Bsczo 303

BSCZO-
BSCZO 303
B. Sc. III YEAR

BIOSTATISTICS, INSTRUMENTATION AND
TECHNIQUES
DEPARTMENT OF ZOOLOGY
SCHOOL OF SCIENCES
UTTARAKHAND OPEN UNIVERSITY
BSCZO-303
BIOSTATISTICS, INSTRUMENTATION AND

TECHNIQUES
DEPARTMENT OF ZOOLOGY
SCHOOL OF SCIENCES
Phone No. 05946-261122, 261123
Toll free No. 18001804025
Fax No. 05946-264232, E. mail [email protected]
htpp://uou.ac.in
MEMBER OF THE BOARD OF STUDIES
Prof.P.D.Pant Prof. B.D.Joshi

Director I/C School of Sciences Retd.Prof.
Uttarakhand Open University Gurukul Kangri University, Haridwar.
Prof. H.C.S.Bisht Prof. H.C.Tiwari

Department of Zoology Retd. Prof. & Principal
DSB Campus, Kumaun University, Haldwani Nainital.
Nainital.
Dr.N.N.Pandey Dr.Shyam S. Kunjwal

Principal Scientist, Department of Zoology
Directorate of Coldwater Fisheries (DCFR) Uttarakhand Open University
Indian Council of Agriculture Research (ICAR) Haldwani (Nainital)
Bhimtal (Nainital).
PROGRAMME COORDINATOR
Dr.Shyam S. Kunjwal
Department of Zoology
School of Sciences
Uttarakhand Open University
Haldwani (Nainital).
Unit Writer Unit no.

1. Dr.Lakhan Singh Unit: 1, 2&3
Assistant Professor
HAPPRC
HNB Garhwal University,
Uttarakhand
2. Dr.Harish Chandra Unit: 4, 5& 6

Assistant Professor
HAPPRC
HNB Garhwal University,
Uttarakhand
COURSE EDITOR
Dr.Anju Thapliyal Dr.Shyam Kunjwal

Department of Zoology Department of Zoology
BGR Campus Pauri Uttarakhand Open University
Course Title and Code : Biostatistics, Instrumentation and Techniques (BSCZO303)

ISBN No. : 978-93-90845-33-0
Copyright : UTTARAKHAND OPEN UNIVERSITY
Edition : 2019
Published by : UTTARAKHAND OPEN UNIVERSITY, HALDWANI, NAINITAL- 263139
Printed by :
CONTENTS
COURSE 1: BIOSTATISTICS, INSTRUMENTATION AND TECHNIQUES
COURSE CODE: BSCZO303 CREDIT: 3
Unit Block and Unit title Page

number number
BLOCK I: BIOSTATISTICS
1 Data collection and presentation 1-40
2 Measures of central tendency 41-74
3 Measures of Variability/ Dispersion 75-109
BLOCK II: INSTRUMENTATION AND

TECHNIQUES
4. Principles and uses of analytical instruments 110-133
5. Microtomy and Microscopy 134-148
6. Separation techniques and cryopreservation 149-169

BIOSTATISTICS, INSTRUMENTATION AND TECHNIQUES BSCZO303
UNIT-1 DATA COLLECTION AND PRESENTATION
CONTENTS
1.1- Objectives
1.2- Introduction
1.3- Definitions of Statistics and biostatistics
1.3.1- Definitions of statistics
1.3.2- Definitions of biostatistics
1.4- Statistical symbol
1.4.1- Statistical symbol
1.4.2- Scope of Biostatics
1.4.3- Applications of biostatistics
1.5- Data and its types
1.5.1- Data
1.5.2- Types of data
1.5.2.1- Nominal data
1.5.2.2- Ordinal data
1.5.2.3- Ranked data
1.5.2.4- Discrete data
1.5.2.5- Continuous data
1.6- Data collection and related terms
1.6.1- Population, sample, sampling unit and sampling frame
1.6.1.1- Population
1.6.1.2- Sample
1.6.1.3- Sampling unit
1.6.1.4- Sampling frame
1.6.2- Principle steps in a sample survey
1.6.3- Sampling and non sampling errors
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1.6.3.1- Sampling errors

1.6.3.2- Non-sampling errors
1.7- Types of sampling schemes
1.7.1- Subjective or purposive or judgment sampling
1.7.2- Probability sampling
1.7.2.1- Simple random sampling
1.7.2.1.1- Selection of a simple random sample
1.7.2.2- Stratified random sampling
1.7.3- Mixed sampling
1.8- Organization and representation of data
1.8.1- Organization of data
1.8.2- Representation of data
1.8.2.1- Bar diagram
1.8.2.2- Pie chart
1.8.2.3- Histogram
1.8.2.4- Frequency polygon and frequency curve
1.8.2.5- Pictograms
1.8.2.6- Line chart
1.8.2.7- Cumulative frequency curve or ogive
1.8.2.8- Scatter diagram
1.9- Summary
1.10- Glossary
1.11- Self assessment question
1.12- References
1.13-Terminal Questions
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1.1 OBJECTIVES
Following are the objectives of this chapter:
1. To know the definitions of Statistics and Biostatistics.
2. To know about some statistical symbols.
3. To know the scope and applications of Biostatistics.
4. To know about data, its collection and collection techniques.
5. To know about organization and representation of data by many graphical techniques

such as histogram, pie chart, frequency polygon etc.
1.2 INTRODUCTION
We welcome the reader who wishes to learn biostatistics. In this chapter we introduce
you to the subject. First of all we define statistics and biostatistics and then examples are
given where bio- statistical techniques are useful. These examples show that biostatistics has
an importance in advancing our biological knowledge; biostatistics helps to evaluate many
life-and-death issues in medicine.
We advise you to read the examples carefully and then think yourself, “What can be
inferred from the information presented?” What would you do with the data after they are
collected? How can it be presented and what you can get from it? We want you to realize
that biostatistics is a tool that can be used to benefit you and society.
There is no royal road to biostatistics. You need to be involved. You need to work
hard. You need to think. If you analyze the actual data, the result will be a powerful tool that
has immediate practical uses. Our main purpose is to develop thought patterns in your mind
that are useful in evaluating information in all areas of your life.
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1.3 DEFINITIONS OF STATISTICS AND BIOSTATISTICS

Much of the joy and pain in life arises in situations that involve considerable
uncertainty. Here we are giving two situations which show that the study of statistics and
biostatistics is necessary.
1. Parents of a child with a genetic defect consider whether or not they should have another
child. They will base their decision on the chance that the next child will have the same
defect.
2. To choose the best therapy, a physician must compare the diagnosis or future course, of a
patient under several therapies. A therapy may be a success, a failure, or somewhere in
between; the evaluation of the chance of each occurrence necessarily enters into the decision.
1.3.1 DEFINITION OF STATISTICS
Statistics is the science which deals with the collection, classifying, presenting, comparing
and interpreting numerical data collected to throw light on any sphere of enquiry- Lovitt.
The science of statistics is a most useful servant, but only of great value to those who
understand its proper use- W.I.King. Statistics provides tools and techniques for research
workers- A.M. Mood. Planning is the order of the day and without statistics planning is
inconceivable- L.H.C. Tippet.
Statistics may be defined as a science of numerical information which employs the process of
measurement and collection, classification, analysis, decision making and communication of
results in a manner understandable and verifiable by other- Cecil H. Meyers
1.3.2 DEFINITION OF BIOSTATISTICS
Biostatistics is the application of statistics methods applied to biological areas.

Biological laboratory experiments, medical research (including clinical research), and health
services research all use statistical methods. Many other biological disciplines rely on
statistical methodology.
There are three reasons for focusing on biostatistics:

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1. Some statistical methods are used more deeply in biostatistics than in other fields. For
example, a general statistical textbook would not discuss the life-table method of analyzing
survival data of importance in many bio-statistical applications. The topics in this book are
adapted to the applications in mind.
2. Examples are drawn from the biological, medical, and health care areas; this helps you
maintain motivation. It also helps you in understanding how to apply statistical methods.
3. A third reason for a book on biostatistics is to teach the material to the audience of health
professionals. In this case, the interaction between students and teacher, but especially among
the students themselves, is of great value in learning and applying the subject matter.
1.4 STATISTICAL SYMBOL
1.4.1 STATISTICAL SYMBOL
Some of the statistical symbols which are useful to biostatistics students are:
f: Frequency of the variate
x : Arithmetic Mean of a given set of values or of a distribution
M e : Median of a given set of values or of a distribution
M o : Mode of a given set of values or of a distribution
σ : Standard Deviation of a given set of values or of a distribution
σ 2 : Variance of a given set of values or of a distribution
Σ : Sum of all the values of a given set
Q.D.: Quartile deviation of a given set of values or of a distribution
M.D.: Mean deviation of a given set of values or of a distribution
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1.4.2 SCOPE OF BIOSTATISTICS
Biostatistics is the application of statistics in different fields of biology. The science

of biostatistics includes the design of biological experiments, especially in medicine,
pharmacy, agriculture, forestry, environmental science, fishery etc; the collection,
summarization, and analysis of data from those experiments; and execute interpretation and
inference from the results. A major branch of this is medical biostatistics, which is
exclusively concerned with health and medical sciences.
In current world, the scope of biostatistics is increasing rapidly. If we discuss about

biostatistics, we see that almost all educational programmes in biostatistics are at
postgraduate level. They are most often found in schools of public health, affiliated with
schools of medicine, forestry, or agriculture, or as a focus of application in departments of
statistics.
In larger universities where both a statistics and a biostatistics department exist, the
degree of integration between the two departments may range from the bare minimum to
very close collaboration. In general, the difference between a statistics program and a
biostatistics program is twofold: (i) statistics departments will often host
theoretical/methodological research which are less common in biostatistics programs and (ii)
statistics departments have lines of research that may include biomedical applications but
also other areas such as industry (quality control), business and economics and biological
areas other than medicine
There is a special need of the subject bio statistics because it related with such areas
as medical , pharmacy, forestry, agriculture, etc, which are very necessary for the betterment
of society.
1.4.3 APPLICATION OF BIOSTATISTICS
The importance and application of statistics in the field of biology is increasing day
by day. Why it is so? The reason is that in biology the interplay of casual and response
variables follow the laws that are not in the classic mold of 19th century physical science. In
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that century, biologists such as Robert Mayer, Helmholtz, and others in trying to show that
biological process were nothing but physicochemical phenomena, helped create the
impression that the experimental methods and natural philosophy that had led to such
dramatic progress in the physical sciences should be imitated fully in biology.
Many biologists even to this day have retained the tradition of strictly mechanistic
and deterministic concepts of thinking, while physicists, as their science became more
refined and came to deal with ever more elementary particles, began to resort to statistical
approaches. In biology most phenomena are affected by many casual factors, uncontrollable
in their variation and often unidentifiable. Statistics is needed to measure such variable
phenomena with a predictable error and to ascertain the reality of minute but important
differences.
A Biostatistics centre could jointly organize working groups, the seminar series,
computing infrastructure and possibly consulting and clinical trials coordinating centre
cervices. The main objective of the centre would be to estimate, collaborate on, and circulate
results of research in a particular subspecialty in the following reasons:
1. Statistical methods for longitudinal studies;

2. Statistical genetics;
3. Foundations of inference;
4. Bayesian biostatistics
5. Biostatistician practice and education.
The most critical short term problem in the field of biostatistics is the information
system. We need to incorporate modern, web-based technologies into the everyday
workings of the department of biostatistics. We need reliable and accessible systems that
are competitive with those available to departments of statistics and biostatistics. We
likely build collaborations with computer science students.
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1.5 DATA AND ITS TYPES
1.5.1 DATA
The information collected from census or surveys or from other sources is called raw data.
The word data means information. The adjective raw attached to data indicates that the
information collected cannot be used directly. It has to be converted into more suitable form
before it begins to make sense to be utilized gainfully. Raw data is like raw rice. Raw rice has
to be cooked properly and tastefully before it is eaten and digested. Similarly, raw data has to
be converted into proper form such as tabulation, frequency distribution form, etc, before any
inference is drawn from it.
There are two ways of statistical data;
1. Primary data 2. Secondary data
Primary data: It is the data collected by some person or organization for his own use from
any primary source. For example the data of census report collected by the centre
government, the data collected by any agency for its own purpose, the gadget of India, etc.
Secondary data: It is the data collected by some other person or organization for their own
use but the investigator also gets it for his own use. For example the data collected by any
medical agency can be used by some other medical institute students.
In other words, primary data are those data which are collected by you to meet your own
specific purpose where as the secondary data are those data which are collected by somebody
else. A data can be primary for one purpose and secondary for the other.
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1.5.2 TYPES OF DATA

The primary data are of the following types:
1.5.2.1 Nominal Data
In the study of biostatistics, we meet many different types of numerical data. The different
types have varying degrees of structure in the relationships among possible values. One of
the simplest types of data is nominal data, in which the values fall into unordered categories
or classes. In a certain study, for instance, males might be assigned the value 1 and females
the value 0. Numbers are used mainly for the sake of convenience; numerical values allow us
to use computers to perform complex analysis of the data. Nominal data that take on one of
two distinct values-such as male and female are said to be dichotomous or binary,
depending on whether the Greek or the Latin root for two is preferred. However, not all
nominal data need be dichotomous. Often there are three or more possible categories into
which the observations can fall. For example, persons may be grouped according to their
blood type, such that 1 represents type O, 2 is type A, 3 is type B, and 4 is type AB.
1.5.2.2 Ordinal Data
When the order among categories becomes important, the observations are referred to as
ordinal data. For example, injuries may be classified according to their level of severity, so
that 1 represents a fatal injury, 2 is severe, 3 is moderate, and 4 is minor. Here a natural order
exists among the groupings; a smaller number represents a more serious injury. A second
example of ordinal data is Eastern Cooperative Oncology Group's classification of patient
performance status.
Status 0: Patient fully active, able to carry on all predisease performance without restriction.
Status 1: Patient restricted in physically strenuous activity but ambulatory and able to carry
out work of a light or sedentary nature.
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Status 2: Patient ambulatory and capable of all self-care but unable to carry out any work
activities; up and about more than 50% of waking hours.
Status 3: Patient capable of only limited self-care; confined to bed or chair more than 50% of
Waking hours.
Status 4: Patient completely disabled; not capable of any self-care; totally confined to bed.
1.5.2.3 Ranked Data
In some situations, we have a group of observations that are first arranged from highest to
lowest according to magnitude and then assigned numbers that correspond to each
observation's place in the sequence. This type of data is known as ranked data. As an
example, consider all possible causes of death in the India. We could make a list of all of
those causes, along with the number of lives that each one claimed in. If the causes are
ordered from the one that resulted in the greatest number of deaths to the one that caused the
smallest and then assigned consecutive integers, the data are said to have been ranked.
1.5.2.4 Discrete Data
For discrete data, both ordering and magnitude are important. In this case, the numbers
represent actual measurable quantities rather than mere labels. In addition, discrete data are
restricted to taking on only specified values-often integers or counts-that differ by fixed
amounts; no intermediate values are possible. Examples of discrete data include the number
of motor vehicle accidents in Dehradun in a particular month, the number of times a woman
has given birth, the number of new cases of tuberculosis reported in the India during a one-
year period, and the number of beds available in a particular hospital. Note that for discrete
data a natural order exists among the possible values. If we are interested in the number of
times a woman has given birth, for instance, a larger number indicates that a woman has had
more children. Furthermore, the difference between one and two births is the same as the
difference between four and five births. Finally, the number of births is restricted to the
nonnegative integers; a woman cannot give birth 3.4 times because it is meaningful to
measure the distance between possible data.
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1.5.2.5 Continuous Data
Data that represent measurable quantities but are not restricted to taking on certain specified
values (such as integers) are known as continuous data. In this case, the difference between
any two possible data values can be arbitrarily small. Examples of continuous data include
time, the serum cholesterol level of a patient, the concentration of a pollutant, and
temperature. In all instances, fractional values are possible. Since we are able to measure the
distance between two observations in a meaningful way, arithmetic operations can be
applied. The only limiting factor for a continuous observation is the degree of accuracy with
which it can be measured; consequently, we often see time rounded off to the nearest second
and weight to the nearest pound or gram. The more accurate our measuring instruments,
however, the greater the amount of detail that can be achieved in our recorded data.
In a study of the effects of maternal smoking on newborns, for example, we might first
record the birth weights of a large number of infants and then categorize the infants into three
groups: those who weight less than 1500 grams, those who weight between 1500 and 2500
grams, and those who weight more than 2500 grams. Although we have the actual measures
of birth weight, we are not concerned with whether a particular child weighs 1560 grams or
1580 grams; we are only interested in the number of infants who fall into each category.
From prior experience, we may not expect substantial differences among children within the
very low birth weight, low birth weight, and normal birth weight groupings. Furthermore,
ordinal data are often easier to handle than continuous data and thus simplify the analysis.
There is a consequent loss of detail in the information about the infants, however. In general,
the degree of precision required in a given set of data depends on the questions that are being
studied.
As we progressed, the nature of the relationship between possible data values became
increasingly complex. Distinctions must be made among the various types of data because
different techniques are used to analyze them. It does not make sense to speak of an average
blood type of 1.8; it does make sense, however, to refer to an average temperature of 4.55°C.
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1.6 DATA COLLECTION AND RELATED TERMS
1.6.1 POPULATION, SAMPLE, SAMPLING UNIT AND SAMPLING
FRAME
1.6.1.1 Population
Population is a collection of units or objects of which some property is defined for

every unit or object. Population may consist of finite or infinite number of units. Population
is also called universe by a number of statisticians and scientists.
The inhabitants of a region, number of wheat fields in a state or district, fruit plants in
a city, number of students in a institution, insects in a field, persons suffering from any
particular disease, workers in a institution, total no of person in city, total households, total
no of students in any university, are a few examples of finite populations. All real numbers,
all stars in the sky are examples of infinite populations. Generally, the population has a large
number of animates and inanimates. Moreover, the units or subjects constituting the
population may vary from survey to survey in the same region of activity depending upon the
aims and objective of the survey.
In brief, one should very well keep in mind that statistical population is not only the human
population which is usually considered in literary sense. It is generally a group or collection
of items specified by certain characteristics or defined under certain restrictions.
1.6.1.2 Sample
A sample is the portion of the population that is examined to make inferences about the
population or a part or fraction of population, which represent it, is known as sample. Sample
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consists of a few items of the popul

population.
ation. In principle a sample should be such that it is a true
representative of the population.
If poll stars are trying to take the pulse of the nation prior to an election their target
population consists of those who will go to the polls and vote, wher
whereas
eas those whose opinions
they actually obtain constitute a sample of that population.
1.6.1.3 Sampling Unit
The constituents of a population which are the individuals to be sampled from the population
and cannot be further subdivided for the purpose of sampling at a time are called sampling
units. For example to know the average income per family, the head of the family is a
sampling unit. To know the average marks in a paper of a class a single student is a unit.
1.6.1.4 Sampling Frame
For accepting anyy sampling procedure it is necessary to have a list or a map

identifying each sampling unit by a number. Such a list or map is called sampling frame. For
example, a list of students of a class, a list of patients of a particular disease a list of workers
inn a factory, a list of voters, a list of staff members of a college, a list of villages in a district,
etc., are a few examples of sampling frame.
Figure- 1.1
.1 Population v/s Sample
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1.6.2 PRINCIPLES STEPS IN A SURVEY
The main steps in the planning and execution of a survey are as follows:
1. Objective of the survey. The first step is to define clearly the objective of the survey.
It is generally found that even the sponsoring agency is not quite clear in its thinking as to
what it wants and how it is going to use the results. The sponsors of the survey should take
care that the objectives should be fulfilled with the available resources presented in the form
of time, money and manpower.
2. Defining the population to be sampled. The population from which the sample is to
be taken should be defined clearly. For example in sampling of farms clear-cut rules must be
framed to define a farm in respect of shape, size, etc., keeping in mind the border- line cases
so as to enable the investigating person to decide whether to include or not a particular farm
in the population.
3. The frame and sampling unit. The sampling units must cover the entire population
and they must be distinct, obvious and non-overlapping in the sense that every element of the
population belongs to one and only one sampling unit. For example, in socio-economic
survey for selecting people in a town, the sampling unit might be an individual person, a
family, a household or a block in a locality.
In order to cover the population decided upon, there should be a list, map or some other
acceptable material, called the frame, which serves as a guide to the to the population to be
covered. The construction of the frame is one of a main problem since it is the frame which
determines the structure of the sample survey. If the frame is not up-to-date, it should be
brought up-to-date before using it.
4. Data to be collected. The data should be collected keeping in view the objective of the
survey. The tendency should not be to collect too many data some of which are never
subsequently examined and analyzed. A practical method is to chalk out an outline of the
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tables that the survey should produce. This would help in eliminating the collection of
irrelevant information and ensure that no necessary data are omitted.
5. The questionnaire or schedule. Having decided about the type of the data to be
collected, the next important step of the sample selection is the construction of the
questionnaire (to be filled by the respondent) or schedule of enquiry (to be filled by the
interviewer) which requires skill, special technique as well as familiarity with the subject
matter. The questions should be clear, brief, non- offending, polite in tone, clear-cut and to
the point so that not much scope of guessing is left on the part of the respondent or
interviewer. Suitable and detailed instructions for filling up the questionnaire or schedule
should also be prepared.
6. Method of collecting information. The two methods commonly used to collect the
sample data are:
(i) Interview method. In this method, the investigator goes from house to house and
interviews the individuals personally. He asks the questions one by one and fills up the
schedule on the basis of the information gained from the individuals.
(ii) Mailed questionnaire method. In this method the questionnaire is mailed to the
individuals who are required to fill it up and returns it duly completed.
Whether the data should be collected by interview method or mail questionnaire method
or by physical observation has to be decided keeping in view the cost, time, accuracy and
money.
7. non – respondents. Due to practical difficulty the data cannot be collected for all the
sampled units. For example the selected respondent may not be available at his place when
the investigator goes to him or he may refuse to give certain information. This is called non-
response problem. Such cases of non-response should be handled with caution in order to
draw unbiased and valid conclusions.
8. Selection of proper sampling design. The size of the sample (n), the procedure of
selection and the estimation of parameters along with their margins of uncertainty are some
of the important problems that should be tackled carefully.
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A number of designs for the selection of a sample are available and a good selection
will guarantee good and reliable estimates but the relative time and money factors should
also be considered for adopting any sampling design.
9. Organization of field work. It is very essential that the investigator should be

trained in locating the sample units, recording the measurements, the methods of collection
of required data before starting the field work. The success of a survey to a great extent
depends upon the reliable field work. It is very necessary to make provisions for adequate
supervisory staff for inspection after field work.
10. Pretest. From practical point of view a small pre-test has been found very useful.
It always helps to decide upon effecting methods of asking questions and results in the
improvement of the questionnaire. In case of large scale surveys it provides the better idea
about the cost and time factor.
11. Summary and analysis of the data. The analysis of the data may be classified
into the following steps:
(i) Scrutiny and editing of the data. An initial quality check should be done by the
supervisory staff when the investigators are in the field. This will help in amending recording
errors or in eliminating data that are inconsistent.
(ii) Tabulation of data. Before carrying out the tabulation of the data, we should
decide the procedure for tabulation of the data which are incomplete due to non-response.
The method of tabulation, hand or machine should be depending upon the size of the data.
(iii) Statistical analysis. A properly scrutinized, edited and tabulated data now
prepared for the statistical analysis. There are different methods of estimation; therefore a
suitable formula should be used for the estimation of the parameters.
(iv) Information for future surveys. Any completed survey helps in providing a
note of caution for designing future surveys. The information in the form of the data means,
standard deviation, the nature of the variability and the cost, time, etc., are important which
are helpful for future surveys. Any completed sample survey is a lesson for future surveys in
recognizing and rectifying the mistakes committed in the post survey.
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Figure-1.2
1.2 Sampling Design Process
1.6.3
6.3 SAMPLING AND NON
NON- SAMPLING ERRORS
The errors in the collection, processing and analysis of the data are of two types:
1. Sampling error 2. Non

Non-sampling error
1.6.3.1 Sampling errors
Sampling errors arise in the collection of a sample and the reas

reason
on is because only a
small part of the population is used for getting the population parameter estimates. Therefore
these are absent in the complete enumeration. The main reasons of these errors are:
1. Faulty selection of the sample;

2. Substitution of the existing
ting unit;
3. Faulty demarcation of the sampling units;
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4. Constant error due to improper choice of the statistics for estimating the population
parameters.
1.6.3.2 Non- Sampling errors
The errors due to the inductive process of inferring about the population on the basis of a
sample, the non-sampling errors arise at the stages of observation, ascertainment and
processing the data and so are present in the complete enumeration and sample survey both.
The reasons of these errors are:
1. Faulty planning or definitions

2. Response errors
3. Non-response errors
4. Errors in coverage
5. Compiling errors
6. Publication errors
1.7 TYPES OF SAMPLING SCHEMES

The technique of selecting a sample is of fundamental importance in the theory of sampling
and usually depends upon the nature of the data and type of enquiry. The procedure of
selecting a sample may be classified into three forms:
1. Subjective or judgment sampling

2. Probability or random sampling
3. Mixed sampling
1.7.1 SUBJECTIVE OR PURPOSIVE OR JUDGMENT SAMPLING
In this scheme of sampling the sample is selected with some definite purpose in mind
of the selector and so the selection of the sampling units depends completely on the decision
of the selector. This sampling suffers from the drawback of favoritism depending the beliefs
and prejudices of the selector and so does not provide a true representative sample of the
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population. For example if the selector wants to give the picture that the standard of living
has increased in Dehradun city, he may take individuals in the sample from the posh colonies
of the city and ignore the colonies where low income group and middle class group live.
Another example, suppose a sample of TB patients has to be drawn. Since, it is not possible
to ascertain a population of TB patients, the persons turning up to TB sanitorium and having
TB are selected in the sample.
This sampling method is seldom used and cannot be recommended for general use.
However, if the selector is experienced and skilled and this sampling is carefully applied,
then judgment sampling may provide useful results. This sampling is used in the selection of-
national players of a national team and opinion surveys.
1.7.2 PROBABILITY SAMPLING
Probability sampling is the scientific method of selecting samples according to some

laws of chance in which each unit of the population has some pre-assigned probability of
being selected in the sample. The different types of probability sampling are:
(i) Where each unit has an equal chance of selection.

