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Summer Research Fellowship Programme of India's Science Academies

Training on various instrumentation techniques used in bionanotechnology laboratory

Kalaiarasan Pichaipillai

Bharathidasan University, Tiruchirappalli 620024

Dr. Anil K. Suresh

Associate Professor, Department of Biotechnology, SRM University, Amaravathi, A. P. 522502

Abstract

How to do research comes before doing research. The foremost essential criteria for any researcher to execute research is to possess adequate knowledge on various instrumentation techniques in terms of the principle involved, why that particular equipment is used, what information does the equipment provide ,eventually analysing the data and coming up with the interpretations. Henceforth, we during the first two weeks of our summer training period as IASc-INSA-NASI fellows were trained for the basic analytical and instrumentational techniques available at biomedical nanotechnology laboratory. Some of the techniques that we can handle and perform independently are digital weighing balance, vortex, pH meter, hot plate with magnetic stirrer, water bath, double distillation unit, hot air oven, digital ultrasonic cleaner, centrifuge, concentrator plus, autoclave, CO incubator, gel rocking, orbital shaker, uv-vis spectrophotometer, agarose gel electrophoresis, SDS-page, laminar air flow chamber, biosafety cabinet, Liquid nitrogen.

Keywords: bio-Instruments

Abbreviations

Abbreviations
nm   Nanometer
 N Normality
 M Molarity
 HCL Hydrochloric acid
 NaOH Sodium hydroxide
 BSL Biosafety level
 HEPA  High-efficiency particulate air or High -efficiency particulate absorbing or High-efficiency particulate arrestance
BSC  Biosafety cabinet
 UVC Ultraviolet germicide wavelength
 RPM  Rotation per minute
 SDS Sodium dodecyl sulfate
 PAGE Polyacrylamide gel electrophoresis 
 EtBrEthidium bromide 
 APS Ammonium persulfate
 TEMED Tetramethylethylenediamine
BPB  Bromophenol blue
 AuNPs Gold nanoparticles

INTRODUCTION

Bioinstrumentation is an application of biology, biotechnology, microbiology, biomedical, nanotechnology field. We are focussing on the devices and mechanics used to measure, evaluate, and treat biological systems. It focuses mainly on the use of multiple sensors to monitor the characterization of chemicals and biological samples. Bio instruments are ubiquitous. They play a significant role in the area of biotechnology, medical and research field. All the biomedical instruments need to be compatible with the biological materials for subjective and perceptive analysis which can accomplished by direct contact or by indirect contact. The technologies, products, and applications enable the scientists and researchers to work with precision for bringing out new advancements. Instruments are used as a tool to analyse the samples for qualitative & quantitative purpose to measure and manipulate accordingly. Including Chemistry, physics, biology and materials science, nanotechnology has a greater impact influencing the methodologies involved in interpreting and analysing the results. Precision of bio instruments and exquisite technologies has opened great avenues enhancing the chances of success within the respective projects of investigators. The lack of instruments on one hand or the ability to upgrade ageing local facilities on the other hand, simply elicits the science done in the future. Many newly developed instruments are important because they enable us to explore phenomena with more precision and speed. The development of instruments maintain a symbiotic relationship with science as whole advanced tools enabling scientists to answer increasingly complex questions, and new findings which in turn enable the development of more powerful and novel instruments.

Objectives of the Research

To learn about the various instrumetation techiniques and its application for further research field.

LIST OF INSTRUMENTS LEARNED

Digital weighing balance, vortex, pH meter, hot plate with magnetic stirrer, water bath, double distillation unit, hot air oven, digital ultrasonic cleaner, centrifuge, concentrator plus, autoclave, CO incubator, gel rocking, orbital shaker, uv-vis spectrophotometer, agarose gel electrophoresis, SDS-page, laminar air flow chamber, biosafety cabinet, Liquid nitrogen.

Digital Weighing Balance

"Richard Salter invented by balance maker around 1770".

OBJECTIVE:

To find out the mass of the given sample by using the sensibility methods.

AIM:

To measure the accurate amount of samples and chemicals by using the digital weighing balance.

PRINCIPLE:

A counteracting force is created to be compare the unknown mass. The weighing pan is attached to an electromagnetic coil, through which electric current is flowing. The coil floats in a magnetic field created by an amplifier.

