Lab on Cell Biology

 

Light microscopes (optical microscope)

Principle of a light microscope (optical microscope)

As mentioned earlier, light microscopes visualize an image by using a glass lens, and magnification is determined by, the lens’s ability to bend light and focus it on the specimen, which forms an image. When a ray of light passes through one medium into another, the ray bends at the interface causing refraction. The bending of light is determined by the refractive index, which is a measure of how great a substance slows the speed of light. The direction and magnitude of the bending of the light are determined by the refractive indexes of the two mediums that form the interface.

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 They use lenses to focus light on the specimen, magnifying it thus producing an image. The specimen is normally placed close to the microscopic lens.

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 Microscopic magnification varies greatly depending on the types and number of lenses that make up the microscope. Depending on the number of lenses, there are two types of microscopes i. e Simple light microscope (it has low magnification because it uses a single lens) and the Compound light microscope (it has a higher magnification compared to the simple microscope because it uses at least two sets of lenses, an objective lens, and an eyepiece). The lenses are aligned in that, they can be able to bend light for efficient magnification of the image.

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 The functioning of the light microscope is based on its ability to focus a beam of light through a specimen, which is very small and transparent, to produce an image. The image is then passed through one or two lenses for magnification for viewing. The transparency of the specimen allows easy and quick penetration of light. Specimens can vary from bacterial to cells and other microbial particles.

Principle of a light microscope

A medium with a lower refractive index such as glass to air normally speeds up the light penetration and makes light bend away from the normal and when light is passed through a medium with a greater refractive index such as air to glass, it normally slows down and bends towards the normal, perpendicularly to the surface.

If an object is put between these two mediums ie between water and air, in this case, a prism, the prism will bend the light at an angle. This is how the microscopic lenses work, they bend the light at an angle. The lens (convex) on receiving the light rays, focuses the rays at a specific point known as the focal point (F-point). The measure of distance from the center of the lens and the focal point is known as the focal length.

A microscope uses lenses whose strength is predetermined, in that, the strength of a lens is directly related to the focal length i.e short focal length magnifies objects more than lenses with a long focal length.

Microscopy works strictly with a factor of resolution whereby resolution is the ability of a lens to be able to differentiate small objects that are closely packed together. The resolution of a light microscope is determined by a numerical aperture of its lens system and by the wavelength of the light it employs; a numerical aperture is a definition of the light wavelengths produced when the specimen is illuminated.

A minimum distance (d) between two objects that distinguishes them to be two separate entities, determined by the wavelengths of the light can be calculated by an Abbe equation using the wavelength of the light that illuminated the specimen (Lambda, λ) and the numerical aperture (NA, n sin Ɵ) i.e. d=0.5 λ/n sin Ɵ

Figure: Labeled Diagram of a Light Microscope.

Types of light microscopes (optical microscope)

The modern types of Light Microscopes include:

1. Bright field Light Microscope

2. Phase Contrast Light Microscope

3. Dark-Field Light Microscope

4. Fluorescence Light Microscope

Brightfield Light Microscope (Compound light microscope)

This is the most basic optical Microscope used in microbiology laboratories which produces a dark image against a bright background. Made up of two lenses, it is widely used to view plant and animal cell organelles including some parasites such as Paramecium after staining with basic stains. Its functionality is based on being able to provide a high-resolution image, which highly depends on the proper use of the microscope. This means that an adequate amount of light will enable sufficient focusing of the image, to produce a quality image. It is also known as a compound light microscope.

Parts of a Bright-field microscope (Compound light microscope)

It is composed of:

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 Two lenses which include the objective lens and the eyepiece or ocular lens.

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 Objective lens is made up of six or more glasses, which make the image clear from the object

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 The condenser is mounted below the stage which focuses a beam of light onto the specimen. It can be fixed or movable, to adjust the quality of light, but this entirely depends on the microscope.

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 They are held together by a sturdy metallic curved back used as an arm and a stand at the bottom, known as the base, of the microscope. The arm and the base hold all the parts of the microscope.

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 The stage where the specimen is placed, allowing movement of the specimen around for better viewing with the flexible knobs and it is where the light is focused on.

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 Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob, found on the microscopes’ arm, which can move the stage or the nosepiece to focus on the image. the sharpen the image clarity.

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 It has a light illuminator or a mirror found at the base or on the microbes of the nosepiece.

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 The nosepiece has about three to five objective lenses with different magnifying power. It can move round to any position depending on the objective lens to focus on the image.

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 An aperture diaphragm also is known as the contrast, which controls the diameter of the beam of light that passes through the condenser, in that, when the condenser is almost closed, the light comes through to the centre of the condenser creating high contrast. But when the condenser is widely open, the image is very bright with very low contrast.

Magnification by Bright field Microscope (Compound light microscope)

During visualization, the objective lens remains parfocal which means, when the objective lens is changed, the image still remains in focus. The objective lens plays a major role in focusing the image on the condenser forming an enlarged clear image within the microscope, which is then further magnified by the eyepiece to a primary image.

What is seen in the microscope as an enlarged clear image of the specimen is known as the virtual image. To calculate the magnification, multiply the objective and eyepiece objective magnification together. The magnification is standard, i.e not too high nor too low, and therefore depending on the magnification power of the lenses, it will range between 40X and 100oX.

Calculation of magnification = Magnification of objective lens/magnification of the eyepiece lens

Applications of the Bright Field Light Microscope (Compound light microscope)

Vastly used in Microbiology, this microscope is used to view fixed and live specimens, that have been stained with basic stains. This gives contrast for easy visibility under the microscope. Therefore, it can be used to identify basic bacteria cells and parasitic protozoans such as Paramecium.

Phase Contrast Microscope

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 This is a type of optical microscope whereby small light deviations known as phase shifts occur during light penetration into the unstained specimen. These phase shifts are converted into the image to mean, when light passes through the opaque specimen, the phase shifts brighten the specimen forming an illuminated (bright) image in the background.

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 The phase-contrast microscope produces high contrast images when using a transparent specimen more so those of microbial cultures, thin tissue fragments, cell tissues, and subcellular particles.

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 The principle behind the working of the phase-contrast microscope is the use of an optical method to transform a specimen into an amplitude image, that’s viewed by the eyepiece of the microscope.

Parts of the Phase Contrast Microscope

The instrumentation of the Phase Contrast Microscope is based on its light pathways from receiving the source of light to the visualization of the image.