(ii) Where sampling units have different chances of selection.
(iii) Where chances of selection of unit is proportional to the sample size.
Some techniques which are commonly used in sampling are as follows:
1.7.2.1 Simple random sampling
This is the basic and most commonly used method of sampling. In this method each
unit of the population has an equal chance of selection in the sample.
In this method, an equal probability is attached to each unit of the population at the first
draw. It also indicates an equal probability of selection for the remaining units at the
subsequent draws.
For example, to draw a simple random sample from an outdoor patient register of the
department of obstetrics and gynecology, each entry would need to be numbered
subsequently. If you want to draw a sample of size 700 out of 3500, a list of 700 random
19 | P a g e
numbers between 1 and 3500 would need to be prepared using one of the known procedures
(described later). The 700 entries made in the register corresponding to 700 random numbers
present in the prepared list would make up the required sample.
There are two ways in simple random sampling, if the unit drawn is replaced back before
the next unit is drawing, the technique is called simple random sampling with replacement and if
the drawing units are not replaced back and the next draws are done without selected units, the
technique is called simple random sampling without replacement.
1.7.2.1.1 Selection of a simple random sample
Mainly two approaches are use to draw a simple random sample:
(a) Lottery system method

(b) Mechanical randomization or random numbers method
(a) Lottery system. This is the simplest method of selecting a random sample. The
process is given below:
Suppose for a survey we want to select (n) students out of a class of (N) students. We
assign the numbers 1 to N; one number to each student and we write these numbers on (N)
identical chits which are same in size shape and color. These chits are put in a bag and
thoroughly shuffled and then (n) chits are drawn one by one. The (n) students corresponding
to the numbers on these chits will make the required sample.
This method is quite independent of the properties of the population. Generally, in place
of chits, cards are used. This is one of the most reliable methods of selecting a random
sample.
(b) Mechanical randomization or random numbers method. The lottery method is

time consuming, if the population is large. In random numbers methods, a randomly
generated numbers’ table known as random number table is used to draw the required
sample. There are many tables of this types prepared by many professors and scientists.
These tables are so constructed that each of the digits 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 appears
same number of times independently of each other. If we want to select a sample from a
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population of size N ( ≤ 99) then the numbers can be combined two by two and we will get
pairs from 00 to 99. Similarly if N ( ≤ 999) or N ( ≤ 9999), and so on, then we combine the
numbers three by three for N ( ≤ 999) and four by four for N ( ≤ 9999), and so on. Since each
of the digits 0, 1, 2,…..,9 occurs equal number of times independently of each other, so does
each of the pairs 00 to 99 or triplets 000 to 999, or quadruplets 0000 to 9999, and so on.
The steps of drawing the random sample are as follows:
(i) Identify the N units in the population with numbers from 1 to N.

(ii) Select at random any page of the random number table and pick up the numbers
row wise or column wise or diagonal wise at random.
(iii) The population units corresponding to these selected numbers form the required
sample.
Some commonly used random number tables are:
1. Tippet’s (1927) random number table. (Tracts for computers No. 15, Cambridge
University Press).
2. Fisher and Yates (1938) Tables (in Statistical tables for Biological, Agricultural
and Medical Research)
3. Kendal and Babington Smith’s (1939) random number tables (Tracts for
computers, No. 24, Cambridge, University, Press).
4. Rand Corporation (1955) random number table. (Free Press, Illinois).
Example1. Draw a random sample (without replacement) of size 15 from a population of

size 500.
Solution First of all we identify 500 units in the population with numbers from 1 to 500.
Then we select at random any page of the random number table discussed above row wise or
column wise or diagonally, we select on by one three digited numbers, discarding the
numbers over 500, until 15 numbers below 500 are obtained. Since we are using simple
random sampling technique, the numbers selected previously will also be discarded. The 15
numbers finally, so selected will constitute the required sample. The following is an extract
from the first set of 40 four-digited numbers in Tippet’s random number tables:
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2952 6641 3992 9792 7669 5911 3170 5624
4167 9524 1545 1396 7203 5356 1300 2693
2370 7483 3408 2762 3563 1089 6913 7691
0560 5246 0112 6107 6008 8126 4233 8776
2754 9143 1405 9025 7002 6111 8816 6446
The table for the selection of 15 units of the sample is as follows:
Table -1
S. No. Random No. Unit selected/ not selected
1. 295 Unit selected. Since, 295 ≤ 500
4. 992 Discard random number. Since, 992 ≥ 500
8. 911 Unit not selected. Since, 911 ≥ 500
11. 244 Unit selected. Since,244 ≤ 500
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Starting with first number and moving row-wise, the units in the population with the
numbers:
295, 266, 413, 279, 317, 056, 244, 167, 415, 451,
396, 353, 300, 269, will be the 15 units selected in the required
sample.
Figure-1.3Simple random sampling
1.7.2.2 Stratified random sampling
Stratified random sampling comes under the category of restricted sampling. When
the population is heterogeneous with respect to some major characteristics, applying simple
random sampling directly is not suitable. In such a situation, first of all the population is
divided into homogeneous groups under certain criteria. These groups are termed as strata or
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stratum. From each stratum independent, independent samples are selected using any of the
known sampling methods. If the sample selection is carried out using simple random
sampling in each stratum, the sample design is called stratified random sampling.
Information about each individual sampling unit is rarely available. Hence, the strata
are formed on some broad basis such as localities in a city, districts in a state, etc. If the
population is heterogeneous, stratified random sampling is more efficient. This is because a
large sample is necessary to get an estimate of a characteristic with the same precision, if we
ignore stratification. To be more specific, if every person in the population has the same
hemoglobin level, and then a sample of even one individual would be enough to get a precise
estimate of the average hemoglobin level. Let us clarify it further: in stratified random
sampling, sapling units within each stratum have similar characteristic (e.g., hemoglobin
levels) but different from those in other strata (e.g., disease status). In such a case, only a
small sample from each stratum may provide a precise estimate of the hemoglobin level for
that stratum. The estimates obtained for each stratum may be combined to get a precise
estimate of hemoglobin levels for the population. A simple random sampling approach to the
entire population without stratification would require comparatively large sample size than
the total of stratum-specific samples to obtain an estimate of hemoglobin level with the same
level of precision.
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Figure-1.4Stratified random Sampling
1.7.3 MIXED SAMPLING
If the samples are selected partly according to some laws of chance and partly
according to a fixed sampling rule, i.e., there is no involvement of probabilities, they are
termed as mixed samples and the technique of selecting such samples is known as mixed
sampling.
1.8 ORGANIZATION AND REPRESENTATION OF DATA
1.8.1 ORGANIZATION
Data collected as such do not give any meaning. This is divided according to its type
explained in 1.5 and then it is consolidated by way of tabulation. Rearrangements and
grouping according to requirement and standards are done, thus summarizing tables from
data tabulation, which give meaning to the information collected. Data are tabulated by (1)
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manual procedure (2) Mechanical procedure (3) Computer feeding. IN preparation of tables
following principles are followed:
(i) A rough draft of the table should be prepared first. Before drawing out the
final table, rough draft should be examined carefully.
(ii) Headings of the rows and columns should be brief and clear.
(iii) Title, note, row and column are made specific, connoting meaning or
expressions.
(iv) Numbers of class intervals are decided as per aims of study which should not
be too small or too big.
(v) Symbols used, should be explained.
(vi) Tabulated data should specify the units of their measurements.
(vii) The sources from which data are obtained should be given.
1.8.2 REPRESENTATION OF DATA
Tabulated data will give some information and also allow for further analysis.
The columns and rows in a table make eye strain and there are chances of poor visual
impression of data presented in a tabular form. Now the well tabulated data can be
represented in the form of picture, diagram or figure which will help in good
comparison through good visual impression. The representation of quantitative data
through charts and diagrams is known as graphical representation of statistical data. A
picture is said to be more effective than words for describing a particular thing or
phenomenon. Main objective of diagram is to help the eye to grasp series of numbers
and to grasp the meaning of series of data and also to assist the intelligence.
There are various types of graphs in the form of charts and diagrams. Some of
them are:
1. Bar diagram, 2. Pie chart, 3. Histogram, 4. Frequency polygon and

Frequency curve, 5. Pictograms, 6. Line chart, 7. Cumulative frequency
curve 8. Scatter diagram
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1.8.2.1 Bar diagram
The simplest type of graph that can be used to represent the categorical data is the bar
diagram. It is also called a columnar diagram. The bar diagrams are drawn through
columns of equal width. In this diagram we show the category of the variable on the X-
axis and the frequencies on the Y-axis on a graph paper. A bar of each category is of the
variable is drawn and the height of the bar represents the frequency of that category.
Since the data is of qualitative nature or quantitative data of discrete type, bars should not
be next to each other and there should be an equal gap between two successive bars.
Following rules were observed while constructing a bar diagram:
(a) The width of all the bars or columns is similar.
(b) All the bars should are placed on equal intervals/distance.
The following types of bar graphs are possible:
(a) Simple bar graph
(b) Double bar graph
(c) Multiple bar graphs
We will illustrate each of these graphs by the following illustrations:
(a) Simple Bar Diagram
A simple bar diagram is constructed for an immediate comparison. It is advisable to

arrange the given data set in an ascending or descending order and plot the data variables
accordingly. In Base hospital has been found patients in OPD in particular disease as
below in year 2012.
Month: Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec
Patients: 285 315 250 289 386 410 452 620 421 186 450 500
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700
600
500
400
300
200
100
0
Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec
Figure
ure -1.5 simple bar diagram
(b) Double Bar Diagram
When two components are grouped in one set of variable or different variables of one
component are put together, their representation is made by a double bar diagram.
diagr In this
method, different variables are shown in a single bar with different rectangles. From
above example, patients were divided in two categories as male and female and the data
is given below:
Month: Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Male 100 250 150 189 270 200 350 275 215 86 300 200
Female: 185 115 100 100 116 210 102 345 206 100 150 300
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Male Female
350
300
250
200
150
100
50
0
Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec
Figur
Figure -1. 6 Double Bar Diagram
(c) Multiple Bar Diagram
Multiple bar diagram shows that the proportion of subgroup between two or more categories
are represented with a bar giving proportion to each of them within the bar. It is also
advisable to make one bar as 100% and each subcategory is given proportion within the
graph.
1.8.2.2 Pie Chart
Pie diagram is another graphical method of the representation of categorical data. Pie
is a mathematical constant defined as the ratio of the circumference of a circle to the
th diameter
and is equal to 22/7. It is drawn to depict the total value of the given attribute using a circle.
In the pie chart, a circle (total 360o) is divided into sectors with areas proportional to the
frequencies or the relative frequencies of the cate
categories of a variable. Dividing the circle into
corresponding degrees of angle then represent the sub
sub– sets of the data. Hence, it is also
called as Divided Circle Diagram
Diagram.
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Example 2. A household with a monthly salary of Rs. 7200 plans his budget for a month as
given below:
Item Food Rent Education Savings Misc. Total
Amount (Rs.) 3000 800 1200 1500 700 7200
Make a pie chart for this data.
Solution. First of all we find the angles of each sector as follows:
Total of data corresponds to 360o. Let xo = the angle at the centre for item A, then for
the data given in above example to draw pie graph, we find the angles of each category.
Calculation of Angles
For Food:
f 3000
Angle at centre = × 360 o = × 360 o = 150o. Here f= Frequency of food and
∑f 7200
∑ f = Total frequency
For Rent:
f 800
Angle at centre = × 360 o = × 360 o = 40o
∑f 7200
Similarly, we can calculate the remaining angles, and the total of angles column should
always come to 360o.
Table-2
Item Amount (Rs.) Angle
Food (A) 300 150
Rent (B) 800 40
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Education (C) 1200 60
Savings (D) 1500 75
Miscellaneous 700 35
Total 7200 360
Figure-1.7Pie chart
1.8.2.3 Histogram
A two dimensional frequency density diagram is called a histogram. A histogram is a

diagram which represents the class interval and frequency in the form of a rectangle. There
will be as many adjoining rectangles as there are class intervals. There are two types of
histograms-
(1) Histogram with equal class intervals

(2) Histogram with unequal class intervals
To draw a histogram, you should follow the steps as stated below:
1. Class intervals must be exclusive. If the intervals are in inclusive form, convert them
to the exclusive form.
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2. Draw rectangles with class intervals as bases and the corresponding frequencies as
heights.
3. If the intervals are equal, then the height of each rectangle is proportional to the
corresponding class frequency.
4. If the intervals are unequal, then the area of each rectangle is proportional to the
corresponding class frequency density.
Example 3. Draw a histogram for the following data showing the class interval and their
corresponding frequencies.
Class interval 0-5 5-10 10-15 15-20 20-25
Frequency 4 10 18 8 6
Figure-1.8 Histogram
Example 4. Following is the distribution of shops according to the number of wage - earners
employed at a shopping complex.
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Table-3 showing the distribution of wage earners
Number of wage earners No. of shops Frequency density
Under 5 18 3.6
5 – 10 27 5.4
10 – 20 24 2.4
20 – 30 20 2.0
30 – 50 16 0.8
Illustrate the above table by a histogram, showing clearly how you deal with the unequal
class intervals.
Solution. When the class intervals are unequal, we construct each rectangle with the class
intervals as base and frequency density as height.
Frequency density = Frequency/ Class width
Figure- 1.9
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1.8.2.4 Frequency Polygon and Frequency Curve
In a frequency distribution, the mid-value of each class is obtained. Then on the graph
paper, the frequency is plotted against the corresponding mid-value. These points are joined
by straight lines. These straight lines may be extended in both directions to meet the X - axis
to form a polygon. If these points are joined by a free hand smooth curve then it is called
Frequency curve.
Example 5. The growth rate of different crops like rice, wheat, birth rates, death rates and
life expectancy are given in the following table. Make a frequency polygon from it.
Table-4 Showing class interval and frequency
Class interval Mid Marks Frequency
40 – 44 42 3
45 – 49 47 10
50 – 54 52 12
55 – 59 57 15
60 – 64 62 7
65 – 69 67 5
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Figure-1.10
1.8.2.5 Pictograms
Pictograph is the use of pictures or images to present data. They will give the quick
idea for the frequency of the characteristics and fraction also marks on pictures, e.g., bus
for transport, man for cases, cot for hospital beds, etc. It is widely used by government
and private organizations. The chief advantage of this method is its attraction.
1.8.2.6 Line chart
It is most widely used in medical science. It shows the trend of times. Data having
some order as age –wise incidence of a disease can be represented by a line chart. It is drawn
by taking one variable on the horizontal X-axis and the other variable on the vertical Y-axis.
This graph shows the effect of one variable on the other variable, e.g., age specific incidence
of cancer among males of Delhi.
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Cumulative frequency curve
If we plot the less than cumulative frequencies rather than frequencies against the
upper limits of the classes, the curve obtained on joining these points by free hand curve is
called less than cumulative frequency curve or ogive or less than ogive and If we plot the
more than cumulative frequencies rather than frequencies against the lower limits of the
classes, the curve obtained on joining these points by free hand curve is called more than
cumulative frequency curve. The advantage of this curve is that it enables us to answer the
queries related to the frequency distribution of the variable.
1.8.2.8 Scatter diagram
It is the simplest way of the representation of bivariate data. Thus for the bivariate
distribution (x, y) ; if the values of the Variable X and Y be plotted as x along X-axis and the
y along the Y-axis respectively in the x y plane, the diagram of dots so obtained is called
scatter diagram.
1.9 SUMMARY
From the study of this chapter the students came to know the definitions of statistics
and biostatistics, the scope and applications of biostatistics. The students studied and learnt
about data. What is data? What are the types of data? The classification of different types of
data provides knowledge to treat different types of data. We learn from the study of this
chapter the different steps necessary for adopting any sampling procedure and the two types
of error involved in the collection of sample and complete census. We learn definitions of
some terms related to sampling as population, sample, sampling frame and sampling unit.
Also we learn many sampling techniques for collecting sample data. In the last of this chapter
we study and learn the organization and many representation techniques of data through
graphs, pictures and chart.
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1.10 GLOSSARY
Data: Collected and recorded information which is either in numerical form otherwise it has
no meaning.
Qualitative data: The data collected on some quality characteristic.
Quantitative data: The data collected on quantitative variable.
Variable: Any attribute, any phenomenon or any event that can have different values.
Tabulation: It is the process to [put the data into different groups or classes.
Sampling Frame: It is the list or map of population units on the basis the selection of a
sample is carried out.
Srswr: Simple random sampling a technique where each unit of the population has equal
chance of selection in the sample and when sampling is done with replacement, i.e., selected
unit is replaced before the next unit selection is made.
Srswor: It is also simple random sampling technique but without replacement, i.e., the unit
selected is not replaced back.
Stratified random sampling: It is the technique of random sample when the population has
heterogeneity by dividing it into homogeneous groups.
Bar diagram: Diagrammatic representation technique of frequency data for nominal classes
by bars in which the length of bars is proportional to the class frequencies.
Cumulative frequency curve: The curve in which the cumulative frequencies rather than
frequencies are plotted against the class intervals. These are of two types less than and more
than types and their intersection represent median.
Histogram: It is a diagrammatic representation of the frequency distribution of a quantitative

data with areas of the rectangles proportional to the class frequency.
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1.11 SELF ASSESMENT QUESTIONS

Question 1. For the given data draw a bar chart.
Year 1978 1979 1980 1981 1982 1983
Rice (in tons) 4500 5700 6100 6500 4300 7800
Question 2. For the data give below make a pie chart.
Blood group A B AB O Total
Frequency 15 25 20 30 90
Question 3. Make a histogram and frequency polygon for the data given below.
Profit per shop 0-100 100-200 200-300 300-400 400-500 500-600
No. of shops 12 18 27 20 17 6
Question 4. What are the different types of data? Explain in brief. Also explain the different
sampling schemes of data collection.
Question 5. The data collected directly by the investigator or his/her team is called
(a) Secondary data (b)Primary data
(c) Population data (d) Sample data
Question 6. The data collected from already published material is called
(a) Secondary data (b)Primary data
(c) Population data (d) Sample data
Question 7. In bar diagram the base line is:
(a) Horizontal (b) Vertical

38 | P a g e
(c) False base line (d) any of the above
Question 8. Less than and more than ogives intersects at;
(a) mean (b) median
(c) mode (d) origin
Question 9. In cases of frequency distribution with classes of unequal width, the heights of
bars in a histogram are proportional to:
(a) class frequency (b) class intervals
(c) frequencies in percentage (d) frequency densities
Question 10. Sampling frame is a term used for:
(a) a list of random numbers (b) a list of voters
(c) a list of sampling units of the population (d) none of the above
Question 11. A selection procedure of a sample having no involvement of probability is:
(a) Purposive sampling (b) Judgment sampling
(c) Subjective sampling (d) all the above
Answers: 5- (b), 6- (a), 7- (b), 8- (b), 9- (d), 10- (c), 11- (d).
1.12 REFERENCES
Agarwal, B.L., Programmed Statistics, New Age International (P) Ltd, New Delhi, (2003).
Arora, P.N. and Malhan, P.K., Biostatistics, Himalaya Publishing House (P) Ltd. Mumbai,
(1996).
Belle, G., Fisher, L., Heagerty, P.G. and Lumly, T.,Biostatistics- A Methodology for the
Health Science, John Willey & Sons, inc. Pub., Hoboken, New Jersey, (2004).
Gupta, S.C. and Kapoor, V.K., Fundamentals of Mathematical Statistics, Sutan Chand &
sons, Daryaganj, New Delhi, (1970).
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Gupta, S.C. and Kapoor, V.K., Fundamentals of Applied Statistics, Sutan Chand & sons,
Daryaganj, New Delhi, (1976).
Pagaro, M. and Gauvreau, K., Principles of Biostatistics, Duxbury Thomson Learning,