Analytical balances are highly sensitive lab instruments designed to accurately measure mass.

PROCEDURE:

1.      Calibration is very important for the accuracy of any balance. A balance needs a true weight to tune itself. 

2.      Calibration is done by giving a base point (ZERO).

3.      Load certain amount of chemical to be measured.

4.      To wave the samples A, B & C. A (BSA) B (starch) C (sucrose).

5.      BSA to wave the 0.1g of BSA, 2mg of starch and 1g of sucrose.

6.      In order to get the accurate reading of the chemical supposed to be measured, weighing balance need to be calibrated at the first.

weight.gif
    Digital weight balance

    USES:

    Ø  Laboratory balances in general measure the mass of an object in the laboratory.

    Ø  They are used to measure solids, liquids, and tissue.

    Ø  They have a wide range of uses in laboratory including clinical research.

    Ø  The digital mass balances used in the General Chemistry labs, life science laboratories are highly sensitive for weighing substances down to the milligram (0.001 g) level using metal containers at room temperature.

    PRECAUTION:

    Ø  Avoid overloading beyond the rated capacity of the balance. Avoid any moisture or water on the balance.

    Ø  Avoid using the balance under direct sun light. This may cause discoloration or malfunctions

    RESULT:

    With the use of digital weighing balance, accurate amount of chemical can be measured which can be used for further research purpose.

    pH Meter

    "1934 Arnold O. Beckman for the first time invented commercially successful electronic pH meter."

    AIM

    To determine the pH concentration of the solution by using the pH meter.

    PRINCIPLE

    The principle of the pH meter is to measure the concentration of hydrogen ions. Acids dissolve in water forming positively charged hydrogen ions (H+). The greater the concentration of hydrogen ions, the stronger is the acid.

    MODE OF ACTION

    Ø  pH meter, an electric device used to measure hydrogen-ion activity (acidity or alkalinity) in solution. Fundamentally, a pH meter consists of a voltmeter attached to a pH-responsive electrode and a reference (unvarying) electrode.

    Ø  Always keep pH electrode moist. The electrodes are kept in a solution of 4 M KCl.

    Because KCl imparts the least chance for ionic imbalance in the used electrolyte in salt-bridge. 

    ADVANTAGE OF pH METER:

    Accuracy: pH meter shows the accurate value of the concentration of hydrogen ions present in the solution.

    Ease to use: pH meter is easy to operate and in deciphering the ionic concentration of hydrogen ions within the solutions.

    Consideration: pH meter is to use a check the pH value of the solution. Once calibrated, it can be used to measure the ionic concentrations of hydrogen in various solutions independently.

    phmeter.jpg
      pH meter

      Example: 1

      Preparation of 0.1N of HCL in 100ml at pH: 6.4.

      Normality is a measure of concentration equal to the gram equivalent weight per litre of solution.

      Solution:

      Required volume: 100ml, Required volume: 0.1N, Equivalent weight: 36.46 of HCL

      Formula : Normality = req. volume × req. con× equivalent weight/1000

      N = 100× 0.1× 36.46/1000 = 0.3646.

      N. FACTOR (AMOUNT OF HYDROGEN IONS) N = 0.3646/ 1 =0.3646ml of HCL in 100ml of D.H2o.

      Use 0.3646ml of HCl to make upto 100ml at pH: 6.4 Followed by calibration using base for further use.

      Example:2

      To prepare the 10M of NaOH solution in 100ml pH: 8.0. 

      Molarity (M) is the concentration of a solution expressed as the number of moles of solute per litre of solution.

      NaOH (Mol. Wt. 40)

      Solution:

      Molarity = Req.volume x Req.con x molecular weight/1000

      = 100 x 10M x 40/1000 = 40g

      i. 40g of NaOH pellets dissolve in 70 ml water by adding.

      ii. 0.1N or 1N HCl buffer solution is used to adjust the pH value 8.0. Making it 100ml by adding deionised water.

      RESULT:

      ü  pH meter helps to get the accurate concentration of hydrogen ions in the solution prepared.

      ü  pH meter helps to check the concentration of hydrogen ions present in the solution.

      1.      Obtaining 100ml of 0.1N of HCL at pH: 6.4.

      2.      Obtaining 100ml of 10M of NaOH at pH: 8.0.

      Vortex

      "The vortex mixer was invented by the Kraft brothers (Jack A. Kraft and Harold D. Kraft)". 

