The document provides a detailed overview of micrometry and karyotype analysis, explaining the use of micrometers to measure microscopic objects and the process of calibrating ocular and stage micrometers. It discusses karyotype analysis, the characteristics of chromosomes, and methods for measuring chromosome length and structure using microscopic techniques. Additionally, specific karyotype categories and their classifications according to Stebbins (1971) are outlined, highlighting the significance of chromosome asymmetry in plant taxonomy.

1. Prof. Ichha Purak
Department of Botany
Ranchi Women’s College, Ranchi
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MICROMETRY AND KARYOTYPE ANALYSIS

2. MICROMETRY / MICROMEASUREMENT
Micrometry is measuement of Microscopic objects. For this unit of measurement
used is micron (µ) m- micrometer which is =1/1000mm .
In order to measure microscopic objects, micrometers are used.
There are two types of micrometers:-
a) Stage micrometer b) Ocular micrometer
The stage micrometer is used to determine the value of one division of ocular for a certain
microscope.
The measurement of any object is possible with the help of ocular inserted into eyepiece of a
microscope.
Once the value of ocular micrometer for a microscope is determined at a given magnification
(Caliberation is done), the measurement of any object (material) is possible with the help of
ocular micrometer only.
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3. DESCRIPTION OF MICROMETERS
Stage micrometer is a simple slide, in its centre a scale is present ,which has
1 mm divided into 100 divisions, therefore I division of stage micrometer is equal to
0.01mm= 10 µm
Has
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10 X
40 X

4. The ocular micrometer is round cover slip like structure . In the center of this round
glass also a scale is present, having 100 divisions.
After every 10 division digits 10,20,30-100 are marked.
The value of 1 division of ocular is unknown and varies from microscope to
microscope and also varies in different magnifications
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5. PROCEDURE of Caliberation
The stage micrometer is placed on the microscope, below the objective and ocular
micrometer is introduced in the eye piece.
By rotating the eye piece, scale of ocular micrometer is brought parallel to scale of stage
micrometer.
The two scales are coincided at any point. The no. of stage division between 10 ocular
divisions is counted at 10x.
10 ocular division = X division of stage micrometer
1 ocular division = X/10 division of stage micrometer
= X/10x .01mm=X/10 x .01 x 1000 µm (micron)= Xµm
At low power 10 ocular division is equal to 15 stage divisions. So least count of the
microscope at low power is 15 micron (um).
In the same way least count is calculated for high power. At high power(40x) 10 ocular
division coincides with only 3.5 stage divisions, so least count is 3.5 micron
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6. Least count calculation of provided microscope/ Caliberation
1div of stage = 0.01 mm. or 1/100 mm.
1mm= 1000 micron = .01 x 1000 =10 micron
1div of stage = 10 micron
IN LOW POWER-
10 div of ocular coincides with 15 div of stage micrometer
1 div of ocular coincides with 15/10 div of stage micrometer
= 15/10 x.01mm = 15/10x.01x1000µm = 15 µm
IN HIGH POWER :-
10 div of ocular coincides with 3.5 div of stage micrometer
1 div of ocular coincides with 3.5/10 div of stage micrometer
= 3.5/10x.01mm = 3.5/10 x .01x1000 µm = 3.5µm
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7. Least count calculation of provided microscope
In Low Power10x,10x
15µm
In high power 10x40x
3.5 µm
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8. For measurement of length and breadth of cells (Spirogyra,Oedogonium,Onion root tip
cells),low power of microscope is used.
The slide containing material is placed below objective and ocular micrometer is introduced
into eyepiece.
By rotating eyepiece ocular scale is brought parallel to length of cell (at least 5 readings are
taken and averaged).
No of ocular divisions multiplied by least account gives length of cell in Micron (µm).
In the same way for measuring breadth, occular is brought parallel to breadth and number of
ocular divisions are counted and multiplied with least account.
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For measurement of the chromosomes a well scattered metaphase plate is focused under
the microscope. By rotating the eyepiece ocular scale is brought parallel to each
chromosome and all chromosomes and their short arm and long arm are measured. To know
the length in micron the number of ocular division is multiplied by least account.

