eChapters

Principle

Monomeric acrylamide (which is neurotoxic) is polymerised in the presence of free radicals to form polyacrylamide. The free radicles are provided by ammonium persulphate and stabilised by TEMED (N’N’N’N’-tetramethylethylene-diamine). The chains of polyacrylamide are cross-linked by the addition of methylenebisacrylamide to form a gel whose porosity is determined by the length of chains and the degree of crosslinking. The chain length is proportional to the acrylamide concentration: usually between 3.5 and 20%. Cross-linking bisacrylamide is usually added at a ratio of 2g BIS 38g acrylamide.

Principle

RNA is negatively charged at neutral pH and when an electric field is applied, it migrates towards the anode. RNA retains much of its secondary structure during electrophoresis unless it is first denatured. The addition of formaldehyde to the agarose gel maintains the RNA in its linear (denatured) form. The following protocol is based on Formaldehyde Agarose Gel Method.

Principle

To retrieve and purify any specific DNA fragment from an agarose gel slice; the expected yield is from 50-75% of the amount in the gel slice.

Time required

Restriction digest - minimum of 2 hours

Electrophoresis - approx. 4 hours

Elution - 2-5 hours depending on fragment size

DNA Purification -2 hours

here are many methods for extracting DNA from gel apart from using commercial kits, some methods however yield cleaner DNA than others.

1. Freeze squeeze

Excise band and place pieces of it in a home-made spin-column (a piece of filter placed in a PCR 0.5ml eppendorf, pierced the bottom of the PCR tube with a needle). Place the PCR tube at -20 °C for 15-20 mins or freeze in liquid N2. Then place the PCR tube into the 1.5 ml eppendorf and spin at top speed in a microfuge for 15 mins, collect DNA solution which can be EtOH ppt or use directly.

2. Powdered silica

Melt excised bands at 70°C cool and add equal volume of TE-buffered phenol and mix. Spin for 5 min, remove aqueous phase, repeat and EtOh ppt. the DNA.

3. Electroelution

Agarose gel electrophoresis is the easiest and commonest way of separating, capable of resolving mixtures of DNA fragment that cannot be separated conveniently by other procedures. The purpose of the gel might be to look at the DNA, to quantify it or to isolate a particular band. The DNA is visualised in the gel by addition of ethidium bromide. This binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light.

Agarose, which is extracted from seaweed, is a linear polymer whose basic structure is shown in figure.

Principle

The principle of Electrophoresis involves the motion of a particle in a fluid medium, where the gravitational attraction (on a flowing liquid) is the sedimenting force, and the frictional force is the opposing force and is proportional to the velocity of the particle. These forces balance each other acting in opposite directions and hence balance each other. This will lead to the uniform motion of the particle in the mobile liquid medium (i.e. the particle moves at constant velocity). In case the external field is an electric field instead of a gravitational field, a macromolecule will respond to the external field in two ways. In case the molecule is charged, it will migrate in an electric field to the electrode of opposite charge. Under this principle the technique of electrophoresis functions.

All electrophoresis operations are governed by the principle:

Principle

Cloning experiments would be very difficult if we do not screen those very few cells that took up the recombinant plasmid DNA, as the transformation efficiency is very low as stated before (usually less than 1 in 10⁵ cells). Hence it is necessary that there should be a selection mechanism (i.e. conditions where cells grow if they express a gene, but die if they do not) to sort out which are transformed cells and which are not make the job easy.

A commonly used selection mechanism is antibiotic resistance. If the E. coli strain used is not resistant to the antibiotic, but the plasmid DNA contains a gene coding for resistance, then only the cells that have taken up the plasmid will be able to grow. Most commonly used plasmids contain a gene for resistance to ampicillin; only cells that have taken up the plasmid will be able to grow and form colonies.

Note: Uptake of plasmid DNA is rare; hence each group of cells visible on a plate represents the offspring of a single cell that will contain a single molecule of plasmid DNA.

Transformation

The uptake of DNA by competent cells is called transformation. Transformation involves:

Mix a small volume of DNA (usually 1-3 µl) with 100 µl of competent cells.

The mixture is incubated on ice for 30 minutes to allow the cells to take up the DNA.