      AIM:

      Preparing the reaction mixture by bringing solute and solvent phase in equilibrium.

      PRINCIPLE:

      Vortex flow meters operate under the vortex shedding principle, where oscillating vortexes occur when a fluid such as water flows past a bluff body. The frequency that the vortexes are shed depend on the size and shape of the body.

      v.jpg
        Vortex mixer

        FUNCTION:

        Ø  To mix the sample at a constant speed of rotation homogenously.

        Ø  The rangeability declines proportionally with an increase in viscosity, decrease in density, or reductions in the maximum flow velocity of the process.

        Ø  Vortex shedding meters are therefore unsuitable for use on high viscosity liquids.

        ADVANTAGES:

        Ø  Low wear (relative to turbine flow meters), Relatively low cost of installation and maintenance

        Ø  Low sensitivity to variations in process conditions, Stable long term accuracy and repeatability

        Ø  Applicable to a wide range of process temperatures, Available for a wide variety of pipe sizes

        Ø  The flow of liquid suspension, General water applications

        Ø  Liquid chemicals & pharmaceuticals

        LIMITATIONS:

        Ø  Not suitable for very low flow rates.

        Ø  Minimum length of straight pipe is required upstream and downstream of the vortex meter

        Ø  Vortex flow meters are suitable for a variety of applications and industries but work best with clean, low-viscosity, medium to high-speed fluids.

        Magnetic Stirrer

        "Hot plate and magnetic stirrer invented by Theodore Hug Attorney on 22nd March 1960."

        PRINCIPLE:

        Ø  A magnetic stirrer is a laboratory device consisting of two bar magnet either a rotating magnet or stationary electromagnets creating a rotating magnetic field. This device is used to cause a stir bar immersed in a liquid to spin very quickly, agitating or mixing the liquid.

        Ø  A magnetic stirrer often includes a provision for heating the liquid. Stirrers are often used in laboratories, especially in the field of biology.

        WORKING METHODOLOGY:

        Ø  Magnetic stirrer working is repulsive for like charges and attractive for unlike charges. Micro motor drives magnet to generate rotating magnetic field to stir the stirring bar in a vessel, making solution conduct completely mixed reaction, stirring speed is adjustable, widely applies to solvent stirring in different viscosity.

        Ø  It equips with the temperature control system, which can heat and control the temperature of the sample according to experiment requirement, maintaining the needed temperature condition and guaranteeing the mixed liquid meets the experiment need.

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          Hot plate with magnetic stirrer.

          ADVANTAGES:

          Ø  The magnetic stirrers are also known as magnetic stir plates and quite commonly used for experiments in chemistry and biology.

          Ø  There are very useful when you need to mix component, either solids or liquids and get a homogeneous liquid mixture. Some of the common samples include bacterial growth media as well as buffer solutions.

          Ø  The main function of a stirrer is to agitate the liquid for speeding up the reactions or improving mixtures. A magnetic stirrer is often used with hot plates.

          DISADVANTAGES:

          Ø  Magnetic stirring cannot be used at very high temperatures, as the protective coating of the stir bars will degrade.

          Ø  Very large volumes of liquids cannot be stirred properly, neither do viscous liquids nor thick suspensions.

          Ø  Note: To calibrate the temperature and rpm at various level.

          Magnetic Stirrer

          "During 1921, water chiller was patented and invented by Willis Carrie".

          PRINCIPLE:

          A water bath is laboratory equipment made from a container filled with heated water. It is used to incubate samples in water at a constant temperature over a long period of time. All water baths have a digital or an analogue interface to allow users to set the desired temperature. 

          FUNCTION:

          A water bath is laboratory equipment made from a container filled with heated water. It is used to incubate samples in water at a constant temperature over a long period of time.

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            water bath

            PRECAUTION:

            Ø  It is not recommended to use a water bath with moisture sensitive or pyrophoric reactions. Do not heat a bath fluid above its flash point.

            Ø  The water level should be regularly monitored, and filled with distilled water only. This is required to prevent salts from depositing on the heater.

            Ø  Disinfectants can be added to prevent the growth of organisms.

            Ø  Raise the temperature to 90 °C for sterlization purpose.

            Ø  Markers tend to come off easily in water baths. Use water-resistant ones.