Therefore its sequentially made up of:

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 Light source (Mercury arc lamp)

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 Collective lens

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 Aperture

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 Condenser

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 Condenser annular

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 Specimen

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 Objective

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 Phase plate

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 Deflected light

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 Phase ring

Applications of Phase-Contrast Microscope

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 Determine morphologies of living cells such as plant and animal cells

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 Studying microbial motility and structures of locomotion

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 To detect certain microbial elements such as the bacterial endospores

Dark-Field Light Microscope

This is a specialized type of bright field light microscope that has several similarities to the Phase-Contrast Microscope. To make a dark field Microscope, place a darkfield stop underneath and a condenser lens which produces a hollow cone beam of light that enters the objective only, from the specimen.

Darkfield Microscope

This technique is used to visualize living unstained cells. This is affected by the way illumination is done on the specimen in that, when a hollow cone beam of light is transmitted to the specimen, deviated light (unreflected/unrefracted) rays do not pass through the objectives but the undeviated (reflected/refracted) light passes through the objectives to the specimen forming an image.

This makes the surrounding field of the specimen appear black while the specimen will appear illuminated. This is enabled by the dark background this the name, dark-field Microscopy.

Applications of the Dark Field Microscope

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 It is used to visualize the internal organs of larger cells such as the eukaryotic cells

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 Identification of bacterial cells with distinctive shapes such as Treponema pallidum, a causative agent of syphilis.

The Fluorescent Microscope

The fluorescent Microscope, the specimen emits light by adding a dye molecule to the specimen. This dye molecule will normally become excited when it absorbs light energy, hence it releases any trapped energy as light. The light energy that is released by the excited molecule has a long wavelength compared to its radiating light. The dye molecule is normally a fluorochrome, that fluoresces when exposed to the light of a certain specific wavelength. The image formed is a fluorochrome-labeled image from the emitted light

Fluorescence Microscopy

Applications of the Fluorescent Microscope

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 Used in the visualization of bacterial agents such as Mycobacterium tuberculosis.

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 Used to identify specific antibodies produced against bacterial antigens/pathogens in immunofluorescence techniques by labeling the antibodies with fluorochromes.

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 Used in ecological studies to identify and observe microorganisms labeled by the fluorochromes

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 It can also be used to differentiate between dead and live bacteria by the color they emit when treated with special stains

CAMERA LUCIDA

Camera Lucida was developed by Wollaston for making drawings of microscopic objects used along with microscope. This is an accessory instrument.

DEFINITION:

It is a simple optical device which enable the observer to make reasonably accurate outline drawings of correct scale and proportions of objects seen under the microscope.

OR

It is an instrument, mounted on the top of the body tube of a compound microscope for making clear, simple and exactly proportionate outline sketches of the objects under study.

CAMERA LUCIDA-PARTS

Mirror, Reflecting prism

Mirror: Adjustable, inclined at 45° both to the desktop and the prism.

Prism: surface is silvered. The particular central area that coincides with eyepiece is not silvered.

PRINCIPLE:

a) The light rays passing through he ocular lens are deflected at 90 by the prism.

b) These rays are further deflected at90 by the mirror.

c) This arrangements give a virtual image on the desktop beside the microscope. If a piece of paper is

kept there, the viewer can see and draw that image.

d) Magnification is due to the angular deviation

Camera Lucida, when attached with a compound microscope, helps drawing microscope images of objects on paper. It works on simple optical principle reflecting beam of light through a prism and a plane mirror.

The microscopic image of the object is reflected by the prism on to the plane mirror and there from the image is reflected on to the plane paper. The observer moves the pencil on the lines of the image and draws a correct and faithful figure of the object on the paper.

There are three main parts of a camera lucida the attachment ring, the prism, and the mirror. The attachment ring attaches the camera lucida with the body tube of the microscope. The prism rests just above the eyepiece when the instrument is attached with the microscope.

The observer now views the image of the object under the microscope through the prism which reflects the image horizontally onto the plane mirror. The plane mirror, attached at the tip of an arm rotates, and is set at an angle of 45° with reference to the prism and the plane paper.

ADVANTAGES

➤Gives accurate sketch of the microscopic objects.

➤The outline sketch is a magnified version of original object on the slide. -Tracing of outline is an easy task

CALCULATION OF MAGNIFICATION

It is computed by multiplying the power of the evepiece and the objective lens used.

1) Focus the stage micrometer at the center of the field of observation.

2) Draw a few division with the help of the drawing prism

3) For more accurate result, take 2 more drawings at the left and right side of the field of observation. (Because the magnification varies according to the position of the stage micrometer, greatest at the left and lowest at the right)

4) Measure the drawn divisions by a mm scale

Calculate magnification as follows:

e.g. 1 magnification stage division= 5 mm (1 mm=1000μ)

i.e., 10μ is magnified to 5x1000μ=5000μ

1μ is magnified5000/10 =500

So, the magnification is 500 times.

MICROMETRY

The field of science dealing with the measurement of microscopic objects using micrometer is called micrometry or morphometrics.

While studying microorganisms, it is often necessary to measure their length, breadth, diameter etc. This can be done with the help of a device called micrometer.

MICROMETER:

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 Greek-micro(u): small, meter: a measure Micrometer is an instrument attached to microscopes for measuring dimensions of small objects.

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 WILLIAM GASCOIGNE invented the micrometer. Robert Hooke improved Gascoigne's micrometer by substituting parallel hairs for the parallel edges.

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 Microscopic measurements were first performed in the late 1600s by Anton Von Leeuwenhoek, who used fine grains of sand as a gauge to determine the size of human erythrocytes.

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 Microscopic measurements are in the range of the average field diameter of wide field eyepieces i.e. 0.2μ -25μ.

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 Measurements below 0.2μ are beyond the resolving power of the microscope.

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 Lengths larger than the field view of wide field eyepiece are usually measured with stereomicroscopes.

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 The measurements are expressed in mm, μm (light microscope) or nm(EM).

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 The parameters that can be measured are: length(linear), thickness(volume), area and angles.

COMPONENTS OF MICROMETER

Micrometry involves measurement of minute or microscopic objects using microscope.The micrometer consists of two components namely a stage micrometer and an ocular micrometer

Stage Micrometer :

This is a glass slide that can be mounted on the stage of the microscope. It is similar to a microscope slide and measures 7.5 cm x 2.5 cm. It has a linear scale of length 1 mm engraved on it. This 1 mm distance is precisely divided into 100 equal divisions. Therefore each small division is equal to 0.01 mm (10 um). Every 5th and 10th divisions are demarkated by longer lines.

Ocular Micrometer

Ocular micrometer is a glass disc with a diameter of 21 mm. While in use, it

is placed on top of the field diaphragm of the eye piece. At the centre of this disc

is engraved an arbitrary linear scale with 100 equal divisions. Every 10th division

is demarkated by longer lines.