The cells are then heat-shocked at 37 to 42°C for 1 minute, and cold shocked on ice for 2 minutes.

The heat shock/cold shock reverses the effect of the competent cell process, by healing the damage inflicted by the competent cell solution.

Add 0.5 ml of LB culture mediumz

Incubate the cells for 10 - 30 minutes at 37°C to aid the healing process.

This cell/medium mixture is then spread onto bacterial culture plates containing agar, LB, and a selection mechanism.

Steps for Cloning

Prepare DNA.

Select restriction enzymes for your insert and vector, and determine the appropriate reaction buffers.

Combine the following in a microfuge tube (30 µL total volume):
2 µg DNA
1 µL Each Restriction Enzyme
3 µL 10x Buffer
3 µL 10x BSA
x µL H₂O (to bring total volume to 30 µL)

Principle

Plasmids requires hosts to replicate, hence it has to be inserted into bacteria. But efficiency of uptake of plasmid DNA by bacteria is low and will not do so without getting degraded. Therefore, to enhance the probability of uptake of the plasmid DNA by bacteria it is necessary to make the bacterial cells “competent” to absorb the plasmid DNA. One of the methods to increase efficiency of cell competence is given below. It should be noted that competent cells are more fragile than normal bacteria. Vortexing the cells, heating the cells above 42ºC or to 42°C for prolonged periods, or exposure of the cells to abusive treatments may kill them

Principle

The rate of joining of DNA fragments is affected by seven parameters:

DNA concentration,

Concentration of compatible ends,

Size of the fragments,

Presence or absence of complementary, single-stranded ends (some nucleases form blunt ends and in these cases special techniques are needed for joining),

G + C content and length of the complementary single strands,

Temperature of the reaction and

Ionic composition of the solution.

Start with a 1:2 molar ratio of vector: insert DNA when cloning a fragment into a plasmid vector. This ratio will vary with other types of vectors, for example, cDNA and genomic cloning vectors. The following example illustrates the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment.

Principle of DNA Ligase Mechanism

The process of connecting two pieces of DNA together is called ligation, and is catalyzed by an enzyme called ligase. The ligase used most often in molecular biology is derived from the T4 bacteriophage, and uses ATP to supply the energy necessary for the reaction. In addition to ATP, T4 ligase requires DNA with a 5´-phosphate group and free 3´-hydroxyl groups.

Applications

Ligation of blunt or cohesive-ended DNA fragments.

Repair of nicks in double-stranded nucleic acids.

Reagents

Restriction enzymes of choice, such as BamH1 and EcoRI

Restriction enzyme reaction buffer,

70 % Ethanol

100 % Ethanol

3 M Sodium acetate (pH 5.2)

Distilled water

Plasmid DNA isolated from bacteria, such as pGFP(R) and pBC(R)

Reagents

Nuclease-Free Water: 14µl

10X Restriction Buffer: 2µl

Acetylated BSA (1mg/ml): 2µl

DNA (~1µg): 1µl

Restriction Enzyme (10u): 1µl

Final Volume: 20µl

Principle

Since the entire DNA sequence of the bacteriophage is known, we know exactly where a restriction enzyme will cleave the viral DNA and the size of the DNA fragments generated. This restriction enzyme digest will create a pattern of specific DNA fragments that will serves as a “DNA ladder”.

Principle

Restriction digestion is one of the most common reactions performed in molecular biology. Restriction endonucleases (RE) are also known as restriction enzymes, which recognize short, specific DNA sequences. There are three types of RE: Type-I, Type-II and Type-III. Type II RE, most commonly used for DNA analysis and genetic engineering, cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. All Type II RE recognises a unique nucleotide sequence at which it cuts a DNA molecule and nowhere else. The recognition sequence is often a six base pair palindromic sequence (the top DNA strand from 5' to 3' is the same as the bottom DNA strand from 5' to 3'), but others recognize four or even eight base pair sequences. Many RE will not cut DNA that is methylated on one or both strands of the recognition site, although some require substrate methylation.