            Ø  If the application involves liquids that give off fumes, it is recommended to operate a water bath in a fume hood or in a well-ventilated area.

            Ø  The cover is closed to prevent evaporation and to help to reach high temperatures.

            Ø  Set up on a steady surface away from flammable materials.

            TYPES OF WATER BATH:

            Circulating water bath: Circulating the water baths are ideal for applications when temperature uniformity and consistency are critical, such as enzymatic and serologic experiments. Water is thoroughly circulated throughout the bath resulting in a more uniform temperature.

            Non-circulating water bath: This type of water bath relies primarily on convection instead of water is uniformly heated. Therefore, it is less accurate in terms of temperature control. In addition, there are add-ons that provide stirring to non-circulating water baths to create more uniform heat transfer.

            Shaking water bath: This type of water bath has extra control for shaking, which moves liquids around. This shaking feature can be turned on or off. In microbiological practices, constant shaking allows liquid-grown cell cultures grown to constantly mix with the air.

            Double Distillation Unit

            "Jabir ibn Hayyan was inventor of double distillation unit".

            AIM:

            To prepare the deionized water through the double distillation unit.

            Principle

            Distillation is the process of separating the components or substances from a liquid mixture by using selective boiling and condensation. Distillation may result in essentially complete separation (nearly pure components) or it may be a partial separation that increases the concentration of selected components in the mixture. In either case, the process exploits differences in the volatility of the mixture's components.

            IN BIOLOGY LABORATORY

            Ø  Fractional distillation must be used in order to separate the components by repeated vaporization-condensation cycles within a packed fractionating column. This separation, by successive distillations, is also referred to as rectification.

            Ø  As the solution to be purified is heated, its vapours rise to the fractionating column. As it rises, it cools, condensing on the condenser walls and the surfaces of the packing material.

            Ø  Here, the condensate continues to be heated by the rising hot vapours. It vaporizes once more.

            Ø  Each vaporization-condensation cycle will yield a purer solution of the more volatile component. In reality, each cycle at a given temperature does not occur at exactly the same position in the fractionating column.

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              Double distillation unit

              ADVANTAGES:

              Ø  It is an efficient method of water softening for smaller purposes.

              Ø  It is relatively cheap. It can also be reused.

              DISADVANTAGES:

              Ø  Some of the unwanted elements may be found in the distilled water. Therefore, separation of these two can be tough.

              Ø  As a process of water softening, distillation requires a keen eye, so that unwanted elements do not mix with water.

              Ø  When distillation is done on a larger scale, a very high amount of energy needed.

              Ø  The distilled water does not contain any oxygen and is also very tasteless water will get.

              Hot Air Oven

              "French Chemist Louis Pasteur in 1864 to develop the techniques utilised in hot air oven."

              AIM:

              To sterilize the laboratory equipment by using the HOT AIR OVEN.

              PRINCIPLE

              Ø  Sterilization by dry heat is accomplished by conduction. The heat is absorbed by the outside surface of the item, then passes towards the centre of the item, layer by layer. The entire item will eventually reach the temperature required for sterilization to take place.

              Ø  Dry heat does most of the damage by oxidizing molecules. The essential cell constituents are destroyed and the organism dies. The temperature is maintained for almost an hour to kill the most difficult of the resistant spores. 

              WORKING METHODOLOGY

              Ø  The Hot Air Oven works on the basis of Hot Air inside the chamber which is created due to the forced air circulation. The circulating fans and fan motor that are equipped with the instrument helps to create even temperature inside the chamber which helps to achieve an optimum level of heat inside the Hot air oven. After heating the specimen in the oven at least for two hours, the test specimens are kept inside the chamber until the temperature comes down and then the sterilized material is removed from the oven.

              Ø  The instrument is widely used to sterilize glassware in pharmaceutical industries such as Petri dishes, pipettes, bottles, test tubes, flasks, pestle, etc.

              Ø  The most common time-temperature relationships for sterilization with hot air sterilizers are170°C(340°F)for30 minutes or 160°C (320°F) for 60 minutes, and 150°C (300°F) for 150 minutes or longer depending up the volume.

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                Hot air oven

                ADVANTAGE OF STERILIZED HOT AIR OVEN

                Ø  Glassware (Petri dishes, flasks, pipettes, and test tubes)

                Ø  Materials that contain oils, Metal equipment (scalpels, scissors, and blades)

                Ø  Glass test tubes can be sterilized using a hot air oven

                Ø  Hot air ovens use extremely high temperatures over several hours to destroy microorganisms and bacterial spores.

