( EXPERIMENT 1.

1 )
AIM: .....o stud }1 the pollen gerniinatto:i on a slide.

REQUIREMENTS
Fresh seasonal flowers, slide, coverslip, microscope, sucrose, boric acid, m-agnesium sulphate,
potassium nitrate, beakers etc.
I -

PROCEDURE
1. Prepare a nutrient solution by dissolving 10 g sucrose, 10 g boric acid, 30 mg magnesium
sulphate and 20 mg potassium nitrate in 100 ml of water.
2. Take a few drops of this solution on a clean slide, and dust a few pollen grains from the stamen
of a mature flower on it.
3. Observe the slide in the microscope after 5 minutes and then observe it regularly for about h alf
an hour.

OBSERVATION
In nutrient medium, the pollen grain germinates . The tube cell enlarges and comes out of the
pollen grain through one of the germ pores to form a pollen tube. The tube nucleus descends to the tip
of the pollen tube. The generative cell also passes into it. It soon divides into two male gametes. Each
male gamete is lenticular to spherical in outline.
 Exme
lntine
Germ
pores

Tube nucleus
Male gametes

Fig. 1. 1. Germ inati on of pollen g.rain s.

PRE CA UTION S
1. Flow ers shou ld be fresh ly pluc ked.
2. Use clea n slide to obse rve th~ polle n grain s.
 Alm
To study the population density of plants by quadrat method .

Materials Required
• M.etrc sca le • Thread
• Fiel.d
• Nails • Paper • Hammer

Theory/Principle
Population
Population is defined as a group of individuals of the same species which inhibit a particular space at a particular
time. The number of individuals in a population never remains constant. It may change (increase or decrease) due l(i
many factors like birth rate, death rate and migration.

Population Density
It represents the number of individuals of the species in any unit area at a specific time. The unit area may be smal)
or large, depending on the size and nature of the plant community under study.
Population density is calculated by counting all the individuals present at a given time in a given space, divided by
the number of units of area of space.
It is calculated as follows:
Total number of individuals of the species in all the sampling units (S)
Density (D) = Total number of sampling units studied (Q)

Quadrat Method
Counting all the individuals in a population is the most accurate method to determine its size. However, thi.s approacb
is not usually feasible, especially for large populations or extensive habitats. Usually, plant population is calculated
by quadrat method. A quadrat is a square that encloses an area within a habitat. For herbaceous plants, a metre square
(1 m x 1 m) quadrat is normally used.

Procedure
• Select a field to study the population density of the plants.
• Measure 1 metre x 1 metre area with the help of the metre scale in the field to make a quadrat.
• Fix four nails at the comers of the quadrat and tie each end of the nails using a thread.
• Similarly, make nine more quadrats randomly in the field of study.
• To make the counting easy, divide the quadrat further to make smaller squares.
• Count and mention the number of plants of a particular species in a square and similarly mention the number
of plants of another species if present.
• No~, count and add .the total number of a particular plant species of all the squares to get the total number of a
particular plant species of the quadrat.
,~·-•---~---• Repeat the same experiment for the other quadrats also.
~
• Record the data in the observation tab le.
1m

~ ,q; + +~ ,q; ~
~ ~ ~
Thread
~
~ ~ ~
~
~~ + ;i::_
~
~
~
~ ~ ~ ~
1m
1m

~ ~ ~~ ~ + ~ ~
~
+ ~ ~
~ ~ ~ ~ ~ ~ ~
~ + ~
~ ~

~
~ ~ ~
1m

~ SpeciesA ~ Species B ~ Species C

+ Species D

A quadrat with sub-units

Observations

Name of Number of plants per quadrat area Total number of Total Population
the plant individuals in all the number of density (D)
species I II Ill IV V VI VD VIII IX X ten quadrats (S) quadrats (Q) D=S/Q

Species A

Species B

Species C

Species D

Result
The population density of a plant species in a given field can be calculated as:
Pop I t· . Total number of plants in ten quadrats (1 m x 1 m)
u a 10n density =
10

~ recautions \
• Measure the quadrat accurately.
• Mark the quadrats close to each other from one field only.
• The vegetation should not be damaged while laying the quadrat.
Aim
To study plant population frequ ency by qu adrat mt:thod.

