by mild oxidizing agents and enzymes. This property is utilized in
detecting and quantitating glucose in biological fluids such as urine or blood.

25
The cuprous oxide formed is brownish in color. For optical quantification, this compound is
reacted with phosphomolybdic acid, to give blue color (whose intensity is proportional to the
concentration of Cu2O and thereby glucose).

The oxidizing agent used here is tartrate complex of alkaline copper (II) sulphate, known as
Fehling’s solution. A precaution needs to be taken when used with blood, since it reacts with blood
proteins and may give false results; blood is deproteinized (by tungstic acid) before use.

Glucose is a primary source of energy in most organisms. It is broken down stepwise (during the
processes of glycolysis and Kreb’s cycle) to give CO2 and water, along with 7300 kCal of energy
per mole. Excess glucose is stored as glycogen in animals and as starch in plants. In man, a constant
level of glucose in maintained in blood by the interplay of hormones such as insulin, glucagon and
epinephrine.
Yet another method of glucose estimation, which is more rapid than the Folin-Wu’s method, is the
GOD/POD method. Here, glucose oxidase (GOD) is the oxidizing agent and peroxidase (POD)
catalyzes the subsequent reaction to produce red coloration.
Glucose determination is mainly useful in diagnosis of diabetes mellitus, the condition in which
blood glucose levels are elevated (hyperglycemia). Other diseases like hyperthyroidism and
hyperpituitarism also leads to hyperglycemia. Hyperglycemia occurs frequently as a result of over
dosage of insulin based anti-diabetic treatment. If untreated, it may lead to coma. In today’s lab,
we’ll be exploring the enzyme activity, which is lactase and it forms glucose as a product

REQUIREMENTS
1. Lactase tablet
2. 15 ml of milk
3. Sucrose 5 gram
4. 10 and 100ml measuring cylinder
5. 500ml beaker
6. Test tubes with stand
7. Distilled water
8. Marker pen
9. Stopwatch
10. Hotplate/Burner

26
 11. Glucose Test strips
12. Glass Rod

PROCEDURE
Preparation of Solutions: Keep following solutions ready before conducting the experiment:
1. Enzyme solution – Add and dissolve the lactase tablet to 200ml distilled water.
2. Skim milk – Milk from which cream has been removed.
3. Sucrose solution – Add 5 gram of table sugar to 100 ml of distilled water. Stir to
dissolve the sugar.
4. Denatured Enzyme Solution –
i. Place 20 ml of enzyme solution into a test tube.
ii. Add 200 ml of water to a 500 ml beaker.
iii. Place the test - tube in the beaker.
iv. Place the beaker along with test – tube on a hot plate or burner.
v. Boil the water in the beaker for 30 min. Cool it down to room temperature.
After all solutions are ready follow the following steps:
5. Label the test – tube as –
a. A. Test – tube with skim milk + Enzyme solution.
VIII-
b. B. Test – tube with skim milk 1+ Water.
c. C. Test – tube with milk + Denatured enzyme solution.
d. D. Test – tube with sucrose solution + Enzyme solution.
6. In test – tube A, Add 2 ml of skim milk and 1 ml of enzyme solution.
7. Wait for 5 min. (or more) and test for glucose with the glucose test strip. Record
your data in the result table.
8. In test – tube B, Add 2 ml of skim milk and 1 ml of water and repeat step 7.
9. In test – tube C, Add 2 ml of skim milk and 1 ml of denatured enzyme solution and
repeat step 7.
10. In test – tube D, Add 2 ml of the sucrose solution and 1 ml of enzyme solution and
repeat step 7.
11. In test – tube E, Add 2 ml of the sucrose solution and 1 ml of water. Repeat step 7.

OBSERVATION
Find out presence and absence of glucose in the test – tube.
Type of solution Glucose result
Positive ‘+’ Negative ‘-‘
Test tube A: Skim milk + Enzyme
Test tube B: Skim milk + water
Test tube C: Skim milk + Denatured Enzyme
solution
Test tube D: Sucrose solution + Enzyme sol.
Test tube E: Sucrose solution + water

PRECAUTIONS
 Denaturing the enzyme is very difficult. Boil for longer period of time.
27
RESULTS
Indicate the presence or absence of glucose in the solutions by ‘+’ and ‘-‘ mark, respectively.

MARKS OBTAINED

SIGNATURE OF THE INSTRUCTOR

28
 EXPERIMENT No. 5

OBJECTIVE
To extract total DNA from bacterial colonies.