9. Measurement of Vegetative cells of Oedogonium
SN Length of Cell Breadth of Cell
No of Oc
divisions
Average Least
Count
Length in
micron
No of Oc
divisions
Average LC Breadth in
micron
1. 15 14 15 210 µ 3 3.4 15 51.0 µ
2 14 4
3 12 3
4 13 4
5 16 3
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10. B
D F
A-10x 10x Least account 15 µm B -10x40x Least account 3.5 µm
Measurement of length and breadth of Oedogonium cells with the help of ocular
micrometer introduced into eye piece of microscope
C D
A
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11. 10X 40X LC 3.5µ
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Photograph of Ocular and stage micrometer at same
magnification

12. KARYOTYPE ANALYSIS
Some features of chromosome like number, size and centromeric position of individual
chromosomes are rather characteristic of a given species. The term karyotype is used for
characteristic chromosome complement of an organism.
Karyotype represents the chromosome constitution of a cell of an individual. It deals with
Number of chromosomes, the length of chromosome, thickness of chromosome, the position
of centromere, presence of secondary constriction and the size of satellite etc. of the somatic
chromosome complement.
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Generally karyotype is prepared from well scattered chromosomes of mitotic metaphase plate.
For this chromosomes are measured with the help of high power magnification of microscope or
by camera lucida (having prism) drawing of metaphase plate made on paper & also of stage
micrometer on same magnification or photo micrograph of metaphase plate and also either of
stage micrometer or ocular micrometer in the same magnification are taken and then
measurement is done.

13. Meiotic prophase and metaphase are also studied to identify homologous pairs, bivalents ,
chaisma formation, their termination ,recombination etc.
Each organism /species has characteristic number of chromosomes
Pisum sativum (Pea) 2n=14 Allium cepa (Onion ) 2n=16
Vicia faba (Baxla) 2n=12 Lens culinaris (Lentil) 2n=14
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Zygote and Somatic cells are diploid (2n) having two sets of chromosomes, which are
contributed during fertilization by male and female gametes which are haploid (n)
Two chromosomes in diploid set are sex chromosomes (Heterosomes) and others are
autosomes
Some genera and species have more than 2 sets of chromosomes showing polyploid series
Triticum or wheat shows diploid count of 14,28 and 42
Triticum monococcum 2n=14 (diploid) Triticum dicoccum 2n=28 (Tetraploid)
Triticum aestivum 2n=42 ( Hexaploid) Basic chromosome number X=7

14. The normal human karyotype contain 22 pairs of autosomal chromosomes and one pair of sex
chromosomes.
Normal karyotype for females contain two X chromosomes and are denoted 46, XX
Normal karyotypes for males have an X and a Y chromosome and are denoted 46,XY.
Any variation from the standard karyotype may lead to development of abnormalities.
HUMAN KARYOTYPE Homo sapiens (human ) 2n=46
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Male XY
Female XX