REs can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a flush or “blunt end”. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called “cohesive ends”, as these single-stranded overhangs could potentially come together again through complementary base-pairing, as shown below:

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Is amyl alcohol

Phenol

Proteinase K

RNase A

Sodium acetate, pH 5.2

Sodium chloride, 5 M
Sodium thiocyanate (NaSCN), 1 M

TE Buffer (Tris-EDTA), pH 7.4

Tween 20

Xylene

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Isoamyl alcohol

Phenol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

RNAse A

Sodium dodecyl sulfate (SDS) solution, 10%

Reagents

EDTA

Ethanol, absolute

Isopropanol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

Sodium Acetate, 3 M pH 5.2 (Molecular Biology Grade)

Sodium chloride (NaCl)

Sodium Dodecyl Sulfate (SDS ), 10%

Tris EDTA (TE), pH 7.4

Tris EDTA (TE), pH 8.0

Tris HCl, pH 8.0

Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, and decant supernatant.

Resuspend cells in 400 µL TE by vortexing, add 50 µL 10% SDS, 50 µL proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37^oC.

Shear DNA by 3-5 passages through a 26 G needle.
Extract twice with 500 µL phenol: chloroform (1:1), and twice with 500 µL chloroform. 
Precipitate nucleic acids by adding 25 µL 5 M NaCl and 1 mL 95% EtOH, vortex, and centrifuge for 10 min, decant supernatant. Dry pellet.

Preparation of Solutions

GTE buffer

50 mM Glucose,

25 mM Tris-Cl,

10 mM EDTA,

pH 8

NaOH/SDS lysis solution

0.2 M NaOH,

1% SDS

Solution Preparation

1. Glycerol mix (1 L)

Weigh 25 grams (liquid weight) of glycerol and

Add dH₂O to 1 Liter (Autoclave).

2. Potassium mix (1 L)

170 mL of 1M KH₂PO₄ (monobasic),

720 mL of 1M K₂HPO₄ (dibasic), and

110 mL of dH₂O (Autoclave)

Principle

The principle is to extract plasmid DNA from bacterial cell suspensions and is based on the alkaline lysis procedure developed by Birnboim and Doly (Nucleic Acids Research 7:1513, 1979). It is a known fact that plasmids are much smaller super coiled DNA molecules than the much larger and less super coiled bacterial chromosomal DNA. This difference in topology enables selective precipitation of the chromosomal DNA and cellular proteins and separates them from plasmids and RNA molecules. The cells are lysed under alkaline conditions, which denature both nucleic acids and proteins, and when the solution is neutralized by the addition of Potassium Acetate, chromosomal DNA and proteins precipitate because it is impossible for them to renature correctly (they are so large). Plasmids renature correctly and stay in solution, effectively separating them from chromosomal DNA and proteins.

Isolation of plasmid DNA from E. coli is a common routine in research laboratories. One can perform a widely-practiced procedure that involves alkaline lysis of cells. This protocol often referred to as a plasmid “mini-prep,” yields fairly clean DNA quickly and easily.

Procedure

Fill a microcentrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge. Dump supernatant and drain tube briefly on paper towel.

Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel.

Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipet.

Principle

Apart from PCR for generating large amounts of DNA, the second method is to use bacteria to replicate the DNA and then purify the replicated DNA from the bacteria. Plasmid DNA is usually the DNA of interest for this method. A plasmid is a double stranded circular DNA molecule that will replicate in an organism. A typical plasmid contains four important features:

The plasmid must be circular, because bacteria generally will not replicate linear ? DNA.

The plasmid must contain a sequence that functions as an origin of replication (ori).

The plasmid must contain a selection mechanism that will force the bacteria to retain the DNA; the most common type of selection mechanism used in bacteria is a gene for resistance to an antibiotic such as ampicillin or tetracycline.

The plasmid must contain a region for the insertion of the experimental DNA. A generic plasmid exhibiting these features is shown at right.

An expression plasmid that allows expression of foreign DNA should have a strong promoter segment for transcription of inserted DNA.

Must have an effective ribosome binding site that will allow efficient translation of the transcribed RNA.