PROCEDURE OF MICROMETRY

1. Standardization of ocular micrometer using stage micrometer.

2. Find the value of 1 ocular division (eg:25μ).

3. Remove stage micrometer and place object slide.

4. Measure the object using ocular scale.

5. Calculate the actual size.

e.g. Suppose mean length and breadth of a cell is 4.2 and 2.5 ocular divisions

Length =4.2×1 ocular value =4.2×25 =105μ Breadth=2.5×1 ocular value =2.5×25 =62.5μ

➡Size of the cell=105μ×62. 5μ

CALIBRATION OF OCULAR MICROMETER

The magnification of a microscope is calibrated using the stage micrometer

and ocular micrometer. This is done by determining the value of each arbitrary

division of the ocular micrometer in terms of millimeters or microns.For calibration, stage micrometer is mounted on the stage of the microscope and focussed until the graduations become visible. Then, the ocular micrometer is introduced on top of the field diaphragm of the eye piece. Once

the stage and ocular micrometer scales become visible, both are aligned parallel

to each other.

The stage micrometer is then moved and positioned so that its

initial division (or any other division near to the initial division) coincides with

the initial division (or any other division near to the initial division) of the ocular

micrometer. The number of divisions on both the scales is counted upto a farthest

point where the two scale divisions again coincide. The value of one ocular

micrometer is calculated as follows:

Micrometric calibration involves finding out the absolute magnitude of each division of the OM scale with respect to the magnitude of the SM division. This value is called micrometer value, or calibration factor. Calibration can be done only after super imposing the graduations of both stage and ocular micrometers.

1 division in ocular = number of divisions on stage micrometer

number of coinciding divisions on ocular micrometer

One stage division=0.01 mm or 10μ

ADVANTAGES OF MICROMETRY

➤ Best method for measuring microorganisms.

➤ Best method for taxonomic measurements.

MITOTIC CEIL DIVISION IN ONION ROOTTIP

ATM

To observe different stages in onion root tip

Apparatus Required

Microscope, slide, coverslip, watch glass.

Reagent Required

Acetocarmine Stain dilute Hal & Carnoy's mixture

Procedure

An onion & placed on the mouth of the bottle in such a way the root tip of onion touches the water in a bottle within 2 or 3 days. Fresh roots will appear. The roots are cut and nearly the length of centimetres from the tip and observed in carnoy's fluid for 24 hours. Before Squash preparation 1 or 2 tip are placed in a drop of lid takes in a watch glass for about 2 minutes. The cells of the root tips become sharper because the act dissolved the cell wall. This treatment facilitated permeability of the stain and also cavy to squash. The root tips are transferred to the 2 drops of acetocarmine stain is taken in watch glass After about 2minutes the root tips are placed in a clean slide and coverslip is put over the root tips. A squash is made by pressing the c coverslip gently.

Observation & Discussion

Different stages have been observed chromosome are noticed clearly is the light power of microscope.

Prophase

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 The prophase is the first stage of mitosis.

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 The nuclear membrane disintegrates and disappears.

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 The chromosomes become shortened and thickened. chromosome is formed of two chromatids. The two chromatids of a chromosome are connected by a centromere.

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 The nucleolus starts to disappear.

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 Spindle fibres appear and they extend pole to pole.

Metaphase

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 The metaphase is the second stage of mitosis.

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 The chromosomes lie at the equatorial plate.

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 Some of the fibers of the spindle attach with the centromere of each chromosome and are known as chromosomal fibers.

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 Some other fibers of the spindle extend from one pole to the other pole. These are known as continuous fibers.

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 The fibers which occur in between the chromosomes are called interzonal fibres.

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 The centromere of each chromosome divides into two, each being associated with a chromatid.

Anaphase

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 Anaphase is the third stage of mitosis.

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 The chromatids of each chromosome are separated and form two chromosomes called daughter chromosomes.

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 The chromosomes are connected by spindle fibres.

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 The daughter chromosomes move towards thee opposite poles of the cell.

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 This migration of the daughter chromosomes is achieved by the contraction of chromosomal fibres and the stretching of the interzonal fibres.

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 The chromosome develop the shape of J or L or V according to the position o the centromere in the chromosome.

Telophase

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 The telophase is the final stage of mitosis.

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 The chromosomes with their centromeres at the poles begin o uncoil and lengthen.

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 They aggregate together to form at the poles.

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 The nucleolus begins to reappear.

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 The nuclear membrane develops around the chromosome from the elements of endoplasmic reticulum. Spindle fibers break down and are absorbed in the cytoplasm.

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 Two daughter nuclei are formed and they attain the appearance of the interphase nuclei.

Significance of mitosis:

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 It helps in maintaining the same number of chromosomes in daughter cells after division.

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 It is responsible for growth and development of multicellular organisms.

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 It helps in repairing of damaged tissues.

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 It helps the cell to maintain proper size.

STAINING AND OBSERVATION OF POLYTENE CHROMOSOME IN

SALIVARY GLANDS OF CHIRONOMUS LARVA

Aim

To observe the giant chromosomes of salivary gland in Chironomus4 larva

Materials Required

Chironomus larva, needle, slide, coverslip, acetocarmine, stain.

Procedure

With the help of two needles the head of the Chironomus larva was detached from the body. The slide was not kept under the dissection microscope. By gentle squaring the salivary gland was removed from the root of the body. The slide than the Salivary gland was stained by acetocarmine was prepared. The slide was kept under the high power of the microscope and observed.

OBSERVATION

The cells of salivary gland contain a large number of chromosomes They appeared as hum worm live thousands of chromonematic glands During the vital stages of development. The polytene chromosome exhibit swellings called balbiani rings the putting can be induced by the mating hormone ecdysone temperature 4 dh

DISCUSSION

Giant Chromosome

These are exceptionally larger chromosomes. They are described as unusual chromosomes by A.M. Winchester. There are two types of giant chromosomes, namely polytene chromosomes and lamp brush chromosomes.

Polytene chromosomes

Polytene chromosomes have the following salient features:

1. It was discovered by Balbiani in 1881

2. It is found in the salivary gland cells of Chironomus larva. Hence it is also called salivary gland chromosome.

3. It is a giant chromosome. It is larger in size. For example, in Drosophila melanogaster it is 1000 times larger than somatic chromosomes.

4. The larger size of the chromosome is due to the presence of many longitudinal strands called chromonemata. Hence, they are also called polytene chromosome.

5. The many strands of the giant chromosome are due to of repeated division of the chromosome without the cytoplasmic division. This type of division is called endomitosis.

6. The polytene chromosome contains two types of transverse bands, namely dark bands and inter bands. The dark bands are darkly stained and the inter bands are lightly stained with nuclear stains. The dark bands contain more DNA and less RNA. The inter bands contain more RNA and less DNA.

