Course Title: Fundamentals of Genetics

Class: B.Sc. (Ag) First Year IInd Semester

Topic: Study of divisional stages in Meiosis

Name of Teacher: Dr. Pratibha Bisen, Assistant Professor (Plant Breeding & Genetics)

College of Agriculture, Balaghat, JNKVV, Jabalpur (MP)

Aim: To observe the different stages of Meiosis using anthers of Tradescantia

Tradescantia is an ideal plant for studying meiosis, because a single flowering stem

contains a lineup of buds, from the smallest at the bottom to the largest at the top. These largest

have already gone through meiosis and produced pollen. Young anthers in buds about six down

from the top usually contain cells undergoing meiosis to yield microspores.

Materials required:

Anthers of Tradescantia

Paper towel

Slide

Beaker

Farmer’s fixative

Aceto-carmine

Coverslip

Preparation of slides and coverslips:

Wipe clean a regular microscope slide with a paper towel and water.

Put the clean slide on a new piece of clean paper towel.

Take a coverslip from the supply and place it on a dry paper towel nearby.

Remove any dust or hair that you can see on the coverslip.

Isolating Anther Cells that are Undergoing Meiosis Work in groups of four.

Each flowering stem of Tradescantia is terminated by a group of flowers, below which

are two leaf-like bracts.

Cut a group of flowers and put it into a beaker of water.

Remove the bract from one side.

Procedure:

Count back six buds from the open flower; choose one of the buds. Push aside the petals

and sepals to reveal the stamens.

Carefully remove the six stamens and put them in a small drop of Farmer’s fixative on

your microscope slide. (The fixative kills everything in the stamens all at once, so that the

chromosomes will be in excellent form for observation.)

Add a small drop of aceto-carmine stain to the anther contents. Tap and poke at the

stamens just as you did the root tips, with the idea of opening up the stamens so that the

cells undergoing meiosis will spill out onto your slide.

Remove all the big chunks once you’re sure that all the anthers are punctured.

Figure 1 Slide Preparation for study of Meiosis cell division

Carefully cover your preparation with a coverslip. You can reduce the number of bubbles

by placing one end of the coverslip in the stain, and letting it fall over the preparation.

Look at your preparation under the medium power of your compound microscope.

Decide which stages of pollen development you have in your flower from the drawing of

pollen development. You may well have more than one stage within a given flower, so be

sure to scan the slide carefully.

If you see good mitosis or meiosis, you may want to do a squash to spread the

chromosomes out. To do a squash, fold your slide between layers of paper towel, place it

on a lab bench, and press directly down on the middle of your slide with the bottom of

your thumb. Do not move your thumb from side to side or you will smudge your

preparation.

Course Title: Fundamentals of Genetics

Class: B.Sc. (Ag) First Year IInd Semester

Topic: Study of divisional stages in Mitosis

Name of Teacher: Dr. Pratibha Bisen, Assistant Professor (Plant Breeding & Genetics)

College of Agriculture, Balaghat, JNKVV, Jabalpur (MP)

Aim: Preparation of onion root tip squash and observation of different stages of mitosis cell

division.

Materials required:

Onion root tips

Glass slides

Cover slips

Watch glass

Blade

Brush

Blotting paper

Spirit lamp

1N HCl

Glacial acetic acid

Carmine

Preparation:

Actively growing onion root tips are required for this activity. Allow at least 2–4 days for

new roots to grow. To grow root tips, obtain 5–6 onion bulbs or green onions. Remove

any dried, old root growth from the bottom of the bulbs. Place each onion bulb into a

plastic cup or jar of water so that only the root portion of the bulb is under water.

Acetocarmine is prepared by dissolving 1g of carmine in boiling glacial acetic acid.

Then, it is filtered through a filter paper to obtain the desired intensity of stain.

Procedure:

Collect 1cm long two to three bits of onion root tips in a watch glass and was wash

thoroughly using distilled water.