                Ø  The ovens use conduction to sterilize items by heating the outside surfaces of the item, which then absorbs the heat and moves it towards the centre of the item.

                DISADVANTAGE OF HOT AIR OVEN

                Ø  Time-consuming method because of the slow rate of heat penetration and microbial killing. High temperature is not suitable for most materials.­

                Digital Ultrasonic Cleaner

                PRINCIPLE

                Ultrasonic sensors emit short, high-frequency sound pulses at regular intervals. If they strike an object, then they are reflected back as echo signals to the sensor, which itself computes the distance to the target based on the time-span between emitting the signal and receiving the echo.

                WORKING METHODOLOGY (Sound waves in a conductive medium)

                Ø  It is actually the method of displacing the sound waves in a conductive method on which the basic technique of Ultrasonic cleaning base.

                Ø  Sound waves are transferred without any blockade when they are passed on into compressible surroundings such as air.

                Ø  When it comes to an ultrasonic fluid, the negative pressure is produced when sound energy increases. The fluid then breaks up because of that negative pressure giving birth to a vast amount of small hydrogen cavitation.

                Ø  On their meeting with the sound waves, these hydrogen cavitation’s start to vibrate. The hydrogen cavitation expands as a result of those vibrations and they start to implode after getting unstable.

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                  Ultra sonic cleaner.

                  ULTRASONIC CLEANING:

                  ADVANTAGES

                  Ø  To use the manual methods can be cleaned using ultrasonic cleaning. In fact to clean every corner and tiniest gaps of an object clean accurate.

                  Ø  Ultrasonic cleaning is not only very effective. This method of cleaning does not damage the object.

                  Ø  Ultrasonic cleaning is very fast as compared to manual methods. To save time consumption

                  Ø  Ultrasonic cleaning remains untroubled by the shape of the object to be cleaned, it saves a huge amount of time.

                  Ø  Most hard, non-absorbent materials (metals, plastics, etc.) not chemically attacked by the cleaning fluid are suitable for ultrasonic cleaning. Ideal materials for ultrasonic cleaning include small electronic parts, cables, rods, wires, and detailed items, as well as objects made of glass, plastic, aluminium or ceramic.

                  Ø  Ultrasonic cleaning does not sterilize the objects.

                  Ø  To remove the strain in automotive, sporting, printing, marine, medical, pharmaceutical, electroplating, disk drive components, engineering, and weapons industries.

                  Centrifugation

                  Benjamin Robins (1707–1751) invented a whirling arm apparatus.

                  WORKING PRINCIPLE:

                  Under Newton’s second law of motion”

                  The gravitational force is proportional to the mass of the particle and the acceleration of the gravitational field”.

                  Ø  The process of separating fractions of systems in a centrifuge.

                  Ø  The most basic separation is to sediment a pellet at the bottom of the tube, leaving a supernatant at given centrifugal force density may be used as a basis for sedimentation in density gradient centrifugation.

                  Ø  A centrifuge is a type of research equipment that spins a liquid suspension at higher rotation rates to separate it into distant layers based on density gradient.

                  centr.jpg
                    Centrifuged sample.

                    WORKING PRECAUTION:

                    1. Inspect the centrifuge tubes and bottle for any damage for before use.

                    2. Cap tubes with the proper lid.

                    3. Wipe the outside of the tube with disinfection before placing it in the centrifuge.

                    4. Clearly label the tubes for identification.

                    5. Balancing is extremely important at higher centrifugation speeds.

                    6. Place the centrifuge on a firm level surface.

                    7. Choose the proper rotor to use at the speed you need.

                    8. Load the tubes opposite each other in the centrifuge.

                    9. Enter the centrifugation speed.

                    10. Keep a safe distance while the centrifuge is running.

                    11. Turn off the centrifuge if it is wobbling.

                    12. Open the lid only after the rotor has completely stopped.

                    13. Remove the tubes carefully after the centrifuge has completed stopped spinning wipe down the rotor and centrifuge after each use.