U.S.A. (2000).
Prabhakara, G.N., Biostatistics, Jaypee Brothers Medical Publishers (P) Ltd, New Delhi,
(2006).
Singh, D. and Chaudhary, F.S., Sample Survey Designs, New Age International (P) Ltd, New
Delhi, (1986).
Sudaram, K.R., Dwivedi, S.N., Sreenivas, V., Medical Statistics, Wolters Kluwer (P) Ltd,
New Delhi. (2010).
1.13 TERMINAL QUESTIONS
1. What is sampling frame?
2. What do you know about histogram?
3. Explain stratified random sampling
4. What is the difference between simple random sampling with and without
replacement?
5. What are different types of graphs or charts for data representation? Explain any
two of them.
6. Explain the difference between sample and population.
7. What is the need of biostatistics?
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Unit-2– MEASURES OF CENTRAL TENDENCY
CONTENT
2.1- Objectives
2.2- Introduction
2.3- Mean
2.3.1- Mean of individual items
2.3.2- Mean in discrete frequency distribution
2.3.3- Mean in continuous Frequency distribution
2.3.4- Short- cut method for mean
2.3.5- Step deviation method of mean
2.3.6- Weighted mean
2.3.7- Combined mean
2.3.8- Corrected mean
2.3.9- Merits, demerits and uses of mean
2.4- Median
2.4.1- Median in individual series
2.4.2- Median in discrete frequency distribution
2.4.3- Median in continuous distribution
2.4.4- Merits, demerits and uses of median
2.5- Mode
2.5.1- Types of mode of a distribution
2.5.2- Mode in individual series
2.5.3- Mode in discrete frequency distribution
2.5.4- Grouping method of mode
2.5.5- Mode in continuous distribution
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2.5.6- Merits, demerits and uses of mode

2.6- Summary
2.7- Glossary
2.9- References
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2.1 OBJECTIVES
From the study of this chapter the students will be able:
1. To know about the measures of central tendency- mean, median and mode.
2. To know the merits and demerits and uses of these measures.
3. To know about different methods of measuring mean, median and mode.
4. To know the situations where which measure is better to use?
5. To know the advantages of short cut methods of computing mean.
2.2 INTRODUCTION
In the previous chapter, we discussed data collection, data organization and data
representation techniques. The data representation techniques such as frequency histograms and
frequency polygons, introduced the concept of the shape of distributions of data. For example, a
frequency polygon illustrated the distribution of body mass index data. We expend chapter 1 on
these concepts by defining measures of central tendency.
Measures of central tendency as the name suggests are numerical measurement of the
central part of the distribution. Measures of central tendency are also called averages or measures
of location because they show the location of the centre of the distribution from which the data
were sampled. According to Professor Bowley, averages are, “statistical constants which enable
us to comprehend in a single effort the significance of the whole.” In other words, these are
numbers that tell us where the majority of values in the distribution are located. For example the
average marks in a distribution of marks of all the students of a class. The averages which are
commonly used in biostatistics are as follows:
1. Mean or arithmetic mean 2. Median 3. Mode.
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2.3 MEAN
Mean or arithmetic mean of a series of data is the ratio of the sum of the observations to
the number of observations. If x1 , x 2 ,......x n are the observations of a series then their arithmetic
mean is given by
n
x + x 2 + .....x n ∑x
i =1
i
x= 1 = (1)
n n
And if the corresponding frequencies, f1 , f 2 ,.... f n of the variables x1 , x 2 ,......x n are given, then
the arithmetic mean is defined as ratio in which the numerator is the sum of products of the
variables with their frequencies and denominator is the sum of the frequencies.
n
f x + f 2 x 2 + ..... f n x n ∑fx
i =1
i i
x= 1 1 = (2)
∑ fi N
where, N = ∑f i = sum of frequencies.
2.3.1 MEAN OF INDIVIDUAL ITEMS
Mean of individual items is given by the ratio of the sum of items to the number of items
as given in formula (1).
Example 1. Find the arithmetic mean of triglycerides present 10 patients in their blood samples
in a hospitalas:
25, 30, 21, 55, 47, 10, 15, 17, 45, 35
Solution. Let x be the average triglyceride value and since these are individual items, their mean
can be computed by formula
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x1 + x 2 + .....x n ∑
xi
i =1
x= =
n n
25 + 30 + 21 + 55 + 47 + 10 + 15 + 17 + 45 + 35 300
= = = 30
10 10
2.3.2 MEAN IN DISCRETE FREQUENCY DISTRIBUTION
If x1 , x 2 ,......x n are the observations in a discrete distribution according to some
characteristic and f1 , f 2 ,.... f n be their corresponding frequencies then the arithmetic mean is
given by the formula (2). The computation procedure for mean can be easily understood with the
help of the example given below.
Example 2. The distribution of marks of 50 students of B.Sc. class in a botany semester

examination is given below. Find the average of marks.
Marks (x) 12 23 25 35 45 15 40
Frequency(f) 3 10 12 10 2 8 5
Solution. Since this is a discrete distribution so the average of marks is given by the formula (2).
For the computation of average marks we prepare the following table:
Table1. For calculation of Mean in Discrete Distribution
Marks (x) Frequency (f) fx
12 3 36
23 10 230
25 12 300
35 10 350
45 2 90
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15 8 120
40 5 200
Total ∑ f = 50 ∑ f x = 1326
f x + f 2 x 2 + ..... f n x n ∑fx
i =1
i i
1326
x= 1 1 = = = 26.52
∑ fi N 50
This is clear that it is not necessary that average will be a number presenting in the
data and also it is not an integer value while the marks in integers.
2.3.3 MEAN IN CONTINUOUS DISTRIBUTION
In case of continuous distribution, there are given class intervals and their
corresponding frequencies. First of all we find the mid values of these classes and treat them
as the variable values. Now we apply the formula (2) for the calculation of arithmetic mean.
The procedure will be clear from the following example.
Example 3. For the data given in the below table on systolic BP of 68 patients, calculate the
arithmetic mean.
Table 2.
Systolic BP (mmHg) Frequency (f) Systolic BP (mmHg) Frequency (f)
90-100 3 140-150 11
100-110 5 150-160 9
110-120 7 160-170 6
120-130 10 170-180 2
130-140 15
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Solution. For the calculation of mean we prepare the following table:
Table3. For calculation of Mean in Continuous Distribution
Systolic BP (mmHg) Frequency (f) Mid Value (x) Fx
90-100 3 95 285
100-110 5 105 525
110-120 7 115 805
120-130 10 125 1250
130-140 15 135 2025
140-150 11 145 1595
150-160 9 155 1395
160-170 6 165 990
170-180 2 175 350
Total ∑f = 68 ∑ fx = 9220
∑fx
i =1
i i
9220
x == = = 135.6mmHg
N 68
2.3.4 SHORT-CUT METHOD FOR MEAN
For the computation of mean short –cut method is applied when the variable values
and their frequencies are large. To make the computations easy we take a middle value in the
given values of x as assumed mean and subtract this assumed mean from all the values of x.
This assumed mean is also called provisional mean. Then the formula for the calculation of
arithmetic mean is given by as follows:
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x = A+
∑d
n (3)
Where A= assumed mean, n= number of observations in the given data
d= x- A= deviation of all the variate values from assumed mean A.
Steps of computation for short- cut method:
Step 1. Take any observation (generally, middle value if we arrange the values in
ascending or descending order of magnitude) of the individual series as assumed mean A.
Step 2. Find the deviation of the values of variate x from assumed mean A, i.e., calculate
the differences d= x- A
Step 3. Find the sum of d and use above formula (3), we find the value of mean.
If the frequencies corresponding to the variate values are given, then we use
the formula for mean as follows:
x = A+
∑ fd
N (4)
Where, N = ∑ f = sum of frequencies. Here we find the product of f and d.

If the data is continuous, we find the mid values as x and then d= x- A. Now
apply the above formula (4). The procedure will be clear from the examples as
give below.
Example 4. The marks of the 7 students of a class in a test are as given below:
12, 15, 22, 25, 35, 40, 45
Find the mean by short-cut method.
Solution. Let us take assumed mean A=25. Now we prepare the table for the computation
of mean as given below:
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Table-4 Mean for individual data by short cut method
X d = x- 25
12 -13
15 -10
22 -3
25 0
35 10
40 15
45 20
Total ∑ d = 19
Arithmetic mean x = A +
∑d = 25 +
19
= 25 + 2.71 = 27.71
n 7
Thus the average of marks of the given 7 students of the class is 27.71
Example 5. Ten patients were examined for uric acid test. The operation was performed
1050 times and the frequencies so obtained for different number of patients (x) are shown
in the table given below. Compute the arithmetic mean by short- cut method.
x: 0 1 2 3 4 5 6 7 8 9 10
f: 2 8 43 133 207 260 213 120 54 9 1
Solution. Let 5 be the assumed mean. Now we prepare the table for the calculation of
mean.
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Table-5 Mean for discrete grouped data by short cut method
X Frequency (f) d = x- 5 fd
0 2 -5 -10
1 8 -4 -32
2 43 -3 -129
3 133 -2 -266
4 207 -1 --207
5 260 0 0
6 213 1 213
7 120 2 240
8 54 3 162
9 9 4 36
10 1 5 5
Total ∑f = 1050 ∑ fd = 12
∑ fd = 5+
12
= 5 + 0.0114 = 5.0114cm
N 1050
Thus the average for uric acid is 5.0114.
2.3.5 STEP DEVIATION METHOD OF MEAN
It can be used in grouped data. When all the classes are of equal width (say h), in
continuous data and the values of x are at equal interval in discrete grouped data then the we may
simplify the calculations by taking d= (x- A)/ h in short-cut method. Now the formula for the
calculation of mean becomes.
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x = A+
∑ fd × h
N
Here, the symbols have the same meaning as in short-cut method above and h is the gap
between the two values of x or class interval.
Example 6. Find the mean by step deviation method for the data of blood pressure of 68 patients
as given in the following table.
BP(mmHg) (x) 90 100 110 120 130 140 150 160 170
Frequency ( f) 3 5 7 10 15 11 9 6 2
Solution. We take assumed mean A= 130 and here interval between any two values of x is 10,
i.e., h= 10. Now prepare the table for the computation of mean.
Table6. Step Deviation Method of Mean in discrete grouped data
BP (mmHg) Frequency (f) x − 130 fd

d=
10
90 3 -4 -12
100 5 -3 -15
110 7 -2 -14
120 10 -1 -10
130 15 0 0
140 11 1 11
150 9 2 18
160 6 3 18
170 2 4 8
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Total ∑f = 68 ∑ fd = 4
∑ fd × h = 130 + 4
× 10 = 130 + 0.588 = 130.588mmHg
N 68
Thus the average for BP is 130.588mmHg.
Example7. For example 3, calculate arithmetic mean by step deviation method.
Solution. For the calculation of mean the table is given below:
Table-7. Mean by Step Deviation Method in Continuous Data
Systolic BP Frequency Mid Value x − 135 fd

d=
(f) (x) 10
(mmHg)
90-100 3 95 -4 -12
100-110 5 105 -3 -15
110-120 7 115 -2 -14
120-130 10 125 -1 -10
130-140 15 135 0 0
140-150 11 145 1 11
150-160 9 155 2 18
160-170 6 165 3 18
170-180 2 175 4 8
Total ∑f = 68 ∑ fd = 4
∑ fd × h = 135 + 4
× 10 = 135 + 0.588 = 135.588mmHg
N 68
52 | P a g e
Thus the average for uric acid is 135.588mmHg.
Example 8. In a study on patients of typhoid fever the following data are obtained. Find the
arithmetic mean.
Age in years 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89
No. of cases 1 0 1 10 17 38 9 3
Solution. This is inclusive type data; first of all we convert it to exclusive type data. The
procedure for converting inclusive type data to exclusive type data is as follows:
We see that the upper limit of the first class is 19 and the lower limit of the second
class is 20 and their difference is 20-19=1. Now subtract half of the difference, i.e., 0.5 from
the upper limit and 0.5 to the lower limit. Also we see that this difference is the same for
each of the class. So the new classes are as 9.5-19.5, 19.5-29.5 and so on.
Now for the calculation of mean any method discussed above can be used. Here we
apply step deviation method.
Table-8. Mean by Step Deviation Method in Inclusive Data
Age Frequency Mid Value x − 44.5 fd

d=
(f) (x) 10
9.5-19.5 1 14.5 -3 -3
19.5-29.5 0 24.5 -2 0
29.5-39.5 1 34.5 -1 -1
39.5-49.5 10 44.5 0 0
49.5-59.5 17 54.5 1 17
59.5-69.5 38 64.5 2 76
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69.5-79.5 9 74.5 3 27
79.5-89.5 3 84.5 4 12
Total ∑f = 79 ∑ fd = 128
∑ fd × h = 44.5 + 128 × 10 = 44.5 + 16.2 = 60.7
N 79
2.3.6 WEIGHTED MEAN
In computation of arithmetic mean some items are more important than the others, in
such cases the weight age should be given to the items according to their importance. For
example if we want to have an idea of the change in cost of living of group of people of a
certain locality, then the simple mean of the prices of the commodities consumed by them
will not do, since all the commodities are not equally important, e.g., wheat, rice and pulses
are more important than cigarettes, tea, confectionery, etc.
If x1 , x 2 ,......x n are the variate values of a distribution and w1 , w2 ,....wn be their

corresponding weights then weighted mean is give by:
xw =
∑w x i i
∑w i
Example 9. The following table gives the platelets count (in lakh/cmm) from the analysis of
the blood samples on five different days in a pathology laboratory. Find the average platelets
count per patient.
Day 1 2 3 4 5
Platelates count 0.50 0.75 1.00 1.50 2.00
(in lakh/cmm) (w)
No. of patients (x) 65 80 95 90 70
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Solution. The table for the calculation of weighted mean is given by:
Table 9. Table for Weighted Mean
Platelets count (x) No. of patients (w) Wx
0.50 65 32.5
0.75 80 60.0
1.00 95 95.0
1.50 90 135
2.00 70 140
Total ∑ w = 400 ∑ wx = 462.5
xw =
∑w x
i i
=
462.5
= 1.156
∑w i 400
Thus, the average platelets per patient are 1.156 lakh/cmm.
2.3.7 COMBINED MEAN
If x1 , x 2 ,......x m are the means of m series of sizes n1 , n2 ,....nm respectively, then their
combined arithmetic meaning x is given by:
x=
∑n xi i
; i = 1,2,......m
∑n i
Example 10. There are 40 male and 10 female employees in a firm. The mean salary of male
employees is Rs.520 and that of female employees Rs. 420. Find the combined average
salary of all the employees.
Solution. Here, n1 = 40, n2 = 10, x1 = 520, x 2 = 420
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Combined mean x =
∑n x i i
=
n1 x1 + n 2 x 2 520 × 40 + 420 × 10 25000
= = = 500
∑n i n1 + n 2 40 + 10 50
Hence, the average salary of all the employees is Rs. 500.
2.3.8 CORRECTED MEAN
Some times there are problems of such type that we used wrong digits while the
actual digits were different, then we replace the wrong digits with the correct digits and now
we can get the correct mean. The procedure will be clear from the example given below.
Example 11. A student calculates the mean of 20 observations as 25.2. Later on he found
that he misread one observation 34 in place of 43, find the correct mean.
Solution. We know the mean of individual series is given by:
x=
∑ x or ∑ x = nx = 20 × 25.2 = 504
n
But he misread 43 as 34. So the correct total of x= 504-34+43=513.
So correct mean=513/ 20 =25.65
2.3.9 MERITS, DEMERIRS AND USES OF MEAN
Merits:
1. Mean is rigidly defined.
2. It can be calculated easily by a non mathematical person also.
3. It is based upon all the observations.
4. Among all the averages, it is affected least by fluctuations of sampling.
5. It is the best measure to compare two or more series.
6. It is easily understandable.
7. It is the most widely used method of central tendency.
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Demerits:
1. It is affected much by extreme values.
2. It cannot be calculated in case of open end classes.
3. It cannot be calculated in case of qualitative data such as intelligence, beauty, etc.
`4. In extremely asymmetrical distribution, mean is not a suitable measure of central

tendency.
5. It cannot be calculated if any observation is missing.
6. It may lead to wrong conclusions if the details of the data are not given. For
example the marks of two students in three successive tests are respectively 30, 40, 50
and 50, 40, 30. We see that average score of both the students is same, we can say that
both students are of same level while first is improving and the second is deteriorating.
Uses of Mean:
1. It is very much used in practical situations.
2. A common man uses it for computing his monthly budget.
3. It is very much used in sampling and inference.
4. A businessman uses it for computing per unit profit, output per person, average
expenditure and average profit per week or per month, etc.
2.4 MEDIAN
Median of a distribution is the middle most value of the variable if the values of the
variable are arranged in ascending or descending order of their magnitude. The median
divides the observations of the variable in such a way that half of the observations of the
variable lie above the median and half below this. Median is thus called a positional average
because it locates at the middle of the observations. But if the number of observations is even
then after arrangement there will be two middle values and the median will be the average of
these two middle values.
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2.4.1 MEDIAN IN INDIVIDUAL SERIES
Arrange the data observations, (say n) in ascending or descending order of magnitude.

Now there can be two cases:
Case 1. If n is odd then middle most, i.e., (n+1/2)th term value is the median.
Case2. If n is even then there are two middle terms (n/2)th and (n+1/2)th, then median
is given by:
n n  
 2 th +  2 + 1thterm
  
Me = 
2
Example 12. The marks of 9 children in a test exam are: 12, 23, 34, 11, 14, 15, 13, 16, 45.
Find the median of the marks.
Solution. Arrange the given observations in ascending order of magnitude, we get
`11, 12, 13, 14, 15, 16, 23, 34, 45
Here the number of observations n =9, i.e., odd.
So the median is the (9+1)/2 th, i.e., 5th term value, i.e., 15.
Example 13. The number of blood LDL (in mg/dl) present the blood samples of 12 patients
are: 5, 19, 42, 11, 50, 30, 21, 0, 22, 52, 36, 27
Find the median of the data.
Solution. On arranging the given observations in ascending order of magnitude, we get,
0, 5, 11, 19, 21, 22, 27, 30, 36, 42, 50, 52
Here number of observations = 12, i.e., even. So median is given by
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n n   12  12  
 2 th +  2 + 1thterm  2 th +  2 + 1thterm
     
Me =  =
2 2
(6th + 7th)term 22 + 27 49
= = = = 24.5
2 2 2
So median is 24.5 mg/dl which does not belong to the data. So in case of even number of
observations median is not present in the data observations.
2.4.2 MEDIAN IN DISCRETE FREQUENCY DISTRIBUTION
characteristic and f1 , f 2 ,.... f n be their corresponding frequencies then for the calculation of
median we calculate the cumulative frequencies. The median is calculated with the help of
the following steps.
Working steps for median
Step1. Arrange the given values in ascending order of magnitude.
Step2. Find the total of frequencies, called cumulative frequency and denoted by c.f.
N
Step3. Find , where N= ∑ f
2 .
N
Step4. Find cumulative frequency just greater than 2 . The value of x corresponding to this
cumulative frequency is the required median.
Example14. Find the median for the following data.
X 21 15 17 9 5 7 8 10
F 2 5 3 4 5 1 6 12
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Solution. For calculating the median we arrange the values of x in ascending order and then
prepare the cumulative frequency table as follows:
Table10. Median in Discrete Distribution
x f c.f.
5 5 5
7 1 6
8 6 12
9 4 16
10 12 28
15 5 33
17 3 36
21 2 38
N = ∑ f = 38
Here N/2 = 38/2= 19 and cumulative frequency just greater than 19 is 28. The value of x
corresponding to cumulative frequency 28 is 10. So the median of the given data is 10.
2.4.3 MEDIAN IN CONTINUOUS FREQUENCY DISTRIBUTION
When the data is in class interval form, the class corresponding to c.f. just greater than
N/2 is called the median class and the median is computed by the following formula:
N 
 −C
Me = L +  ×h
2
f
Where L= lower limit of the median class
C= cumulative frequency just before the median class

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N= Total of frequency
f = frequency of the median class
h = magnitude of the median class
Example15. The following table gives the distribution of weights of 100 persons. Find the
median of this data.
Weight 40-45 45-50 50-55 55-60 60-65 65-70 70-75 75-80 80-85 85-90
Frequency 1 3 6 10 15 25 15 10 11 4
Solution. For computing the median we prepare the following table:
Table11. Median in Continuous Distribution
Weight (in kg) (x) Frequency (f) Cumulative