Must have high copy number, i.e., the host bacterium should have many copies (usually >100)

PCR Principles and Procedure

Polymerase chain reaction (PCR) is a technique that allows the generation of large amounts of a single DNA sequence from a mixture of sequences; the fragment generated can be designed to contain specific starting and ending positions based on the needs of the experiment. PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. These components include:

Cell Genotypes

A large variety of bacterial strains, like E. coli, are frequently used in molecular biological experiments. Students should choose the strain or strains required for an experiment on the basis of genetic properties of the strains. It is thus pertinent that a sound knowledge of the genetic makeup of the E. coli stains is acquired.

The genetic makeup of the strains of E.coli is distinctly different from the wild type (K12 or B strains) strains from which they are derived and contains genes that can be modified. Genes are supposed to be mutated when they are not listed in a genotype. In most cases, the genes listed are mutated to the point of inactivity. In a few cases, a “-” (indicating inactive or absent or deleted), “+” (indicating active). , The genotype is a factor in choosing a bacterial strain. Chosen strains should be ascertained for their utility for the specific experiment.

Small Scale Cultures

Fresh culture from a freshly streaked plate should be taken while experimenting with E. coli cells. For preparing a small scale culture of E. coli , prepare 3-5 ml of LB medium/broth (or appropriate broth include antibiotic if the culture contains a plasmid) in two sterile 50 ml tubes. Inoculate one tube with a single colony from a fresh plate. The second tube is used as a broth control. Incubate both tubes at 37°C, shaking vigorously overnight. Inspect the tubes the next morning. The broth control should be clear and the inoculated culture should be very turbid. Make a note of any debris found in the tubes and only incubate longer if the culture is not dense. Never allow cells to overgrow. For some applications, cells can be stored at 4°C for short periods prior to use.

Storage

Nucleic acids are very sensitive to contaminations hence care should be taken while processing and storing DNA and RNA. The following properties of reagents and conditions need to be taken care of:

Heavy metals cause breakage of phosphodiester linkage. One can use EDTA which is an excellent heavy metal chelator.

UV rays cause extensive damage to nucleic acids. UV light at 260 nm causes breakage of thymine dimers and cross-link, including loss of biological activities.

Ethidium bromide results in photo oxidation of DNA. Oxidation products can cause phosphodiester breakage.

Nucleases are found on human skin; therefore, it is advisable to avoid direct or indirect contact between nucleic acids and fingers.

Most DNAes are not very stable; however, many RNAses are very stable and can absorb to glass or plastic and remain active.

At -20°C: extensive single and double strand breaks results. Hence for long-term storage it should be stores at -70°C. ? It is best to store DNA in high salt ( >1M) in the presence of high EDTA (>10mM) at pH 8.5, for long-term storage.

Storage of DNA in CsCl with ethidium bromide in the dark is an excellent storage medium. There is about one phosphodiester break per 200 kb of DNA per year.

Storage of ë DNA in the phage is better than storing the pure DNA.

Autoclave Operating Procedures

Place all material to be autoclaved in an autoclavable tray. All items should have indicator tape. Separate liquids and solids should be autoclaved separately. Ensure that the lids on all bottles are loose. Do not put a large number of items in tray, to ensure that all items reach the appropriate temperature. Also allow sufficient air/steam circulation.

Steps in autoclave

Pressure should read ‘O’ when you open the pressure chamber of the autoclave.

Place items to be autoclaved in the autoclave and close the door. Some autoclaves require that you also lock the door after it’s closed.

Set time - typically 20 minutes.

Temperature should be set at 121°C already, but double-check and change if necessary.

Set cycle: If liquid, set “liquid cycle” or “slow exhaust”. If dry, set “dry cycle” or “fast exhaust” + dry time.

Start the cycle. On some autoclaves, the cycle starts automatically at step 5. On others, turn to “sterilize”

Several substrates, like proteins (enzymes), are very sensitive to alteration in the concentration of the solution they are placed in. Hence is the importance of a buffer which controls the chemical properties of the medium. Literally a “buffer” has the ability to resist changes in the hydrogen ion concentration. Although, buffers do contain other molecules, that might, in addition to the hydrogen ion concentration, influence the ionic strength, the activity of enzymes, and other parameters of the experiment.

