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(1) Simple microscope : It is also known as magnifying glass and consists of a convergent lens. Leeuwenhoek (1683) designed a primitive microscope and discovered cells with it. It was the first tool ever used to observe biological objects. Its magnification power was 14 – 42 times only, so it is considered as simple microscope. (2) Compound microscope or Light microscope : The first compound microscope was assembled by Zacharias Janssen and J. Janssen, the Dutch spectacles makes in 1590. The compound microscope was prepared by Kepler and Galileo in 1611. However, it was not used for laboratory study. It is simplest, widely used microscope having three lens i.e., condensor, which collects the light rays and precisely focuses them on the objects; objective lens, which magnifies the image by three objective lenses, i.e., low power (10x), high power (45x) and oil immersion lenses. In a compound microscope an object can be magnified upto 1000 times and the magnification is independent of intensity of light, size of microscope and numerical aperture. The light microscope is also called bright field microscope because it forms the image when light is transmitted through the object. (3) Fluorescent microscope : It was developed by Coons (1945). It is observed that when ultraviolet light is irradiated on certain chemical substances, they absorb it and emit visible light. These chemical substances are called fluoro-chromes. The fluorescent substances e.g., quinine sulphate, rhodamine and auramine are used to stain the cellular objects and these objects are easily visible as fluorescent areas when illuminated with ultraviolet light. (4) Polarizing microscope : It was invented by Tolbart. In this microscope the plane polarised light is used as a source of illumination. Unlike the ordinary light, plane polarised light vibrates only in one direction and the cellular objects are easily visible as they appear bright against the dark ground. Polarizing microscope is helpful in studying the spindle fibres in the cells. (5) Ultraviolet microscope : It was invented by Caspersson. In this microscope the source of illumination is ultraviolet radiations having shorter wavelengths \[(1500\text{ }{AA}\text{ }\text{ }3500\text{ }{AA})\] as compared to ordinary visible light. In this microscope, the lenses are made of fluoride, lithium fluoride or quartz instead of glass. Ultraviolet microscope is helpful in quantitative determination of all those cell components which absorb ultraviolet rays. (6) Phase contrast microscope (i) Discovered by Dutch man Fredericke Zernicke (1935). (ii) Source of illumination is visible light. (iii) It is used to study living cells and tissues without staining and effect of chemical and physical agents on the living cells. (iv) It is also used to study spindle formation, pinocytosis, karyokinesis, cytokinesis etc. (v) It is draw back is low magnification power so subcellular organelles smaller than \[0.2\,\mu ,\] like ribosomes, lysosomes, ER, cannot be visualised. (7) Interference microscope (Morten et.al.) (i) It’s principle is similar to that of the phase contrast microscope and gives / studies quantitative data. (ii) Nomarski interference contrast microscope is useful to study mitosis /cell components in living state. (iii) more...

They are unstable isotopes which function like normal elements but emit positive or negative particles, e.g., \[^{3}H\](Tritium), \[^{14}C\](Carbon), \[^{32}P\](Phosphorus), \[^{35}S\](Sulphur), \[^{42}K\](Potassium), \[^{131}I\](Iodine). Radioactivity is recorded in different parts by Geiger counter or scintillation counter or autoradiography to know regions of use and transport. The tracers have been used for knowing pathway of mineral transport (Stout and Hoagland, 1939), organic solute transport (Vernon and Aronoff, 1952), carbon assimilation (Calvin, 1955). Where radioactive elements are not available, heavy isotopes are used, e.g., \[^{15}N{{,}^{18}}O.\] Their fate is recorded by mass spectroscopy and density gradient centrifugation. Meselson and Stahl (1958) studied DNA replication and Ruben et al (1941) evolution of oxygen (photolysis of water) in photosynthesis by using heavy isotopes.

Some of the biological molecules undergo changes after their synthesis. We can cite here the case of RNA. The transcription of hnRNA from DNA ultimately leads to the formation of m-RNA. These changes can be studied through pulse-labelling technique.