7. The bands of polytene chromosomes become enlarged at certain times to form swellings called puffs or Balbiani rings. The formation of puffs is called puffing.

In the regions of puffs the chromonemata uncoil and open out to form many loops. Thus, puffing is caused by the uncoiling of individual chromomeres in a band. The puffs indicate the site of active genes when mRNA synthesis takes place.

PREPARATION AND IDENTIFICATION OF DIFFERENT STAGES OF MEIOSIS IN

GRASSHOPPER TESTIS CELL

Aim:

To observe the different stage OF Meiosis in Grasshopper testis follicule.

Material Required:

Compound Microscope, Dissection Microscope, slides, coverslip, Male Grasshopper etc.

Reagents Required:

Acetocarmine stain.

Identify the Male grasshopper and make a cut between thorax and abdomen with fine blade separate the abdomen and given a gentle procedure to remove the testicular Follicle Collect the follicle in Water glass Containing distilled water. Transfer a few Follicles to a slide and add Few drops of acetocarmine Stain Leave it for 10 minutes to get Stained. place a coverslip gently on Preparation the Follicle and press it. The preparation is observed under high power the Microscope.

Observation:

* Various stage of Meiosis is observed

* Meiosis Cell division is dived to in two phases Meiosis I and Meiosis

* Meiosis I is divided into four phases Prophase. I, Metaphase I, Anaphase I and Telophase I.

Prophase I

prophase is divided into five sub stages

i) Leptotene

ii) Zygotene

il) Pachytene

iv) Diplotene

v) Diakinesis.

Leptotene

1) In the Leptotene stage the Chromosome become a long thread like shape.

(2) The Centriole duplicates and each daughter Centriole Migrates towards opposite pole of the Cell.

(3) On reaching the polar each Centriole duplicates and their each pole of the cell possess two Centrioles of a single diplosome.

Zygotene:

1) The Homologous Chromosome which Come from the over and by the sperm and attached towards each other and their pouring taken place.

2) The Synapsis begin at one or more points along the longer of the homologous chromosome.

Pachytene:

1) The Chromosome become fruited spirally around each other and cannot be distinguished separately.

2)Each homologous chromosomes splits lengthwise to form two chromatids.

Diplotene:

1) Un pairing homologous chromosome take place

2) Chromatid of each tetrad are Cleanly visible.

3) Chromatid of the paired homologous Chromosome physically joined at one or more discrete points called chiasmata.

Diakenesis:

1) Bivalent Chromosome more contest and evenly distributed in the nucleus

2) The nucleolus detaches from the nuclear organizer and ultimately disappear.

3) Nuclear envelop break Down.

Melaphase I

Metaphase -I Consist of Spindle filter attachment to Chromosome and chromosomal elignment at the equator.

Anaphase -I

1) Homologous are free from each other due to shortening at chromosomal Filter.

(2) Each Homologous chromosome with its two chromatid and it divided Centrosome more towards the opposite slide pole of the Cell.

Telophase-I

1) The arrival of haploid set of Chromosome at each pole define the onset of Telophase - I During which nuclei are reassembled.

2) The Endoplasmic reticulum form the nuclear envelop around the chromosome.

3) The Chromosomal become Uncoil

4) The nucleolus reappear and thus two daughter chromosomes are formed.

The Second Mitotic division is actually the Mitotic which divides the haploid Mitotic division includes prophase - Ⅱ, Metaphase II, Anaphase II, Telophase-II

Prophase- II

1) Each Centriole divides into two and thus two pairs of Centriole are formed.

2) Each pair of centrioles migrate to the opposite pole.

3) The Nuclear Membrane and the nucleolus are disappeared.

Metaphase II

The Chromosome get arranged on the equator of the Spindle. The Chromosome centromere divides into two and thus each Chromosomes produce two daughter Chromosomes.

Anaphase

1) The daughter chromosome moves towards the opposite pole due to the shortening of Chromosome Microtubule and stretching of interzonal Microtube and Spindle.

2) The nucleolus disappears.

Telophase

1) The Endoplasmic reticulum form the nucleus envelope around the Chromosome.

2) The Nucleolus reappear due to the synthesis of RNA and accumulation of ribosomal protein.

Significane of Meiosis:

1) Meiosis maintains a constant number of chromosomes the organisms.

2) By Crossing over it provided on opportunity for gene exchange and cause material of the evolutionary process.

MOUNTING OF BUCCAL EPITHELIUM AND USING VITAL STAINS

Aim

To observe Squamous epithelium Cells.

Material Required:

Compound microscope, slide, Coverslip, tongue cleaner.

Reagent Required : Eosin

Procedure:

1) The Mucous from the roof of the buccal cavity is dropped in the centre of the slide with the help of the clean tongue Cleaner.

2) The Mucous is washed with water.

3) A drop of Eosin is added o it.

4) After two Minutes, excess of eosin is Washed With Water.

5) The Cover slip is part on it and focussed Under the Microscope.

Inference:

1) Squamous epithelium a mass is a tissue. It is mass of similar cells carrying out similar function.

2) It is continues larger of tissue covering the body externally or internal surface.

Eg: Inner lining of skin of Buccal Cavity, Outer skin of frog.

3) The Epithelial tissue covering the external body surface is called external epithelium.

Eg Outer skin of frog.

4)The epithelium tissue covering the internal surface is called inner epithelium.

5) It is simple epithelial tissue as it made up of single layer of cells.

6) The cells are flat, thin a tile like.

7) The cell is in close content with one another.

8) These are no intercellular spaces.

9) They are situated on a basement membrane.

10) They have no Vascularization but they contain nerve fibres.

11) The cells are hexagonal in shape.

PREPARATION OF HUMAN BLOOD SMEAR

AIM :

To find out the various kinds of human blood smear.

Apparatus Required:

Slide, sterilized needle, microscope

Procedure :

A tip of index finger is punctured by a sterilized needle and large drop of blood is allowed to collect and is placed in one and of the clean slide, with the help of another slide uniform smear is prepared the blood smear is allowed dry air. Add few drops of leismann's stain and dry the Slide in air. Then few drops of its allowed, now an Istria colour appear, on the surface of the blood. Air is blown through a pipette over. The Slide so that water and stain are an examined under the microscope. Different types of leucocytes can be observed.

OBSERVATION

White blood cells (leucocytes)

Leucocytes are colourless, amoeboid, nucleated cells devoid of haemoglobin and other pigments. Approximately 6000 to 8000 per cubic mm of WBCs are seen in the blood of an average healthy individual.

Depending on the presence or absence of granules, WBCs are divided into two types, granulocytes and agranulocytes. Granulocytes are characterised by the presence of granules in the cytoplasm and are differentiated in the bone marrow. The granulocytes include neutrophils, eosinophils and basophils.

Neutrophils

Neutrophils are also called heterophils or polymorphonuclear (cells with 3-4 lobes of nucleus connected with delicate threads) cells which constitute about 60%-65% of the total WBCs. They are phagocytic in nature and appear in large numbers in and around the infected tissues.

Eosinophils

Eosinophils have distinctly bilobed nucleus and the lobes are joined by thin strands. They are non-phagocytic and constitute about 2-3% of the total WBCs. Eosinophils increase during certain types of parasitic infections and allergic reactions.

Basophils

Basophils are less numerous than any other type of WBCs constituting 0.5%- 1.0% of the total number of leucocytes. The cytoplasmic granules are large sized, but fewer than eosinophils. Nucleus is large sized and constricted into several lobes but not joined by delicate threads. Basophils secrete substances such as heparin, serotonin and histamines. They are also involved in inflammatory reactions.

Agranulocytes

Agranulocytes are characterised by the absence of granules in the cytoplasm and are differentiated in the lymph glands and spleen. These are of two types, lymphocytes and monocytes.

Lymphocytes

Lymphocytes constitute dif 28% of WBCs. These have large round nucleus and small amount of cytoplasm. The two types of lymphocytes are B and T cells. Both B and T cells are responsible for the immune responses of the body. B cells produce antibodies to neutralize the harmful effects of foreign substances and T cells are involved in cell mediated immunity.

Monocytes

Monocytes (Macrophages) are phagocytic cells that are similar to mast cells and have kidney shaped nucleus. They constitute 1-3% of the total WBCs. The macrophages of the

central nervous system are the 'microglia, in the sinusoids of the liver they are called 'Kupffer cells and in the pulmonary region they are the 'alveolar macrophages.

PREPARATION OF FROG BLOOD SMEAR

AIM

To observe different types of cells of frog

REQUIREMENTS:

Frog slides, needle microscope, Leishman's stain

PROCEDURE

A dop of blood of frog is taken by opening the viscera and placed at one end of the slide. By means of another. Slide the drop of blood drawn across the first Slide. So as to take thin film the smear is allowed to dry in the air. A few drops Leishman’s stain is added and kept it for 5 min, then a few drops of distilled water is added on the slide and it is mined with the stain and water, thoroughly and keep it for 3 min. Then the slides are washed under the running tap water & it is observed under high microscope

OBSERVATION:

Blood is a liquid tissue and red in colour. It has two components namely liquid component cellular component. The liquid component is called plasma, the cellular component is Corpuscles. The Corpuscles are a types namely red blood corpuscles, white blood corpuscles and platelet. The RBC’s are red colour. They are nucleated oval in shape. They are called eythrocytes. They carry o2 and Co2. The WBCs are colourless. They are 3 types

i) Polymorphonuclear leucocytes.

ii) Macrocytes

iii) lymphocytes.

They platelets are smaller in size. They help in blood clotting.

PLANT CELL

Plant Cell Structure

Plant cells are eukaryotic cells that vary in several fundamental factors from other eukaryotic organisms. The plant cell is rectangular and comparatively larger than the animal cell. Plant cell structure includes various components known as cell organelles that perform different functions to sustain itself. These organelles include:

Cell Wall

It is a rigid layer which is composed of polysaccharides cellulose, pectin and hemicellulose. It is located outside the cell membrane

The primary function of the cell wall is to protect and provide structural support to the cell.

Cell membrane

It is the semi-permeable membrane that is present within the cell wall. It is composed of a thin layer of protein and fat.

The cell membrane plays an important role in regulating the entry and exit of specific substances within the cell.

Nucleus

The nucleus is a membrane-bound structure that is present only in eukaryotic cells. The vital function of a nucleus is to store DNA or hereditary information required for cell division, metabolism and growth.

1. Nucleolus: It manufactures cells’ protein-producing structures and ribosomes.

2. Nucleopore: Nuclear membrane is perforated with holes called nucleopore that allow proteins and nucleic acids to pass through.

Plastids

They are membrane-bound organelles that have their own DNA. They are necessary to store starch and to carry out the process of photosynthesis. It is also used in the synthesis of many molecules, which form the building blocks of the cell.

Leucoplasts

They are found in the non-photosynthetic tissue of plants. They are used for the storage of protein, lipid and starch.

Chloroplasts

It is an elongated organelle enclosed by phospholipid membrane. The chloroplast is shaped like a disc and the stroma is the fluid within the chloroplast that comprises a circular DNA ,with green coloured pigment called chlorophyll required for the process of photosynthesis. The chlorophyll absorbs light energy from the sun and uses it to transform carbon dioxide and water into glucose. Chromoplasts

They are heterogeneous, coloured plastid which is responsible for pigment synthesis and for storage in photosynthetic eukaryotic organisms. Chromoplasts have red, orange and yellow coloured pigments which provide colour to all ripe fruits and flowers.

Central Vacuole

It occupies around 30% of the cell’s volume in a mature plant cell. Tonoplast is a membrane that surrounds the central vacuole.

Golgi Apparatus

They are found in all eukaryotic cells, which are involved in distributing synthesised macromolecules to various parts of the cell.

Ribosomes

They are the smallest membrane-bound organelles which comprise RNA and protein. They are the sites for protein synthesis, hence, also referred to as the protein factories of the cell.

Mitochondria

They are the double-membraned organelles found in the cytoplasm of all eukaryotic cells. They provide energy by breaking down carbohydrate and sugar molecules, hence they are also referred to as the “Powerhouse of the cell.”

Lysosome:

Lysosomes are called suicidal bags as they hold digestive enzymes in an enclosed membrane. They perform the function of cellular waste disposal by digesting worn-out organelles, food particles and foreign bodies in the cell. In plants, the role of lysosomes is undertaken by the vacuoles.

Animal Cell

Animal cells range in size from a few microscopic microns to a few millimetres. The largest known animal cell is the ostrich egg, which can stretch over 5.1 inches across and weighs about 1.4 kilograms. This is in stark contrast to the neuron in the human body, which is just 100 microns across.

The shape of animal cells also varies, with some being flat, others oval or rod-shaped. There are also more intriguing shapes such as curved, spherical, concave and rectangular. Most of the cells are microscopic in size and can only be seen under the microscope.

Animal Cell Structure

Animal cells are generally smaller than plant cells. Another defining characteristic is its irregular shape. This is due to the absence of a cell wall. But animal cells share other cellular organelles with plant cells as both have evolved from eukaryotic cells.

A typical animal cell comprises the following cell organelles:

Cell Membrane

A thin semipermeable membrane layer of lipids and proteins surrounding the cell. Its primary role is to protect the cell from its surrounding. Also, it controls the entry and exit of nutrients and other microscopic entities into the cell. For this reason, cell membranes are known as semi-permeable or selectively permeable membranes.

Nucleus

It is an organelle that contains several other sub-organelles such as nucleolus, nucleosomes and chromatins. It also contains DNA and other genetic materials.

Nuclear MembraneIt is a double-membrane structure that surrounds the nucleus. It is also referred to as the nuclear envelope.

Centrosome

It is a small organelle found near the nucleus, which has a thick centre with radiating tubules. The centrosomes are where microtubules are produced.

Lysosome

They are round organelles surrounded by a membrane and comprising digestive enzymes which help in digestion, excretion and in the cell renewal process.

Cytoplasm

A jelly-like material which contains all the cell organelles, enclosed within the cell membrane. The substance found within the cell nucleus, contained by the nuclear membrane is called the nucleoplasm.

Golgi Apparatus

A flat, smooth layered, sac-like organelle which is located near the nucleus and involved in manufacturing, storing, packing and transporting the particles throughout the cell.

Mitochondrion

They are spherical or rod-shaped organelles with a double membrane. They are the powerhouse of a cell as they play an important role in releasing energy.

Ribosome

They are small organelles made up of RNA-rich cytoplasmic granules, and they are the sites of protein synthesis.

Endoplasmic Reticulum (ER)

This cellular organelle is composed of a thin, winding network of membranous sacs originating from the nucleus.

Vacuole

A membrane-bound organelle present inside a cell involved in maintaining shape and storing water, food, wastes, etc.

Nucleopore

They are tiny holes present in the nuclear membrane which are involved in the movement of nucleic acids and proteins across the nuclear membrane.

Bacteriophage

Bacteriophage is a bacterial virus. It is a virus living inside bacterial cells. Bacteriophage means bacteria eating agent. It was first described by Twort in 1915.

The common bacteriophage is T, bacteriophage. It is parasitic on human colon bacteria Escherichia coli. It is also known as coliphage.

The T, phage is tadpole-shaped. It consists of 3 parts, namely a head, a neck and a tail The head is polyhedral. It is covered by a protein coat called capsid. The capsid is made up of about 2,000 protein sub units called capsomeres.

A double stranded DNA is present inside the head. The DNA is highly coiled and tightly packed. It contains more than 75 genes.The neck is very short and it contains collar. It connects the head with the tail.

The tail consists of a central hollow core tube. Through this core tube the DNA of the phage can pass into the bacterial cell. The core tube is covered by a sheath or tube made up of about 144 protein contractile subunits.

The free end of the core tube has a hexagonal end plate. The end plate has 6 spikes and 6 fibres. The spikes are used for penetration and the fibres are used for attachment on the host. The bacteriophage has a complex symmetry. It exhibits a combination of icosahedral and helical symmetry. The head is icosahedral in symmetry and the tail is helical in symmetry.

The bacteriophage has 2144 capsomeres. Of these, 2000 capsomeres are present in the head and 144 capsomeres are in the tail. The 2000 capsomeres of the head are arranged in the form of an icosahedron having 20 triangular sides called facets and 12 corners called vertices. There are two types of capsomeres, namely pentons and hexons. Pentons are 12 in number and are located on the head. The hexons are located on the facets.The 144 capsids of the tail are helically arranged to form the core tube.

Structure of E.coli

• E.coli is Escherichia coli. It is a colon bacterium. It lives in the colon of man. It is the indigenous flora of intestine. • It is a prokaryote.

• It is rod-shaped. Hence it is a Bacillus. It is 1.5μm broad and 6.5μm long.

• It is Gram negative. It appears red in Gram staining.It is a facultative anaerobe.

• It is peritrichous having flagella all over the body.It is motile.

• It is chemo-organotrophic in nutrition. 37°C temperature is the optimum for its growth.

• It is covered by a capsule. Hence it is a capsulated bacterium.

• Below the capsule there is cell wall. The cell wall is composed of peptidoglycan. Below the cell wall, there is a plasma membrane.

• The cytoplasm contains a circular double stranded DNA. It is the bacterial chromo- some. It does not contain a nuclear membrane with 70S ribosomes.

• One or more membranous vesicles are attached to plasma membrane. They are called mesosomes.

• Small circular double stranded DNAs are also found in the cytoplasm. They are called

plasmids. The surface of bacteria contains numerous flagella used for locomotion.

• In between the flagella, there are short appendages called pili.

• It reproduces asexually and sexually. E.coli reproduces asexually by binary fission and endospore formation. Sexual reproduction includes conjugation, transformation an transduction.

• It can ferment glucose and produce acid and gas. • It is harmless. But occasionally it attains virulence and produces enterotoxin. • It produces diseases like diarrhoea, septicaemia meningitis and urinary tract infections.



 A ribosome is made from complexes of RNAs and proteins and is, therefore, a ribonucleoprotein. Around 37 to 62% of RNA is comprised of RNA and the rest is proteins.



 Each ribosome is divided into two subunits:

1. A smaller subunit which binds to a larger subunit and the mRNA pattern, and

2. A larger subunit which binds to the tRNA, the amino acids, and the smaller subunit.



 Prokaryotes have 70S ribosomes respectively subunits comprising the little subunit of 30S and the bigger subunit of 50S.



 Their small subunit has a 16S RNA subunit (consisting of 1540 nucleotides) bound to 21 proteins.



 The large subunit is composed of a 5S RNA subunit (120 nucleotides), a 23S RNA subunit (2900 nucleotides) and 31 proteins.



 Eukaryotes have 80S ribosomes respectively comprising of little (40S) and substantial (60S) subunits.



 The smaller 40S ribosomal subunit is prolate ellipsoid in shape and consists of one molecule of 18S ribosomal RNA (or rRNA) and 30 proteins (named as S1, S2, S3, and so on).



 The larger 60S ribosomal subunit is round in shape and contains a channel through which growing polypeptide chain makes its exit.



 It consists of three types of rRNA molecules, i.e., 28S rRNA, 5.8 rRNA and 5S rRNA, and 40 proteins (named as L1, L2, L3 and so on).



 The differences between the ribosomes of bacterial and eukaryotic are used to create antibiotics that can destroy bacterial infection without harming human cells.



 The two subunits fit together and work as one to translate the mRNA into a polypeptide chain during protein synthesis and multiple ribosomes are attached to the same mRNA strand, this structure is known as polysome. after the synthesis of polypeptide the two sub-units separate and are reused or broken up.

Structure of Mitochondria



 The mitochondrion is a double-membraned, rod-shaped structure found in both plant and animal cell.



 Its size ranges from 0.5 to 1.0 micrometre in diameter.