In any biological experiment, appropriate buffer must be selected based on their effect on the experiment. Ideal buffers control pH and ionic strength without interacting adversely with the experiment undertaken. For example, although phosphate is a common buffer, it may not be suited when phosphate is a substrate or product of the reaction being studied. Also, some proteins interact poorly with some buffers. For example, Tris is not ideal as it exhibit high pKa and the large change in pKa when temperature changes, although it is cheap and most proteins are stable in Tris buffers, moreover it seldom reacts with reaction substrates hence, Tris buffer is commonly used in biochemical experiments.

To prepare a series of dilutions of the original more concentrated stock solution, one need to consider both the required final concentration and required volume of the diluted material. The dilution can be readily calculated by the equation:

A spectrophotometer is an analytical instrument that measures the absorbance of a test sample (solution). The term “spectroscopy” comes from the word “spectrum”. “Spectroscopy” therefore implies the use of multiple wavelengths of light. Spectrophotometers have the ability to specifically measure absorbance at specific wavelengths. The most commonly used method to allow this involves a “monochromator”, a device (either a prism, or more commonly, a diffraction grating) that splits the incident light into its component wavelengths, and allows only light of the desired wavelength to reach the sample. The ability to measure absorbance at different wavelengths is very useful, because the extinction coefficient of a compound varies with wavelength. In addition, the absorbance spectrum of a compound can vary depending on the chemical composition of the compound, and depending on the nature of the solvent around the compound.

Absorbance is a useful quantity and follows the Beer-Lambert law. The extinction coefficient of a molecule at a given wavelength can be calculated using the Beer-Lambert equation from absorbance measurements for solutions of known concentration, which states that:

In molecular biology and biochemistry, the ability to accurately and reproducibly measure and transfer small volumes of liquids is critical for obtaining useful results. For volumes less than 1 ml, the most common method for measuring liquid volumes involves the use of a device known as a pipetman or micro-pipette.

The pipetmen can be both fixed and variable volumes. Commonly used pipetmen are: P1000, P200, P20, P10 etc. P1000 are useful for volumes from 200 to 1000 µl. P200 are useful for volumes from 20 to 200 µl. P20 are useful for volumes from 0.5 to 20 µl. For beginners fixed volume pipetmen are more useful. It is important to ascertain whether the correct pipetman is chosen for measuring the volume one needs.

This book is designed to introduce to the students some of the most widely used experimental procedures in biotechnology, including DNA/RNA isolation, manipulation, cloning, protein purification and characterization. The students will also get familiarized with some of the equipments frequently used in biochemistry and molecular biology.

The biotechnology laboratory course is an exploration of procedures. Hence, in order to get full benefit from the course, the students need to read the manual, which is self explanatory. The more effort they put into the course work, the more they will learn. Before the start of actual lab works, one needs to spend some time reading the Laboratory Manual. This reading will provide background information and an outline of the procedures to be performed.

The biotechnology laboratory is conducted as a “directed” research project. This means that although the general procedures are well established, the overall goal of each experiment is the acquisition of new information. Because of the nature of scientific research, predicting the outcome of experiments that have not previously been performed is difficult. It may therefore be necessary to design new experiments based on the results of previous ones, or to repeat experiments that yielded uninterruptable or ambiguous results.

Major Families

This chapeter deals with indigenous knowledge on use of various plant species in traditional medicine, food, dye, wood and in tanning etc.

Malvaceae

The family Malvaceae is also known as Mallow family, characterised by mainly shrubs and herbs. The plants of this family frequent have stellate trichomes, persistant sepals, epicalyx present, stamens numerous and monadelphous, anthers monothecous, carpels 5 or more.

Abroma angusta (Devil’s Cotton)

Paste of the leaf is applied to heal wounds and to relieve joint pain or back pain. Young shoots are used as food. Roots are used to cure itching.

Abutilon indicum (Kanghi)

The whole plant is used to make brooms. Plant also has medicinal value and its sap is used against ulcer, leprosy, piles, dysentery and fever.