Microscopy (Gk. Micros = small ; skopein = to see) It is practice of using microscopes for the study of finer details of small objects including cells and tissues. Microscope are instruments consisting of lenses (made of glass / Lithium fluoride / electromagnetic lens) which magnify and resolve small objects not visible to unaided eye for the study of their details. The term microscope was coined by Faber in 1625. Magnification : Is the power of enlargement, which is the ratio of \[\text{Magnification}=\frac{\text{Size of the image with}\,\text{the instrument}}{\text{Size of the image with unaided eye}}\] Magnification of a microscope is roughly equal to the multiple of magnifying power of objective lens and ocular lens (eye piece) e.g., if the magnification power of an ocular lens is \[10\,X\] and of the objective is \[40\,X,\] then the total magnifying power of a microscope is \[10\times 40=400\,X\] (the magnification power of a microscope is represented by the symbol 'X'). Resolving power : It is the ability of a system to distinguish two close objects as two distinct objects. Its values is calculated by Abbe equation - \[{{L}_{m}}=\frac{0.61\lambda }{NA}\] Here, \[\lambda -\] is wavelength of used light, \[NA-\] Numerical Aperture, \[(NA=n\sin \theta )\] Numerical aperture is multiple of refractive index of medium (n) and \[\sin \theta \], which is sine of angle substended by optical axis and outer ray covered by objective. The value for best objective \[\sin e\,70{}^\circ =0.94.\] The resolving power of human eye is 100mm or microns (0.1 mm). This means that two points less than 100mm apart appear as one point to our eyes. Father of microscopy is Leeuwenhoek. He built first 270 X magnification microscope in 1672.

Mitochondria (Gk. Mito = thread ; chondrion = granule) are semi autonomous having hollow sac like structures present in all eukaryotes except mature RBCs of mammals and sieve tubes of phloem. Mesosomes of prokaryotes (bacteria) is analogous to mitochondrion in eukaryotes. Mitochondria are also called chondriosome, chondrioplast, plasmosomes, plastosomes and plastochondriane. Discoveries (1) These were first observed in striated muscles (Voluntary) of insects as granules by Kolliker (1880), he called them “sarcosomes”. (2) Flemming (1882) called them “fila” for thread like structure. (3) Altman (1890) called them “bioplast”. (4) C. Benda (1897) gave the term mitochondria. (5) F. Meves (1904) observed mitochondria in plant (Nymphaea). (6) Michaelis (1898) demonstrated that mitochondria play a significant role in respiration. (7) Bensley and Hoerr (1934) isolated mitochondria from liver cells. (8) Seekevitz called them “Power house of the cell”. (9) Nass and Afzelius (1965) observed first DNA in mitochondria. Number of mitochondria : Presence of mitochondria depends upon the metabolic activity of the cell. Higher is the metabolic activity, higher is the number e.g., in germinating seeds. (1) Minimum number of mitochondria is one in Microasterias, Trypanosoma, Chlorella, Chlamydomonas (green alga) and Micromonas. Maximum numbers are found (up to 500000) in flight muscle cell, (up to 50000) in giant Amoeba called Chaos – Chaos. These are 25 in human sperm, 300 – 400 in kidney cells and 1000 – 1600 in liver cells. (2) Mitochondria of a cell are collectively called chondriome. Size of mitochondria : Average size is \[0.51.00\,\,\mu \,m\] and length up to \[110\,\,\mu \,m.\] Smallest sized mitochondria in yeast cells \[(1\,\mu \,{{m}^{3}}).\] and largest sized are found in oocytes of Rana pipiens and are \[2040\,\,\mu \,m.\] Ultrastructure : Mitochondrion is bounded by two unit membranes separated by perimitochondrial space (6 – 10nm wide). The outer membrane is specially permeable because of presence of integral proteins called porins. The inner membrane is selective permeable. The inner membrane is folded or convoluted to form mitochondrial crests. In animals these are called cristae and in plants these folding are called tubuli or microvili. The matrix facing face is called ‘M’ face and face towards perimitochondrial space is called ‘C’ face. The ‘M’ face have some small stalked particles called oxysomes or \[{{F}_{1}}\] particle or elementory particle or Fernandez – Moran Particles (\[{{10}^{4}}{{10}^{5}}\] per mitochondria). Each particle is made up of base, stalk and head and is about 10nm in length. Oxysomes have ATPase enzyme molecule (Packer, 1967) and therefore, responsible for ATP synthesis. These elementary particles are also called \[{{F}_{0}}\text{ }{{F}_{1}}\] particles. The \[{{F}_{1}}\]  particle is made up of five types of subunits namely \[\alpha ,\,\beta ,\,\gamma ,\,\delta \] and \[\varepsilon .\] of these \[\alpha \] is heaviest and \[\varepsilon \] is lightest. \[{{F}_{0}}\] particles synthesize all the enzymes required to operate Kreb’s cycle.       Semi-autonomous nature of mitochondrion : Mitochondria contain all requirements of protein synthesis : (1) 70 S ribosomes. (2) DNA molecules more...

The concentration of various ions in different parts is now studies by using a glass microelectrode. It has silver wire dipped in KCl solution. This technique is used for studying the movement of ions through ions channels. The ions channels are intrinsic membrane proteins. For studying this passive transport of ions through ion channels Neher and Sakman developed a Patch clamp technique for which they are awarded Nobel prize in 1991.

It is an another technique of separation. In which patricles of different charges and sizes are separated under the influence of electric field. e.g., nucleic acids, proteins, amino acids, nucleotides can be separated by this method. The technique was discovered by Russian physicist Alexender Reuss in 1807. In immunoelectrophoresis antibodies coupled with radioisotopes, specific enzymes or fluorescent dyes are used in detection of particular proteins. The technique is highly sensitive. It can separate molecules in picogram and nanogram quantities and distinguish proteins which differ from each other in only one amino acid.

A number of dyes or stains are known to colour specific parts. Certain dyes can be used even in case of living materials. They are called vital stains, e.g., neutral red, methylene blue. Fuelgen or Schiff?s reaction was developed by Fuelgen and Rossenbeck (1924). Identification and localization of chemical compounds  of a cell is studies in cytochemistry. Some important cytochemical stains
Stain Used for staining Final colour
Acetocarmine Chromosomes Pink
Acid fuchsin Cortex, cellular walls, mitochondria Magenta
Aniline blue Fungal hyphae Blue
Basic fuchsin Nucleus Magenta red
Crystal violet more...
Discovered by Michael Tswett (1906). This technique is used to separate the molecules of different substances present together. Mixture of molecules is run over an adsorption medium. Chromatography may be following types. Adsorption or Column chromatography : The stationary phase consists of a column of charcoal, silica, alumina, calcium carbonate or magnesium oxide. The solution is made to percolate through this column when different chemicals get absorbed at various levels. The technique is useful for separation of tissue lipids. Thin layer chromatography : The stationary phase consists of a thin plate of cellulose powder or alumina. As a few drops of mixture are poured over it, the different chemicals spread to different distances. The method is useful in separation of amino acids, nucleotides and other low molecular weight products. Paper chromatography : A paste of mixture is applied near one end of a chromatographic paper (or Whatman 1). The lower end below the paste is dipped in a solvent. As the solvent rises in chromatographic paper, the different chemicals of the mixture spread to different distances. The paper can be rotated to obtain two dimensional chromatogram. Types : (a) Ascending (b) Descending (c) 2-D chromatography. Ion exchange chromatography : Beads of cellulose and other materials having negative and positive charges are placed in a column. The mixture (mobile phase) is poured over the column. As the mixture passes through the column, its constituents separate according to their charges. The technique is used in purification of insulin, plasma fractionation and separation of proteins. Gel fractionation / Gel filtration chromatography (Molecular sieve chromatography) : The stationary phase consists of gel forming hydrophilic beads which contain pores, e.g., sephadex (cross-linked dextran). As the mixture is poured over the gel, larger molecules pass out unimpeded while small molecules are trapped in the pores. The technique is used in separation of proteins. It is also employed in determining their molecular weight by calibrating the column with proteins of known molecular weight. Affinity chromatography : Satationary phase consists of column of ligands (molecules that bind to other specific molecules at particular sites). Mixture is allowed to pass through the column. Chemical linkages are established between ligands and their specific chemicals. Others pass out of the column. The technique is used in separation of enzymes, immunoglobulins, mRNA, etc.

In isotonic medium cells components are separated, it is two step process. Homogenisation : Cell products are separated  in isotonic medium (0.25 M sucrose solution) either with the help of homogeniser of ultrasonic vibrations kept at 0 – 4°C. A homogenised cell is called homogenate. Differential centrifugation : Homogenisation product is rotated (centrifuged) at different speeds. The sediment or pellete of each speed is collected. e.g., nuclei at \[1000\times g\](g= force of gravity) for 10 minutes, chloroplast and mitochondria at \[10,000\times g\] for 15 minutes. The particle settle according to their sedimentation ratios. Sedimentation coefficient is expressed in svedberg unit ‘S’ related with molecular weight of the particles. For the detail study of mitochondria it is the best technique. 'S' is measured by analytical centrifugation. The various cell organelles and macromolecules sediment in the following order. \[Nucleus\to Chloroplast\to Mitochondria\to Ribosome\to DNA\to mRNA\to tRNA\]


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