THEORY
DNA is in the nucleus of almost every cell. The length of DNA per cell is about 100,000 times as
long as the cell itself. However, DNA only takes up about 10% of the cell’s volume. This is
because DNA is specially packaged through a series of events to fit easily in the cell’s nucleus.
The structure of DNA, the double helix, is wrapped around proteins, folded back onto itself, and
coiled into a compact chromosome. It is of interest to know that the DNA in the largest human
chromosome (i.e. chromosome number 1 consists of 220 million base pairs and would be 8.5 cm
lond if straightened.
Chromosomes can be studied using microscopes, but the double helix of a chromosome is so thin
that it can only be detected through procedures like X-Ray Crystallography, Infra Red and Nuclear
Magnetic Resonance spectroscopy. Chromosomal DNA from a single cell is not visible to the
naked eye. However, when chromosomal DNA is extracted from multiple cells, the amassed
quantity can easily be seen and it looks like strands of mucous-like, translucent cotton. DNA is a
macromolecule that is one of four necessary molecules for life (sugars, proteins, fats, and nucleic
acids). DNA is a polymer of nucleotides including 4 nitrogenous bases, deoxyribose sugar and
phosphate (Fig. 1- DNA Diagram). These nucleotides are linked by
a. Covalent bond between phosphates and sugars
b. Hydrogen bond between complementary base pairs.
DNA provides the blueprint for transmission of genetic information. The strands of DNA inside
the nucleus are directions for creating all other components of the cell necessary for living.
Detergents solubilize and break down the lipids and proteins that form the primary cell
membrane and disrupt the bonds that hold the membrane together. The cell contents, including the
nucleus, are thus released and become available for further treatment or isolation. Sodium lauryl
sulphate (SDS) is an active ingredient in detergents. The final step in the present procedure requires
alcohol. The solubilized DNA comes in contact with the
alcohol where the two liquid layers meet (Interface). The
alcohol dehydrates and precipitates the DNA, as DNA is
insoluble in the alcohol. If the procedure is done properly, fine,
long strands of DNA will form at the interface and can be easily
spooled onto a stirring rod.

Fig. 1 Structure of a DNA molecule

 Two chains run in opposite directions.

 Two chains form a helix
 Sugar-phosphate backbone provides framework for bases
(A, C, G, T)
 Hydrogen bonds strengthen structural bases (A with T,
C with G) to hold chains together
 To extract DNA, individual cells must be lysed to release
DNA in the solution

29
 Other macromolecules including proteins and carbohydrates must be removed using proteases
to purify DNA.
 Plasmid is a circular extra chromosomal double stranded DNA present in some bacteria. It is
widely used in recombinant DNA applications to clone gene(s) of interest and produce ample
copies of the gene(s). In order to be of use in cloning it should possess (i) an origin of
replication to allow its independent replication within E. coli (ii) a selectable marker, usually
antibiotic resistance (iii) one or more unique restriction sites where gene of interest can be
added.
 Plasmids can be isolated from bacteria by breaking open the bacterial cells using detergents
like sodium dodecyl sulfate (SDS) and a strong base/alkali like sodium hydroxide. The
detergent solubilizes the phospholipid bilayer and denatures the protein. The alkali aids the
breaking of the cell wall and hydrogen bonding between the DNA base-pairs. EDTA is used
in the solution to chelate divalent cations like Ca2+ and Mg2+ essential for bacterial cell-wall
integrity and DNase activity. Presence of EDTA facilitates rupturing of the cell and prevents
the degradation of plasmid by the bacterial DNases. Sucrose maintains the isotonic
osmolarity and Tris maintains the desired pH. Potassium chloride (KCl) facilitates separation
of SDS bound protein and genomic DNA structures. Potassium replaces sodium from SDS
forming KDS which leads to formation of hard pellet leaving the plasmids in the supernatant.

REQUIREMENTS

1. Antibiotic containing Luria Bertani agar plate

2. Bromophenol blue solution (0.4% w/v)
3. EDTA (0.5 M, pH 8.0)
4. Ethidium bromide (10 mg/ml)
5. KCl (4 M)
6. NSS solution (0.2 N NaOH, 0.5% SDS, 20% sucrose). Precaution: NSS is to be prepared
fresh for each use

PROCEDURE

1. Grow the bacteria containing plasmid, on rich agar medium like Luria Bertani agar containing
the appropriate antibiotic (in our case it’s ampicillin). The size of the colony should be approx.
2-3 mm in diameter (It takes approx. 18-24 hrs at 37°C for most bacterial strains).

2. Using a sterile toothpick or loop pick a colony and suspend it in 50 µl of sterile 10 mM EDTA
(pH 8.0) in a microfuge tube.

3. Now add 50 µl of a freshly made solution of NSS (0.2 N NaOH, 0.5% w/v SDS, 20% w/v
sucrose). Close the top of the tubes and then mix their contents by vortexing/tapping the tube
for 30 sec.

30
4. Incubate the tube at 70°C (in water bath) for 5 min. and then allow it to cool to room
temperature.

5. Add 3µl of a solution of 2 M KCl. Vortex the tube for 30 sec. and then incubate the tube on
ice for 5 min.
6. Centrifuge the tube at maximum speed (13000 rpm) for 3 min at 4°C to remove bacterial
debris.
7. Collect the supernatant in fresh microfuge tube and add 0.5 µl of a solution containing 0.4%
bromophenol blue. Label your tube well with your group name/number and date and store this
tube at 4°C.
Next week, you’ll be using same supernatant to run on Agarose gel for electrophoresis
experiment.

OBSERVATIONS

RESULT

MARKS OBTAINED

SIGNATURE OF INSTRUCTOR

31
 Experiment-6
OBJECTIVE

Agarose gel electrophoresis to separate the plasmid and visualize the separated bands.