Frequency (c.f.)
40-45 1 1
45-50 3 4
50-55 6 10
55-60 10 20
60-65 15 35
65-70 25 60
70-75 15 75
75-80 10 85
80-85 11 96
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85-90 4 100
Total N = ∑ f = 100
Here N/2 = 10/2= 50 and cumulative frequency just greater than 50 is 60. The class
corresponding to cumulative frequency 60 is 65-70. So class 65-70 is the median class. Now
median is given by:
N 
 −C
Me = L +  ×h
2
f
here L= lower limit of the median class= 65
C= cumulative frequency just before the median class=35
N= Total of frequency =100
f = frequency of the median class=25
h = magnitude of the median class=5
M e = 65 +
(50 − 35) × 5 = 65 + 15 × 5 = 65 + 3 = 68
25 25
So median of the given data is 68kg
2.4.4 MERITS, DEMERIRS AND USES OF MEDIAN
Merits:
1. Median is rigidly defined.
2. It is not affected at all from extreme values.
3. It is easy to understand and to calculate.
4. In case of individual series data it can be located merely by inspection.
62 | P a g e
5. It can be calculated in case of open end classes.
6. Its graphical representation is also possible.
7. It can be computed even if the classes are of unequal interval.
8. In case of qualitative data, e.g., beauty, honesty, intelligence, etc. it is the best
measure of central tendency.
Demerit:
1. It is not amenable to algebraic treatment.
2. It is a positional average and is based only on the middle term. It does not use all
the observations of the data.
3. In case of irregular distribution, it is not a good measure.
4. In case of even number of observations it cannot be determined exactly, it can be

estimated only by the average of the two middle terms.
5. In comparison to mean it is affected much by fluctuations of sampling.
Uses:
1. It is a good measure if numerical measurements are not possible.
2. In case of qualitative data where the observations cannot be determined

quantitatively, it is the only average.
3. It is generally used in studying the average intelligence or average honesty of a

group of people.
2.5 MODE
Mode is the most frequent item of the series, i.e., in a given set of observations a item
or observation which is repeated maximum number of times an all other observations cluster
around this, is called mode. For example, the average height of an Indian male is 5 feet 6
63 | P a g e
inch; the average size of the shoes of an Indian male is number 7, etc. Mode is also known as
norm.
2.5.1 TYPES OF MODE OF A DISTRIBUTION
Unimodal: If the data of a distribution has only one mode then the distribution is
called unimodal.
Bimodal: If we find that there are two items in a distribution which have the same
number of repetitions, then these two items are the modes and the distribution is
called bimodal.
Trimodal: Similarly, in a distribution, if there are three such items that they have the
same frequency then these three items are called the modes of the distribution and the
distribution is called trimodal.
Ill- defined mode: If there exists more than one mode in a distribution, then mode is
called ill-defined.
2.5.2 MODE IN IDDIVIDUAL SERIES
In case individual series mode is the most frequent observation. It is clear from the
following example.
Example 16. Find the mode of the series given below:
2, 3, 4, 7, 9, 3, 2, 1, 5, 3, 6, 3, 8, 3
Solution. In the given series the observation 3 is repeated maximum number of times (5) so
the mode of the given series is 3.
2.5.3 MODE IN DISCRETE FREQUENCY DISTRIBUTION
In case of discrete frequency distribution, mode is the value of the variable which has the
maximum frequency. Consider the following example:
Example 17. Find the mode of the following frequency distribution:
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Variable (x) 2 5 7 9 11 25 35 43 52
Frequency (f) 1 3 4 8 25 12 11 10 8
Solution: Here we see that in the given distribution, the variable 1 has the maximum
frequency 25. So the mode of this distribution is 11.
2.5.4 GROUPING METHOD OF MODE
When the distribution is irregular, the frequencies are increasing and decreasing in
An irregular pattern or the difference between the maximum frequency and the frequency
succeeding or proceeding to it is small and the observations are concentrated on either side,
in such a situation mode cannot be determined merely by inspection. In such a case, we apply
the grouping method for the computation of mode. The procedure of grouping method will
be clear from the following example.
Example 18. Find the mode of the following distribution.
Variable (x) 2 3 4 5 6 7 8 9 10 11 12 13
Frequency (f) 1 3 4 5 7 10 11 10 9 14 7 5
Solution. Here we see that initially the frequencies are increasing from 1 to 11 and then
decreasing but the frequency 14 of the variable value 11 is again increasing and then
decreasing up to frequency 5. This distribution shows an irregular pattern. So for the
calculation of mode we apply the grouping method of mode. For this we prepare a table and
the procedure of preparing the table is explained below the table.
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Table12. Table for grouping the frequencies
Variable Frequency(f)
(x)
Column Column Column Column Column Column
(i) (ii) (iii) (iv) (v) (vi)
2 1
3 3
}4
4 4
}7 }8
}9 }12
5 5
}12 }16
6 7
}17 }22
7 10
}21 }28
8 11
}21 }31
9 10
}19 }30
10 9
}23 }33
11 14
}21 }30
12 7
}12 }26
13 5
Prepare a table from the frequencies of the distribution. In column (i), we have the
original frequencies. Mark bold type the maximum frequency in this column. Column (ii) is
prepared by adding the frequencies two by two as 1+3 = 4; 4+5 = 9 and so on. Mark bold
type the maximum frequency in this column also. Column (iii) is prepared by adding the
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frequencies two by two leaving the first frequency. Column (iv) is prepared by adding the
frequencies three by three. Column (v) is prepared by adding the frequencies three by three
leaving the first frequency and column (VI) is prepared by adding the frequency three by
three leaving the first two frequencies. In each column make bold type the maximum
frequency. The table is given above:
Now to find the mode we prepare the following analysis table:
Table13. ANALYSIS TABLE
Column number Maximum frequency Value(s) of x related to the
(1) (2) maximum frequency (3)
i 14 11
ii 23 10, 11
iii 21, 21 7, 8, 11, 12
iv 30 8, 9, 10
v 33 9, 10, 11
vi 31 7, 8, 9
In the analysis table column number (1) shows the columns serially from the above table 12,
column number (2) shows the maximum frequency from the same table 12 and column
number (3) shows the value of x related to the maximum frequency or the values of x which
contributes in the maximum frequency. Finally, in column number (3) of the analysis table
we see that the value 11 is repeated maximum number of times. So 11 is the mode of the
above distribution.
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2.5.5 MODE IN CONTINUOUS FREQUENCY DISTRIBUTION
In case of grouped continuous frequency distribution the maximum frequency shows

that the related class is the modal class and for the computation of mode we use the following
formula:
Mo = L +
( f1 − f0 ) ×h
(2 f1 − f0 − f2 )
Where L= lower limit of the modal class
h= magnitude of the modal class
f1 = frequency of the modal class
f 2 = frequency of the class succeeding the modal class
f 0 = frequency of the class preceding the modal class
For a moderately asymmetrical distribution the mode can be calculated by a formula

given by Karl Pearson as follows:
Mode = 3 Median – 2 Mean
Example 19. Following table shows the blood pressure and the frequency related to it. Find
the mode of this distribution.
Table 14.
C.I. Frequency C.I. frequency
70-80 2 110-120 32
80-90 4 120-130 28
90-100 14 130-140 12
100-110 35 140-150 5
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Solution. From the table it is clear that maximum frequency is 35 and the related class is the
100-110. So 100-110 is the modal class. Now to compute the mode we use the following
formula:
Mo = L +
( f1 − f0 ) ×h
(2 f1 − f0 − f2 )
Here L= lower limit of the modal class= 100
h= magnitude of the modal class= 10
f1 = frequency of the modal class= 35
f 2 = frequency of the class succeeding the modal class= 32
f 0 = frequency of the class preceding the modal class= 14
M o = 100 +
(35 − 14) × 10 = 100 +
210
= 100 + 8.75 = 108.75
∴
(70 − 14 − 32) 24
So mode of the given distribution is 108.75.
2.5.6 MERITS, DEMERITS AND USES OF MODE
Merits:
1. Mode is easy to understand and to calculate.
2. It is not affected by extreme values.
3. It can be determined graphically.
4. In some cases it can be located by inspection only.
5. It can be computed for the distributions of unequal class intervals provided the
modal class; the class preceding the modal class and succeeding the modal class are of
equal width.
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6. It represents the most frequent value of the distribution, practically it is very

useful.
Demerits:
1. It is not based upon all the observations.
2. It is not subjected to algebraic treatments, i.e., we cannot compute the combined

mode if we have the modes of the two series.
3. In some cases mode is ill defined. In some cases it is not possible to find a clear
mode. Some series have two modes and some more than two modes.
4. As compared to mean, mode is affected much by fluctuations of sampling; it is an

unstable measure of central tendency.
5. If the modal class or the class preceding or succeeding the modal class are of
unequal width, it cannot be determined.
6. There are different formulas for the calculation of mode.
Uses:
1. It is used to find the ideal size; it is very useful in business forecasting.
2. It is very useful in ready-made market, e.g., shoes, shirts, jeans etc.
3. It is very useful in commercial management.
2.6 SUMMARY
The study of this chapter provides us the knowledge of central tendency and measures
of central tendency. From the study of this chapter we came to know the definitions of the
measures of central tendency as mean, median and mode. We studied and learnt different
methods of computing mean. We learnt about weighted mean and combined mean. We learnt
how we can calculate the mean, median and mode in case of individual series, in case of
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discrete distribution and continuous distribution. We studied the grouping method of mode.
We studied the merits, demerits and uses of mean, median and mode also. From the study of
these methods, merits, demerits and uses we came to know the situations where which
method is suitable and also which measure is suitable for the particular situation? Over all we
learnt a lot about measures of central tendency.
2.7 GLOSSARY
Measures of central tendency: These are mean, median and mode and they provide
us the most important knowledge about the distribution. All these give the study of central
part of the distribution.
Inclusive class: A class in which its upper and lower both limits are included is called
inclusive class.
Exclusive class: A class in which only one limit (generally lower) is included and other limit
(upper) is not included in the class is called exclusive class.
Arithmetic Mean: Arithmetic means or simply mean is ratio of sum of values to the number
of values.
Median: On arranging the values according to magnitude, the middle most value of the
arranged data is the median.
Mode: The most frequent item in the series is called mode.
Multimodal Distribution: If a distribution has more than one mode, it is called multimodal
distribution.
Ill-defined Mode: If in a distribution there exists more than one mode, mode is called ill-
defined mode.
Cumulative frequency Table: A table got on adding the successive frequencies is called
cumulative frequency table.
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Question 1. Find the arithmetic mean of the following series:
2, 13, 4, 12, 13, 5, 16, 17, 11, 22, 32, 42, 44
Question2. Find the median and mode of the following series:
12, 32, 2, 3, 15, 16, 12, 13, 23, 43, 35, 12
Question 3. Find the mean of the following distribution:
x: 12 22 13 14 15 17 24 25
f: 2 1 3 10 8 5 3 2
Question4. Find the median for the given data.
I.Q.: 92 65 77 72 110 89 112 98
f: 3 5 4 10 6 2 1 5
Question5. Find the mean, median and mode for the data given in the following table:
I.Q. Frequency I.Q. Frequency
90-100 11 130-140 43
100-110 27 140-150 28
110-120 36 150-160 16
120-130 38 160-170 1
Question6. Which of the following is not a measure of central tendency?
a. Mean b. variance c. median d. none of these
Question7. If a constant 10 is added in each observation of a set of data, the effect on mean
is:
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a. Mean is decreased by 10. b. Mean is increased by 10
c. Mean is multiplied by 10 d. Mean is not affected
Question8. The best measure of central value in case of qualitative data is:
a. Mean b. mode c. Median d. None of these
Question9. The measure which is affected much by extreme observations is:
a. Mean b. mode c. median d. Variance
Question10. If we add 3 in each observation, the effect on median is:
a. Median is decreased by 9 b. Median is increased by 3
c. median is not affected d. Median is multiplied by 3
Question11. The most frequent item in the series is called:
a. median b. mean c. mode d. none of these
Question12. The average of 3 numbers is 2 and the average another 4 numbers is 5. What is
the combined average?
a. 7 b. 10 c. 3 d. 3.71
Question13. For a group of 100 students, the mean score in a test was found to be 40. Later
on it it was found that a value 45 was misread as 54. The correct mean will be:
a. 40.50 b. 39.85 c. 39.80 d. 39.91
Question14. Arrangement of observations according to magnitude is carried out in:
a. mean b. mode c. median d. none of these
Question15. Grouping method is used for:
a. mean b. mode c. median d. all of these
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Question16. Which measure of central tendency is most widely used?
a. mean b. mode c. median d. all of these
Question17. For asymmetrical distribution, the relation between the measures of central
values is:
a. mean= mode- 2 median b. mode= 2 median- 3 mean
c. mode= 3 median- 2 mean d. mode = 3 mean- 2 median
Question18. Which measure is affected least by fluctuation of sampling is:
a. mode b. mean c. median d. none of these
Question19. The measure which cannot be calculated with open end classes is:
a. mean b. mode c. median d, all of theses
Answers: 1. 17.92, 2. 14, 12, 3. 16.24, 4. 77, 5. 126.4, 126.84, 122.86, 6. b, 7. b, 8. c, 9.

a, 10. b, 11. c, 12. d, 13. d, 14. c, 15. b, 16. a, 17. c, 18. b, 19. a.
2.9 REFERENCES
(1996).

U.S.A. (2000).
74 | P a g e
(2006).
New Delhi. (2010).
Question1. Define arithmetic mean and write down its merits and demerits
Question2. Explain step deviation method of mean by taking an example.
Question3. Which measure is the best in qualitative data? Give its merits and demerits.
Question4. Which measure cannot be calculated with open end classes and why?
Question5. Which measure is best for a industry and why?
Question6. What is the importance of mean?
Question7. Explain grouping method of mode.
75 | P a g e
Unit-3 MEASURES OF VARIABILITY/ DISPERSION
CONTENTS
3.1- Objectives
3.2- Introduction
3.3- Measures of Variability
3.3.1- Range
3.3.1.1- Merits and demerits of range
3.3.2- Interquartile Range
3.3.2.1- Interquartile range for individual series
3.3.2.2- Interquartile range for discrete distribution
3.3.2.3- Interquartile range for continuous distribution
3.3.2.4- Merits and demerits of interquartile range
3.3.3- Mean deviation
3.3.3.1- Mean deviation for individual series
3.3.3.2- Mean deviation for discrete distribution
3.3.3.3- Mean deviation for continuous distribution
3.3.3.4- Merits, demerits and uses of Mean deviation
3.3.4- Standard deviation
3.3.4.1- Standard deviation for individual series
3.3.4.2- Short-cut method
3.3.4.3- Standard deviation for discrete distribution
3.3.4.4 -Standard deviation for continuous distribution
3.3.4.5- Merits, demerits and uses of standard deviation
3.4- Variance and coefficient of variation.
3.4.1 Variance
3.4.2 Coefficient of variation
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3.5- Summary
3.6- Glossary
3.8- References
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3.1 OBJECTIVES
From the study of this chapter the students will be able:
1. To know about the measures of variability such as range, interquartile range, mean
deviation and standard deviation.
2. To know the advantages and disadvantages of these measures.
3. To know about the differences of these measurement.
4. To know the situations where which measure is better to use?
5. To know why the coefficient of variation is the best to comparing two or more series.
3.2 INTRODUCTION
In the previous chapter we study various measures of central tendency and the
methods of measuring them. The measures of central tendencies give us only an idea of the
central part of the distribution. Although these measures provide us important information
about the population, i.e., they are necessary but not sufficient for explaining a distribution
properly. These measures do not provide the information about the variability of the
observations. Variability is an important factor in nature. There is slight variability in heights
of a region of persons but there is more variation from the heights of the persons of other
region. To understand a distribution more clearly we should study about variability.
Let us consider the example of distribution of marks 5 students of three sections of a

class. The marks of section A are 5, 5, 5, 5, 5; the marks of section B students 3, 4, 5, 6, 7 ;
and the marks of section C students are 0, 1, 5, 9, 10. We see that the total score of all the
three section students is 25 and the average score of all the three section students is also same
5. But we see that there is no variability in section A marks; it is a constant series. There is
less variability in marks of section B; but there is more variability in section C students
marks. It is clear from the example that the distribution of marks of the three sections is quite
different while the average is same.
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The entire concept of statistics is based on variation. If there is no variation and all
individuals are alike, there is no need for collecting any sample data because one individual
can provide all the information of the complete data which we want to study on the basis of
the complete data set. Bur variation in any phenomenon is inherent and statistical techniques
help us to explain this variation. Thus we have to know dispersion. Dispersion means
scatteredness and this tells us about the homogeneity and heterogeneity of the distribution.
To know a distribution more clearly we study the variability or scatteredness.
3.3MEASURES OF VARIABILITY
Commonly used measures for variability are as follows:
1. Range
2. The interquartile range
3. Mean deviation
4. Standard deviation
3.3.1 RANGE
The simplest measurement of variability is range. It is defined as the difference

between the extreme observations of the distribution, i.e., the difference between the largest
and the smallest observation of the distribution. This uses the two extreme observations and
does not provide the information on the middle values of the distribution. It is simple
measurement of variability but its results are misleading if the two extreme observations are
unusual.
There are four situations of occurrence in range value.
i. There may be no variability at all (see table A Below).

ii. There may be less variation between the extreme observations (see table B
below) .
iii. There may be very much variation between the extreme observations (see
table C below).
iv. None of the observation is representative of the mean (see table D below).
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Table1. Situations in range value
A B C D
7 6 2 49
7 6 5 0
7 8 6 0
7 7 7 0
7 8 8 0
7 6 9 0
7 8 12 0
Mean= 7 Mean= 7 Mean= 7 Mean= 7
Range= 5-5 Range= 6-8 Range= 2-12 Range= 0-49
Variation= 0 Variation= 2 Variation= 10 Variation= 49
3.3.1. Merits and Demerits of Range
Merits:
1. It is simple understand and to calculate

2. Unit of measurement is same as the unit of variable under study.
3. It does not require mathematical calculation.
Demerits:
1. It uses only two extreme observations and does not use all other information so
there is no importance for collection of all other data observations. A good
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measure should use all the data observations for providing better estimate of
variability.
2. It is not an ideal measure.
3. It cannot be calculated if the distribution contains open-end classes.
4. It is not amenable to further mathematical treatment.
3.3.2 INTERQUARTILE RANGE
It provides more knowledge about the distribution as it includes middle 50%

observations of the distribution. It is defined as the difference between the first and third
quartile of the distribution. The third or upper quartile ( Q3 ) divides the distribution in
such a way that 75% observations lie below it and 25% observations of the distribution
above it. It’s just opposite first quartile or lower quartile ( Q1 ) divides the distribution in
such a way that 25% observations of the distribution lie below it and 75% observations
above it. Thus, interquartile range gives us information about 50% central observations of
the distribution. If interquartile range is high, it means that the middle 50% observations
are a long distance and if it is low, it means that middle 50% observations are closely
near to each other. Quartiles divide a series into four equal parts.
There are different methods for calculating interquartile range, depending upon the type
of distribution. For calculating interquartile range we have to calculate the two quartiles.
3.3.2.1 Interquartile Range for Individual Series
Let the variable under study X takes the values x1 , x 2 ,......x n , then for the calculation
of interquartile range we apply the following steps;
Step1. Arrange the given observations in ascending or descending order of their

magnitude.
Step2. Now calculate first quartile ( Q1 ) and third quartile ( Q3 ) by the method:
 n +1
Q1 = Value of   th term in the arranged series.
 4 
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 n +1
Q3 = Value of 3   th term in the arranged series.
 4 
Step 3. The value of Q3 – Q1 is the interquartile range.
Example1. Find the interquartile range for the following series:
2, 12, 23, 4, 34, 25, 21, 17, 14
Solution. Arrange the given observations in ascending order of magnitude, we get,
2, 4, 12, 14, 17, 21, 23, 25, 34
Here n= 9
 n +1
∴ Q1 = Value of   th term in the arranged series.
 4 
 9 +1
= Value of  th = 2.5 th term in the arranged series.
 4 
= Value of 2nd term + ½ (3rd term - 2nd term)
= 4+ ½ (12 – 4) = 4+ 8/2 = 4+4 = 8
 n +1
Q3 = Value of 3   th term in the arranged series.
 4 
= Value of 3(2.5)th term = value of 7.5th term
= Value of 7th term + value of ½ (8th term – 7th term)
= 23+ ½ (25 – 23) = 23+ 1 = 24
Interquartile range = Q3 – Q1 = 24 – 8 =16
This shows that 50% observations of the given series lie between 8 and 24.
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3.3.2.2 Interquartile Range for Discrete Distribution
characteristic and f1 , f 2 ,.... f n be their corresponding frequencies then for the calculation of
quartiles, we calculate the cumulative frequencies. The interquartile range is calculated with
the help of the following steps.
Working steps for interquartile range
Step1. Arrange the given values in ascending order of magnitude.
Step2. Find the total of frequencies, called cumulative frequency (denoted by c.f)
N
.Step3. Find for first quartile, where N= ∑ f
4 .
N
Step4. Find cumulative frequency just greater than . The value of x, corresponding
4
this cumulative frequency is the value of first quartile.
N
Step5. Find 3   for third quartile.
4
N
Step6. Find cumulative frequency just greater than3 . The value of x,
4
corresponding to this cumulative frequency is the value of third quartile.
Step7. For interquartile range, find Q3 – Q1 .
Example2. Find the interquartile range for the following data.
X 21 15 17 9 5 7 8 10
F 2 5 3 4 5 1 6 12
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Solution. For calculating the interquartile range, first of all we calculate first and third
quartile and for this, we arrange the values of x in ascending order and then prepare the
cumulative frequency table as follows:
Table2. Quartiles in Discrete Distribution
x F c.f.
5 5 5
7 1 6
8 6 12
9 4 16
10 12 28
15 5 33
17 3 36
21 2 38
N = ∑ f = 38
N 38
Here = = 9.5 . The cumulative frequency just greater than 9.5 is 12 and the value of
4 4
x, corresponding to cumulative frequency 12 is 8. So the first quartile is 8.
N 38
Here 3 =3 × = 28.5 . The cumulative frequency just greater than 28.5 is 33 and the
4 4
value of x, corresponding to cumulative frequency 33 is 15. So the third quartile is 15.
Interquartile range = Q3 – Q1 = 15 – 8 =7.
This shows that 50% observations of the given series lie between 8 and 15.
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3.3.2.3 Interquartile Range for Continuous Distribution
N
is the first quartile class and the value of first quartile is given by:
4
N 
 −C
Q1 = L +  ×h
4
f
Where L= lower limit of the first quartile class
C= cumulative frequency just before the first quartile class
f = frequency of the first quartile class
h = magnitude of the first quartile class
N
3 is third quartile class and the value of third quartile is given by:
4
 N 
3 − C 
Q3 = L +  ×h
4
f
Where L= lower limit of the third quartile class
C= cumulative frequency just before the third quartile class
f = frequency of the third quartile class
h = magnitude of the third quartile class
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Example3. The following table gives the distribution of weights of 100 persons. Find the
interquartile range for this data.
Weight 40-45 45-50 50-55 55-60 60-65 65-70 70-75 75-80 80-85 85-90
Frequency 1 3 6 10 15 25 15 10 11 4
Solution. For calculating the interquartile range, first of all we calculate first and third
quartile and for this we prepare the cumulative frequency table as follows:
Table3. Quartiles in Continuous Distribution
Weight (in kg) (x) Frequency (f) Cumulative

Frequency (c.f.)
40-45 1 1
45-50 3 4
50-55 6 10
55-60 10 20
60-65 15 35
65-70 25 60
70-75 15 75
75-80 10 85
80-85 11 96
85-90 4 100
Total N = ∑ f = 100
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N 100
Here = = 25 . The cumulative frequency just greater than 25 is 35 and the
4 4
corresponding class is 60 – 65. So 60 – 65 class is the first quartile class and the first quartile is
given by:
N 
 −C
Q1 = L +  ×h
4
f
Where L= lower limit of the first quartile class = 60
C= cumulative frequency just before the first quartile class = 20
N= Total of frequency = 100
f = frequency of the first quartile class = 15
h = magnitude of the first quartile class = 5
N 
 −C
Q1 = L + 
4  × h = 60 + (25 − 20) × 5 = 60 + 1.66 = 61.66
∴
f 15
N 100
Here 3 =3 = 75 . The cumulative frequency just greater than 75 is 85 and the
4 4
corresponding class is 75 – 80. So 75 – 80 class is the third quartile class and the third quartile is
given by:
 N 
3 − C 
Q3 = L +  ×h
4
f
Where L= lower limit of the third quartile class = 75
C= cumulative frequency just before the third quartile class = 75
N= Total of frequency =100
f = frequency of the third quartile class = 10
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h = magnitude of the third quartile class = 5
 N 
3 − C 
Q3 = L + 
4  × h = 75 + (75 − 75) × 5 = 75
∴
f 10
So the third quartile is 75.
Interquartile range = Q3 – Q1 = 75 – 61.66 =13.34.
This shows that 50% observations of the given series lie between 61.33 and 75.
3.3.2.4 Merits and Demerits of Interquartile Range
Merits:
1. It is better measure than range.
2. It provides information on 50% observations of the distribution.
3. It is not affected from extreme observations
4. It can be calculated in case of open- end classes.
5. Unit of measurement is same as the unit of variable under study.
Demerits:
1. It provides information only on middle 50% observations and does not give
information on rest of the 50% observations. A good measure should use all the
data observations for providing better estimate of variability.
2. It is not an ideal measure.
3. Its value is quite variable from sample to sample.
4. It is not amenable to further mathematical treatment.
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3.3.3 MEAN DEVIATION
Arithmetic mean, the measure of central tendency plays an important role in the
measure of variability also. A measure of variability in the data can be obtained as the
average deviation of observations from the arithmetic mean. This is called mean deviation. A
measure of variability can be obtained by taking the deviations of the observations from their
mean, ignoring the signs. This deviation can be taken from any mean but usually arithmetic
mean, median and mode is used for this. Mean deviation is least when deviation is taken
from median.
Let us consider the marks of 5 students of a class. The marks are 6, 7, 9, 10, and 18.
The arithmetic mean of the marks is 10. The deviations of marks from their arithmetic mean
are – 4, – 3, –1, 0, 8. We see that the sum of deviations is zero. This is the property of
arithmetic mean, i.e., the sum of deviations taken from mean is zero. So in calculating mean
deviation come across two problems:
1. Which average should be taken for deviation?

