Date:

Experiment No.
and study mitotie cell division and cell cycle phases.
Aim: To pertom squash technique
Requirements:

I. lem long Moong (Vigna

radiata)/Onion (Allum copa) root tip.
and Methanol,
2, xative (1:3) Glacial acetie acid
3, 01%buter Touidine blue or
Geimsa stain.

$. Sides, coverslips ete.

6, Dissetion box

Procedure:
DTake lmm long oot tip of moong secd sprout.
UFix the tip ìn 1:3 Acetomethanol fixative for 15 minutes.
becomes soft.
Remove fixative and keep in 0.5N HCI for 4-5minutes till the tissue
UStain for 15minutes in 0.1% toluidine blue or geimsa stain and then give a wash
with distilled water for apprOx. Imìn.
DReep the tip on a clean slide.
O Placeacoverslip and squash the tissue gently.
DObserve the slide under 10X and 40X of microscope.

Discussion:
Mitosis is divided into five phases viz. Prophase, Prometaphase, Metaphase, Anaphase and
Telophase.

Prophase:

UProphase is marked by the appearance of thin, thread like condensing chromosomes.

0 Each prophase chromosomes is composed of two coiled filaments, the chromatids
which are the result of replication of DNA during S-phase.

DAs proplhase progresses the chromatids becomes shorter and thìcker and the two
sister chromatids of each chromosome are held together by a special DNA
containing region called centromere or primary constriction.
DThe nuclear envelope dissolves spindle fibers that start
disappears. appearing and nucleolus
 Experiment No. 1
Aim: To perform squash technique and study mitotic
Date:

cell division and cell

19-03- 19
cycle phases.
Requirements:
1.
lcm long Moong (Vigna radiata)/ Onion
(Alium cepa) root tip.
2. Fixative (1:3) Glacial acetic acid and Methanol.
3. 0.1% buffer Toluidine blue or Geimsa stain.
4. 0.5N HCI

5. Slides, coverslips etc.

6. Dissection box

Procedure:
D Take lmm long root tip of moong seed
sprout.
U Fix the tip in 1:3
Acetomethanol fixative for 15 minutes.
DRemove ffxative and
keep in 0.5N HCI for 4-5
minutes till the tissue becomes sof
p Stain for 15
minutes in 0.1% toluidine blue or geimsastain and
with distilled water for approx. I min. then give a wash
D Keep the tip on a clean slide.
0 Place a coverslip and squash the tissue
gently.
O Observe the slide under 10X and 40X of microscope.

Discussion:
Mitosis is divided into five phases viz. Prophase,
Telophase. Prometaphase, Metaphase, Anaphase and

Prophase:
UProphase is marked by the appearance of thin, thread like condensing chromosomes.
J Each prophase chromosomes is composed of two coiled filaments, the chromatids
which are the result of replication of DNA during S-phase.
UAs prophase progresses the chromatids becomes shorter and thicker and tneDNA
two
sister special
chromatids of each chromosome are held together by a
containing region called centromere or primary constriction.
appearingandnucleolus
The nuclear envelope dissolves spindle fibers that start
disappears. 69
 I Sotenphau i

k l wall
l-mebane
mmbrane ngtoplan

IL

fcell-aL
l membiare
nuelea Kondne chornatin
nemban

cntiiole
-Apindle fibree
metaphise plate
Experiment No.
Date:
19-0- \9

Prometaphase:

envelope which enables sk

I It ischaracterized by the breakdown of the nuclear
chromosomes.
mitoticspindles to interact with the
D Thus, sister chromatids become attached by their kinetochores to opposite poles.
Balanced bipolar forces fold chromosomes on the metaphase plate.
Metaphase:

D It is characterized by shortest and thickest chromosomes.

DTheir centromeres occupy the equatorial plane of the mitotic apparatus, althouoh she
chromosomal arms may extends in any direction.

D At this stage, the sister chromatids are still held together by theirs
the kinetochores of two sister chromatids face centromeres and
opposite poles. This would
proper separation in the next phase. (Anaphase)
permit the

Anaphase:
DIt begins abruptly with the splitting of each chromosome into sister
chromatids
called daughter chromatids.
During anaphase there is a movement of chromatin
towards the poles and senaration
of the poles themselves is accomplished by
elongation of the polar microtubule.
Telophase:
O Itbegins when two sets of
chromosomes reach opposite poles.
D Anew nuclear
membrane is forms d around each set of chromosomnes forming two
daughter nuclei.
0 The mitotic apparatus
except the centriole disappears, the nucleoli start
reappearing
Results:
The stages observed are:

In the prophase, centriole of interphase is

divided and the chromosome become Shortel,

70
mataptaue
 Date:
Experiment No. (4-07-19
thicker, double identical parts of centromere.

this stage and are situated at equatorial

In metaphase nuclear spindle fibers are formed at
plane.

begin to shift towards the poles.