THEORY

Gel electrophoresis, a standard laboratory procedure is used for separating DNA based on its
molecular size (length in base pairs) and shape. An electrical field is applied to the gel matrix
into which the DNA/RNA sample is loaded. Being negatively charged the DNA/RNA (owing to
the phosphate groups in their structure) moves through the gel matrix toward a positive electrode
(anode). Shorter and compact DNA/RNA molecules will migrate faster than the longer and
bulkier ones. The approximate length of the DNA fragments can be determined by running
alongside a DNA ladder (a collection of DNA fragments of known lengths).

The gel is usually made up of agarose, a polysaccharide obtained from the red algae Gelidium
and Gracilaria species, by boiling the powder in appropriate buffer. On cooling down, the
solution solidifies into a porous matrix, a result of hydrogen bond formation between the agarose
monomers. The percentage of agarose used in gel making decides the pore-size of the matrix.
Hence depending on the size of the DNA molecules to be separated using electrophoresis one
decides the percentage of agarose to be used (refer table 1). During electrophoresis the gel is
submerged in buffer to avoid heating and subsequent melting of the gel. Buffer also helps in
maintaining the pH, that may result out of hydrolysis of H2O molecules during the
electrophoresis. Change in pH can affect mobility of the DNA as pH variation can cause
variation in the negativity of the DNA molecule. Tris-acetate-EDTA (TAE) is a commonly used
buffer.

Table 1

Recommended Optimum Resolution for Linear DNA

% Agarose

0.5 1,000–30,000bp

0.7 800–12,000bp

1.0 500–10,000bp

1.2 400–7,000bp

1.5 200–3,000bp

2.0 50–2,000bp

32
REQUIREMENTS

6X loading dye: 0.25 % bromophenol blue, 0.25% Xylene cyanol, 30% glycerol
Methylene Blue 0.1% (w/v)
DNA marker/ladder
1X TAE buffer: 40 mM Tris, 20 mM acetic acid and 1 mM EDTA (final pH: 8.3-8.6).
Agarose Powder

CAUTION
Use of lab coats is advised for this lab.

PROCEDURE
1. Add required amount of agarose to 1 X TAE buffer. For example, add 0.25 gm agarose to 25
ml of TAE to get 1% gel. Heat the mixture in microwave to melt the agarose. (Occasionally stir
to achieve uniform gel)
3. Pour the mixture into a sealed casting tray, place the comb on the caste to obtain wells in the
gel. Let it set for 20-30 minutes. (Avoid formation of any air bubbles as it interferes with the
current and hence the movement of DNA)
4. Gently remove the comb and transfer the gel to the electrophoresis apparatus filled with 1X
TAE buffer. The level of TAE should be 2 – 3 mm above the gel.
5. Load 30 µl of isolated plasmid (from previous experiment) mixed with the loading dye (final
concentration: 1X) into the well. Add marker (5 µl of 100 ng/µl) to one of the wells. (Avoid
overloading to prevent smearing of the bands)
6. Apply voltage 1-5V/cm (calculate final voltage based on the distance between the electrodes).
7. After approx. 30 min. the bromophenol blue dye would have migrated two-thirds to three-
fourths the length of the gel. Stop the current.
8. Take the gel out carefully, without breaking and put it in the staining box. Rinse the gel with
deionized water. (Immerse gel into water and decant the water).
9. Add 0.1% methylene blue solution to the gel for nearly 10-15 minutes. Slowly in your hands,
shake the box with gel and methylene blue.
10. Decant the methylene blue solution and store it in a bottle. It is reusable.
11. Add water to the gel to destain the gel, this is to enhance the contrast in order to visualize the
bands better.
OBSERVATIONS

1. No. of bands seen in the gel:_________________________

2. Plasmid: band number ______________
3. RNA: band number ______________
4. Genomic DNA: band number ______________
RESULTS

MARKS OBTAINED

SIGNATURE OF INSTRUCTOR

33
 Experiment -7

OBJECTIVE
To study the phenomenon of plasmolysis in onion peel.

THEORY
The process, where the osmotic loss of water, due to the solute concentration difference inside
and outside of a cell, occurs in plant cells to result in pushing the plasma membrane away from
the cell wall, is termed as plasmolysis. It occurs when a cell is kept in hypertonic solution.
However, when a cell is kept in hypotonic solution, the cytolysis occurs, where the outer osmotic
pressure becomes higher to cause a net flow of water into the cell. Through plasmolysis, we can
determine the ionic nature of the cell's vicinity/environment, and also we can find the rate of
solute transfer inside or outside the cell membrane.

When placed in hypertonic solution, the plant cell loses the water and it becomes flaccid (termed
as wilting). Even further loss of water causes plasmolysis and the cell protoplasm is pulled out
of the cell to create a gap between the cell wall and cell membrane. Lastly the cytorrhysis occurs,
where the complete cell wall is crumpled. Although difficult, but still plants have developed
some ways to prevent the excessive water loss, like an excessive water gain. Plasmolysis can be
completely reverted back, when a cell is kept in a hypotonic solution. When plant cell is placed
in hypotonic solution water enters in causes the cell to swell. However, presence of cell-wall
prevents the cell from bursting. In this state the plant cell is considered to have become turgid.
Turgidity is the reason why plants stand erect. The same is not true for animal cells. The animal
cell would burst out when placed in hypotonic solution. Stomata also helps the plant cells, as
they store the water and doesn't allow the cell to be dried out. Yet another way is the utility of
wax in the plant. Wax helps retain water in the plant cells. Albeit these evolved multiple ways,
plant cell rarely employs them, as plasmolysis occurs only in extreme conditions, to be very rare
in nature.