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16. For karyotype analysis some of following parameters can be undertaken
 Total Length of individual chromosome ( TLIC ) in micron (µm )
 Arm ratio(r) or index to centromeric position
 Centromeric Index
 Index to relative length of chromosome
 Relative Chromosome Length
 Thickness of chromosome
 Average chromosome length (ACL)
 Total Chromatin Length of Haploid Complement (TCL)
 Total Chromatin volume of haploid complement(TCV)
 F Percentage
 Total Form Percentage (TF%)
 Symmetry (S) percentage
 Karyotype category(KC)-Stebbins(1971)
 Karyotype formula(KF)
 Idiogram/ Karyogram
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17. Total Length of Individual Chromosome (TCIL)
It is measured in micron ( µm) and includes length of both arms and centromeres
(from one telomere to another telomere).The chromosomes are grouped
variously on the size of the chromosome.
A B C D
1)5.01-10 3.01-5.00 1.01-3.00 0.25-1.00
Large Median Small Minute
2)7.5-10.1 5.01-7.5 2.5-5.00 Less than 2.5
micron
Large Median Small Minute
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18. Arm Ratio -It is the ratio of long arm & short arm of a chromosome. Classification of
chromosome on arm ratio (r) is made as per Levan et.al(1964).
Value of r(arm ratio ) can range from 1 (if SA=LA) to  (limit for SA=0)
Arm ratio( LA/SA) Symbol Position of centromere
1.00 M Absolute median
1.01-1.7 m Median
1.71-3.00 Sm Sub-median
3.01-7.00 St Sub-terminal
More than 7.01/∞ T Terminal
Arm ratio(r) or index to centromeric position
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19. Continued : Index to centromeric position
S/L first proposed by Battaglia (1955), it is reciprocal to the arm ratio (r ) . Its values can range
from 1 (if S = L) to 0 (if S = 0). It is the ratio of short arm to long arm of a chromosome,
as proposed again by Sawa(1965) in Characeae
Arm Ratio
(S/L)
Short arm/Long
arm
Symbol used Position of
centromere
Type of
chromosome
1.00 m median Metacentric
0.5-0.99 sm submedian Submetacentric
Less than 0.49 St Sub-terminal Acrocentric
0.00 t terminal Telocentric
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20. Short arm (S)= P arm
Long arm (L)= Q arm
Centromeric index = S/(L+S)
It is the proportion of short arm in respect with the whole chromosome.
Its values can range from 0.5 (if S = L) to 0 (if S = 0).
Complementary to Centromeric Index=L/(L+S)
it is the proportion of long arm in respect with the whole chromosome.
Its values can range from 0.5 (if S = L) to 1 (if S = 0).
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21. Index to relative length Types of chromosomes and the symbols
Less than 0.75 Small (s)
0.75-1.00 Medium small (M1)
1.01-1.25 Medium large (M2)
More than 1.25 Large(L)
Index to relative length of chromosome
It is ratio of length of each chromosome to mean length of complement.
Relative Chromosome Length (RCL) -It is ratio in percentage between total length of
individual chromosome and total chromatin length of haploid complement
RCL= TLIC/TCL x100
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22. Thickness Of Chromosome
Chromosomes are classified in to 3 categories on the basis of thickness of
chromosome
TYPE RANGE OF THICKNESS
Slender 0.1 to less than 1 micron
Moderately Thick 1.00 micron
Thick more than 1.0 micron
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23. Average Chromosome Length (ACL )- Average chromosome length is the ratio
between total chromatin length of haploid complement and total number of
chromosomes in haploid set.
Total Chromatin Length (TCL)- TCL is sum total of length of all chromosomes of a
complement (haploid set)  (CL)
Total Chromatin Volume (TCV)- Total chromatin volume of haploid complement is
sum total of chromatin volume. (CV=πr2l)
F Percentage- is the ratio in percentage of short arm length to entire chromosome
length
F% = Short arm length of chromosome / Length of chromosomes x100
.
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24. Symmetry (S) percentage – It is ratio in percentage of length of the shortest chromosome to
length of longest chromosome of the complement and is calculated as follows
S%=G I=Gradient Index
Total Form Percentage (TF%)- It is a ratio in percentage of total sum of short arm length to total
chromatin length of the complement as advocated by Huziwara (1962)
TF% = Total length of short arm in a chromosome set /Total length of chromosome set x 100
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25. Karyotype Category (KC) - A karyotype exhibiting large differences between smallest and
largest chromosome and fewer metacentric chromosomes is asymmetric, which is believed to
be an advance feature over symmetric karyotype ,in which less difference between largest
and smallest chromosome and more chromosomes are with metacentric centromere
Karyotypes have been classified into 12 categories (1A- 4C) as per Stebbins(1971) taking into
account both the degree of difference between the largest(LC) and Smallest(SC) chromosome
of complement and position of centromere on chromosome
He established these by recognizing three degrees of difference (A-C) between the largest
and smallest chromosome of the complement and four degrees (1-4) with respect to the
proportion of chromosomes which are metacentric with an arm ratio of less than 2:1
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26. .
KARYOTYPE CATEGORIES AS PER STEBBINS(1971)
LC/SC
P=No of chromosomes with sm centromere /No of total chromosomes
Proportion of chromosome with arm ratio< 2:1
0.00 0.01-0.50 0.51-0.99 1.00
<2:1
1A(most primitive)
2A 3A 4A
2:1-4:1 1B 2B 3B 4B
>4:1 1C 2C 3C 4C(most
advanced)
LC= longest chromosome
SC= Smallest chromosome
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27. The Karyotype of 1A category of Stebbins (1971) are most primitive having less difference
between largest and smallest chromosome and less number of chromosomes with asymmetric
centromeric position ( Sm,St,t) and more with M & m centromeric position (Levan et. al 1964)
where as 4C category of Stebbins(1971) is most advanced having large difference between
largest and shortest chromosome and more number of chromosomes with Sm,St and t
centromeric position.
Karyotype asymmetry- The concept of karyotype symmetry Vs asymmetry was developed by
Levitzky(1931)
The information about karyotype asymmetry has been known to be significant in plant taxa
where association occur between increasing asymmetry of karyotype and other characteristics
such as chromosome number, plant morphology or plant habit.
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According to Stebbins (1971) , increasing asymmetry occurs through pericentric inversion,
changing median centromeric position to subterminal one
A B C D Eᵒ F G H I J Median A B C D H G Fᵒ E I J Sub-terminal

28. Karyological features of Lens culinaris L-4149 n=7,2n=14
Chr.no Long
Arm(LA)
(micron)
Short
Arm(SA)
(micron)
Satelite Total Length of
Chromosome
In micron
LA/SA Chr. Category on the basis of
Centromeric
position
size
1 4.25 2.55 - 6.80 1.67 m B
2 4.25 1.70 - 5.95 2.50 sm B
3 3.00 1.70 - 5.10 2.0 Sm B
4 2.55 1.70 - 4.25 1.5 m C
5 2.55 1.70 - 4.25 1.5 m C
6 2.55 1.70 - 4.25 1.5 m C
7 2.55 0.85 - 3.40 3.00 sm C
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Karyotypic Formula (KF):- It illustrates types of chromosomes on size and index of centromeric
position of the chromosome compliment.
Karyotypic Formula = (1m2sm)B + (3m1sm)C

29. The chromosome complement reveals that
chromosome length ranges from 3.40 µm (SC) to 6.80 µm (LC)
Karyotype reveals 4 pairs with median and 3 pairs with submedian centromere
Total chromatin length (TCL) of diploid set is 68 .0µm & of haploid set = 34.0 µm
Average chromosome length – 4.86 µm
LC/SC= 6.80/3.40= 2.0
P Ratio- No of chromosomes with Sm centromere/total no of chromosomes
= 3/7= 0.42
Karyotype Category as per Stebbins (1971) - 2B
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30. Idiogram/ Karyogram:- It can be presented graphically by arranging the chromosome in
descending order of length in a straight line. The longest chromosome is placed on right
side and smallest on left side.
All chromosomes in a complement (Karyotype) do not bear the centromere at the same
position while in graphical representation either the position of centromere is fixed at a
point or are located on different points and one telomere of chromosome is fixed .
Sat chromosomes are represented by a small break on the arm which belongs it and a
scale is taken ( 1cm=1µm ) so a chromosome having 4 µm length will cover 4 cm
(4 squares of graph paper) and if it has median centromere then 2cm above and 2cm
below ,the gap represents the centromere.
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31. Metaphase plate
Outline drawing
Idiogram (haploid set) n=7
Position of centromere is fixed
2m
Karyological features of Lens culinaris L-4149,2n=14
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Ideogram (haploid set) n=9
Position of one telomere is fixed
3m

32. Pisum sativum (Pea) n=7,2n=14
Vicia faba ( Baxla) 2n=12
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Idiogram
Haploid set
n=6