Drain off the distilled water and add 1 drop of 1N HCl plus nine drops of acetocarmine.

Warm the watch glass gently for 5mins keep it aside for cooling.

Take a bit of the tip on a clean slide, place enough drops of acetic acid to it to place a

cover slip.

Figure : Squash of onion root tip showing different stages of mitosis

Cover the glass slide with blotting paper and apply pressure with the help of a thumb to

get squash and proper spreading of cells.

Remove the blotting paper, clean the slide with tissue paper, observe under microscope

and report the stages observed by you.

Course Title: Fundamentals of Genetics

Class: B.Sc. (Ag) First Year IInd Semester

Topic: STUDY OF MICROSCOPE

Name of Teacher: Dr. Pratibha Bisen, Assistant Professor (Plant Breeding & Genetics)

College of Agriculture, Balaghat, JNKVV, Jabalpur (MP)

AIM: To study the compound microscope.

Definition: Microscope is an instrument, used to see the magnified images of very small objects,

which cannot be seen with a naked eye. A good microscope not only provides high magnification

(enlargement of the image of the object), but also gives fair resolution (differentiation of

neighbouring point as separate entities). Commonly used microscope in biological experiments is

the compound microscope where magnification takes place at two places.

Types of Microscopes

Depending on the source of illumination, microscopes can be divided into two categories. They

are:

Light Microscopes: These use light rays to illuminate objects. e.g. Dissection

microscopes and compound microscopes.

Electron Microscopes: These illuminate objects with a beam of highly charged

electrons. e.g. Transmission electron microscope (TEM) and scanning electron

microscope (SEM). These microscopes provide better magnification than light

microscopes.

Description:

It consists of 3 parts.

Support system-It comprise of base, stage and body tube.

Illumination system- It throws light on the object for proper viewing. It comprises of

light source, mirror, iris, diaphragm and condenser. The light source may be a plain or

concave mirror or electrically illuminated by a tungsten filament lamp or a halogen lamp.

Mirror and electric light source are generally interchangeable.

Magnification system- This includes a set of lenses aligned in such a manner so that a

magnified real image can be viewed. The objective is a set of lenses placed near the

object. It partially magnifies the object, which can be observed through the eyepiece in a

more magnified form.

Illumination system used in microscopes:

Plain mirror- Use plain mirror when a fixed source of light is used

Concave mirror- When sky light is used Concave mirror helps to converage the beam on

to the condenser.

Substage lamp interchangeable with mirror- Where there is no electricity or battery,

mirror can be used.

Built in stage lamp(tungsten filament or Halogen lamp) with intensity adjustment

Light adjustment in a microscope:

While viewing an object, sometimes the objects has to brightly illuminated, on other

occasions, less light is needed

Less light rays on the object can be altered in 2 ways by means of condenser. Condenser

can be moved upwards with the knob so as to make the object more brighter.Condenser

can be moved downwards to make the objects less brighter.

Light illuminating the object may be adjusted with Iris Diaphragm. The diaphragm may

be opened or closed to increase or diminish the light falling on the condenser here

making the object more or less bright.

PARTS OF MICROSCOPE

Eye piece -To observe the magnified image of the objective.

Draw tube -To fit the eye piece inside.

Coarse adjustment knob -To bring the objective roughly.

Fine adjustment knob -To bring the fine adjustment of the specimen.

Body tube -Holds the objective at the bottom and eye piece at the top.

Revolving nose piece -Holds the objectives, which can be revolved.

Arm -To hold microscope while handling.

Mechanical stage -Rectangular/square in shape, aperture at the center help in holding the

specimen with spring clips provided.

Objectives -The magnification lenses showing the real inverted image. It is available in

6x, l0x, 45x & l00x.

Condenser –Condenses the light and allows parallel beam of light.

Mirror -Reflects the light through the condenser and aperture

Base - Foundation of the microscope.