India, the land of oldest civilization known through out the world for its vibrant cultures, faiths, lifestyle, traditions, rituals etc. which are interwoven with each other since time immemoral. Here people of different ethnic groups live together who speak different languages and dialects according to their religion and choice. Altogether, 68 million inhabitants belonging to 227 ethnic groups, comprising of 573 tribal communities exists in different part of the country. These people constitute 8.08% of the total population of India (1991 Census of India) and speaks <750 dialects that can be classified into one of the following three language families: Austro-Asiatic (AA), Dravidian (DR), and Tibeto-Burman (TB). When come to think of different tribal community’s inhabiting India, they can be easily divided into six major racial lineages i.e. - Negroid, Mongoloid, Proto-Australoid/ Austrics, Mediterranean or Dravidian, West Brachycephals and Nordic Aryans

Plants have played an important role in many mythologies and religions of the world and have been known for deep and sacred meanings throughout the ages. It is the symbolic representation of the old cultural traditions in the form of a tree across the world. Christmas tree in the Christianity and Bodhi tree in Buddhism are the example of tree featured in mythology. In most of the folklore, folktales and folk religion trees are often said to be homes of tree sprits. If history is traced, we will found that Druidism and Germanic paganism appear to have involved in religious conservational practices of sacred groves. Druidism is a nature based religion that was taught by Druids about 2000 years ago. The term druid itself possibly derives from the religeous word for oak. Therefore, the Druidism reconnects us with nature, our ancesters and ourselves by working with plants or trees.

Trees are honored in Indian culture for their different beneficial characters like medicinal properties, food, fodder, maintaining hydrological cycle, moderate the climate etc. There are some trees that have attained more than a special benevolence in Hindu mythology. In Indian mythology number of plants have been given sacred value and worshipped by different cultures. Some important plants of sacred value have been discussed here.

Before carry out any ethnobotanical work, first define the goal of the assignment or study before selecting the approach, which best suits the interests, budget and schedule. Most of the methods used in ethnobotanical studies are time taking and costly because several field trips are required in several seasons for covering different ritual occasions and seasonal farming activities which are continue for a number of years for completion of an assignment. In this way, an assignment may be very long or short as per selection approach of an ethnobotanist. Long-term assignment or projects may be continue for several years and they require sufficient financial input. When sufficient financial add and time does not available to accomplished long term assignments, in that case a Rapid Ethnobotanical Appraisal (REA) can be done.

Indigenous culture

Indigenous means originating where it is found. If knowledge originates from, and is bound to local experiences, taking its local world not perhaps as the only one, but as the most relevant of all they know may be called indigenous. Indigenous knowledge is human lifeexperience in a distinct natural and cultural amalgamation within a unique local and timely setting. It is an authentic appropriation of being, meaning that this very process happens exclusively in a given locality at a given time. In the field of medicine, Chinese culture (indigenous) developed Acupuncture technique. An acupunturist places needles into the skin to balance out the flow of chi which is believed to be the energy that flows through the human body. In theory, Chi runs along 14 pathways called meridains and when a patient is injured, stressed, or ill, their chi is supposedly being blocked. By placing needles in the Meridians the patients chi is thought to be put back into balance. Like Chinese culture, indigenous culture of different places/countries occurs and have their own belief and methods.

Indigenous cultures speak in various ways, for example we could speak from the perspective of our so-called ‘civilizations’ but how do we know that this perception is not contrary to the truth. We could speak on the basis of our traditional religious concepts but again these concepts may in reality be the opposite of what we believe. If we speak about the indigenous from the point of view of an anthropologist we arrive at a cold and empty language, a study that only pays attention to external and superficial matters, in the same way an anatomist analyses our inner organs or our bones but they are still a long way from knowing the reality. We believe that it is better to speak about indigenous cultures on the basis of their own reality, in relation to how they were as much as how they are now because in the course of time from yesterday to today many things have happened that are worth examining.

In the starting of twentieth century, the ethnobotany progressed from the untested compilation of knowledge to a bigger method and abstract reorientation. Nowadays, the ethnobotany practices need a spread of skills such as:

• Training on the identification and preservation of plant specimens
• Training on linguistic and sociology to understand local language and their family structure
• Training on the psychology and cultural or traditional about plants
• Training on pharmecology and medicobotany

Ethnobotanists interact in a very broad array of analysis queries and practices, that don’t lend themselves to straightforward categorization. However, the subsequent headings arrange to describe a number of the key areas of recent ethnobotanical study.