33. A- Photomicrograph of metaphase plate(n=56) B- Outline drawing of metaphase plate
A B
Karyological features of Chara
fibrosa var.fibrosa
f. tylacantha(Nordst.)
R D W
(Purak and Noor ,2018)
C- Karyogram showing 56 chromosomes
KARYOTYPIC FORMULA
( 8M 6m) B + ( 10 M 22m 4sm 4 t) C + (2 t) D
C
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34. Metaphase Allium cepa 2n=16
Metaphase Vicia faba 2n=12
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Some Metaphase Plates

35. CVCL (Paszko,2006) –is a ratio between standard deviation and mean of a sample x 100, ranges
from 0 (no variation ) to 100 ( a measure of interchromosomal asymmetry)
Coefficient of variation of chromosome length = CV CL = (SCL/XCL) X 100 =A2 X 100
MCA (Mean Centromeric Asymmetry), where Centromeric Asymmetry of a single chromosome is
given by the formula (L-S)/(L+S).Accordingly, MCA = A × 100
= Mean (L-S)/(L+S) X100= A X 100 (a measure of intrachromosomal asymmetry ) Peruzzi
and Eroglu (2013)
Coefficient of variation of the Centromeric index = CVCI = ( SCI/XCI) X 100
(a measure of centromere position heterogeneity in the karyotype ) precisely assesses the
relative variation in centromere position in a complement. Paszko (2006)
These 3 parameters have the potential to display even minor karyotypic variations.
KARYOTYPE ASYMMETRY : 3 ADDITIONAL PARAMETERS
After Stebbins (1971) formulation of 12 categories of karyotype, some additional parameters
were later on added related with Karyotype asymmetry
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36. Battaglia E (1955) Chromosome morphology and terminology. Caryologia 8: 179–187
Huziwara Y (1962) Karyotype analysis in some genera of Compositae. VIII. Further studies on the
chromosome of Aster. American Journal of Botany 49: 116–119.
Levan A, Fredga K, Sandberg AA (1964) Nomenclature for centromeric position on chromosomes.
Hereditas 52: 201–220.
Levitsky GA (1931) The karyotype in systematics . Bulletin of Applied Botany,Genetics and Plant Breeding
27: 220-240
Paszko A (2006) A critical review and a new proposal of karyotype asymmetry indices. PlantSystematics
and Evolution 258: 39–48.
Peruzzi L and Eroglu H E ( 2013) Karyotype asymmetry : again how to measure and what to measure ?
CompCytogen 7(1) ; 1-9
Sawa T (1965) - Cytotaxonomy of the Characeae: karyotype analysis of Nitella opaca and Nitella flexilis.
Amer. J. Bot., 52 (9): 962-970
Stebbins GL (1971) Chromosomal evolution in higher plants. London, UK: Edward Arnold (Publishers) Ltd.
Watanabe K, Yahara T, Denda T, Kosuge K (1999) Chromosomal evolution in the genus Brachyscome
(Asteraceae, Astereae): Statistical tests regarding correlation between changes in karyotype and habit
using phylogenetic information. Journal of Plant Research 112:145–161.
Zarco R ( 1986) A new method of estimating karyotype asymmetry . Taxon 35: 526-530
REFERENCES
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37. Some computer aided programs (SOFTWARES ) have been developed from time to time for measurement
of chromosomes (short and long arm ) with the help of images uploaded on computer
1.Micromeasure can be downloaded from http://www.biology.colostate.edu/MicroMeasure
2.KaryoType can be downloaded from (http://mnh.scu.edu.cn/soft/blog/karyotype/)
3.IdeoKar: an ideogram constructing and karyotype analyzing software
This software and related documentation is freely available on the website of the University of Kurdistan at:
http://agri.uok.ac.ir/ideokar/index.html.
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REFERENCES
Aaron Reeves (2001) MicroMeasure: A new computer program for the collection and analysis of cytogenetic
data.Genome 44: 439–443
Altinordu F et. al. (2016) A tool for the analysis of chromosomes: KaryoType TAXON : 7 pp
Ghader Mirzaghaderi & Karim Marzangi (2015) IdeoKar: an ideogram constructing and karyotype analyzing
software, Caryologia, 68:1, 31-35

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THANKS