In anaphase, chromosomes
soe
constricting from the middle and the nucleus is
In telophase, the cell membrane starts
prominent.

71
 - e p i t h e i ac e l o

Bas
body

Buccal cels
 26-19
Experiment No. 2 Date:

Aim: To isolate Buccal epithelial cells; make smear and study Barr body.

Requirements:

1. 0.1% Toluidine Blue.

2. Clean slides.
3. Fixative ( 1:3 GIacial acetic acid and methanol)
4. Dissection box

Procedure:

DGargle mouth thoroughly to remove bacteria.

i Scrap inside ofthe cheek with the clean slide.
D Prepare an even smear by keeping another slide at 45 angle over the specimen slide.
0 Fix it immediately in fixative for 10 minutes.
n Put the smear to semidry.
Put the slide in the stain for Sminutes.
0 Wash it with distilled water, drop by drop.
D Dry it and then, observe the slide under 10X
and 40X of microscope.

History:

In 1994. a scientist M.L. Barr and

Bertrans first discovered the flakes inside the nuclear
membrane of female cells (Nerve cell of cat). Hence these flakes were named Barr body.
Another scientist Unho in 1959 found that the Barr
body is the condensation of X
chromosomes. Later on 1972 M.F. Lyon also found then in
Klinefelter male &she gave a
hypothesis which is known as Lyon's hypothesis. The
in female cell is due to dosage
condensation of extra chromosome
compensation in mammalian homogamative female n
males only one X-chromosome is present while in
the case of female there are twO X
chromosome present as double dosage which shows hyper activity so, to
prevenl
expression, one extra X-chromosome under goes hetero chromatinzation and form Bar
body at the periphery of the nucleus this is
known as dosage compensation.

72
I Nsmal Female

mclew Canbodey
K-ce
(buses)

Nma Male

ucleous ele
Experiment No. 2
Date:
26-0 -|9
DOSAGE COMPENSATION:

same even if o
from gene expression in female is
This final products that is the proteins .4
chromosomes as in super female. Because of all the
has (48 XXXX that is)14 extra X ataates
thin strand of chromatin sometime
XXX will be inactivated by heteropycnosis. A leucocvtes
known as drum stick in
to the nucleus present close to the nuclear is

IMPORTANCE:

The sex chromatin of foetus can also be detected by barr body technology, after 3
month of pregnancy by amniocentesis.
n It's also helps in detecting the abnormality that is present in number of chromosomes

RESULTS:

DThe buccal epithelial cells appeared cuboidal with protoplasm enclosed by the cell
membrane the cytoplasm was slightly stained blue with appearance of granules and
centrally located.
O It showed dense nucleolus at the periphery of the nuclear membrane.
O Most cells showed dense darkly stain flakes
which was the Barr body the smear was
of a normal female.

73
otochcoradia

Qlot:

S
Dicat
Wasa
(stanch
lay)
-Epdevis
bundle
 Fodevsis,
(gtorsch lasyp

Nascla

-averchuma
tochsralio aloct: 1s. Dicat
 +olsovalinn

Motochmalia

in
pant
iua

dortion laimdl
ddlleo
u tach
teludine -Selaurchama -Pectin -digin
 Date: 9-3-19
Experiment No. y

components.
mucous, mast cells, granules,
U The most important metachromatic tissue is epithelial
amyloids, muco polysaccharides.
group and creates a color
UThe dye possessing metacromatia property has a thiazine
intermediate dimers
contrast from blue to red. The polymeric from is red, while the
and trimers are violet and purple.

site specific test for muco

DLison and Fizle, described metachromatia as a
polysaccharides. After staining areolar connecting fibroblast, collagen fibers are
mast cells which have muco
generally stained blue, while macrophages and
polysaccharides show pink or reddish color.

Result:

- Pect'o is stained redoish pple red

is stauneo blue.
appeored
coupounds
OHher pwnol'c
blwsh
geen usll be reddsh pnpe
ponendy ma
elemen and sdoren Ony
Trachery
o be
pupl
Siere
mdolle Uouela is red o pple
Shanch remas uwstaned

78