14.1 Introduction

In this chapter we shall describe various simple and easy to fabricate fiber optic sensors. Most of these sensors are based on intensity modulation. Intensity modulated optical fiber sensors have been extensively studied in comparison to sensors based on other modulation techniques. This is because of their several advantages such as simplicity, reliability, flexibility and relatively low cost. Intensity modulated sensors make use of multimode fibers. Multimode fibers have large core and high numerical aperture that make the coupling of light into the fiber easier. The large core is also better suited to propagate high power laser beams. Further, the plastic-clad fused silica multimode fibers are available. Plastic-clad silica multimode fibers have an additional advantage. Their plastic cladding can be removed easily permitting access to the core. The evanescent field in the unclad part has been used for various kinds of chemical and biochemical sensors (see chapter 5). Above all, these fibers are cheap. This chapter is devoted to simple and easy to make both intrinsic and extrinsic type sensors. Sensors utilizing bare-ended fibers have also been described.

13.1 Introduction

Distributed optical fiber sensors are those sensors which monitor a single measurand at a large number of locations or continuously over the path of the optical fiber. The measurand can be a physical or a chemical quantity. The distributed optical fiber sensor generally consists of a common light source, optical fiber to carry light to the sensing probes and a return fiber to carry modulated light to a photodetector. These sensors can utilize any kind of light modulations described earlier. The advantages associated with distributed sensing are reduced number of components and hence the cost, reduced weight and cabling, and electrical passivity. The applications include the measurements of temperature, pressure, strain, current and chemical concentration. If the measurand is monitored continuously along the fiber length then the sensor is called intrinsic distributed sensor. If it is measured at a finite number of locations then the sensor is called quasi-distributed sensor. The quasi-distributed sensor has a number of advantages over intrinsic distributed sensor. In quasi-distributed sensor, spatial resolution is not as important as in intrinsic distributed sensor provided that individual sensors can be clearly resolved. Further, in quasi-distributed sensing, the sensor elements can be tailored in terms of responsivity and range to meet the specific needs of the application. In this chapter we shall describe various kinds of distributed sensing approaches and few optical fiber distributed sensors.

12.1 Introduction

Among a variety of optical evanescent wave techniques, spectroscopies based on the resonant excitation of surface plasmons have matured into a widely accepted surface analytical method used in different fields. Liedberg et al¹ were the first who demonstrated the exploitation of surface plasmon resonance (SPR) for chemical sensing. Since then the SPR sensing principle has received much attention. In this chapter we shall first describe the principle of this technique and then few sensors that have utilized this phenomenon.

11.1 Introduction

In previous chapters we have described fiber optic sensors based on intensity and phase modulations. These sensors have some problems in practical applications that need to be solved. Some of these are fluctuation of light source power, loss in couplers, scattering and absorption effects that affect measurement performance, temperature drifts and vibrations. In comparison to intensity and phase modulated sensors fiber Bragg grating (FBG) sensors have following advantages:

High resolution

Insensitive to intensity fluctuation because the signal obtained from an FBG sensor is encoded directly in wavelength domain.

Provision of well localized sensing region

Linear output as a function of the measurand

The capability of multiplex many sensors along one fiber

10.1 Introduction

There are very few frequency modulated fiber optic sensors. This is because frequency modulation of light occurs under a limited range of physical conditions. The principal one is based on Doppler effect. There are few other circumstances under which the frequency of light is modulated. These include luminescence and Raman scattering. Luminescence has been included in chapter 7under fluorescence based sensors. In this chapter we shall focus on the sensors based on Doppler effect and Raman scattering. In particular, we shall discuss the measurement of blood flow velocity, colloidal particle motion and Raman spectrum using optical fibers.

9.1 Introduction

Many physical parameters such as pressure, temperature, electric current etc. can be measured using polarization-modulated sensors. In these sensors modulation of state of polarization of light in the sensing region by an external stimuli takes place. A variety of physical effects influence the state of polarization of light. These phenomena are Faraday effect, electro-optic effect and photo-elastic effect. Polarization modulated sensors, in some cases, offer significant advantages not only over conventional sensors but also over widely used fiber optic interferometric sensors. These sensors require control over the state of polarization of light entering the sensing region of the fiber. Thus, for these sensors, highly birefringent fibers or single mode fibers are used. In this chapter we shall first discuss magneto/electro-optic effects and then shall describe sensors for the measurement of electric current/ magnetic field, voltage, pressure, temperature etc. utilizing polarization modulation.