REQUIREMENTS

1. Onion, cut into slices approximately 1 cm wide, 1or 2

2. Microscope
3. Microscope slides,1per specimen
4. Cover slips, 1 per specimen
5. Distilled water
6. Salt solution (sodium chloride) 5% w/v
7. Rubber bulb
8. Pipettes
9. Forceps
10. Filter paper

34
PROCEDURE

1. Cut a 1 cm square of onion. Then peel off a single layer of the red cells from an inner fleshy
leaf of the onion.
2. Place the strip on a slide. Cover it with
a drop or two of distilled water. Add a
cover slip.
3. Look at the cells through a microscope,
starting with the low power lens.
4. Take another strip of cells from your
plant material. This time mount the
cells with a couple of drops of 5%
sodium chloride solution.
5. Examine through the microscope and
compare the cells to those mounted
with distilled water.
6. After a few minutes draw out the
sodium chloride solution with a piece
of filter paper placed at the edge of the
coverslip. Replace it with distilled water
added at the other side of the coverslip.
7. See what happens to the cells.

OBSERVATIONS

A. Cells mounted in presence of water

General Features

Diagram

B. Cells mounted in presence of Sodium Chloride

35
General Features

Diagram

RESULTS:

MARKS OBTAINED:

SIGNATURE OF INSTRUCTOR

PRECAUTIONS

1. Onion may irritate some students’ eyes to the point of discomfort. It may be dipped in
water to avoid it.
2. Take care with microscope slides and (especially) cover slips which are fragile and break
easily. Ensure students know how to deal with broken glass.
3. Sodium chloride is described as ‘low hazard’ on Hazard 47B.

36
 Experiment No. 8

OBJECTIVE
(i) Preparation of temporary mount of leaf epidermis to study the structure of stomata
(ii) Measurement of transpiration rate.

THEORY
The epidermis is the outer layer of cells covering the leaf. It forms the boundary separating the
plant’s inner cells from the external world.
The epidermis serves the following functions:
a. Protection against water loss by way of transpiration.
b. Regulation of gas exchange, secretion of metabolic compounds.
c. Absorption of water (in some species)
Most leaves show dorso-ventral anatomy: The upper (adaxial) and lower (abaxial) surfaces have
different construction and may serve different functions. The epidermis is usually transparent as it
lacks chloroplast and is coated on the outer side with a waxy cuticle that prevents water loss. The
cuticle is in some cases thinner on the lower epidermis than on the upper epidermis, and is
generally thicker on leaves from dry climates as compared with those from wet climates.
The epidermal tissue includes several differentiated cell types: epidermal cells, epidermal hair cells
(trichomes) cells in the stomate complex : guard cells and subsidiary cells. The epidermal cells are
the numerous, largest, and least specialized type of cells and form the majority of the epidermis.
These are typically more elongated in the leaves of monocots than in those of dicots.
The epidermis is covered with pores called stomata. The part of a stomatal complex consisting of
a pore is surrounded on each side by chloroplast containing guard cells, and two to four subsidiary
cells that lack chloroplasts. The inner walls of guard cells are thicker than the outer walls and they
contain prominent chloroplast and nuclei. These cells are responsible for opening and closing of
stomata. Opening and closing of the stomata complex regulates the exchange of gases and water
vapour between the outside air and the interior of the leaf and plays an important role in allowing
photosynthesis without letting the leaf dry out. In a typical leaf, the stomata are more numerous
over the abaxial (lower) epidermis than the adaxial (upper) epidermis and more numerous in plants
from cooler climates.

Fig 1: Structure of stomata in monocot plants Fig2: Structure of stomata in dicot plants

REQUIREMENTS
1. Slides
2. Filter paper
3. Brush
4. Coverslip
5. Needles
37
 6. Water
7. Aloe Vera / Lily leaf / any other leaf from which a peel can be obtained easily
PROCEDURE
1. Takethe leaf. Cut it into smaller pieces of about 6cm2 .
2. Wash it with water
3. Fold the leaf on it’s upper surface to break it such that it still remains attached.
4. Gently pull the broken end apart.
5. You will find the lower epidermis separating from the rest of the leaf.
6. Take a fine pair of scissors and cut a small regular piece of the peel and transfer it into a petridish
containing water.
7. Take a clean slide. Put a drop of water in it’s centre and transfer the peel from the petridish to the
slide with help of a brush. Place the coverslip.
8. Remove the extra water of the slide with a folded filter paper.
9. Examine the slide first under low power and then under high power.
10. Record your observations

OBSERVATIONS
Draw and label as seen under the microscope.

RESULTS
The total count of stomata in an visible area seen through the eye piece is ___________.