Materials Required
• Field • Metre scale • Th read
• Nail s • Paper • !lammer

Theory/Principle
Frequency
Determining plant population :frequency using quadrat method has become popular primarily because it is relatively
simple and objective. Frequency represents the number of times a plant species i s present within a given number uf
sample quadrats. It is measured by noting the presence of a species in random sample areas which are di stributed a,
w idely as possible throughout the area of study. Once analyzed , the sample data e nables us to calculate population
frequency of the entire population by the following method:
% Frequency = Number of sampling units in which the speci es occur x 100
Total number of sampling units employed for the study

Quadrat Method
Calc.u\ate the plant population frequency using the quadrat method. A quadrat is a sample plot of a specific size
used for the study of population or a community. It is used in many different scientific disciplines like vegetati on
assessment, including plant density, plant frequency and plant biomass . Frequency is highly influenced by
the size and shape of the quadrats used. The area that is chosen for study must not be so big that it cannot be
sampled adequately, or so small that the habitat is difficult for sampling. For herbaceous plants, a metre square
'( \ m x l m) quadrant is normally used.
n
procedure
• Select a field to study the populat i on frequen cy of the plants .
make a quadrat.
• Measure I m x I. m area with th e he lp of metre scale in the field to
s using a thread.
• Fix four nails at th e comers of the qu adrat and tie each end of th e nail
• Similarly, make nine more quadrat s random ly in th e fi e ld .
• Select the plant species for study of th e populat ion frequen cy.
the table .
• Observe the presenc e of speci es 'A' in the first quadrat and mark it in
ive ly and record the data in the tabl e.
• Similarly, check for the presenc e of species ' A' in other quadrat s respect
in the table.
• Repeat the same procedu re for species ' B ' and 'C' and reco rd th e data
given below:
• Calculate the percent age frequen cy of occurre nce usi ng th e formula
occu r x 100
Percent age frequen cy = Numbe r of samplin g units in which the species
Total number of samplin g units employ ed fo r the study
1m ~Nail

~ ~ + +~ ~~ ~ Thread
~ ~ ~
;f-
~ ~ ~

~ ~
~~ + ;f- ~
;f- ;f-
~ ~ ~ 1m
1m

~~
~
~ ~~ ~ +
~ ~
+ ~
~ ~ ~ ~ ~
~ ~
~ + ;f-
~ ;f-
~
~ ~ ~
1m

~ SpeciesA ;f- Species B ~ Species C

+ Species D

A quadrat with sub-units

Observations

Name of Numbe r of quadra ts employ ed in the study (Q) Numbe r of quadra ts .Percen tage of
the plant in which the species is freque ncy
species I II m IV V VI VII VIII IX X presen t (N) F=N/Q x 100
~ ;

Species A
~

Species B
~

Species
,___
C

Species D
..___

, P.RACTICAL SKILLS IN BIOLOG Y-12 63

 I
Resu lt Total number of quadrat s in which species occur
Total number of quadrat s studied x 100
Freque ncy percen tage for a species =

4,- Preca ution s \

• Measu re the quadrat accurately.
• Mark the quadrat s close to each other from one field onJy.
• The vegetat ion should not be damage d wbiJe laying the quadrat.
,Core Experiments ~45

( EXPERIMENT 6. 1 )
vf onion 1.i;, to fitudv '.tt:l 1 ioui;
/AJM: To prepm ·e . tc.~p orary an.. toca·. ,minc staine d mmm !
'f",>01:

stages of '1nton s.

REQU IREMENTS
scissors, forcep s, needles,
Onion bulbs, conical flasks /glass bottle s, corked vial/tu be, petrid ishes,
ed water, spirit lamp, micro scope ,
methyl alcohol, acetic acid, hydro chlori c acid, acetoc armin e, distill
.slides, coverslips, blotti ng paper etc.