8.1 Introduction

Interferometric or phase modulated sensors have received special attention because these are much more sensitive than intensity modulated sensors and can be used over a much larger dynamic range. The sensors rely on the interferometric technique. Generally, the sensor employs a coherent light source such as a laser and two single mode optical fibers. Light is split and coupled into both the fibers. If the measurand perturbs the optical properties of one of the fibers relative to the other, a phase shift occurs. The change in phase can be measured very precisely. For the measurement a sophisticated electronic processing equipment is needed. Thus the interferometric sensors are expensive compare to intensity modulated sensors. There are four kinds of interferometer configurations: Mach-Zehnder, Michelson, Fabry-Perot and Sagnac interferometers. The Mach-Zehnder, Michelson and Sagnac interferometers are the two beam interferometers while in the case of Fabry-Perot interferometer multiple beam interference occurs. In this chapter we shall first describe interference phenomenon and then different kinds of fiber optic interferometers. In the end we shall describe a number of sensors based on interference phenomenon or phase modulation.

7.1 Introduction

There are numerous wavelength dependent phenomena that may be used to modulate the wavelength of incident light source. These are wavelength dependent absorption, luminescence, dispersion, interference and scattering. Wavelength modulated sensors use a broadband source, a wavelength modulator or measurand (i.e. analyte), a form of spectrometer (using prisms, gratings or interference filters), a detector (single photodiode or array) and signal processing unit. Silicon photodiode is cheap and can be used for detection but it limits the detection wavelengths to the region 400 to 1050 nm. The sensitivity or the resolution of the wavelength modulated sensor depends on the characteristics of the detector, the inherent resolution of the spectrometer, mechanical stability of the modulation unit and the spectrometer, and the capability of the signal processing unit. However, wavelength modulated sensors do not require any form of referencing like intensity modulated sensors. In intensity modulated sensors, loss in intensity occurs due to connectors and splices, microbending, mechanical creep and misalignment of light sources and detectors due to environmental effect. Further the fluctuations in light source can affect the performance of the sensor. Thus, for intensity modulated sensors one needs referencing to cancel out these losses. In some intensity modulated sensors two wavelengths are used. One wavelength is used to cancel out all of the losses while the second is used to sense the measurand.

Wavelength detection is relatively easy if the spectral changes result from changes in chemical indicator dyes, fluorescence, phosphorescence, or black-body radiation. In these cases the incident and emitted wavelengths are far apart and therefore detection is easy. If the spectral change occurs due to mechanical movement, then the wavelength filtering mechanisms must have sufficient resolvable spectral points. The reasons for using fluorescence in the fiber optic sensors are well known. Fluorescence is intrinsically more sensitive than absorbance and is more flexible in that a great variety of analytes and physical parameters are known to change the emission of particular fluorophores. The fluorescence based sensors utilize the change in the intensity, lifetime or colour of fluorescence as the affected parameter by the measurand. That means, any interpretable observation of fluorescence can be made the basis of a sensor. In this chapter we shall first describe luminescence phenomenon and then various physical and chemical fiber optic sensors based on wavelength modulation and fluorescence phenomenon.

6.1 Introduction

There are a variety of intrinsic and extrinsic fiber-optic sensors which use absorption spectroscopy indirectly for the detection of various chemical species. These include pH, metal ions, oxygen in water etc. In such sensors a suitable reagent is immobilized on a solid support. The support can be the core of the fiber or some optical material attached at the end of the fiber. Further it is optically transparent to allow transmission of the light signal from the interface and should remain inert to the chemical reaction being analyzed. The analyte (or the chemical species) when comes in contact of the reagent affects the absorption properties of the reagent. In other words, in these sensors, the reagent acts as a chemical transducer for analytes that are not directly measurable by optical techniques. Following conditions for the use of reagents in sensors must be met: