Chapter 1 - CELL STRUCTURE AND FUNCTIONS

Cell

A cell is the smallest unit of living matter. According to German biologist Rudolf Virchow pronunciation: Firkoh) "every cell comes from a pre-existing cell".

Properties:

1. A cell is also the functional unit of the organism.
2. Cells can take in nutrients, break them down to release energy, and get rid of wastes.
3. They can reproduce, react to stimuli, and maintain internal environment different from their surroundings.

Note: Organelle is a specialized subunit within a cell that has a specific function.

Cell Theory

By the middle of the nineteenth century, biologists clearly recognized that all living things are composed of cells. This is known as cell theory. The cell theory is one of the unifying concepts of biology.

TECHNIQUES USED IN CELL BIOLOGY

To study the structure and functions of cell and its organelles following techniques are used:

1. Cell fractionation
2. Centrifugation
3. Differential staining
4. Microdissection
5. Tissue culture
6. Chromatography
7. Electrophoresis
8. Spectrophotometry

Cell Fractionation

It is a common approach for studying functions of a cell. A particular cell organelle is isolated from other cell components and allowed to perform its normal functions in a test tube.

Generally cells are broken apart as gently as possible. A common procedure is to grind up i.e. to homogenize cells in a suitable medium (with correct pH, ionic composition and temperature). This is done with a homogenizer (food mixer). The mixture is then centrifuged.

Centrifugation 

It is the process to separate substances on the basis of their densities. It is done by the machine called centrifuge (see Figure). Contents are kept in tubes that are much like the test tubes. This machine can spin the tubes. Spinning the tubes exerts a centrifugal force on the contents. As the number of revolutions per minute increase so does the centrifugal force increase.

A Centrifuge 

Differential Staining

Most biological structures are transparent. Differential Staining is the most common method used to differentiate between different structures. Certain stains when used in low concentrations are non-toxic to

living tissues and can therefore be used on living material. These are called vital stains e.g. methylene blue.

• When only one stain, such as borax carmine is used it is called single staining. 
• When two stains, one that will stain nucleus e.g. haematoxylin and other that will stain cytoplasm e.g. eosin are used, the process is called double staining or differential staining.

Microdissections

When dissection is made under microscope, it is called microdissection. It is done to remove tumour or granules from delicate tissue or cells like, brain, heart and nerve cells. These days the image is seen on large TV screen or monitor while dissecting.

Tissue Culture

Cell cultures are a suspension of cells in a liquid medium. Tissue cultures are small pieces of plant or animal organ grown in liquid or on solid medium. 

In plant tissue the root and shoot tips are taken and cultured in a suitable medium to see any infection. Phloem tissue of plants is removed and placed in a germ free medium containing adequate food supply, mineral salts and growth substances. The cell will develop into a new plant, which will flower and produce seeds.

Animal tissue culture is usually set up by growing individual cells to form a single layer of cells over the surface of a glass container. Tissue cultured cells are used to see any abnormality in the cell e.g. cancer etc.

Chromatography

It is a procedure through which various substances in a mixture are separated from each other and identified. Separation involves the use of two phases, one of which is stationary and the other is mobile. Separation depends upon the differential movement of the test substances between two phases. Paper chromatography is a simple and most widely used technique.

Chromatography Chamber

1. Column Chromatography
2. High performance liquid Chromatography (HPLC)
3. Gas Chromatography (GC)
4. Ion-exchange Chromatography
5. Size exclusion Chromatography
6. Thin layer Chromatography (TLC)
7. High performance thin layer Chromatography(HTLC)
8. Paper Chromatography
9. Affinity Chromatography

Electrophoresis

It is a laboratory procedure that separates molecules according to their size, shape, molecular weight and surface charge whether (+) or (-). Macromolecules such as nucleic acids or proteins can be separated in a

mixture. Often the gel is sandwiched between glass or plastic plates to form a viscous slab (see Figure). The two ends of the slabs are suspended in two salt solutions that are connected by electrodes to a power source. When voltage is applied to the apparatus, the molecules present in the gel migrate through the electric field according to their individual charge and they move away from one another in the gel. The negative charged molecule will move towards the positive pole and the molecule having positive charge will move towards the negative pole. Later on the molecules can be pin pointed by staining the gel.

Gel Electrophoresis

Spectrophotometry

A spectrophotometer (see Figure) is an instrument that measures the amount of light that passes through the sample and from this it can be calculated how much light was absorbed. The amount of light absorbed at

each wavelength is plotted in a graph and the result is what we call the absorption spectrum. The procedure is called spectrophotometry.

• It can be used to determine the wavelengths of light that take part in photosynthesis. 
• It is also used to know the turbidity or cloudiness. The more cells e.g. microorganisms are in suspension the greater will be turbidity.

spectrophotometer 

Resolution and Magnification in Microscopy

Most animal cells and plant cells are between 10 µm and 30 µm. The unit of microscopic measurement is micrometer (preferred to micron). The correct symbol is µm i.e. the µm is the abbreviation for micrometer (1 µm = 1/1000 mm).

Resolution is the minimum distance that two points can be separated and be distinguished as two separate points by an optical instrument.

One way to increase resolution, is to increase magnification to make small object seen larger. The increase in the apparent size of an object is called magnification. 

Graticule 

Graticule is a photographically produced gird, cemented between two glasses. In order to use the graticule for measurement it must be calibrated so that we know what each square represents when a particular object

is used. 

Micrometre

Measurement of microscopic objects is called micrometry. This can be done using specially designed scales or micrometer. There are two types of micrometers: Ocular graticule or micrometer and stage micrometer.

Ocular Micrometer

Ocular micrometer is also known as eyepiece micrometer (see Figure). It is a circular glass piece. It can be

put between the two lenses of the eyepiece. 

As this micrometer is put between the two lenses of the ocular so, it is called ocular micrometer. The micrometer acts as ruler and its scale is used for direct measurement of the object.

Stage Micrometer

As this scale is placed on the stage of the microscope, so it is called stage micrometer.