PRECAUTIONS
1. Do not press the coverslip hard as it is very thin.
2. Always hold the slide by its edges to avoid making the slide dirty.
3. Always use a brush to transfer the peel from petridish to the slide.
4. The peel should be cut to a proper size and its curling must be avoided.
5. The microscope should be handled with care.

b) Measurement of the rate of transpiration using potometer

THEORY

Movement of Water in a Plant

Water enters a plant through the root hairs, passes through the tissues of the root into the xylem
and travels up through the xylem vessels into the leaves. Transpiration, the evaporation of water
from the leaves, is the major factor that pulls the water up through the plant. When water enters

38
the roots, hydrogen bonds link each water molecule to the next so the molecules of water are pulled
up the thin xylem vessels like beads on a string. The water moves up the plant, enters the leaves,
moves into air spaces in the leaf, and then evaporates (transpires) through the stomata (singular,
stoma).
There are hundreds of stomata in the epidermis of a leaf. Most are located in the lower epidermis.
This reduces water loss because the lower surface receives less solar radiation than the upper
surface. Each stoma allows the carbon dioxide necessary for photosynthesis to enter, while water
evaporates through each one in transpiration.

REQUIREMENTS
1. Potometer
2. Twigs from a mesophytic plant

PROCEDURE

Estimation of transpiration rate

1. Take 3 % cobalt chloride solution from beaker and pour into the Petri dish.
2. Take some filter paper strips and dip them in the cobalt chloride solution. Keep the strips
in the solution for 3-5 minutes. They become pink in colour when wet.
3. Remove the strips from the solution using forceps.
4. Place the strips on the wire gauze to allow them to dry. The filter paper becomes blue in
colour on drying.
5. Select one healthy leaf and clean the leaf to remove the water droplets using a filter paper.
Take the dry pieces of cobalt chloride paper from the wire gauze.
6. Place the dried strips of cobalt chloride paper: one on the upper and the other on the lower
surface of a leaf of the potted plant.
7. Take two glass slides and place one over the upper and the other over the lower side of the
leaf.
8. Clip the slides together using binder clips.
9. Note the time taken by the cobalt chloride paper to change its blue colour to pink.

RESULT

MARKS OBTAINED

SIGNATURE OF THE INSTRUCTOR

39
 EXPERIMENT- 9

OBJECTIVE
(i) Measurement of Mitotic Index and duration of mitosis in the given plant tissue
(ii) Observation of various stages of Mitosis through readymade slides.
THEORY
Mitosis is somatic cell division. The cell division takes place in two stages; karyokinesis (division
of cell nucleus and cytokinesis (division of the cytoplasm contents). The karyokinesis is divided
into four phases- prophase, metaphase, anaphase and telophase.
In any population of mitotically active cells, only some cells are in dividing phases (karyokinesis)
at any one time, while other cells are in interphase (non-dividing phase). The fraction or percentage
of dividing cells is defined as the mitotic index (MI). MI is important to determine the duration of
mitosis in the cell cycle of growing tissue/cells and also for scaling up of cell culture for various
biotechnological purposes.
We have already learned the working principles and parts of microscope, emphasizing the idea
that microscope is used for visualizing the objects, which cannot be seen with the naked eye. Now,
in order to be visible through a microscope an object must possess a certain degree of contrast with
its surrounding medium. This contrast is a result of the fact that less light is transmitted through
the object than through the medium. This decreased light transmission is caused by two factors-
light absorbed by the object and light refracted out of the optical path of the microscope by a
difference in the refractive index between the object and the surrounding medium. Contrast can be
greatly increased by staining procedures: treatment with dyes that bind selectively either to the
whole cell or to certain cell components, thus producing a much greater absorption of light.
Thus in addition to magnification, resolution; and contrast is the third important factor for
microscopy.

OBJECTIVE- I
To determine the ‘Mitotic Index’ and duration of mitosis in the given plant tissue.
REQUIREMENTS
Reagents:
1. 10% Hydrochloric Acid- Take 9 ml water and add 1 ml HCl to it slowly.
2. Acetocarmine Stain – Mix 90 ml Acetic Acid with 110 ml distilled water (45% acetic acid).
Add 2 gm carmine to 100 ml 45% acetic acid and boil for 30 minutes. Cool and make up
the volume of filtered stain to 100 ml by adding 45% acetic acid and store.
3. Farmer’s Fixative – Mix absolute alcohol and glacial acetic acid in 3:1 ratio.
4. Glycerin/ Liquid paraffin.

40
Materials:
1. Microscope with 40X and 100X objectives.
2. Microslides.
3. Coverslips no. 1 (Square/ round)
4. Tooth picks/ matchsticks.
5. Tissue paper/ filter paper.
6. Root tip of Alium cepa (Onion
7. Slides of various stages of cell division

PROCEDURE
1. Cut the root tip from onion bulbs, about 3-4 mm from the tip (at the base of the meristem)
and rinse briefly in distilled water.
2. Place the tip in 10% HCl (v/v) for 5 minutes at room temperature and rinse again in distilled
water.
3. Put the tip on acetocarmine solution for 5 minutes and rinse again in distilled water.
4. Place the stained tip in a drop of water on a microslide and cover with a coverslip.
5. Gently tap the root tissue with the flat end of a glass rod/ tooth pick/ match stick to produce
a squash having homogeneous cell suspension. Do not tap hard on the coverslip, it may
break the coverslip.
6. Remove the excess liquid from under the coverslip by placing tissue paper/ filter paper
over the coverslip and press gently with index finger. Seal edges with glycerin/ liquid
paraffin.
7. Examine under the microscope.

OBSERVATION
Count the number of mitotic and interphase cells under the microscope at 10 X 40 magnification
from at least three different places.

No. of cells

Cell Type Site I Site II Site III Total

Mitotic Cells

Interphase Cells

CALCULATION

41
 No. of mitotic cells
Mitotic Index = = Frequency of mitotic cell
Total no. of cells counted
Duration of Mitosis = Mitotic Index x Duration of Cell Cycle

(Given that cell cycle duration = 19 hours)

RESULTS
Mitotic Index =
Mitotic Duration =

OBJECTIVE II
To observe various stages of mitosis through readymade slides.

MARKS OBTAINED

SIGNATURE OF THE INSTRUCTOR

42
 Experiment- 10
OBJECTIVE
(i) Measurement of Hemoglobin content in the human blood
(ii) Determination of Blood group and Rh status

THEORY
The blood serves as principal transport medium of the body, carrying oxygen, nutrients and
chemical messages to the tissues and waste products and synthesized metabolites away. It carries
the hormones for activation of various tissues, aids in defense of the body, maintenance of osmotic
pressure, pH, and temperature equilibrium in tissues of the body. Thus blood plays important role
in coordinating the individual cells into a whole complex organism.
The amount of oxygen that can be carried in simple solution is small, however and many highly
organized animals (vertebrates almost without exception) have blood that can bind large quantities
of oxygen reversibly, thus greatly enhancing the amount of oxygen carried.
In mammalian blood, the amount of physically dissolved oxygen is about 0.2 ml O2 per 100ml
blood, and the amount bound reversibly to hemoglobin; the red respiratory pigment, is up to some
100 times as great, about 20ml O2 per 100ml blood.
Hemoglobin is the most widespread and best known respiratory pigment. It consists of a protein
molecule associated with a four-member ring structure known as a porphyrin.
The iron that each hemoglobin molecule contains is bound to porphyrin with one atom of divalent
iron (Fe2+) attached to each porphyrin unit.

All mammalian hemoglobin’s have molecular weights of approximately 65KDa, and are tetramers
of peptide chains, to each of which is bound a heme. Normal human beings synthesize and
incorporate into hemoglobin four distinct but related polypeptide chains as α, β, γ and δ. But with
few exceptions, hemoglobin molecules are constructed by combining two α chains with two β, γ
or δ chains.

43
When the hemoglobin in blood is treated with N/10 HCl, the hemoglobin is converted into
hematinic acid (brown in color) which is compared with standard hematin by the hemometer. The
percentage of hemoglobin in the RBC under normal conditions is almost constant. In a few diseases
such as anaemia, the haemoglobin percentage declines due to loss of haemoglobin from the RBC
or by disintegration of RBC themselves. In men the haemoglobin is about 15.8 gms while in
women it is approximately 13.7 gms in 100ml of blood.

REQUIREMENTS
1. Sahils hemoglobinometer or Gower’s Haldane hemoglobinometer or hemometer.
This instrument consists of –
a) Two tubes which have standard color for comparing the tinge of human blood hemoglobin
solution or acid hematin inside the hemoglobin tube. The hemoglobin tube is graduated on two
sides. On one side it indicates percentage on other side it gives gm percentage.
b) The pasture pipettes which is marked at 20 cm almost in the middle and is connected to a
sucking tube.

2. Dropper
3. N/10 HCl solution
4. Distilled water
5. Fresh blood

PROCEDURE
1. All the apparatus should be clean and dry before use for the estimation of hemoglobin.
2. Take hemoglobin tube; fill it by N/10 HCl upto 2 cm mark on gram percentage side.
3. Prick your left hand finger by a sterilized needle /lancet after cleaning with spirit or 70%
alcohol and suck the blood in Pasteur pipette upto 20 cm mark, wipe off the excess blood
attached to tip of the pipette.
4. Dip the pipette tip in the N/10 HCl of hemoglobin tube and discharge the blood into it.
5. Shake it thoroughly with a glass rod. Observe the color of the solution brown.
6. Add distilled water drop by drop to solution until the color of the blood solution resembles
perfectly with the color of the standard tubes. The color can be compared by keeping the
hemoglobin tube in the hemometer between two standard color tubes (space provided).
7. Remove the hemoglobin tube from the hemometer and note the reading in gram percentage
or percentage directly.
8. Handling of blood should only be done by the instructor and as per norms prescribed by
the institute.
OBSERVATION
The reading should be taken only when the color of the blood solution resembles perfectly with
the color of standard tubes of hemoglobinometer.

RESULT
Hemoglobin content of the blood sample is ___________ gm /dl.

44
 Human ABO Blood Typing

THEORY
The biological uniqueness that each individual attains is frequently noted in the reactions that occur
when it receives biological material from other organisms. In animals the tissues that are removed
from one individual and grafted to another one are frequently sloughed off or rejected because of
incompatibility between the introduced material and host. ABO blood typing is an excellent
example of the serological principle of agglutination. Around the turn of century it was determined
by Karl Landsteiner that there were four different immunological human blood types. This theory
was based on the fact that two distinct antigens (agglutinogens), A and B, could be present on the
surface of RBC’s. Depending on the presence or absence of either or both the antigens, blood types
were established: A, B, AB or O. They make up the ABO classification system as illustrated in the
table-

RBC antigen Plasma antibodies Blood group

(agglutinogen) (agglutinins)
A Anti –B A

B Anti -A B

A and B None AB

None Anti –A and Anti-B O

Of medical importance is the fact that the fluid portion of the blood, the plasma may contain
antibodies (agglutinins). If present these antibodies are not reactive against the individual’s own
RBC’s. When mixed with the RBC antigens of a different blood type, however as during the
course of a blood transfusion, a violent, incompatible agglutination reaction may result. Thus
ABO blood typing is a routine prerequisite to blood transfusions.

Rh FACTOR IN HUMANS
K. Landsteiner and S. Weiener discovered the Rh factor in 1940 from rabbits immunized with
the blood of the monkey Macaca rhesus. The resulting antibodies were found to agglutinate not
only the RBC’s of monkey but those of the high percentage of human populations also.
Individuals whose blood cells react with Rh antibody are termed as Rh positive; those who do
not react are termed Rh negative. The symbol Rh came from the first two letters of the species
name of the monkey. A test for Rh incompatibility is accomplished by placing a drop of blood
from the subject on a slide and introducing anti-Rh serum. Agglutination of erythrocytes
indicates incompatibility, whereas an even distribution of erythrocytes indicates no reaction.
The original antigen, now symbolized Rh is highly antigenic to humans. Thus, cross matching
of Rh factor, as well as ABO types of donor and recipient blood is now used to avoid
incompatibility agglutination reactions following transfusions. Blood is frequently exchanged
between the mother and the fetus during childbirth. Thus, Rh negative mothers are often

45
immunized by blood from Rh positive fetuses (which may result when fathers are Rh positive)
to which they gave birth. Usually no ill effects are associated with the exposure of the mother to
the Rh- positive antigen during the first childbirth (unless the mother has been exposed to Rh
antigen by transfusion). Subsequently Rh- positive children carried by the same mother against
the Rh antigen, which are carried across the placenta in blood serum. Such children may develop
symptoms of hemolytic jaundice and anemia, a condition referred to as erythroblastosis fetalis.
The symptoms may be mild or severe, even resulting in the death of the fetus or new born infants
if appropriate steps are not taken by the physician.
In the experiment to follow, we need to perform an ABO typing procedure by separately mixing
a drop of their blood with anti-A, anti-B and anti-D-sera on a glass slide. The determination of
the blood type is made by observing for agglutination on the slide preparation as illustrated in
the figure

Anti A Anti B Anti D

Group A+

REQUIREMENTS
1. Anti-A, Anti-B and Anti-D blood typing sera and 70% alcohol.
2. Microscopic slides, sterile blood lancets, absorbent cotton, wooden applicator sticks and
wax pencils.

PROCEDURE
1. Using a wax pencil, divide a microscopic slide in half. Label one as anti-A and the other
one as anti-B.
2. Take another slide and mark it as anti-D.
3. Place one drop of each antiserum on the appropriately labelled section of the slides.
4. Using a piece of absorbent cotton moistened with 70% ethyl alcohol, wipe the tip of the
middle finger.
5. Using a sterile bold lancet, prick the disinfected area of the finger.
6. Allow one drop of blood to flow into each of antiserum on the slides.
7. With separate applicator sticks, mix each drop of blood with its respective antiserum.
8. Rock the slide between your fingers in a to and fro motion and observe both mixtures
for one minute for clumping (agglutinations).
9. Observe the slides under the microscope.

NOTE: Handling of blood should only be done by an authorized instructor and as per norms
prescribed by the institute. Appropriate care should be taken for waste disposal.

46
OBSERVATION
In the following diagram, draw the observed antibody response against antigens A, B and D.
Anti-A Anti-B Anti-D

RESULTS
1. Determine and indicate the ABO blood type
2. Indicate the agglutinogen present
3. Indicate the agglutinin present
4. Determine and indicate the Rh type

MARKS OBTAINED

SIGNATURE OF INSTRUCTOR

47
 Experiment - 11

OBJECTIVE
To study the factors affecting the rate of photosynthesis in living leaves

THEORY
Photosynthesis fuels ecosystems and replenishes the Earth’s atmosphere with oxygen. Like all
enzyme-driven reactions, the rate of photosynthesis can be measured by either the disappearance
of substrate or the accumulation of product (or by-products).The general summary equation for
photosynthesis is
2 H2O + CO2 + light → carbohydrate (CH2O) + O2 + H2O
What could you measure to determine the rate of photosynthesis?
• Production of O2 (How many moles of O2 are produced for one mole of sugar synthesized? OR
• Consumption of CO2 (How many moles of CO2 are consumed for every molecule of sugar
synthesized?
In this experiment, you will use a system that measures the accumulation of oxygen.

Because the spongy mesophyll layer of leaves

(shown in Figure 1) is normally infused with
gases (O2 and CO2), leaves or disks cut from
leaves normally float in water. When immersed
in water, oxygen bubbles are usually trapped in
the air spaces of the spongy mesophyll in the
plant leaf. By creating a vacuum in this
experimental procedure, the air bubbles can be
drawn out of the spongy mesophyll, and the
space is refilled by the surrounding solution. This
allows the leaf disks to sink in the experimental
solution. If the leaf disk is placed in a solution
with an alternate source of carbon dioxide in the
form of bicarbonate ions, then photosynthesis can occur in a sunken leaf disk. In presence of
bicarbonate ions and enough light, the leaf disk will begin to produce sugars and oxygen through
the process of photosynthesis. Oxygen accumulates in the mesophyll layer of leaf as
photosynthesis progresses, causing the leaf disks to float again. The time it takes for leaf disks to
float again is a measure of the net rate of photosynthesis.