The answer to this problem is that any of the averages – mean, median or mode
can be used for deviation. But generally the arithmetic mean is used.
2. What should be the sign of the deviations?
The answer to this problem is that we should use the positive sign for the
deviations, i.e., the absolute deviations should be taken. This is done because the
sum of deviations taken from mean is zero. This is clear from the example of
marks of 5 students taken in the above paragraph.
3.3.3.1 Mean Deviation for Individual Series
For an individual series if x1 , x 2 ,......x n are the values of the variable x, then the mean
deviation is given by the following formula:
1
Mean Deviation (M.D.) =
n
∑ xi − A
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When A is taken as arithmetic mean, it is called mean deviation about arithmetic

mean. When A is taken as median, it is called mean deviation about median and when A is
taken as mode, it is called mean deviation about mode.
Example4. Find the mean deviation about mean for the following series:
2, 4, 5, 7, 10, 13, 9, 15, 25
Solution. For finding the mean deviation about mean first of all we find the arithmetic mean
of the given series.
x=
∑ x = 90 = 10
n 9
Now we form the following table:
Table4. Mean deviation in Discrete Series
x x−x
2 8
4 6
5 5
7 3
10 0
13 3
9 1
15 5
25 15
∑ x = 90 ∑ x − x =46
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1 1
n
∑ x−x =
9
× 46 = 5.11
Hence the mean deviation of the given series is 5.11
3.3.3.2 Mean Deviation for Discrete Distribution
characteristic and f1 , f 2 ,.... f n be their corresponding frequencies then the mean deviation
about mean is given by the following formula:
1
N
∑ f x−x , where N = ∑ f = Total frequency
Example5. Ten patients were examined for uric acid test. The operation was performed
1050 times and the frequencies so obtained for different number of patients (x) are shown in
the table given below.
x: 0 1 2 3 4 5 6 7 8 9 10
f: 2 8 43 133 207 260 213 120 54 9 1
Compute the Mean deviation about arithmetic mean.
Solution. Let 5 be the assumed mean. Now we prepare the following table for the
calculation of mean deviation about mean.
Table5. Mean Deviation for Discrete Distribution
X Frequency d = x- 5 fd x−x f x−x

(f)
0 2 -5 -10 5.01 10.02
1 8 -4 -32 4.01 32.08
2 43 -3 -129 3.01 129.43
3 133 -2 -266 2.01 267.33
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4 207 -1 --207 1.01 209.07
5 260 0 0 0.01 2.6
6 213 1 213 0.99 210.87
7 120 2 240 1.99 238.8
8 54 3 162 2.99 161.46
9 9 4 36 3.99 35.91
1 1 5 5 4.99 4.99
0
∑f = 1050 ∑ fd = 12 ∑ f x − x = 1302.56
∑ fd = 5+
12
= 5 + 0.0114 = 5.01cm
N 1050
1 1
Now, Mean deviation (M.D.) =
N
∑ f x−x =
1050
× 1302.56 = 1.24cm
3.3.3.3 Mean Deviation for Continuous Distribution
In case of continuous distribution, there are given class intervals and their
corresponding frequencies. First of all we find the mid values of these classes and treat them
as the variable values. Then we calculate mean and then mean deviation about mean. The
procedure will be clear from the following example.
Example 6. For the data given in the below table on systolic BP of 68 patients, calculate the
mean deviation about arithmetic mean.
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Systolic BP (mmHg) Frequency (f) Systolic BP (mmHg) Frequency (f)
90-100 3 140-150 11
100-110 5 150-160 9
110-120 7 160-170 6
120-130 10 170-180 2
130-140 15
Solution. For the calculation of mean deviation we prepare the following table:
Table6. For calculation of Mean in Continuous Distribution
Systolic Frequency Mid fx x−x f x−x

BP (f) Value
(mmHg) (x)
90-100 3 95 285 40.6 121.8
100-110 5 105 525 30.6 153
110-120 7 115 805 20.6 144.2
120-130 10 125 1250 10.6 106
130-140 15 135 2025 0.6 9.0
140-150 11 145 1595 9.4 103.4
150-160 9 155 1395 19.4 174.6
160-170 6 165 990 29.4 176.4
170-180 2 175 350 39.4 78.8
Total ∑f = 68 ∑ fx = 9220 ∑ f x − x = 866.6
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∑fx i i
9220
x == i =1
= = 135.6mmHg
N 68
Mean deviation about mean (M.D.) is given by
1 1
N
∑ f x−x =
68
× 866.6 = 12.74mmHg
Hence the mean deviation about mean of the systolic BP is 12.74mmHg.
3.3.3.4 Merits, Demerits and uses of Mean Deviation
Merits:
1. Mean deviation is easy to understand and to calculate.
2. Mean deviation is least when taken from median.
3. Mean deviation is less affected from extreme values as compared to range and
standard deviation.
4. Mean deviation is better measure of variability than range and interquartile range as
it uses all the observations of the data.
Demerits:
1. It is rarely used in social sciences.
2. Its results are not accurate because it is least when taken from median but median
itself is not a good measure of central value when the variations in the series are
large.
3. It is not suitable to further algebraic treatment as the negative signs of the

deviations are also taken as positive.
Uses:
It is used in studying the economic problems.

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3.3.4 STANDARD DEVIATION
For describing the scatteredness of the data values the best measure of variability is the
standard deviation. It is denoted by σ . If standard deviation in a data is small, it means there
is high degree of homogeneity in the data values and vice versa if the value of standard
deviation is large, it means there is a large heterogeneity in the data values.
It is defined as the positive square root of the arithmetic mean of the deviations of values
when the deviations are taken from their arithmetic mean.
3.3.4.1 Standard Deviation for Individual Series:
Let the variable under study X takes the n values x1 , x 2 ,......x n , their standard deviation
is given by the following formula:
∑ (x − x) ∑d
2 2
σ = i
σ= where d = xi − x
n or n
The steps of the procedure are as follows:
Step1. Compute the arithmetic mean of the given series.
Step2. Compute the deviations of the series values from the mean, i.e., compute
d = xi − x
Step3. Compute the square of the values got in step 2, i.e., compute d 2 = ( xi − x )
2
.
Step4. Find the sum of values got in step 3 and divide it by the number of values, i.e.,
∑d ∑ (x − x)
2 2
i
compute = .
n n
Step5. Take the square of the value got in step 4. This is the required value of the
standard deviation.
The procedure will be clear from the example given below:
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Example7. Compute the standard deviation of the following series:
12, 15, 17, 21, 28, 27,
Solution. For the computation of standard deviation we prepare the following table:
Table7. S.D. for Individual Series
x d = (xi − x ) d 2 = ( xi − x )
2
12 -8 64
15 -5 25
17 -3 9
21 1 1
28 8 64
27 7 49
∑ x = 120 ∑ d 2 = ∑( xi − x ) = 212
2
x=
∑ x = 120 = 20
Arithmetic mean n 6
∑d
2
212
σ = = = 35.33 = 5.94
Standard deviation n 6
Hence the standard deviation of the given series is 5.94
3.3.4.2 Short-Cut Method of Standard Deviation
This method is applied when mean is in fractional form because in that case the
deviations and their squares make the calculations difficult. So in this case we take the
deviations of the values from an assumed mean.
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Let d = x – A, here A is the assumed mean, then in this case the formula for standard
deviation is given by as :
∑d ∑d 
2 2
σ = − 
n  n  Where n is the number of observations.
 
We follow the following steps for the commutation of S.D. in this case:
Step1. Take any value of the series as assumed mean A.
Step2. Compute the deviations of the series values from the assumed mean, i.e.,
Compute d = xi − A
Step3. Find the total of step 2 values, i.e., find total of d, i.e., Σ d.
Step4. Divide the value of step 3 by number of values ‘n’ and find its square, i.e,
 ∑d 
2
 
 n  .
 
Step5. Compute the square of the values got in step 2, i.e., compute d 2 = ( xi − A)
2
.
Step5. Find the sum of values got in step 5 and divide it by the number of values, i.e.,
∑d ∑ (x − x)
2 2
i
Compute = .
n n
Step6. Subtract the value of step 4 from value of step 5 and then take its square root.
This is the required value of the standard deviation.
The procedure will be clear from the example given below:
Example8. Find the standard deviation in the above example 7 by short- cot method.
Solution. Let us take 21 as assumed mean A. Now we prepare the following table for the
computation of standard deviation.
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Table8. For Short –Cut Method of S.D.
x d = (x − 21) d2
12 -9 81
15 -6 36
17 -4 16
21 0 0
28 7 49
27 6 36
Total ∑ d = −6 ∑ d 2 = 218
∑d  ∑d 
2 2
218
∴ σ = −  = − 1 = 35.33 = 5.94
n  n  6
 
3.3.4.3 Standard Deviation in Discrete Frequency Distribution
characteristic and f1 , f 2 ,.... f n be their corresponding frequencies then standard deviation can
be calculate with the help of these methods:
1. Actual mean method
2. Assumed mean method
3. Step deviation method
The procedures of the three methods will be clear with the help of the examples.
1. Actual mean method:
For this we use the following formula:
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∑ f (x − x )
2
σ =
N
Where N = ∑ f =Total frequency
Example9. Calculate the standard deviation of the distribution of marks of the B.Sc.
botany class students. The data is given below:
x: 12, 18, 17, 15, 20, 25, 32, 42
f: 2, 1, 3, 5, 1 2 10 1
Solution. For the calculation of standard deviation we prepare the following table:
Table9. S.D. for Discrete Frequency Distribution by Actual Mean
X f fx d = (x − x ) ( x − x )2 f (x − x )
2
12 2 24 -12 144 288
18 1 18 -6 36 36
17 3 51 -7 49 147
15 5 75 -9 81 405
20 1 20 -4 16 16
25 2 50 1 1 2
32 10 320 8 64 640
42 1 42 18 324 324
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N = 25 ∑ f x = 600 ∑ f ( x − x ) = 1858
2
Arithmetic mean x =
∑ f x = 600 = 24
∑ f 25
∑ f (x − x )
2
1858
Standard deviation σ = = == 74.32 = 8.62
N 25
Hence the standard deviation of the given distribution is 8.62
2. Assumed Mean Method:

In this method we take a middle vale of x as the assumed mean A and the apply the
following formula:
∑ fd  ∑ fd 
2 2
σ = −  ; where symbols are in their usual meaning.

N  N 
 
For the procedure of this method we take a example below:
Example10. For the above example 9 apply assumed mean method for computing the
standard deviation.
Solution. Let us assume A= 20. Now for the calculation of standard deviation we prepare the
following table:
Table10. S.D. for Discrete Frequency Distribution by Assumed Mean
X f d = ( x − A) fd fd 2
12 2 -8 -16 128
18 1 -2 -2 4
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17 3 -3 -9 27
15 5 -5 -25 125
20 1 0 0 0
25 2 5 10 50
32 10 12 120 1440
42 1 22 22 484
N = 25 ∑ fd = 100 ∑ fd 2 = 2258
∑ fd  ∑ fd 
2 2 2
2258  100 
Standard Deviation σ = −  = − 
N  N  25  25 
 
= 90.32 − 16 = 73.68 = 8.62
Hence the standard deviation of the given distribution is 8.62
3. Step Deviation Method:

This method is applied when the values have some common interval (say h), we
divide the deviations by this common interval and apply the following formula:
∑ fd  ∑ fd 
2 2
σ = −  ×h
N  N 
 
The procedure of the method will be clear from the example given below:
Example11. Daily high blood pressure of a patient on 100 days is given below:
Bp (mmHg): 102 106 110 114 118 122 126
No. of days: 3 9 25 35 17 10 1
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Calculate the standard deviation of the above data.
Solution. Let us take the assumed mean A= 114. Here common interval h= 4. Now we
prepare the following table for the calculation of standard deviation.
Table11. S.D. for Discrete Frequency Distribution by Step Deviation
BP f
d=
(x − 114 ) fd fd 2
(mmHg) 4
102 3 -3 -9 27
106 9 -2 -18 36
110 25 -1 -25 25
114 35 0 0 0
118 17 1 17 17
122 10 2 20 40
126 1 3 3 9
Total N = 100 ∑ fd = 100 ∑ fd 2 = 2258
∑ fd  ∑ fd 
2 2 2
  154  − 12 
S.D.= σ = − ×h = −  ×4
N  N  100  100 
 
= 1.54 − .0144 × 4 = 1.235 × 4 = 4.94 MmHg
3.3.4.4 Standard Deviation in Continuous Frequency Distribution
In case of continuous distribution we find the mid values of classes and treated them
as the variable values x. In this case we can apply all the three methods discussed in previous
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section. But generally step deviation is applied. The formula is the same as in case of discrete
distribution discussed. The procedure is described in the example given below.
Example12. Calculate the standard deviation for the following table giving the age
distribution of 542 persons of a city.
Age in years: 20 – 30 30 – 40 40 – 50 50 – 60 60 – 70 70 – 80 80 – 90
No. of members: 3 61 132 153 140 51 2
Solution. For the calculation of standard deviation, let us take d = ( x − 55) / 10 . Here we let
assumed mean A = 55 and common interval (h) = 10. Now we prepare the following table:
Table12. S.D. in Continuous Distribution by Step Deviation Method
Age Mid- Frequency fd fd 2

group value (f) d=
(x − 55)
(x) 10
20-30 25 3 -3 -9 27
30-40 35 61 -2 -122 244
40-50 45 132 -1 -132 132
50-60 55 153 0 0 0
60-70 65 140 1 140 140
70-80 75 51 2 102 204
80-90 85 2 3 6 18
Total N=542 ∑ fd = −15 ∑ fd 2 = 765
∑ fd  ∑ fd 
2 2
S.D.= σ = −  ×h
N  N 
 
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2
765  − 15 
= −  × 10 = 1.334 × 10 = 11.55
542  542 
Hence the standard deviation of age of the given distribution is 11.55 years.
3.3.4. Merits, Demerits and Uses of Standard Deviation
Merits:
1. It is rigidly defined.
2. It uses all the observations of the data in calculation.
3. It is used in correlation.
4. It is affected least by fluctuation of sampling.
5. It is suitable for further mathematical treatments.
6. It is the best measure of variability.
Demerits:
1. Its calculation is difficult in comparison to other measures of dispersion.

2. It is sensitive to extreme values.
3. It is not easily understandable for a common person.
Uses:
1. It is best measure of comparison of variability.

2. It is used in partitioning between groups and within groups in analysis of
variance and design of variance.
3. It is used with mean in normal distribution for finding the areas.
4. It shows best dispersion of values from the mean.
5. It is very much used in medical field.
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3.4 VARIANCE AND COEFFICIENT OF VARIATION
3.4.1 VARIANCE
It is just the square of the standard deviation. It is denoted by σ 2 . In other words

variance is the arithmetic mean of the squares of the deviations, when deviations are
taken from their arithmetic mean.
3.4.2 COEFFICIENT OF VARIATION
It is the best measure of the comparison of variability of the two series or populations.
The units of measurement of the two populations may be different. This comparison is
possible because it is a unit free measure. It is presented in percentage and
is expressed as:
σ
Coefficient of variation (C.V.) = × 100 ; where notations have their usual meaning.
x
A series having lesser c.v. is called more consistent or more homogeneous, i.e., the
values of the series are closer to the mean of the series and if the c.v. of a series is larger,
it is called more variable or in other words more heterogeneous series, i.e., the values of
the series far apart from the mean of the series.
Example13. Calculate the coefficient of variation of the distribution of marks of the

B.Sc. botany class students. Given the following information:
Average marks Standard deviation of marks
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x = 24 σ =6
σ 6
Solution. Coefficient of variation (C.V.) = × 100 = × 100 = 25%
x 24
Hence the C.V. of the marks is 25%.
Example14. The following data shows the mean and standard deviation on systolic BP
and weight of 10 persons as:
BP Weight
Mean S.D. Mean S.D.
120 15 60 4.5
Compare the two characteristics.
Solution. For comparison of the two characteristics we find the C.V. of these
characteristics.
σ 15
C.V for BP = × 100 = × 100 = 12.5%
x 120
σ 4.5
C.V for Weight = × 100 = × 100 = 7.5%
x 60
We see that the coefficient of variation of BP is more than the coefficient of variation
of weight so BP is more variable than the weight of the given persons.
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3.5 SUMMARY
The study of this chapter provides us the knowledge of dispersion or variability and
measures of variability. From the study of this chapter we came to know the definitions of
the measures of variability - range, interquartile range, mean deviation and standard
deviation. We learnt the different method for calculating interquartile range, mean deviation
and standard deviation for individual series, in case of discrete distribution and continuous
distribution. We studied the merits, demerits and uses of these measures. From the study of
these methods, merits, demerits and uses we came to know the situations where which
method is suitable and also which measure is suitable for the particular situation? Coefficient
of variation is the best measure for comparison on the basis of variability as it uses all the
values and is a unit free measure.
3.6 GLOSSARY
Range: It is the difference between the largest and the smallest observation of the data.
Interquartile Range: It is based on the first and third quartile of the distribution and it
express the limit for the middle 50% observations of the data.
Dispersion: It shows the spread of the data values around the central value of the
distribution.
Mean Deviation: It is the average of the absolute deviations of the data values. Generally
deviations are taken from mean.
Variance: It is the arithmetic mean of squares of the deviations of the values when
deviations are taken from their mean.
Standard Deviation: It is the positive square root of the variance.
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Coefficient of Variation: It is the best measure of comparison on the basis of variability of

two or more series. It can also be used if the units of measurements are different because it is
a unit free measure.

Question1. Find the range of the following series:
1, 23, 32, 24, 45, 42, 35, 37
Question2. Find interquartile range for the given series:
2, 4, 3, 5, 7, 10, 12, 13, 15, 14, 9, 1, 8, 6, 11, 16
Question3. Find the standard deviation of erythrocyte sedimentation rate (ESR) of the data 3,
4, 5, 4, 2, 4, 5, 3 found in 8 normal persons.
Question4. Calculate the mean and standard deviation of the following data on the length of
fishes (in cm) .
Length: 10 20 30 40 50 60 70
No. of Fishes: 4 6 10 15 20 15 10
Question5. A firm has selected a random sample of 100 from its production line and has
obtained the data shown in the table below:
Class interval Frequency Class interval Frequency
130- 134 3 150-154 19
135- 139 12 155-159 12
140- 144 21 160-164 5
145-149 28
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Question6. Compute the standard deviation for data of hypo B.P. (in mmHg).
B.P. : 50 60 70 80 90 100 110 120
No. of Patients; 14 40 54 46 26 12 6 2
Question7. The C.V. of two series of observations are 20% and 25% with S.D. 5 and 8
respectively. What are their arithmetic means?
Question8. Which of the following is not a measure of dispersion?
a. Range b. Mean deviation c. median d. standard deviation
/Question9. The easiest measure of dispersion is:
a. Mean deviation b. S.D. c. interquartile range d. range
Question10. The range of the series 2, 3, 7, 9, 12, 17, 21 is:
a. 2 b. 21 c. 19 d. None of these
Question11. Which measure of dispersion is the best?
a. Range b. mean deviation c. C.V. d. S.D.
Question12. For comparison of two different series, the best measure of dispersion is:
a. Range b. S.D. c. mean deviation d. C.V.
Question13. Mean deviation is least when taken about:
a. Median b. mean c. zero d. mode
Question14. The mean of a series is 10 and C.V. 40%. Its S.D is:
a. 4 b. 16 c. 40 none of these
Question15. If all the values of a series are 4, the S.D. of this series is:
a. Zero b. 16 c. 4 d. 2
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Answers: 1. 44, 2. 8, 3. 0.97, 4. 14.79, 5. 7.21, 6. 15mmHg, 7. 25, 32, 8. c, 9. d.

10. c, 11. d, 12. d, 13. a, 14. a, 15. a.
3.8 REFERENCES
(1996).

U.S.A. (2000).
(2006).
New Delhi. (2010).
Question 1. Define mean deviation and write down its merits and demerits
Question 2. Explain standard deviation and write down its merits and demerits.
Question 3. Which measure of dispersion is the best and why?
Question 4. Which measure is used if measurement units are different?
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UNIT4: PRINCIPLES AND USES OF ANALYTICAL
INSTRUMENTS
CONTENT
4.1- Objectives
4.2 Introduction
4.3. Principles and uses of analytical instruments
4.3.1 pH meter
4.3.2 UV-visible spectrophotometer
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4.1- OBJECTIVES
In this chapter you will learn about the basic or common instrument used in the
Laboratory such as pH meter, UV-visible spectrophotometer, Centrifuges (clinical,
High-speed and ultracentrifuge), Geiger Muller and scintillation counters
4.2. INTRODUCTION
In biological science there is basic requirement of having knowledge of instrument.

Instrument play important role in laboratories such as soil analysis lab, diagnostic lab,
molecular biology lab, microbiology lab, chemistry lab, physics lab and diagnostic lab.
We cannot imagine the laboratories without basic instrument such as pH meter,
centrifuge and spectrophotometer. These instruments are very necessary for analysis.
Instrument not only speedup the analysis but also have accuracy, specificity and
sensitivity. pH meter is one of the important instruments used in basic laboratory to
measure pH of water, soil and food etc. Centrifuge is used to separate the mixture of two
liquid depending on the density of liquid. UV spectrophotometer is generally used in
analytical chemistry for the quantitative determination of different analyses, such as
transition metal ions, highly conjugated organic compounds, and biological
macromolecules. G-M counter and Scintillation counter is used for the detection of
radioactivity.
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4.3. PRINCIPLES AND USES OF ANALYTICAL INSTRUMENTS
4.3.1 pH METER
pH meter is generally used to determine the pH of soil, water and culture medium used
for the cultivation of fungi and bacteria. There is presence of electrode which very
sensitive to detect the change in H ion concentration. The electric circuit measure the
electromotive force developed across the electrode pair. pH is abbreviation of “Pondus
Hydrogenii”and it was proposed by Sorenson in 1909 in order to express the small
concentration of hydrogen ions. pH is a unit of measure that describe acidity and
alkalinity of solution. It is measured on a scale 0 to 14 and defined as the negative
logarithm of hydrogen ion activity.
pH= -log [H+]
The pH value of a substance is directly related to the ratio of the hydrogen ion and
hydroxyl ion concentrations. If the H+ concentration is higher than OH- the material is
acidic. If the OH- concentration is higher than H+ the material is basic.
Figure 4.1. Digital pH meter
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Principle of pH meter
The pH electrode consist of the pH-sensitive electrode which is a thin glass membrane
whose outside surface contacts the solution to be tested. The inside surface of the glass
membrane is exposed to a constant concentration of hydrogen ions (0.1 M HCl) (Fig.8).
Inside the glass electrode assembly, a silver wire, coated with silver chloride and
immersed in the HCl solution known as Ag/AgCl electrode. This electrode carries current
through the half-cell reaction. The potential between the electrode and the solution
depends on the chloride ion concentration, but, since this is constant (0.1 M), the
electrode potential is also constant. To complete electrical circuit reference electrode is
needed. So, Ag/AgCl electrode is immersed in an 0.1 M KCl solution which makes
contact with the sample through a porous fiber which allows a small flow of ions back
and forth to conduct the current. The potential created at this junction between the KCl
solution and the test solution is nearly zero and nearly unaffected by anything in the
solution, including hydrogen ions. A voltmeter in the probe measures the difference
between the voltages of the two electrodes. The meter then translates the voltage
difference into pH and displays it on the screen .
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Figure 4.2: Working of pH meter

Calibration of pH meter
1. Switch on the pH meter and move the knob from stand by to pH.
2. Rinse the electrode with double distilled water and wipe out the tip gently with tissue
paper,
3. Place the electrode into the solution of pH 7.0 and adjust the pH by adjustment given in
pH meter.
4. After adjusting again wash the tip with distilled water and wipe out with tissue paper.
5. Dip the electrode in to solution of pH 4.0 (if your solution is acidic) and pH 9.0 (when
your solution is basic)
6. Turn on the knob to slope position till it shows 4.0 or 9.0.
7. After reading the pH of sample. Again wash the electrode with distilled water.
8. Immerse the electrode in to the distilled till used for the next time.
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4.3.2- UV-visible spectrophotometer
Spectroscopy is the measurement of (spectrum of light) electromagnetic radiation, absorbed,

scattered, or emitted by atoms, molecules, or other chemical species. Each chemical species has
unique energy states; spectroscopy can be used to identify the interacting species separately.
Moreover; interaction of light with different compounds offers several possibilities of qualitative
and quantitative measurements. Electrons in an atom move around the nucleus in orbitals and
possess characteristic energy also known as ground state. Energy transfer to these electrons
energizes them substantially to let them leave their orbital to jump over the next level of energy
orbital or level; this is called as excited state. Generally, on ceasing the energy supply, electrons
emit the absorbed energy in the form of radiation giving rise to the atomic spectrum or simply
“line spectrum”, which is represented as graph of the amount of energy absorbed or emitted by a
system against wavelength or similar electromagnetic parameters. Visible light forms a part of
the electromagnetic spectrum with γ rays at one end having wavelength of the order of 10-14 m,
and radio waves at the other end having wavelength 3 × 103 m or greater. As atoms, each of the
electrons in a molecule usually occupies the available lowest energy level (ground state).
Electrons in a molecule change their energy level only after the absorption or emission of the
distinct quanta (particular energy radiation) of radiation by the molecule. Depending upon the
absorption and emission of energy quanta, there occur two viz., absorption and emission spectra
respectively. For molecules, each ground and excited state is subdivided into a number of
vibrational and rotational energy sublevels, molecular spectra is therefore seen as “band
spectra”.
Types of spectra
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Electronic Spectra
Electronic spectra arise due to the outer electrons of atoms changing between major electronic
energy levels. Such spectra occur in the visible and ultraviolet regions and are usually
accompanied by changes in the rotational and vibrational energy levels. These spectra are used
routinely in biochemistry. Fluorescence spectra may also arise owing to these transitions.
Vibration – Rotation Spectra
Vibration spectra caused by changes in the vibration energy levels. They occur in the near infra-
red region and may be accompanied by changes in the rotational energy levels. Such spectra are
sometimes used in studies of the detailed structure of biological macromolecules in non-aqueous
environments.
Electron Spin Resonance (ESR) Spectra and Nuclear Magnetic Spectra
These spectra arise due to changes in the directions of the spins of electrons and nuclei
respectively in a magnetic field. These two types of spectra are valuable for studying the
structure of biological macromolecules.
Molecular Band Spectra
Molecular band spectra may be resolved into a number of very close line spectra, corresponding
to the vibrational and rotational energies of the electrons only at extremely high resolution.
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3.0.1 Absorption
Photons of UV and Visible light may sometimes impart their energy to materials by interaction
with individual atoms or molecules. Energy is imparted to the atoms or molecules, causing the
excitation of valence electrons. Molecules with excited electronic states represent an unstable
state will relax by allowing their electrons to fall to the ground state as soon as possible (10-16
sec.).
How much radiation is absorbed?
The absorbance (A) is defined as the log10 of the ratio of the incident to transmitted light
intensities:
= ( ) - The intensity is measured in the unit of power; therefore the absorbance is a
unit less quantity. The intensity of light can be detected by photo-multiplier tubes. The collision
of a photon of appropriate energy with the suitable molecule results in absorption of light.
Beer-Lambert Law or Law of absorption
The Beer-Lambert law relates the absorption of most molecular species to the concentration (Cn),
the path length (l) and the molar absorptivity (ɛ).
A = ɛCnl
The wavelength of maximum absorption is known as λ max and is usually used as the
wavelength for molar absorptivity (ɛ). ɛ is also sometimes known as extinction coefficient and is
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measured in dm3mol-1cm-1.. The Beer-Lambert law is sometimes expressed in terms of

transmittance T as:
T = 1/A
Pathlength of 1 cm is normally chosen to simplify the calculation of the absorbance or molar
absorptivity. All modern spectrophotometers are designed to comply the Beer-Lambert law. A
plot of absorbance versus wavelength is known as UV-Visible Spectrum and is measured by a
UV-Visible Spectrophotometer.
3.0.2. Spectrometers /Spectrophotometer
A spectrometer or spectrophotometer is a device which measures the absorbance by molecules in
a given sample. Spectrometers are a monochromator equipped with a photo-transducer. Single
and double beam spectrophotometers are readily available in the market whereas double beam
spectrophotometers are commonly used in scientific laboratories because of the ease of function
and precision in readings of absorbance.
Figure 4.4. Schematic of Double Beam Spectrophotometer
spectrophotometer comprised of several devices (figure 3.1) such as monochromatic for

monochromatic light of particular wavelength, grits, photo-detectors, and a processor that send
signals to the interfaced computer. To understand and to define a spectrophotometer, it is
recommended to study each and every component in details.
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Light Source
The irradiation of a sample for UV and Visible spectroscopy requires a light source with constant
output intensity. Tungsten (W) filament lamp are used for UV and visible light (320 – 2500) nm
while hydrogen and deuterium lamps UV radiation. The light source should also be sufficiently
intense so as to allow sufficient transmitted radiation to be detected when the absorption falls
within a range of 0 -2. In more advanced spectrophotometers, tungsten-halogen lamps are
frequently used. Such lamps contain small quantity of iodine and the lamp is enclosed in quartz
housing. The iodine (a halogen) raises the temperature of lamp to 3500K, which permits the
intensity of the output radiation down to ~ 190 nm (UV range). Quartz is permeable for UV
radiation whereas glass blocks the UV radiation.
Hydrogen and deuterium lamps produce high intensity UV radiation. Electrical excitation of
hydrogen atoms at low pressure may produce two hydrogen of either low energy with the high
energy photon or higher energy with low energy photons. As a result of this unequal uniform
output the deuterium or hydrogen lamps give rise to the outputs of radiation over a wavelength
range of 160 – 375 nm.
Monochromator
A monochromator is the most commonly used device to select a wavelength of light for the
irradiation of a sample. Monochromators use a
series of lenses, mirrors, slit and windows together
with either prisms and /or diffraction gratings to
isolate a narrow band of wavelengths. It is
impossible to select one wavelength range. The
wavelength of the radiation source will tend to
follow a Gaussian distribution around a mean
value of wavelength known as the nominal
Figure 4.5 Effective Bandwidth wavelength. The effective bandwidth is defined as
the wavelength range which corresponds to the half peak height width of
The wavelength distribution profile. There are two common types of monochromators viz.,
Monochromators based on refracting prisms and Monochromators based on diffraction grating
120 | P a g e
are used in the modern age spectrophotometers. A monochromator with a bandwidth of less than
~ 0.5 nm is perfectly acceptable.
Figure 4.6 Simplified Schematic of a Prism Monochromator
In refracting prism monochromators, the white light enters via a slit passing through a collimator
into the monochromator. A collimator is equipped with collimating lens, produce a parallel beam
of radiation as shown in Figure 6. The light then passes through a refraction prism that disperses
the light into its component wavelengths. The light is focused by another lens on the focal point
where another slit is placed. By rotating prisms, radiation of different frequency is selected.
Diffraction grating based monochromator
White light passes through an entrance slit and is
focused on a diffraction grating through a concave
mirror. The diffraction grating disperses the light
into its component wavelengths and reflects the
light onto a second concave mirror. The grating can
be rotated by a stepper motor. Light is then focused
on the exit slit by this second concave mirror. By
rotating the grit, light of different wavelengths can
Figure4. 7. Optics of Diffraction Based
be selected. Monochromator
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Light Detector
The absorbance of an analyte is performed by a photon tube or light detector which measures the
intensity of transmitted light. A photo-multiplier tube or photon tube works on the principle of
photo-emission or electronic transition involving the counting of photons enabling the
monitoring of the intensity of light. Figure 3.5 show a photon tube where by the photons enter
into the tube to emit electrons
from the large cathode that are
captured by a wire anode placed in
front of the cathode. The
entire circuit is encased in
quartz housing under high
vacuum. The circuit gets
completed and the current
generated is directly
Figure4. 8. A photo-multiplier tube
Proportional to the intensity of light entering into the photon tube. Photon tube operates as photo-
multiplier tube whereby a cascade effect is produced, which enables the collection of 106 – 107
electrons per photon entering into the tube.
4.4 CENTRIFUGE
Centrifugation is based on the principle of centrifugal force in which liquid are subjected to
high speed to separate solid from liquid or liquid from liquid depending on the density. In
centrifugation heavy particle settled down and light particle will rises to the top. The
substance which is settled down is called “Pellet” and the remaining fluid or overlying fluid
is called “Supernatant”. Centrifugation process separates two substance of different density.
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Basic principle of centrifugation

When the tube is filled with fluid and allowed to spin, as the rotor spin the apparent
centrifugal force act on the sample of fluid and the analyte inside the fluid is pushed both
radially outwards to the side of centrifuge tube.
Relative centrifugal force F = m ω2r
Where,
F= Relative centrifugal force
m= Mass of particle
r= radius of the circular motion of the centrifuge (unit meter).
ω= angular velocity of the centrifuge (unit radian per sec)
The rate of sedimentation depends upon the applied centrifugal field (G), which depend upon
the radical distance of the particle from the rotation axis and square of the angular velocity.
G= ω2r
Where angular velocity (ω) = revolution/min (one revolution is equal to 2 radian)
Putting the value in G , therefore
G=
The relative centrifugal force (RCF) in g units (g=9.8065 M/sec2 or 980 cm /sec 2)
RCF = = 1.11 x 10-5 (revolution min-1)2 r
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The rate of settling of particle depends upon (a) the relative centrifugal force (b) the
density of the specimen, (c) the density of the fluid, (d) frictional forces, and (e) the size
and shape of the specimen
Consider if the suspending particle is spherical, then the rate of settling of spherical
particle can be expressed by following equation.
( )
R=
Where,
R= Rate of settling
r= Radius of the particle
g= acceleration due to gravity
dp= Density of the particle
df=Density of the fluid
n=viscosity of the fluid
The rate of sedimentation depend on the applied centrifugal field i.e. RCF in g units (980
cm/sec2).
Types of rotor
The centrifuge is equipped with two types of rotor
1. Fixed angle rotor: In this type of rotor, the centrifuge tube containing samples are placed
in the shield in the rotor at fixed pre-set angle. The rotor holds the centrifuge at fixed
inclination i.e. 35 degree to the vertical. The solute during centrifugation forced against
the side of the tube resulting in faster separation of solute from the suspension. The
disadvantage of this rotor is that there are chances of abrasion due to striking the particle
to the wall of centrifuge tube. Another disadvantage of this rotor is there is formation of
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smear like sedimentation than clear pellet formation. This type of rotor has short run time
(Fig 9).
Figure 4.9: Fixed angle rotor and formation of pellet on side wall
2. Swinging bucket rotor: In this type of rotor the sample centrifuge tube are placed
vertically and when the machine is started the bottom of the tube swing outward
(horizontally) as the shaft rotates. This rotor has advantage than fixed rotor having clear
pellet at the bottom of the tube (Fig.10).
Figure 4.10: Working of swing bucket rotor before and after centrifugation
3. Vertical Rotor: In this type of rotor angle of placement of rotor is fixed but not at the
slanting position it is vertical i.e. perpendicular to centrifugal field and the rotation axis as
shown in Fig. 4.11. Band separate across the diameter of the tube rather than down the
length of tube.
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Figure 4.11: Working of vertical rotor before and after centrifugation
Types of centrifuge
Bench centrifuge: The most common type of centrifuge which generally used for
common purposes such as separation of serum, plasma from blood sample required for
serological reaction. It is not used for the fine substance such as organelle etc. The
maximum speed of this type of centrifuge is 4000-5000 rpm and they operate is at
ambient temperature. Now a day’s small microfuge are available which can easily put
into refrigerator to keep temperature cool for sedimentation to prevent denaturation heat
sensitive substance such as Protein. .
Refrigerated centrifuge (Large capacity):
Refrigerated centrifuge have inbuilt cooling system for rotor such as compressor. It has
speed of 6000 rpm with relative centrifugal field 6500g. Refrigerated centrifuge is used
for the heat sensitive substance. This type of centrifuge can be run with fixed type rotor
or swinging bucket type rotor. The balancing and placing of sample is very important in
centrifuge. Always keep balance by placing centrifuge tube opposite to each other so that
the load is distributed equally around the axis of rotor as shown in Fig. The sample tubes
or centrifuge tube of 10, 50 and 100 ml can be used for centrifugation. The machine
comes with changeable rotor according to the requirement of researcher (Fig. 4.12).
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High Speed refrigerated Centrifuge: As name indicates this type of centrifuge work on
high speed i.e. 25000 rpm generating RCF of about 60000 gg.. It is also having flexibility
of using both types of rotor i.e. fixed rotor or swinging bucket type rotor. It is used for
separating bacteria, cell organelles and precipitated proteins. It is not used for the
sedimentation of viruses, and small organelle
organelless such as ribosome, for this purpose
ultracentrifuge is required having maximum speed.
Figure 4. 12: Working and parts of Refrigerated centrifuge
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Ultracentrifuge: Centrifuge which is used to sediment viruses and small organelles like
ribosome have speed of 80000 rpm with a relative centrifugal field of upto 600000 g. it
was developed by Svedberg in 1929 and demonstrated the subunit of proteins.
Ultracentrifuges are of two types:
a. Preparatory ultracentrifuge:. Preparatory centrifuge is generally used for the
centrifugation or separation of cellular organelles such as mitochondria, ribosome,
microsome and viruses. It is also used for the gradient separation of solution containing
increasing concentration of dense substance. For e.g. sucrose solution is used for the
separation of cell organelles. Caesium salt is used for the separation of nucleic acid. In
this centrifugation the sample is run at high speed and then rotor is allowed to come to
smooth stop and gradient is taken out to isolate the separated component.
b. Analytical centrifuge (AUC): A typical analytical centrifuge generate centrifugal field of
2500000 g. Analytical centrifuge is used for the determination of the purity of
macromolecule, determination of relative molecular mass of solute in their native state,
for the examination changes in molecular mass of super molecular complex. It consists of
optical detecting system to monitor the material during sedimentation for concentration
distribution in the sample at any time during centrifugation using ultra violet light
absorption and optical refractive index sensitive system. Measurement of sample
concentration at wavelengths from 200 to 800 nm detection of macromolecules
containing strong chromophores. It is used to analyse protein, polysaccharide, nucleic
acid, drug, ligands, gases, organelles and viruses.
When the machine switched on or rotor moves the image of analyte (for e.g cells or
protein) are projected by an optical system on to film or computer. The concentration
of the solution at various points in the cell is determined by absorption of light of the
appropriate wavelength and them measuring the degree of blackening of photographic
film. This will facilitate to observe the separation of sample concentration versus the
axis of rotation due to applied centrifugal force. AUC are generally used for the two
types of experiment
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a. Sedimentation equilibrium experiment has aim to determine the total time course of
sedimentation and report on molar mass and size distribution of dissolved
macromolecule
b. Sedimentation velocity experiment is dealing with the final steady state of the
experiment where sedimentation is balanced by diffusion opposing the concentration
gradient
1.4.1- GEIGER MULLER COUNTERS (GM COUNTER)
The GM counter was named after its inventor Hans Geiger and Walther Muller. This
instrument was first developed by Geiger in 1908 with Ernest Rutherford and name
Geiger counter. At that time this device has limited used i.e. it can detect only alpha
particle. Later on the research scholar of Geiger, Walther Muller improved this device for
detecting more types of ionization radiation. Now a day this instrument is known as
Geiger- Muller Counter which is used as particle detector that measure ionization
radiation i.e. beta particle and alpha particles except gamma particle because it does not
ionize the gas.
Figure 4.13. Working principle of G-M counter
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GM counter consist of a metal tube covered with glass which acts as cathode and along
the axis there is thin wire made up of tungsten which acts as anode. The tube is filled
with inert gas such as Argon, Helium, Neon with halogen added at low pressure. There is
window at one end where mica sheet is fitted, from this window radiation enter into the
tube. A high potential difference of about 1000 V is applied between the electrodes
through a high resistance R of about 100 mega ohm. (Fig.4.13)
Working of G-M counter

It is based on the principle of ionization as the charged particle passing through the gas
medium present in the tube ionizes the atoms of gas by energy transfer.
High potential difference i.e. 1000 V is applied across the anode and cathode of the tube
so that a high radial electric field nears the central wire is obtained (Fig.13). Due to the
formation of high electrical field electrons generated by ionizing collisions between a
high-speed particles entering the tube and the inert gas atoms are accelerated towards the
anode wire by the strong electric field and acquire within a very short distance a high
speed of their own. Because of this speed, they too can ionize other atoms and free more
electrons. The object of the counter is to produce a single pulse for each particle entering
the tube. This can only be achieved if spurious pulses due to secondary electrons released
from the cathode surface by the bombardment of ions are completely suppressed so that
the tube can recover as quickly as possible to be in a state when it is able to record the
next entering particle. A quenching gas (halogen or organic vapours) introduced into the
tube is to serve this purpose. The idea is to allow the inert gas ions on their way to the
cathode to collide with the heavy molecules thereby transfer
Their charges to the molecules and become neutralized - a process known as quenching.
The molecular ions thus produced move slowly to the cathode and on reaching there,
capture electrons from the cathode surface to become neutral molecules. The
multiplication of charges repeats itself in rapid succession producing within a very short
interval of time an avalanche of electrons. The electron avalanche is concentrated near
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the central wire while the positive ions, being much heavier, drift slowly toward the
cathode. The number of electrons striking the wire can be measured to detect the
presence of radioactive emission.
1.4.2- SCINTILLATION COUNTERS
Scintillation counter is important instrument generally used for the detection and
measurement of radiation. The scintillation counter in which radioactive materials
exposed to atoms within the detector that temporarily absorb the radiated energy. These
excited atoms return to their unexcited state and emit photons that are detected by the
scintillation counter.
Working of Scintillation Counter

In scintillation counter there is lining of phosphor in one end of the photomultiplier tube.
Its inner surface is coated with photoemission called photocathode. It acts as negative
terminal. There is presence of several numbers of electrodes called dynodes which are
arranged in the tube at increasing positive potential. When charge particles reach and
strike the photocathode in photomultiplier tube releasing an electron. These electrons
accelerate toward the first dynode and strike it. More numbers of secondary electron are
emitted which accelerates towards the second dynode so on. Finally the chain continues
multiplying the affect of the first charged dynode. Due to this process there is release of a
voltage pulse across the external resistance. This voltage pulse is amplified and electronic
counter (Fig.4.14).
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Fig.4.14 Scintillation Counter
Scintillation counter consist of two parts

1. Scintillator: Scintillation is a material may be solid, liquid (organic or inorganic) or
gaseous which give luminescence or exhibit scintillation when struck by ionization
radiation. The NaI crystal is the most widely used scintillation material which under UV
light glows blue.
2. Photo multilplier Tube: PMT is generally used to creates strong electrical output from a
weak signal developed by striking of photon on photocathode. This electron accelerates
toward the first dynode and strikes it. More numbers of secondary electron are emitted
which accelerates towards the second dynode so on. Lastly electron hits the anode and
creates current which flow to ground through a resistor where it creates a voltage drop
that can be counted.
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4.5. SUMMARY
Modern laboratories of biological sciences should equipped with basic instrument like pH
meter, UV-Vis spectrophotometer, centrifuge etc. pH meter is generally used to
determine the pH of soil, water and culture medium used for the cultivation of fungi and
bacteria. There is presence of electrode which very sensitive to detect the change in H ion
concentration. The electric circuit measure the electromotive force developed across the
electrode pair. Spectroscopy is the measurement of (spectrum of light) electromagnetic
radiation, absorbed, scattered, or emitted by atoms, molecules, or other chemical species.
Each chemical species has unique energy states; spectroscopy can be used to identify the
interacting species separately. Centrifugation is based on the principle of centrifugal force
in which liquid are subjected to high speed to separate solid from liquid or liquid from
liquid depending on the density. In centrifugation heavy particle settled down and light
particle will rises to the top. Geiger- Muller Counter which is used as particle detector
that measure ionization radiation i.e. beta particle and alpha particles except gamma
particle because it does not ionize the gas. Scintillation counter is important instrument
generally used for the detection and measurement of radiation. The scintillation counter
in which radioactive materials exposed to atoms within the detector that temporarily
absorb the radiated energy.
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1.6. GLOSSARY
Bandwidth: The frequency span where constant amplitude input will produce a meter
reading within a specified limit (usually 3db). In controllers, the region around the set point
where control occurs.
Controller: A device capable of receiving a signal from a process and regulating an input to
that process in order to maintain a selected operating condition
Electromotive force (emf): An electrical potential difference which produces or tends to

produce an electric current.
Double Beam: In a double beam spectrophotometer the beam from the light source is split in
two. One beam illuminates the reference cell holder and the other illuminates the sample.
4.7. SELF ASSESSMENT QUESTIONS AND POSSIBLE ANSWERS
Multiple Choice Questions:
1. Which filament lamp is used for UV and visible light:
(a) Tungten (b) Argon
(c) Platinum (d) Lithium
2. G-M Counter is used to measure the
(a) RBC (b) WBC
(c) Intensity of the radioactive radiation (d) intensity of visible light
3. Centrifugation is based on the principle of
(a). Gravitational force (b) Centrifugal force

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(c) Frictional force (d) Vander wall force
4 pH scales ranges from
(a) 0 to 6 (b) 0 to 7
(c) 0 to 10 (d) 0 to 14
4.8. REFERENCES
1. Bair, E.J. (1962). Introduction to chemical Instrumentation, McGraw hill, New York
2. R.C.Dubey and D.K. maheshwari.(2012). Text book of Microbiology. S. Chand &
company.
Q1. Write principle, procedure and working of pH meter.