PROCEDURE
its base by means of a sharp
1. Take a mediu m sized bulb of onion and trim off the old roots from
blade.
its base touchi ng the water.
2. Place the onion on a conica l flask/ glass bottle full of water, with
Keep it for a week to grow the roots.
a mixtu re of 1 : 3 acetic acid
3. Cut 5 mm off the tips of roots and put them into a vial contai ning
(Cutting of root tips should be
·and metha nol. Keep for one hour. This proces s is called fixation .
er and between 9.30 a.m. to
done in the morning betwe en 7.00 a.m. to 8.00 a.m. during the summ
11.30 a.m. during the winter).
1 N hydrochloric acid fo r
4. Remove 2 or 3 root tips and hydro lyse them by warm ing to 60°C in
15 minutes.

Onion

Bottle
i.=.;;~- Onion root
---
---
--- ---
--
-------
---
---
Water ---
---
--- ---
---
--- --
--
---
Beaker
---
-

---
--- --- --
--
------
---
---
--
-- - ---
-~-
----
- -------
---
--
Water
---
--
-- -
- -----
---- --
--
--
--- ---
Fig. 6.1. Metho d of growing onion root tips.

5. Remove the root tips and wash them thorou ghly in water.
a drop and place a coverslip
6. Place a drop of acetoc armin e on a slide; Put one hydrolysed root tip in
on the root.
of a pencil or need.le until
7. Gently squash the root by tappin g the coverslip with the blunt end
the cells separa te and spread out into a very thin layer.
Make sure that there are no air bubbl es under the coverslip.
8.
Gently warm the slide over a flame for a few seconds.
9. dividi ng cells. Exami ne the
Observe first under the low power of the microscope to locate the
d'ff
1
erent stages of mitosi s under the high power of the micro scope .

- ..,.._ ,...._.I'(-:>, ......,.,.....__ ~

..............
"" ,~....
OBSERVATIONS .th ink nucl eus are seen scat tere d. Un
.
Under low power of the m1croscope, recta ngul ar cells
di nn
W1 p
· ct . (Figs . 6 .2 and 6 .3)
·
high power of the microscope fOll owm tages beco me s ·
gs

MaturaUon zone ~

Zone of elongation -
Prophase

Meristematic zone -

Telophase

Anaphase

lnterphase
Fig. 6.2. Different stages of mitosis in the onio n
root tip.

1. Inte rpha se
(i) It is a non-dividing phas e of the cell cycle
betw een two successive cell divis ions .
(ii) Chromatin fibres appe ar in the form of a
netw ork within the nucl eus.
(iii) Nuclear envelope and nucleolus are disti
nct.
2. Prop hase
(i) Chromatin mate rial shor tens and cond ense
s into thre ad like struc ture s calle d chromosom~
(ii) Each chro moso me cons ists of two chro mati
ds, join ted at a poin t calle d cent rom ere.
(iii) Nuclear mem bran e and nucleolus star t disin
tegra tion and disa ppea r at the end of prophast
3. Met aph ase
(i) A bipolar spin dle develops in the cell. Chro
mos ome s beco me thick and two chromatids d.
each chro mos ome beco me clear.
(ii) Chro mos ome s beco me arra nged at the equa
tor of the spin dle.
(iii) Each chro mos ome get attac hed to the spin
dle fibres at its cent rom ere.
4. Anapha se
(i) The two siste r chro rnati ds of each chro mos
ome sepa rate from the cent rom ere and mo111
towa rds the oppo site poles.
(ii) The daug hter chro mos ome s (sep arate d chro
mati ds) appe ar V, J, L and I shap es, dependini
upon the posi tion of cent rom ere.
5. Telo pha se
(i) The spin dle disa ppea rs and the daug hter
chro mos ome s uncoil to form chro mati n fibres t
the two poles. 3

(ii) Nuclear mem bran e and nucl eolu s reap

pear s and two daug hter nuclei appe ar at oppositf
poles.
(iii) Cytoki.nesis occurs by cell plate form ation
betw een the two daug hter nuclei.
 Nuclear
membrane