FACTS AND FIGURES ABOUT MICROSCOPE:

Magnifying power(MP): M.P- magnification of objective x magnification of eye piece.

eg. if you are observing an object on a slide using 10x objective and 5x eye piece then

MP=10x5=50. Thus the object viewed is magnified 50 times.

Resolving power of objective(R.P): Resolving power of an objective is defined as the

ability to separate distinctly 2 small elements of an object which are situated at a short

distance apart R.P. can be measured by Numerical aperture (N.A) of an objective greater

the N.A greater is the resolving power.

Working distance: The distance between the object and the objective is known as

working distance. The working distance decreases with increasing magnification. This

means higher the power of objective, lesser the working distance.

Focusing: Focusing an object while viewing through an eye piece means adjustment of

working distance this is done with the help of coarse adjustment and fine adjustment

knob, coarse adjustment knob is rotated to bring the object in field of view and the fine

adjustment knob is rotated to get a sharp image.

Field of view: The area of the object which one can view through the eye piece is the

field of view. The field of view narrows as magnification increases.

Objectives: Different objectives used in microscopy are

4x- Also known as scanner, which is very low power and is used mainly, to bring a

particular part of the object in the field of view.

10x- Low power objective, with the help of this one identifies the part to be observed in

high power, this does not reveals much details.

40x-This is a high power objective to reveal finer details of the object. This is spring

loaded, which means spring is fitted between the front and back lenses of the objective.

This is to protect the front lens as the working distance is low in high magnification and

the lens may touch the slide while focusing. The spring does not allow pressure on the

front lens when it touches the slide.

100x-Oil immersion lens-This also is a spring loaded objective, requiring very low

working distance and will give an image only when the object is immersed in Cedar

wood oil. This oil is used as it has high refractivity and allows very high resolving power.

INSTRUCTIONS FOR THE USE OF MICROSCOPE

Microscope should be handled carefully as it is valuable instrument.

Keep the microscope free from dust. Avoid touching the instrument with wet fingers.

Do not allow any liquids to get on to the lenses or the stage.

Place the microscope in an upright position at a convenient height facing the source of

light being used.

Ensure that the eyepiece, objectives, mirror and condenser are clean, removing any dust

with a soft brush and lens tissue.

Ensure that the coarse and fine adjustments and condenser focusing are working properly.

If a mechanical stage provided, get acquainted with the extent and directions of its

motion. Adjust the mirror to reflect a good light into the body tube. If there is much more

light in one part of the field than another, tilt the mirror gently until the most even

illumination is obtained.

Illuminate with the plane mirror when a sub stage condenser is used and with the concave

mirror, when it is not in used.

Confirm that the slide with the object to be examined and the cover slip are clean and dry.

Place the slide on the stage with the specimen to be examined below the objective.

While lowering the objective, watch from the side to avoid breaking the slide or

damaging the objective. Using the low power, bring the object in to focus with the coarse

adjustment. Finally, turn the fine adjustment slowly till the focus is perfect and the

object is seen clearly. Readjust the mirror slightly, if necessary, to secure maximum

illumination. Rotate the nose piece and bring the high power objective in to position

without touching the coarse adjustment. With the help of the fine adjustment, bring the

object in to sharp focus. If a sub stage condenser is used, it must be accurately focused.

Do not use the high power unless the object is covered with a cover slip, care must be

taken to ensure that the lens does not touch the specimen at any time.

Examine the slide carefully before drawing. Keep your note book on the right side of the

microscope or at your left if you are left handed.

After use, rotate the nose piece son that an empty position is over the stage. Clean the

lenses with lens tissue or a piece of clean soft leather. A soft cloth dipped in xylol may be

used to remove dirt, which is not easily wiped off with a cloth.

Be sure always to clean the microscope before putting it aside. Always keeps an eye

piece in the microscope to prevent dust getting into the body tube. Keep the microscope

covered when not in use.