Q2. Draw labeled diagram of GM counter.
Q3. Write different types of centrifuge used in laboratory
Q4. Write short note on
a. Bench centrifuge
b. Cooling centrifuge
c. Ultra centrifuge
Q5. Describe the working and principle of Scintillation counter?
Q6. Explain Beer-Lambert law?
Q7. Write principle and working of UV-Vis spectrophotometer
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Unit 5: MICROTOMY AND MICROSCOPY
CONTENT
5.1 Objective
5.2 Introduction
5.3 Types of Microscope
5.4 Summary
5.5 Glossary
5.6 Self assessment Question and Possible Answer
5.7 Reference
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5.1OBJECTIVES
In this chapter, students will able to learn how tissue or cells are fixed and stain to study the
histopathology. The chapter also deals with the different types of microscope used for the
study of the microscopic world.
5.2 INTRODUCTION
At the beginning of science, there was no sophisticated and advanced instrument available to
explore the world of an invisible entity. It was made possible by the development of a
microscope in which the microscopic cell, bacteria, fungi, and other microscopic things was
seen and detailed structure studied. As the science progress, there was the development of
Microbiology has been progressed and now we studied the ultramicroscopic microorganism
such as viruses and bacteriophages. Due to the development of the microscope it is possible
that we can differentiate between different types of cells. The importance of the microscope
in the histopathological investigation cannot be ignored. The abnormal changes in the cell
and tissue of human being can be easily seen with the help of the microscope. There are
different types of microscope has been developed these days according to the need of
researchers and scientists i.e. Compound microscope, Stereo microscope, phase contrast
microscope, Bright field microscope, Dark field microscope, Transmission electron
microscope, Scanning electron microscope.
The morphological changes in the cell or tissues can be visualized through microscope when
it is correctly processed and stained. This chapter focuses on different types of microscope
used in the laboratories for various investigations.
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5.3MICROTOMY
The trimmed block of paraffin is then fixed on a block holder. This block holder is then
secured to the microtome and oriented appropriately with respect to the knife. With each
revolution of the microtome handle, the specimen moves through the blade and a section of
desired thickness is produced. Each successive section adheres to the preceding are forming a
continuous ribbon. The ribbon is cut into small piece and transferred onto clean albumenized
slides. The ribbon is flattened by putting a few drops of water. These floating sections are
stretched using a hot plate or warming table.
Staining
Next, the paraffin is removed with xylene or another appropriate solvent and the specimen is
rehydrated. It is then stained, dehydrated, cleared (made transparent) with xylene, covered
with a suitable mounting medium, and topped with a cover-slip. Various stains are available
to the histologist. Hematoxylin and eosin (H&E) is a frequently used combination of stains.
Haematoxylin imparts a purple colour to substances, but must be linked to a metallic salt
called a mordant before it can function effectively. This combination, called a lake, carries
positive charges and behaves as a basic (cationic) stain. The lake combines electro statically
with negatively charged radicals such as phosphate groups of nucleoproteins. Substances that
become colored by a basic stain are said to be basophilic. Methylene blue, toluidine blue, and
basic fuchsine are basic stains. Unlike haematoxylin, these stains have molecules that carry a
positive charge of their own and do not require a mordant. Acidic (anionic) stains carry a
negative charge and colour cell or tissue components that bear positive charges. Eosin is an
acid stain. It imparts orange or red colorto acidophilic substances. Other commonly used acid
stains are orange G, phyloxine, and aniline blue.
In addition to the widely used H & E staining procedure, numerous other stain combinations
and techniques are available. Some are especially useful for identifying certain tissue
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elements. For example, trichrome procedures such as Mallory’s and Masson’s specifically
stain collagenous fibers within connective tissue. Orcein and Weigert’sresorcinfuchsin are
stains used to color elastic fibers, providing a means of distinguishing them from other
fibrous elements. Reticular fibers and nervous tissue components such as neurons, myelin,
and cells of the neuroglia can be stained by procedures employing the use of silver. There are
also special histochemical and immune histochemical procedures that make possible the
localization of various carbohydrates, lipids, and proteins found in tissue. Stains such as
Wright’s and-Giemsa’s (Romanovsky stains) are available for differentiating the various
cells found in blood and bone marrow.
Fig 5.1Microtomy
Fixation
As discussed in the preceding chapter, fixation of a sample is the first and most important
step in microscopical examination. After being removed from an animal/plant, a tissue or
organ is cut into pieces. These are placed in a fixative for suitable time at room temperature
or at 4oC in a refrigerator. Commonly used fixatives are 4-10% formalin, aqueous or
alcoholic Bouin, acetic alcohol formalin, Comoy’s fluid. The purpose of fixation is to
preserve normal morphology of the tissue and prepare it for further processing.
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Dehydration
After fixation, the next step is to remove the fixative. The aqueous fixative may be removed
by washing the fixed tissue in running water. Alcoholic fixative is removed by washing the
tissue in 70% alcohol several times.
Removal of fixative is followed by dehydration. Dehydration is performed by transferring the

sample through a series of alcohols of increasing concentrations upto 100% alcohol.
Clearing
Dehydration is followed by clearing. Several clearing agents such as xylene, toluene and
benzene can be used. These agents are miscible with alcohol as well as paraffin wax. This
intermediate step is essential before infiltrating the dehydrated tissue with paraffin because
alcohol and paraffin do not mix.
Embedding
Cleared tissue is embedded in a suitable embedding medium. Paraffin wax is the most
preferred medium. Several plastics are also available nowadays and they offer better sections.
Paraffin embedding is carried out in an oven at a temperature just above the melting point of
the paraffin. When infiltration is complete, the specimen is transferred to an embedding mold
of fresh paraffin which is allowed to harden. Then the mold is removed and excess paraffin is
trimmed away.
5.4. TYPES OF MICROSCOPES
The type of microscope was designed as per the requirement and nature of the specimen. So,
there exist huge variations in microscopes required as per the desired magnification and
visualization. However; some most common types of microscopes include compound (having a
combination of lenses) light microscopes, bright and dark field, phase contrast, fluorescence, and
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electron microscopes. The modern optical science of microscopy is based on the designs of
German physicist Ernst Abbé. The applicability of various microscopes depends upon the most
important feature, the resolution power.
RESOLUTION OF MICROSCOPES
To ascertain the distinguished minimum distance between two closely located objects with the
help of a lens is called resolution which can be measured using the Abbé equation:
0.5 !
=
"#$"%
Where, λ is the wavelength of the light used to illuminate the sample and nSin% is the numerical
aperture (NA). Angular aperture (%) as in figure 1 is ½ the angle of the cone of light
(illuminating a specimen) entering the lens of an objective piece of the microscope.
Figure 5.2Angular aperture % of the cone formed on lens by specimen
The numerical aperture is nSin%. NA depends upon the working distance which influences the
angular aperture. For higher values of NA, working distance should be less for achieving higher
angle of cone resulting in the higher resolution. It should be remembered that angle of the cone
depends on the refractive index (n) of the medium and the optical material of lens. The refractive
index of air is 1.0 and the maximum value of angle is 900. Sin90 is 1 therefore; lenses working in
air cannot have a numerical aperture greater than 1.0. To achieve higher resolution greater angle
of cone is required for which immersion oil (colorless) having a refractive index
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Equal to that of medium can be used. It also helps to collect the otherwise dispersing light (from
the edges of specimen) on the objective lens.
LIGHT MICROSCOPE
In the modern age of advanced scientific endeavor, sophisticated microbial examinations are
being deliberated. All the modern age microscopes are compound and light microscope is the
simplest compound microscope, which is very well understood by students of science. Several
variants of light microscopes viz.Bright field, dark field, phase contrast, fluorescence and many
others are available for microscopic examination. A simple compound light microscope has two
sets of lenses i.e., an objective which form magnified image, which is further enlarged by
another lens. All the light microscopes have a source of white light.
BRIGHT FIELD MICROSCOPES

This is an ordinary microscope, commonly used by students of preliminary science. The bright
field microscope forms a dark image against a bright field. Two types of lens sets viz., objective
(commonly 4 in number of different magnifying power) and eyepiece (ocular) are used (figure
2). The total magnification power of microscope using particular objective is calculated by
multiplying the power of objective to the power of eyepiece for e.g., 45X objective and 10X
ocular yield 450X magnification.
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The objective lens forms an enlarged real image which is further magnified by eyepiece and the
image is virtual formed 25 cm beyond the stage. Sub-stage condenser is also used some times for
the purpose to increase the resolution. The specimen can be brought in focus of objective lens by
fine and coarse adjustment knob (figure 2). It should be remembered that a microscopes are par
focal i.e., even if the objective is changed the specimen remain in focus. To visualize by bright
field microscopes, staining of the specimen is often required and it is very difficult to observe
living cells through this microscope.
DARK FIELD MICROSCOPE

Dark field microscopes are used to magnify the images of living and unstained living
microorganisms. In fact this is also technically and structurally similar as bright field
microscopy, only difference is the addition of a dark field stopper between light source and
condenser (figure 3). This light stopper (figure 4) let the light to pass from the open space and
eventually condensers form a hollow cone of light and focused on the specimen to illuminate.
The light that is reflected and
refracted by the
specimen forms
an image, rest
of the part
around the
Figure 5.4. Dark Field Stopper
Figure 5.3. Optics of a Dark Field microscope
Specimen appears black. Since the background of the specimen image is dark, the microscopy is
called dark field.
PHASE-CONTRAST MICROSCOPE
Phase-contrast microscopes produce images as a result of slight differences in refractive index
and cellular density. The images so formed let the observer to have idea of the thickness of the
organism under study. Like dark field, phase-contrast microscopes also have an annular stop disk
with a slight difference of thin transparent ring which produces a hollow cone of light. As the
cone passes through a living organism (bacterial cell or any other living cell) some light rays are
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bent because of the density variations and refractive index. The light wavelength gets retarded by
¼. This deviation focuses to form an image of specimen. The un-deviated light strike a phase
Figure 5. 5 Optics of a phase-contrast microscope
ring in a phase plate (figure 5), a disk placed in the objective. The deviated light (from the
specimen) miss the phase plate and pass through the rest of the plate. The deviated and un-
deviated lights have ½ and ¼ wavelengths of the light from the source. The un-deviated passing
through the plate cancels each other during the formation of image.The un-deviated light render
a bright background and deviated light form dark image giving a sharp contrast resulting in the
peculiar image of specimen. This is dark-phase-contrast microscope.
FLUORESCENCE MICROSCOPE
Usually, light source is required to illuminate the specimen, which is the basis of image
formation by microscopes. However; some objects emit light giving a base for fluorescence
microscopy. In fact, some molecules reach to an excited state by absorbing radiant energy and
re-emit the same energy in the form of light of lower wavelength than what was absorbed earlier,
this is called fluorescence.
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In fluorescence microscope, usually specimens are stained with certain dye molecules called
fluorochromessuch as acridine orange, Lucifer yellow, ethidium bromide, TOTO – 1(vital stain),
DAPI (diamidino – 2 – phenylindole a DNA specific stain),which are excited by intense light
produced by mercuryvaporarc lamp.
Since the light intensity is very high hence an infrared filter is usually used to avoid likely
damages to the microscope itself. Like dark field microscopes, a dark field condenser renders a
dark background to the image so that fluorescence coming out from the specimen could be seen.
Light of specific wavelength excite the stained specimen, the image is formed from the light
emitted. A barrier filter is also placed to remove the ultraviolet light that could otherwise damage
the viewer’s eye.
ELECTRON MICROSCOPY
In practical naked eyes can resolve only up to 10-3 m (thickness of hairs) while light microscopes
can resolve up to 10-7m, although the objects having lesser size exist and cannot be visualized by
light microscopy. In the year 1931 German physicist Ernst Ruska and electrical engineering Max
Knoll constructed the first prototype of electron microscope. Modern age electron microscopes
have magnification power 10,000,000X and resolution up to 50 Pico-meters because electron
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beam having wavelength about 100,000 times shorter than visible light is used to illuminate the
specimen. Two basic type viz., transmission and scanning electron microscopes are commonly
used these days.
TRANSMISSION ELECTRON MICROSCOPE (TEM)
In the transmission electron microscopes, an electron beam is generated by a heated tungsten

filament positioned in the electron gun. This electron beam is focused on the specimen, which
scatters the electron as per the density. Since the electrons cannot pass through the glass lens
therefore magnetic lenses are used to focus. The entire set of magnetic lenses and specimen are
worked under high vacuum as the air inside the column may get charged and moisture along with
other molecules may scatter the electron beam. The electrons passing through the specimen are
scattered, the dense part of the specimen scatter more as compared to the thin region. Thus the
image formed by the scattered electrons will be dark indicating the density and the brighter
region indicate the electron transparent regions (thin regions).
Fig 5.7 Transmission electron microscopes

The scattered electrons form enlarged image on the fluorescent screen and on the photographic
plates, which can be used for reporting purposes.
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Scanning Electron Microscope (SEM)

Scanning electron microscopes are the recent advancement in electron microscopy facilitating
ease in the microbiological and other life science related examinations. Principally same as TEM
except having advancements such as specimen preparation is easy, more well defined images,
3D direct viewing and photography and ease of working. Images in SEM are formed by the
secondary electron produced as a result of shower of primary condensed electron beam under
high vacuum. Figure 7 present the optics of a SEM. An Electron beam is produced by an electron
gun so positioned that primary electrons are focused on the shielded specimen (metal coated). As
soon as electron beam strikes the surface secondary electrons are produced (more from elevated
and fewer from depressed region of the specimen surface), which are detected by a detector as
lighter (indicating elevation because of more secondary electron) and darker (indicating
depression because of few secondary electron production) regions. The electronic signals are
sent to the photomultiplier which is further amplified. A cathode ray tube receives signals from
the photo-multiplier and forms a picture. The image can be seen just as a computer monitor or
television and can be photographed.
SPECIMEN PREPARATION FOR ELECTRON MICROSCOPY

TEM
Since electrons are rapidly absorbed and scattered by solid matter, therefore, extremely finely cut
slices of 20 to 100 nm irrespective of material can only be observed by TEM. The slicing of the
object intended to be observed through TEM need technical practice whereby specimen are fixed
with the fixer chemical for e.g., osmium tetra oxide followed by rigorous dehydration with
organic solvents such as ethanol, acetone etc. Usually, paraffin or any other liquid epoxy plastic
are used to soak the specimen and then solidified to form a block. The thin slices are usually cut
using ultra microtome’s. It should be remembered that prior to use electron microscopes,
complete dehydration of the specimen is a must.
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SEM
The specimen preparation for SEM is easy however; it involves some of the basic requirement as
that of TEM for e.g., dehydration and preservation. In scanning electron microscopes the entire
procedure occurs at high vacuum condition, this may also damage the specimen and since
secondary electrons are required for image formation, specimen are shielded with a thin layer of
metal which prevent the damage and also helps emission of secondary electron from the
specimen surface.
5.5 SUMMARY
The secret of the microscopic world cannot be deciphered until the microscope has not been
discovered. The microscopy is a very useful instrument for the study of materials and can be
used to gain valuable information about a large variety of specimens such as bacteria, fungi,
protozoa etc. Some knowledge of the material and the information that is required is
essential to determine the best techniques to employ when preparing and examining
specimens. In Microscopy sample preparation is the most important of microscopy, as this
determines the quality of the images produced. Disadvantage of the optical microscope is its
resolution. This limitation is overcome by a scanning electron microscope (SEM).In addition,
for 'transparent' specimens; in particular those required for motility, polarized light
microscopy is best method for this type sample.
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5.6. GLOSSARY
Analyzer: A analyzer is used with a polarizer to provide polarizing light.
Achromatic Lens: A lens that helps to correct the misalignment of light that occurs when
it is refracted through a prism or lens.
Abbe Condenser: A lens that is specially designed to mount under the stage and which
typically moves in a vertical direction.
Eyepiece: The eyepiece is the lens nearest to your eye.
Compound Microscope:Originally used to describe a microscope with more than one

objective lens, a compound microscope is now generally understood to be a high power
microscope with multiple, selectable objective lens of varied magnifications.
Condenser:A lens that concentrates the light on a specimen and increases the resolution.
Found in or below the stage on compound microscopes.
Cover Slip:A thin, square piece of glass or plastic placed over the specimen on a
microscope slide. It flattens out liquid samples and helps single plane focusing.
Electron Microscope:A type of microscope that uses electrons rather than light to create
an image of the target.
Darkfield Microscopy: A technique used to enhance the contrast in unstained specimens.

It works on the principle of illuminating the sample with light that will not be collected
by the objective lens, so not form part of the image.
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Monocular Head: A microscope head having a single eyepiece lens.
Nosepiece: The part of the microscope that holds the objective lenses also called a
revolving nosepiece or turret.
Numerical Aperture (N.A.): This is a number that expresses the ability of a lens to
resolve fine detail in an object being observed.
5.7. SELF ASSESSMENT QUESTIONS AND POSSIBLE ANSWERS

1. Numerical aperature is:
(a) nSin& (b) nTan&
(c) nSec& (d) nCos&
2. In fluorescence microscope, usually specimens are stained with certain dye molecules
(a) Simple stain (b) Crystal voilet
(c) Alberts Stain (d) Acridine orange
3. Transmission Electron Microscope magnify upto
(a). 100X (b) 1000X
(c) 400,000X (d) 400X
4 3D image of specimen was obtained with
(a) Compound Microscope (b) SEM
(c) TEM (d) Phase contrast Microscope
5. Image can be seen in SEM
(a) Fluorescent Microscope (b) Anode
(c) As Graph (d) Phosphorescent screen
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5.8. REFERENCES
1.. R.C.Dubey and D.K. maheshwari.(2012). Text book of Microbiology. S. Chand &
company.
2. P.K.Bajpai. (2006). Biological instrumentation and Methodology. S.Chand and
Company.
3. Bruce Alberts. (2014). Molecular Biology of the Cells.W. W. Norton & Company;
Sixth edition
Q1. Write principle, procedure, and working of Light Microscope.

Q2. Draw labeled diagram of a compound microscope.
Q3. Write principle and working of Fluorescent Microscope
Q4. Write short note on
d. Resolution
e. Numerical apertures
f. Magnification
Q5. Describe the working and principle of the Transmission Electron Microscope?
Q6. Write principle and working of Scanning Electron Microscope?
151 | P a g e
Unit 6: Separation techniques and cryopreservation
CONTENT
6.1 Objective
6.2 Introduction
6.3 Chromatography
6.3.1 Chromatography
6.3.2 Column Chromatography
6.3.3 Gas Chromatography
6.4 Electrophoresis
6.4.1 Isoelectric Point
6.5 Cryopreservation
6.6 Summary
6.7 Glossary
6.8 Self assessment Question
6.9 References
152 | P a g e
6.1. OBJECTIVE
In this chapter you will learn different techniques which are related to purification and
separation of desired molecules and develop a basic understanding of Electrophoresis theory
and also become familiar with the procedure involved in agrose gel electrophoresis to
visualize DNA.
This chapter also deals with the preservation of cells, organelle, tissue, organs and microbes
to very low temperature i.e. cryopreservation.
6.2. INTRODUCTION
As the biological sciences progress there is need of some sophisticated, accurate and
sensitive method used for the purification and separation of desired component from the
complex or mixture of two or more chemicals. The chromatography techniques have two
parts i.e. mobile phase and stationary phase. In a chromatography sample mixture is placed in
liquid or gas called mobile phase and the mobile phase carries the sample through a solid
support called the stationary phase. In case of Thin Layer Chromatography (TLC) the
stationary phase is silica coated on glass plate. These techniques have disadvantage such as it
does not have long stationary phase so the length of separation is limited and another
problem with this method it operates in open system so the chromatogram may influenced by
temperature and humidity. Now days, there is some modern methods which is based on the
principle of TLC such as HPLC, GC, HPTLC and Electrophoresis. HPLC is one of the
important instruments used in the pharmaceutical industry for the analysis of drug, plant
metabolite, and toxins and in medical science it is also use for the detection of hazardous
chemicals in blood. Electrophoresis is most commonly used technique in molecular biology
for the analysis of nucleic acid and protein. It is based on method of separating molecules on
153 | P a g e
the basis of charge and molecular weight when applied electrical field. On the basis of
molecular weight of nucleic acid we can predict the nucleic acid of unknown microorganism.
6.3CHROMATOGRAPHY
6.3.1CHROMATOGRAPHY
Chromatography is another chemical procedure to find out and to separate the contents of a
mixture of two or more chemicals. The literal meaning of chromatography is the study of colors
first developed and described by Russian scientist Mikhail Tswett in 1900 which was further
developed by Martin and Synge in early 1950s. In fact the chromatography is a separation
technique based on the “partition or distribution coefficient or more appropriately partition
constant or partition ratio (Kd) that describe the way in which a compound distribute itself
between two immiscible phases”.
Kd = concentration of compound in phase A / concentration of compound in phase B
The term effective distribution coefficient is defined as the total amount, as distinct from the
concentration, of substance present in one phase divided by the total amount present in the other
phase. It is in fact the distribution coefficient multiplied by the ratio of the volumes of the two
phases. If the Kd of a compound between two phases e.g., A & B; is 1 and if the compound
distribution is of 10 cm3 and 1 cm3 respectively, the concentration is the two phases will be same
however; the total amount of the compound in phase A will be 10 times the amount in phase B.
There are two basic and essential phases’ viz., stationary and mobile in chromatography.
Stationary phase remain stable and do not move at all and provide base matrix for eg., solid, gel,
liquid solid / liquid mixtures etc. on which compounds are separated. Mobile phase are either
liquid or gaseous which flows over or through the stationary phase. If base matrix support is
polar (e.g., paper, silica etc.) it is forward phase or if the matrix is non-polar (C-18) it is reverse
154 | P a g e
phase chromatography. The choice of stationary and mobile phase is made so as to have different
distribution coefficient for the mixture sample. This is also achieved by setting up following:
1) Adsorption equilibrium between stationary and mobile phase
2) Partition equilibrium
3) Ion exchange equilibrium
4) An equilibrium between a liquid phases trapped inside the pores of a stationary porous
structure and a mobile liquid phase as in permeation or molecular exclusion
chromatography.
5) Equilibrium between a stationary immobilized ligand and a mobile liquid phase.
Chromatography may be preparative (separating the components of mixture or in advanced terms
“purification”) or analytical (measuring the relative proportion of analyte in a
mixture).Furthermore, there exist two basic types of chromatographic viz., column
chromatography and planar chromatographic techniques.
6.3.2. COLUMN CHROMATOGRAPHY
Column chromatography is a separation technique in which the stationary bed is within a tube
(glass, plastic or metal). The stationary phase comprised of various polar or non-polar porous
materials. The filled column is packed by a liquid mobile phase carrying the mixture to be
separated through the column. There are two types of column used: 1) packed column which is
characterized by the complete filling of the volume of column (figure 1), 2) open tubular column
characterized as stationary phase concentrated along the column wall giving an unrestricted path
to the mobile phase.
155 | P a g e
Fig: Packet Column