 The structure comprises an outer membrane, an inner membrane, and a gel-like material called the matrix.



 The outer membrane and the inner membrane are made of proteins and phospholipid layers separated by the intermembrane space.



 The outer membrane covers the surface of the mitochondrion and has a large number of special proteins known as porins.

Cristae

The inner membrane of mitochondria is rather complex in structure. It has many folds that form a layered structure called cristae, and this helps in increasing the surface area inside the organelle. The cristae and the proteins of the inner membrane aid in the production of ATP molecules. The inner mitochondrial membrane is strictly permeable only to oxygen and ATP molecules. A number of chemical reactions take place within the inner membrane of mitochondria.

Mitochondrial Matrix

The mitochondrial matrix is a viscous fluid that contains a mixture of enzymes and proteins. It also comprises ribosomes, inorganic ions, mitochondrial DNA, nucleotide cofactors, and

organic molecules. The enzymes present in the matrix play an important role in the synthesis of ATP molecules.

Functions of Mitochondria

The most important function of mitochondria is to produce energy through the process of oxidative phosphorylation. It is also involved in the following process:

1. Regulates the metabolic activity of the cell

2. Promotes the growth of new cells and cell multiplication

3. Helps in detoxifying ammonia in the liver cells

4. Plays an important role in apoptosis or programmed cell death

5. Responsible for building certain parts of the blood and various hormones like testosterone and oestrogen

6. Helps in maintaining an adequate concentration of calcium ions within the compartments of the cell

7. It is also involved in various cellular activities like cellular differentiation, cell signalling, cell senescence, controlling the cell cycle and also in cell growth.

Structure of Golgi Apparatus



 It is referred to as the manufacturing and the shipping center of the cell.



 Golgi apparatus was discovered in the year 1898 by an Italian biologist Camillo Golgi.



 Under the electron microscope, the Golgi apparatus is seen to be composed of stacks of flattened structures that contain numerous vesicles containing secretory granules.



 The Golgi apparatus is morphologically very similar in both plant and animal cells.



 Typically, however, Golgi apparatus appears as a complex array of interconnecting tubules, vesicles, and cisternae.

A. Cisternae



 It is the simplest unit of the Golgi apparatus is the cisterna.



 Cisternae (about 1 μm in diameter) are central, flattened, plate-like or saucer-like closed compartments that are held in parallel bundles or stacks one above the other.



 In each stack, cisternae are separated by a space of 20 to 30 nm which may contain rod-like elements or fibers.



 Each cisterna is bounded by a smooth unit membrane (7.5 nm thick), having a lumen varying in width from about 500 to 1000 nm.



 The margins of each cisterna are gently curved so that the entire dictyosome of the Golgi apparatus takes on a bow-like appearance.



 The cisternae at the convex end of the dictyosome comprise proximal, forming or cis-face and cisternae at the concave end of the dictyosome comprise the distal, maturing or trans-face.

B. Tubules



 A complex array of associated vesicles and anastomosing tubules (30 to 50 nm diameter) surround the dictyosome and radiate from it. In fact, the peripheral area of the dictyosome is fenestrated (lace-like) in structure.

C. Vesicles

The vesicles (60 nm in diameter) are of three types:

(i) Transitional vesicles are small membrane limited vesicles which are thought to form as blebs from the transitional ER to migrate and converge to cis face of Golgi, where they coalesce to form new cisternae.

(ii) Secretory vesicles are varied-sized membrane-limited vesicles that discharge from margins of cisternae of Golgi. They, often, occur between the maturing face of Golgi and the plasma membrane.

(iii) Clathrin-coated vesicles are spherical protuberances, about 50 μm in diameter and with a rough surface. They are found at the periphery of the organelle, usually at the ends of single tubules, and are morphologically quite distinct from the secretory vesicles.

Functions of Golgi Apparatus

1. Golgi vesicles are often, referred to as the “traffic police” of the cell. They play a key role in sorting many of the cell’s proteins and membrane constituents, and in directing them to their proper destinations.

2. In animals, the Golgi apparatus is involved in the packaging and exocytosis of the following materials :



 Zymogen of exocrine pancreatic cells;



 Mucus (=a glycoprotein) secretion by goblet cells of the intestine ;



 Lactoprotein (casein) secretion by mammary gland cells (Merocrine secretion) ;



 Secretion of compounds (thyroglobulins) of thyroxine hormone by thyroid cells;



 Secretion of tropocollagen and collagen ;



 Formation of melanin granules and other pigments; and



 Formation of yolk and vitelline membrane of growing primary oocytes.

Structure of Endoplasmic Reticulum

The structure of the endoplasmic reticulum is shaped like a sac. Since ER is of two types, each has its own distinguishing features:

Rough Endoplasmic Reticulum Structure



 The rough endoplasmic reticulum is named so because of its appearance.



 It is a series of connected flattened sacs having several ribosomes on its outer surface, hence the name.



 Rough ER is prominent in cells where protein synthesis happens (such as hepatocytes)

Smooth Endoplasmic Reticulum Structure



 The smooth endoplasmic reticulum, on the other hand, does not have ribosomes.



 Smooth ER has a tubular form.It participates in the production of phospholipids, the chief lipids in cell membranes and are essential in the process of metabolism.



 Smooth ER transports the products of the rough ER to other cellular organelles, especially the Golgi apparatus.

Functions of Endoplasmic Reticulum

The endoplasmic reticulum is categorised into two types, and both these types of ER perform specific functions:

Smooth Endoplasmic Reticulum Function:



 Smooth ER is responsible for the synthesis of essential lipids such as phospholipids and cholesterol. It is also responsible for the metabolism of carbohydrates.



 Smooth ER is also responsible for the production and secretion of steroid hormones,store and releases calcium ions.

Rough Endoplasmic Reticulum Function:



 The majority of the functions of rough ER is associated with protein synthesis.



 The second most important function after protein synthesis and protein folding is protein sorting.

Giant Chromosomes

The giant chromosomes are the chromosomes which are very large in size as compared to normal chromosomes.

They are also called as mega chromosomes.

These chromosomes are very long and thick in appearance during metaphase than normal chromosomes. Hence, they are called as Giant chromosomes.

The Giant chromosomes are of two types-

1. Lampbrush chromosome.

2. Polytene chromosome.

Lampbrush chromosome



 Lampbrush chromosomes were first observed by Walther Flemming in 1882 in the oocytes of Salamander and described by Ruckert in shark oocytes in 1892.



 This is a giant chromosome which is found in the oocytes or immature egg of most of the vertebrates such as fishes, amphibians, reptiles and birds also in some insects.



 They are generally found in oocytes as the DNA content is very high in these cells. They are formed in response to the active synthesis of mRNA molecules for egg cells.