Chromatin
.. ·:'
,•
: fibres
. ••,,
·:•··:·
Nucleolus

Cell membrane

lnterphase stage

Cell wall Nuclear

Nuclear membrane
membrane Disappearing
Nucleolus nucleolus

Chromosom es Chromosomes
Cell wall

Early prophase
Late prophase

,R- Daughter
, ,,,~,\ Spindle
chromosomes
.
, ' ,' 1 • \ ..:.,~ -- +--- fibres

~ Chromatids

''
'\ ,,,,/,
I I,'
, o'
Early anaphase
Metaphase stage

Daughter cells
Cell wall
_;
I ·; . Daughter nuclei
Chromosom es I, ._._~ I
I I I I I I I \
(Chromatids)
\ I
.
- ...I,,,, I' I' '
I I
,- ,,~-+•rr"
\
,,,,,
·
/
Cell plate

I '-+--
"-;__ Nuclear
membrane

Late anaphase T elophase stage

Fig. 6.3. Various.stages of mitosis in onion root tip cells.

PRECAUTIONS
1.
The base of the onion bulb should be i·n contact of water while growing the roots.
2.
Root tip h ould be fixed in the morning between 8 to 10 A.M.
3, ss
. . 1amp.
The slide sh0 uld be warmed gently much above the flame of t h e sp1nt
 DN A from ava ilab le pla nt mat eria l suc h as spin ach leav es, gree n pea seeds'
AIM: To isol ate
pap aya etc.

RE QU IRE ME NT S
pestle,
t mat eria l (suc h as spin ach leav es, gree n pea seed s or gree n papa ya), mor tar and
Plan eriz er or
tube s, liqu id dete rgen t, non -iod ised sod ium chlo ride , dist illed wate r, mea t tend
bea kers , test
le juic e, 95% etha nol, spoo l etc.
pap ain solu tion /jui ce of pap aya /pin e app

PR EP AR AT ION OF SO LU TIO NS
ed
tion is prep ared by add ing 10 mL liqu id dete rgen t and 10 g of non-iodis
• Det erg ent salt solu
er.
sod ium chlo ride to 90 mL of dist illed wat
led
ing 5 g of tend eriz er (enz yme ) to 95 mL of distil
• Me at tend eriz er solu tion is prep ared by add e for
thro ugh mus lin·c loth can be used as subs titut
wat er (Jui ce of pap aya /pin e app le, filte red
mea t tend eriz er). mL of
ng 5 g of non -iod ised sodi um chlo ride in 100
• 5% NaC l solu tion is prep ared by diss olvi
dist illed wat er. zer over
ping 95% etha nol in plas tic bott le in the free
• Chi llin g of etha nol mus t be don e by kee
n igh t.
core Experiments 55

PROC EDURE
• Take 5 g of the plant tissue (spinach leaf/gree n pea seed/gree n papaya) and grind it in the
mortar by adding 10 mL detergen t, salt solution and filter it through muslin cloth.
• Take 10 mL of the filtrate, add 3-4 mL tenderize r/ papaya juice and swirl the test tube by holding
the tube between the two hands to mix the contents.
• Pour 10 mL chilled ethanol carefully down the side of test tube to form a layer on the top of
the content; let it stand undisturb ed for about 3 minutes.
• Using the glass rod stir gently through interface of the two layers to collect the precipita te of
DNA and place it in a test tube with 5% NaCl or distilled water.
• The quantity of DNA present in the given ' plant material can be estimated through
spectrop hotomete r.

- --
--=--"l=

---
--:.-:"

Fig. 8.1. DNA that separates out can be removed by spooling (spool = reel for winding yarn).

OBSERVATION
The addition of ethanol to the solution causes DNA to precipitat ion. The DNA fibres appears as
white precipitate of very fine threads on the glass spool.

PRECAUTIONS
1. The plant material should be washed throughly with distilled water to remove any dust and
dried by blotting before weighing.
2. All the glassware s used must be thorough ly cleaned and dried.
3. The chemicals and enzymes used for the experime nt must be of standard quality which should
be manufact ured by standard pharmace uticals.