MATERIALS
Sodium bicarbonate, Liquid soap (~ 5 mL 250 mL of water), 2 plastic syringes without needle (10
mL), Living leaves (spinach etc.), Hole punch, beakers, Timer, Light source

PROCEDURE
1. Prepare 300 mL of 0.2% bicarbonate solution for each experiment. The bicarbonate will
serve as a source of carbon dioxide for the leaf disks while they are in the solution.

48
 2. Pour the bicarbonate solution into a clear beaker to a depth of about 3 cm. Label this beaker
“With CO2.” Fill a second beaker with only water to be used as a control group. Label this
as “Without CO2.”
3. Using a pipette, add one drop of a dilute liquid soap solution to the solution in each beaker.
The soap acts as a surfactant or “wetting agent” — it wets the hydrophobic surface of the
leaf, allowing the solution to be drawn into the leaf and enabling the leaf disks to sink in
the fluid.
4. Using a hole punch, cut 10 or more uniform leaf disks for each beaker. Avoid major leaf
veins. (The choice of plant material is perhaps the most critical aspect of this procedure.
The leaf surface should be smooth and not too thick.)
5. Draw the gases out of the spongy mesophyll tissue and infiltrate the leaves with the sodium
bicarbonate solution by performing the following steps:
a. Remove the piston or plunger from both syringes. Place the 10 leaf disks into each
syringe barrel.
b. Replace the plunger, but be careful not to crush the leaf disks. Push in the plunger until
only a small volume of air and leaf disk remain in the barrel (<10%).
c. Pull a small volume of sodium bicarbonate plus soap solution from your prepared beaker
into one syringe and a small volume of water plus soap into the other syringe. Tap each
syringe to suspend the leaf disks in the solution. Make sure that, with the plunger
inverted, the disks are suspended in the solution. Make sure no air remains. Move the
plunger to get rid of air from the plunger before you attempt Step d.
d. Now create a vacuum in the plunger to draw the air out of the leaf tissue. Create the
vacuum by holding a finger over the narrow syringe opening while drawing back the
plunger. Hold this vacuum for about 10 seconds. While holding the vacuum, swirl the
leaf disks to suspend them in the solution. Now release the vacuum by letting the plunger
spring back. The solution will infiltrate the air spaces in the leaf disk, causing the leaf
disks to sink in the syringe. If the plunger does not spring back, you did not have a good
vacuum, and you may need a different syringe. You may have to repeat this procedure
two to three times in order to get the disks to sink.
6. Pour the disks and the solution from the syringe into the appropriate clear beaker. Disks
infiltrated with the bicarbonate solution go in the “With CO2” beaker, and disks infiltrated with
the water go in the “Without CO2” beaker.
7. Place both beakers under the light source and start the timer. At the end of each minute, record
the number of floating disks. Then swirl the disks to dislodge any that stuck against the side of
beakers. Continue until all of the disks are floating in “With CO2” beaker.
8. Record your observation.

49
OBSERVATION

RESULT

MARKS OBTAINED

SIGNATURE OF INSTRUCTOR

50
 BIBLIOGRAPHY

1. Cappuccino, J.G. and Sherman, N. 1983. Microbiology: A laboratory Manual, Addison –

Weslay Publishing Company, California.
2. Enger, E.D., Ross, FC. And David B.B. 2011. (13th Edition- BITS Pilani Custom Edition)
Concepts in Biology, McGraw Hill Publishers, New York.
3. Simon, E.J., J.L.Dickey, K.A. Hogam and J.B. Reece. 2016. (5th Edition). Essential
Biology with Physiology. Pearson India Education Sercices Pvt. Ltd., Noida.
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Publishers, USA.
6. Moore, T.C. 1974. Research Experiments in Plant Physiology – A Laboratory Manual,
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7. Plummer, D.T. 1982. An introduction to Practical Biochemistry (2nd Edition,), Tata
McGraw Hill Publishing Company Ltd., New Delhi.
8. Raven, P.H. and Johnson, G.B. 1999, Biology (5th Edition), WCB/McGraw Hill Publishers,
USA
9. Sadava, D.E. 1993, Cell Biology- Organelle Structure & Function, Johnes and Barlet
Publishers.
10. Salisbary, F.B. and Ross, C. 1969, Plant Physiology, Wadsworth Publishing Company,
California
11. White, A., Handler, P. and Smith, E.L. 1973, Principles of Biochemistry (5th Edition)
McGraw – Hill Kogakusha Ltd., London
12. Witham, F.H., Blaydes, D.F. and Devlin, R.M. 1971, Experiments in Plant Physiology.
Van Nostrand Reinhold Company Ltd., Canada.
13. Godkar, P.B. and Godkar,D.O. 2005 (2nd Edition). Textbook of Medical Laboratory
Technology. Bhalani Publishing House, India.
14. Wilson, K. and Walker, J. (5th Edition) Practical Biochemistry: Principles and Techniques.
Cambridge University Press.

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NOTES

52