The separation is done in the column shown in the figure normally to separate the components of
the mixture as for e.g., in the plant extract and not to quantify them. The packed column is gently
handled following by the repeated washing with the solvent used as mobile phase. Sample is
poured in to the column carefully and packed with the liquid mobile phase as in figure 2. The
components of sample mixtures immediately get separated depending upon their partition
coefficient (stationary to mobile) in the column forming band of different color having negligible
differences therefore extra care is needed to separate each component. Separated components are
called as eluent. The washing of column with the solvent to remove the components is called
elution.
Separation of component from mixture sample through column chromatography
156 | P a g e
GAS CHROMATOGRAPHY
Gas chromatography is a column chromatography where the mobile phase is a carrier gas,
usually inert gasses such as helium and/or nitrogen. As shown in figure 3, a GC typically consists
of a gas tank providing inert carrier gas, the flow of which is controlled by a flow controller
connected with the column oven. The column oven is a chamber is a metal casing wherein a
column made of glass or metal is connected at one side with the carrier gas tubing and sample
injection chamber and to the detector at the other end.
The column oven provides temperature for the fugitive diffusion of gas inside the column
containing microscopic layer either of polymer or liquid as solid bed. The separated compounds
are detected by the detector. Detectors may be of various types whereas; flame ionization and
electron capture detectors are commonly used in most of the researches of life sciences and
environmental sciences.
COLUMN OF GC
The heart of a gas chromatography is the column performance of which sets limit to the
separation attainable and determines time of analysis. Two main type of columns viz., 1) packed
column, first introduced by Martin in 1952 and 2) capillary packed column introduced by Halasz
in 1968 are used these days. Pressure factors such as column inlet pressure, required speed of
sample introduced, required sample size (detection limit) are largely determined by properties of
the selected type of column.
157 | P a g e
Figure 6.1. Schematic of a gas chromatograph
Factors determining gas chromatographic performance are directly linked to the column
efficiency. These factors are:
(a) Analysis time: The residence time of the peak maxima in a column, the retention time tR
can be calculated by
'( = ' ( + *)
Where t0 is gas hold up time, r is the retention time of an unreacted component such as air, k is
the capacity ratio. k can be derived from the partition coefficient Kd by multiplying the
volumetric ration of stationary to mobile phase in the column.
./0120-
+ = ,- ( )
.345
(b) Efficiency: Efficiency is expressed as the plates (n), the degree of band broadening a
solute undergoes in a given column. This can be derived from the chromatogram:
678
"=
92
Where σ is the standard deviation (time units) is equal to half width at 0.607 of the height of a
Gaussian peak.
The plate number (n) or the peak (bands) is related to the length of column and height H
equivalent of a theoretical plate. H = L/n
158 | P a g e
H is further dependent on the linear velocity (u) of the carrier gas as in Figure 4.
Figure 6.2 Dependency of Plate Height (H)

and linear velocity (U) of carrier gas
(c) Resolution: (R) express the degree of separation of two components leaving the column
shortly after each other
<678 − 678 >
;=
9
Taking the values of tR:
(∝ −1)+
;= √3
(+ + A)
Where ∝ is relative of two components
The separation is almost complete at R = 6.0 and just acceptable for R = 4.0.
(d) Speed of sample: The capacity of the column, which determines the most practical
parameter, the sample size. Quantity of sample can be introduced in a GC either as a
narrow band of high concentration or as a plug of correspondingly lower concentration.
If the sample is fed into the column as a band of high concentration the Kd will remain
concentration dependent and each part of solute band will move at different speed resulting in
the asymmetric peaks. If the sample is fed as plug of lower concentration, it goes under dilution
with carrier gas as soon as it enters the column. This causes additional symmetric band
159 | P a g e
broadening. The variance σ 2(sec2) of an eluting peak may be considered to be composed of the
variance of the sample at the inlet σ12and the variance σ02due to column processes.
9D = 9D + 9D
Best resolution can be obtained at σ12 = 0 (infinitely narrow sample band).
Carrier gas selection
The selection of the carrier gas depends upon the type of analyte, column temperature and
detectors. For e.g., helium is used in discharge ionization detectors (DID), nitrogen is used in
electron capture detector (ECD) and in flame ionization detector (FID) (in FID hydrogen is used
as fuel gas). In general helium, nitrogen, argon, hydrogen and air are used as carrier gases in
chromatography.
Detectors
All though advancements have been made in the detector technology and currently number of
detectors viz., ECD, FID, DID (as above), PID (photo-ionization detector), PDD (pulse discharge
ionization detector), MSD (mass selective detector) etc. are available for the analysis of variety
of environmental and medical samples. However; ECD and FID are the most employed detectors
worldwide discussed here in details.
Electron Capture Detector (ECD)
Figure 6.3. Schematic of Electron capture detector (A) high electron

density in carrier gas (B) reduced electron density in sample + carrier gas
160 | P a g e
Electron capture detector (ECD) is a device used to analyze halogenated, nitriles and nitro
compounds and is particularly important for the environmental, pharmaceutical, and forensic
studies. ECD was first invented by James Lovelock in 1957. The functioning of ECD is based on
a radioactive beta particle (electron) emitter 63Ni (~ 10 millicurie or 370 mBq) which remains in
a metal foil holding inside a chamber. 63Ni emits electron that move towards anode so as to make
the circuit complete resulting in to the generation of a current. Nitrogen is commonly used for
analysis by ECD because of its low excitation energy enabling more electron density in the
detector chamber generating current of high magnitude. When the sample analyte enters along
with the carrier gas, the molecules of analyte absorb / capture some of the electrons and reduce
the current. while a background signal of current generated by the carrier gas is also distinctly
visualize in a chromatogram. The reduction in the current due to the analyte is directly
proportional to the analyte concentration.
The flame ionization detector (FID) is the industry standard method of measuring hydrocarbons
(HC) concentrations. The sample gas is introduced in to a hydrogen flame inside the flame
chamber. Any hydrocarbon in the sample will produce ions when they are burnt. Ions are
detected using a metal collector (high voltage ion collector) which is based on a high DC
voltage. The current across this collector is thus proportional to the rate of ionization which in
turn depends upon the concentration of HC in the sample gas. Remember FID use hydrogen as
fuel gas (Fig.6). If hydrogen flow is on and column is connected to the detector inlet fitting,
hydrogen gas can flow in to the column oven and create an explosion hazard. Hydrogen flow
inside the FID should be standardized and must not exceed 30 mL/min, while the carrier gas (N2)
flow should be 25 mL/min.
The analytical efficiency of a GC depends upon the appropriate column choice. There is variety
of basic types of column (as discussed earlier in this section) present in the market out of which
wall coasted open tubular (WCOT) and porous layer open tubular (PLOT) columns are widely
used. PLOT columns are of three viz., 1) molecular sieve; 2) Divinylbenzene (DVB) and 3)
161 | P a g e
Alumina (Al2O3) types are quite common for wide range of hydrocarbon analysis. The accuracy
in the sample analysis by a GC has been developed to an advanced level, and gas
chromatography mass spectrophotometer (GCMS) is one of them. The detection limit increases
to ppt (parts per trillion) level. Since GCMS is more sophisticated and an expensive device, extra
care is required while analyzing the samples.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Gas chromatography required the volatilization of the sample, however; HPLC is the method to
identify and quantify the analyte that cannot be converted into the gas phase. The unique
retention time of the analyte in to the column and its characteristic peak as calibrated with
standard is the principle of HPLC analysis. The time for a substance to pass through the column,
termed the retention time which is related to the identity of the compound. Quantitative
information is obtained from the area or height of the peak produced by the detector.
By the choice of appropriate solute (mobile phase) usually methanol and acetonitrile and the
column (packed solid material) efficient separation can be achieved. In an HPLC the sample
(20µl) is injected which is then carried by the solutes. The separation of analyte depends upon
the interaction of the analyte and mobile phase with the stationary phase. High pressure are
required to force a liquid through a tightly packed column filled with small particle material and
the availability of high pressure solvent delivery systems is directly responsible for the “high
performance”.
The detectors used for the identification and quantification in HPLC are complex, in fact no
single universal detector system has yet been developed. More than one detector having unique
detection property can be used in a single HPLC in series. The most commonly employed
detectors include bulk property detector (compare over all change in physical property of the
mobile phase with and without an eluting solute); solute property detectors (respond to a
physical property of the solute that is not exhibited by the pure mobile phase. These detectors are
100 times sensitive and detect sample on a nano-gram level; and ultra-violet photometers (most
commonly used detector).
162 | P a g e
6.4. ELECTROPHORESIS
6.4.1 ELECTROPHORESIS
Electrophoresis is an analytical method frequently

used in molecular biology and life sciences. It is
applied for the separation and characterization of
proteins, nucleic acids and subcellular-sized
particles like viruses and small organelles. The
working principle of electrophoresis is that the
charged particles migrate towards their
corresponding electrode in an applied electrical
field. If conducted in solution, samples are
separated according to their surface net charge density. The most frequent applications, however,
use gels (polyacrylamide, agarose) as a support medium. The presence of such a matrix adds a
sieving effect so that particles can be characterized by both charge and size. Protein
electrophoresis is often performed in the presence of a charged detergent like sodium dodecyl
sulfate (SDS) which usually equalizes the surface charge and, therefore, allows for the
determination of protein sizes on a single gel. Additives are not necessary for nucleic acids
which have a similar surface charge irrespective of their size. The resistance entrusted by the
electrophoretic units also play significant role in producing migration related variations. The
resistance of an electrophoresis unit depends on its size, gel thickness, amount of buffer, buffer
conductivity and temperature. This resistance will normally decrease in time due to a slowly
increasing temperature. Electrophoresis units which have a resistance below the minimum load
resistance of a power supply will trigger an alarm.
For a comprehensive study and to achieve better separations, one should have knowledge of
basics of electrophoresis. This section deals with such information.
163 | P a g e
Isoelectric point (pI)

The isoelectric point is the pH at which a particular molecule or surface carries no net electrical
charge. Biological amphoteric molecules such as proteins contain both acidic and
basic functional groups. Amino acids which make up proteins may be positive, negative, neutral
or polar in nature, and together give a protein its overall charge. At a pH below their pI, proteins
carry a net positive charge; above their pI they carry a net negative charge. Proteins can thus be
separated according to their isoelectric point (overall charge) on a polyacrylamide gel using a
technique called isoelectric focusing.
Isoelectric focusing (IEF)
Isoelectric focusing is an electrophoretic method in which proteins are separated on the basis of
their pIs. It makes use of the property of proteins that their net charges are determined by the pH
of their local environments. Proteins are positively charged in solutions at pH values below their
pI and negatively charged above their isoelectric points. Thus at pH values below the pI of a
particular protein, it will migrate towards the cathode during electrophoresis. While moving
towards the electrode (cathode or anode) proteins lose some protons and their net charge drops
and pH changes, this retard the protein movement, finally it stops to move as the pH become
equals to the pI. Protein diffuses in to a pH higher than its pI, the protein will become negatively
charged and will be driven towards the anode. In this way, proteins condense or focus, into sharp
bands in the pH gradient at their individual pI values. Narrow pH range and high applied voltage
gives excellent resolution in IEF.
Buffers of Electrophoresis
A buffer should be chosen with a pKa that is very close to the desired pH, preferably within a
half point. The buffer will have the greatest capacity both to absorb and/or release protons with
the acid and the base form well represented in solution. It should be noted that pKa is not
constant for all conditions but is a function of the total ionic strength and the temperature, so the
stoichiometry should be modeled after actual running conditions. In general, TAE (tris acetic
acid with EDTA) and TBE (tris borate with EDTA) buffers are used in electrophoresis
technique. TAE buffer provides optimal resolution of fragments >4 kb in length, while for 0.1 to
164 | P a g e
3 kb fragments, TBE buffer should be selected. TBE has both a higher buffering capacity and
lower conductivity than TAE and therefore should be used for high-voltage electrophoresis.
Additionally, TBE buffer generates less heat than TAE at an equivalent voltage and does not
allow a significant pH drift.
Gel Concentration
Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5
to 3%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy
to prepare. It is also non-toxic.Agarose gels have a large range of separation, but relatively low
resolving power.Polyacrylamide is a cross-linked polymer of acrylamide. The length of the
polymer chains is dictated by the concentration of acrylamide used, which is typically between
3.5 and 20%. Polyacrylamide gels are significantly more complex to prepare than agarose gels.
Because oxygen inhibits the polymerization process, they must be poured between glass plates.
Polyacrylamide gels have a rather small range of separation, but very high resolving power.
6.5. CRYOPRESERVATION
Cryopreservation or Liquid Nitrogen
Cryopreservation is the method in which very low temperature is used to preserve living cell,
organs, microorganism, sperm etc for longer period. Polge et al in 1949 was credited for
cryopreservation of the first mammalian cell i.e. spermatozoa. This technique is devised to
keep the cells genetically stable, viable and metabolic inert. The disadvantage of low
temperature preserving cell, tissue, organ and microorganism was formation of ice crystal
which eventually disrupts the cell membrane resulting the death of cell. The main objective
of cryopreservation is to replace water with other material that will not form ice crystals to
overcome the freezing there is addition of antifreeze agent such as Glycerol and DMSO
(Dimethyl Sulphoxide).
Cryopreservation media generally consists of a base medium, protein source, and a

cryopreservative. The cry0preservative both protects the cells from mechanical and physical
165 | P a g e
stress and reduces the water content within the cells, thus minimizing the formation of cell-
lysing ice crystals. The protein source, often fetal bovine serum (FBS), also protects the cells
from the stress associated with the freeze-thaw process. Cells are frozen slowly, at
1C/minute, using programmable coolers or by techniques outlined below. Generally, the
optimum cell density to freeze per 1mL of cell suspension depends on the type of cell.
Mammalian cells are usually frozen between 106 cells/mL to 107 cells/mL. The
cryopreservation media may differ slightly for adherent and suspension cell types.
The following is procedure for cryopreservation of cells
1. Expand culture to allow for adequate cell density for the desired volume to freeze. The
cell culture to be cryopreserved must be in the log phase of the growth cycle
(approximately 2-4 days after subculturing). Determine the cell count for number of
viable cells and the total cell concentration.
2. Centrifuge cells at approximately 200 to 400 x g for 10 minutes, allowing the cells to
form a pellet. During centrifugation, determine the amount of freezing media to prepare.
For example, if using 1 mL cryovials, divide the total cell concentration by the desired
cell density. Example: A 4x107 cell suspension will yield a total of ten 1 mL alliquots at
4x106 cells per alliquot.Prepare 10 mL of freezing medium to easily suspend the pellet at
the correct cell density.
3. Prepare the necessary volume of cryopreservation media (determined above) using the
following guidelines:
4. Resuspend cell pellet(s) using the cryopreservation media, triturating to ensure a single-
cell suspension with as few cell clumps as possible.
5. Dispense into the desired number of vials for cryopreservation.
6. Immediately transfer the vials to a freezer with a minimum temperature of -20 oC for one
hour.
7. Transfer the vials to a -80oC freezer for 24 hours. Alternatively, a dry ice/methanol slurry
using or other insulated box with a cover or lid may be used if a -80oC freezer is not
available.
8. After 24 hours at -80oC, cells may be transferred to a liquid nitrogen storage (-196 oC)
166 | P a g e
6.6. SUMMARY
As the technology and techniques are developing day by day, which made the analysis and
detection faster than earlier conventional method used. This chapter deals with some modern
technique which is now used in research laboratories. Chromatography is chemical procedure
to find out and to separate the contents of a mixture of two or more chemicals. Column
chromatography is a separation technique in which the stationary bed is within a tube (glass,
plastic or metal). The stationary phase comprised of various polar or non-polar porous
materials. The filled column is packed by a liquid mobile phase carrying the mixture to be
separated through the column. Gas chromatography is a column chromatography where the
mobile phase is a carrier gas, usually inert gasses such as helium and/or nitrogen. As shown
in figure 4.2, a GC typically consists of a gas tank providing inert carrier gas, the flow of
which is controlled by a flow controller connected with the column oven. Gas
chromatography required the volatilization of the sample, however; HPLC is the method to
identify and quantify the analyte that cannot be converted into the gas phase. The unique
retention time of the analyte in to the column and its characteristic peak as calibrated with
standard is the principle of HPLC analysis. Electrophoresis is an analytical method
frequently used in molecular biology and life sciences. It is applied for the separation and
characterization of proteins, nucleic acids and subcellular-sized particles like viruses and
small organelles. Cryopreservation is the method in which very low temperature is used to
preserve living cell, organs, microorganism, sperm etc. for longer period.
6.7. GLOSSARY
Absorption: In chromatography, absorption signifies the process by which a solute partitions

into a liquid-like stationary phase.
Mobile Phase: The eluate moving through the column. In gas chromatography (GC) this will be
a gas, and in liquid chromatography (LC) a liquid.
167 | P a g e
Stationary Phase: The substance that remains in one place in the column. In GC this will be a
liquid of high-viscosity, which clings to the inner walls of the column; in LC it will be some sort
of packing, either solid or gel-based.
Eluate: The mobile phase exiting a column.
Eluent: The mobile phase entering a column.
Elution: The passage of the mobile phase through the column to transport solutes.
Flow Rate: The amount of mobile phase that has passed through the column per unit time. The
units are millilitres per second (mL/sec) or, more commonly, millilitres per minute (mL/min).
Isoelectric focusing (IEF): Electrophoresis technique that separates proteins according to
their isoelectric point (pI)
Isoelectric point (pI): pH value at which a molecule carries no electrical charge, or at which
the negative and positive charges are equal.
PAGE: Polyacrylamide gel electrophoresis, a common method of separating proteins
Polyacrylamide gel electrophoresis (PAGE): Electrophoresis technique that uses

polyacrylamide as the separation medium
Rf value: Relative distance a protein has traveled compared to the distance traveled by the
ion front. This value is used to compare proteins in different lanes and even in different gels.
It can be used with standards to generate standard curves, from which the molecular weight
or isoelectric point of an unknown may be determined.
Adsorption: The process of retention in which the interactions between the solute and the
surface of an adsorbent dominate.
168 | P a g e
Retention Time: The elapsed time between sample injection and the appearance of the
chromatographic peak apex
Isocratic: Chromatographic conditions in which a constant composition eluent is used.
Ion Chromatography: An ion-exchange technique in which low concentrations of organic

and inorganic anions or cations are determined using ion-exchangers of low ion-exchange
capacity with dilute buffers.
Degassing: The practice of removing dissolved gases from the eluent. It can be achieved by
helium sparging, applying vacuum to the eluent, ultrasonification or heating.
Isoelectric Point: The pH point at which a molecule no longer has a net charge.
Effective distribution coefficient: It is defined as the total amount, as distinct from the
concentration, of substance present in one phase divided by the total amount present in
the other phase.
SDS-PAGE: Separation of molecules by molecular weight in a polyacrylamide gel

matrix in the presence of a denaturing detergent, sodium dodecyl sulfate (SDS). SDS
denatures polypeptides and binds to proteins at a constant charge-to-mass-ratio. In a
sieving polyacrylamide gel, the rate at which the resulting SDS-coated proteins migrate
in the gel is relative only to their size and not to their charge or shape.
Running buffer: Buffer that provides the ions for the electrical current in an
electrophoresis run. It may also contain denaturing agents. The running buffer provides
the trailing ions in discontinuous electrophoresis.
169 | P a g e
Sample buffer: Buffer in which a sample is suspended prior to loading onto a gel. SDS-
PAGE sample buffer typically contains denaturing agents (including reducing agents and
SDS), tracking dye, and glycerol.
6.8. SELF ASSESMENT QUESTION
1. The Stationary phase in Reverse phase chromatography is made of

A. Polar B. Non Polar
C.Both D. None of the above
2. TLC is based on the principle of

A. Electrical mobility B. Partition chromatography
C.Ion exchange D. Adsorption
3. In isocratic method in HPLC the composition of solvent is

A. Remains Variable B. Remains constant
C.both A and B D. None of the above
4. In gradient method in HPLC the composition of solvent is

B. Remains Variable B. Remains constant
C.both A and B D. None of the above
5. HPLC stands for

A. High Performance Liquid Chromatography B. High Peak Liquid Chromatography
.C.High Pressure Liquid Chromatography D. All of the above
6. Antifreeze agent is
170 | P a g e
A. Glycerol B. DMSO
C.Both A and B D. Methanol
6.9. REFERENCES
1. Harold M. McNair, James M. Miller.2009 Basic Gas Chromatography, 2nd Edition, John
Wiley.
2. Elsa Lundanes, Leon Reubsaet, Tyge Greibrokk.2013. Chromatography: Basic Principles,
Sample Preparations and Related Methods. John Wiley
3. G. Lunn and N. Schmuff, John Wiley & Sons, 1997, HPLC Methods for Pharmaceutical
Analysis.
6.10. TERMINAL QUESTIONS
Q1. Describe the principle and procedure of Thin Layer Chromatography.
Q.2. Write short notes on following
a. Flame Ionisation detectoe
b. Electron capture detector
Q.3. Write the principle, procedure and working of HPLC with suitable diagram.
Q.4. Write the principle, procedure and working of Gas Chromatography with suitable
diagram.
Q.5. Write the principle, procedure and working of Electrophoresis.
Q.6. Write short notes on:

A. Isoelectric point
B. Isoelectric focusing (IEF)
C. Cryopreservation
171 | P a g e

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B. Sc. III YEAR

BIOSTATISTICS, INSTRUMENTATION AND

Prof.P.D.Pant Prof. B.D.Joshi

Prof. H.C.S.Bisht Prof. H.C.Tiwari

Dr.N.N.Pandey Dr.Shyam S. Kunjwal

Unit Writer Unit no.

2. Dr.Harish Chandra Unit: 4, 5& 6

Dr.Anju Thapliyal Dr.Shyam Kunjwal

Course Title and Code : Biostatistics, Instrumentation and Techniques (BSCZO303)

COURSE 1: BIOSTATISTICS, INSTRUMENTATION AND TECHNIQUES

COURSE CODE: BSCZO303 CREDIT: 3

Unit Block and Unit title Page

1 Data collection and presentation 1-40

2 Measures of central tendency 41-74

3 Measures of Variability/ Dispersion 75-109

BLOCK II: INSTRUMENTATION AND

4. Principles and uses of analytical instruments 110-133

5. Microtomy and Microscopy 134-148

6. Separation techniques and cryopreservation 149-169

UNIT-1 DATA COLLECTION AND PRESENTATION

1.6.3.1- Sampling errors

Following are the objectives of this chapter:

1. To know the definitions of Statistics and Biostatistics.

2. To know about some statistical symbols.

3. To know the scope and applications of Biostatistics.

4. To know about data, its collection and collection techniques.

5. To know about organization and representation of data by many graphical techniques

1.3 DEFINITIONS OF STATISTICS AND BIOSTATISTICS

1.3.1 DEFINITION OF STATISTICS

1.3.2 DEFINITION OF BIOSTATISTICS

Biostatistics is the application of statistics methods applied to biological areas.

There are three reasons for focusing on biostatistics:

1.4 STATISTICAL SYMBOL

1.4.1 STATISTICAL SYMBOL

f: Frequency of the variate

x : Arithmetic Mean of a given set of values or of a distribution

M e : Median of a given set of values or of a distribution

M o : Mode of a given set of values or of a distribution

σ : Standard Deviation of a given set of values or of a distribution

σ 2 : Variance of a given set of values or of a distribution

Σ : Sum of all the values of a given set

Q.D.: Quartile deviation of a given set of values or of a distribution

M.D.: Mean deviation of a given set of values or of a distribution

1.4.2 SCOPE OF BIOSTATISTICS

Biostatistics is the application of statistics in different fields of biology. The science

In current world, the scope of biostatistics is increasing rapidly. If we discuss about

1.4.3 APPLICATION OF BIOSTATISTICS

1. Statistical methods for longitudinal studies;

1.5 DATA AND ITS TYPES

There are two ways of statistical data;

1. Primary data 2. Secondary data

1.5.2 TYPES OF DATA

1.5.2.1 Nominal Data

1.5.2.2 Ordinal Data

1.5.2.3 Ranked Data

1.5.2.4 Discrete Data

1.5.2.5 Continuous Data

1.6 DATA COLLECTION AND RELATED TERMS