 These chromosomes are not found in mammals.



 The characteristic features of Lampbrush chromosomes is that it include fine lateral loops that originate from the chromomeres in the diplotene stage of prophase first of meiosis.



 These chromosomes are found in both animal species, vertebrates and invertebrates. So, they can be seen only in the prophase first's diplotene stage of meiosis.



 They are generally smaller in size in case of invertebrates than vertebrates. Its lateral loops give it its characteristic lamp brush like appearance.



 Biological significance



 The loops present in case of lampbrush chromosomes are used in chromosome mapping, particularly those loops that appear at a constant location in the chromosome.



 It provides evidence for eukaryotic gene amplification that is needed in the growth phase of oocytes.



 This can be used for the analysis in hybridization processes.

Structure of Polytene Chromosome

In insects, polytene chromosomes are commonly found in the salivary glands.

As their presence in salivary gland, they are also called as "salivary gland chromosomes".

The polytene chromosome contains two types of bands namely- Dark bands and Interbands (light bands).

The dark bands are darkly stained and the inter bands are lightly stained with nuclear stains.

The dark bands contain more DNA and less RNA and interbands contain more RNA and less DNA. The bands of on chromosomes become enlarged at certain times to form swellings called puffs.

The formation of puffs in case of polytene chomosomes is called puffing.

In the regions of puffs, the chromonemata get uncoil and open out to form many loops. The puffing is caused by the uncoiling of individual chromomeres in a band.

The puffs indicate the site of active genes where mRNA synthesis takes place. The chromonemata of puffs give out a series of many loops laterally.

The chromonematal loops appear as rings, they are called Balbiani rings

Biological significance

The banding patterns in polytene chromosomes were originally used to help map chromosomes, identify small chromosome mutations, and in taxonomic identification.

They are now used to study the function of genes in transcription.

Nucleus

A nucleus is defined as a double-membraned eukaryotic cell organelle that contains the genetic material.

Structure Of Nucleus



 Typically, it is the most evident organelle in the cell.



 The nucleus is completely bound by membranes.



 It is engirdled by a structure referred to as the nuclear envelope.



 The membrane distinguishes the cytoplasm from the contents of the nucleus



 The cell’s chromosomes are also confined within it.



 DNA is present in the Chromosomes, and they provide the genetic information required for the creation of different cell components in addition to the reproduction of life.

Nucleus Function



 It contains the cell’s hereditary information and controls the cell’s growth and reproduction.



 The nucleus has been clearly explained as a membrane-bound structure that comprises the genetic material of a cell.



 It is not just a storage compartment for DNA, but also happens to be the home of some important cellular processes.



 First and foremost, it is possible to duplicate one’s DNA in the nucleus. This process has been named DNA Replication and produces an identical copy of the DNA.



 Producing two identical copies of the body or host is the first step in cell division, where every new cell will get its own set of instructions.

DNA Structure

The DNA structure can be thought of as a twisted ladder. This structure is described as a double-helix, as illustrated in the figure above. It is a nucleic acid, and all nucleic acids are made up of nucleotides. The DNA molecule is composed of units called nucleotides, and each nucleotide is composed of three different components such as sugar, phosphate groups and nitrogen bases.

The basic building blocks of DNA are nucleotides, which are composed of a sugar group, a phosphate group, and a nitrogen base. The sugar and phosphate groups link the nucleotides together to form each strand of DNA. Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) are four types of nitrogen bases.

These 4 Nitrogenous bases pair together in the following way: A with T, and C with G. These base pairs are essential for the DNA’s double helix structure, which resembles a twisted ladder. The order of the nitrogenous bases determines the genetic code or the DNA’s instructions.

Components of DNA Structure

Among the three components of DNA structure, sugar is the one which forms the backbone of the DNA molecule. It is also called deoxyribose. The nitrogenous bases of the opposite strands form hydrogen bonds, forming a ladder-like structure.

DNA Structure Backbone

The DNA molecule consists of 4 nitrogen bases, namely adenine (A), thymine (T), cytosine (C) and Guanine (G), which ultimately form the structure of a nucleotide. The A and G are purines, and the C and T are pyrimidines.

The two strands of DNA run in opposite directions. These strands are held together by the hydrogen bond that is present between the two complementary bases. The strands are helically twisted, where each strand forms a right-handed coil, and ten nucleotides make up a single turn.

The pitch of each helix is 3.4 nm. Hence, the distance between two consecutive base pairs (i.e., hydrogen-bonded bases of the opposite strands) is 0.34 nm.

tRNA Structure

The tRNAs are generally 76-90 nucleotides long. Each amino acid has specific tRNAs. Stop codons are not recognised by any tRNAs. The secondary structure of tRNA looks like a clover leaf and tertiary structure is like an inverted ‘L’ shape. The folded structure is formed due to hydrogen bonding between complementary bases.

Clover leaf structure of tRNA

The secondary folded structure of tRNA has three hairpin loops, which give it an appearance of three-leafed clover. The main constituents of tRNA are:

Acceptor arm

It is formed by the base pairing of 7-9 nucleotides of 5’ terminal and 3’ terminal. The 5’ terminal has a phosphate group and the 3’ ends with a specific sequence of CCA or CCA tail. The amino acid attaches to the 3’ hydroxyl group of the acceptor arm.

The aminoacylation of tRNA or charging of tRNA is the first step of the translation process. The enzyme aminoacyl tRNA synthetases catalyse the reaction.

DHU Loop

D arm has a stem of 3-4 base pairs and it ends in a loop called D loop as it generally contains dihydrouridine, a modified nucleotide.

Anticodon Loop

It has a 5 base pair long stem. It has an anticodon loop, which contains the complementary codon (3 nucleotides sequence) present on mRNA for the amino acid it carries. These unpaired bases of anticodon loop pair with the mRNA codon. Each codon is identified by a specific tRNA.

TΨC Loop

The T arm consists of a stem of 4-5 bp and a loop containing pseudouridine, modified uridine.

Variable Loop

It is present between the TΨC loop and the anticodon loop. Its size varies from 3-21 bases. It helps in the recognition of the tRNA molecule.

tRNA Function



 tRNA plays an important role in protein synthesis.



 It acts as an adapter molecule for linking amino acids to its specific codon present in mRNA.



 Aminoacylation of tRNA is the first step in protein synthesis.



 tRNA is specific to each amino acid and carries them during the translation process in the ribosomal subunits.



 The tRNA transfers the amino acid to the growing polypeptide chain in the ribosomes, which has three binding sites for tRNA, namely A, P and E, which correspond to aminoacyl, peptidyl and exit, respectively.



 This decoding of codons of mRNA by specific tRNAs continues until the entire sequence for a polypeptide chain is translated.