                    USES:

                    1.      To separate the sample based on the density gradient.

                    2.      To use in Biology, Microbiology, Molecular Biology, Biotechnology, Biochemistry & Research laboratory.

                    3.      Chemical industry, pharmaceutical industry, etc.

                    4.      Various type centrifuge is used based on the rpm (slow speed – high speed). Range: 4000- 150,000rpm

                    PROTOCOL:

                    Ø  Collect the 10ml of two of gold nanoparticle at equal volume on separate falcon tube.

                    Ø  Tube A contain AuNPs (Rod). Tube B contain AuNPs (sphere).

                    Ø  To centrifuge the sample at 7500rpm for 15mins at 18֯c.

                    Ø  Finally to get the nanoparticle in AuNPs (sphere) pellet collect and use further study.

                    RESULT:

                    To get the separated nanoparticle by using the centrifuge. It helps to separate the biomolecules based on the density gradient. In this instrument very useful in a biological laboratory.

                    Concentrator Plus

                    "Centrifugal evaporators were invented in the 1960s by Savant Inc".

                    PRINCIPLE:

                    The speed-vac is used to concentrate on small-volume samples. Under vacuum (very low pressure), the vapour-liquid equilibrium of the solvent is shifted towards the gas phase, while your sample (DNA, peptide, etc.) remains primarily in the solid phase.

                    WORKING OF VACUUM:

                    Ø  Using a vacuum you can easily remove solvent with very little stress on your solute, leaving you with a dry, solid sample.

                    Ø  The speed-vac lid should always be closed, and the rotor should always be spinning. The Drying Rate switch on the front of the rotor controls the heat. This is usually set at Low (no heat).

                    Ø  Freeze samples on dry ice prior using in speed-vac to prevent loss of material from bumping. If using a screw-cap Eppendorf tube, loosely place the cap on the tube. If using a normal snap-cap tube, poke a holes two or three.

                    Ø  Close the cap firmly before placing the tube in the speed-vac. The trap should be cleaned at least once a month.

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                      Concentrator plus.

                      USING THE VACUUM SPEED 

                      Ø  Before opening the lid to the speed-vac, you must release the vacuum. This is done by turning the bleed valve perpendicular to the line (closed position).

                      Ø  Place your samples in the speed-vac. Remember to counter balance!

                      Ø  Close the lid. Make sure that the rotor is spinning before you reapply the vacuum. Turn the bleed knob back into the parallel position (open position) so that the speed-vac is connected to the pump.

                      Ø  If the vacuum is connected, you should hear the pump "gurgle" and the lid should be suctioned shut.

                      Ø  Sign your name, time, and sample type on the log beside the speed-vac. Also, indicate if you do not want your samples exposed to heat or light.

                      Ø  Remember to turn the speed-vac back on after you remove your samples.

                      Ø  If you remove samples from the hot speed-vac always check your gloves to make sure the inside of the speed-vac is not hot.

                      USES OF CONCENTRATOR PLUS:

                      Concentrators plus used in proteomics, genomics, cell biology, microbiology, and drug development heat or gas blow down to evaporate the liquid and concentrate DNA, RNA, nucleotides, and other proteins.

                      Autoclave

                      "The autoclave was invented by Charles Chamberland in 1884."

                      AUTOCLAVES WORKING PRINCIPLE:

                      Autoclaves use pressurized steam as their sterilization agent. The basic concept of an autoclave is to have each item sterilized whether it is a liquid, plastic ware, or glassware-come in direct contact with steam at a specific temperature and pressure for a specific amount of time.

                      MODE OF ACTION:

                      Ø  Moist heat destroys microorganisms by the irreversible denaturation of enzymes and structural proteins. The temperature at which denaturation occurs varies inversely with the amount of water present. Sterilization in saturated steam thus requires precise control of time, temperature, and pressure.

                      Ø  Pressure serves as a means to obtain the high temperatures necessary to quickly kill microorganisms. Specific temperatures must be obtained to ensure the microbicidal activity. Minimum sterilization time should be measured from the moment when all the materials to be sterilized have reached the required temperature throughout.

                      Ø  The recommendations for sterilization in an autoclave are 15 minutes at 121°C.

                      QUALITY CONTROL CHECK THE AUTOCLAVE

                      Ø  The effectiveness of steam sterilization is monitored with a biological indicator using an envelope containing spores of Bacillus stearothermophilus.

                      Ø  After sterilization is over inoculated microbe into tryptone soya broth and incubated at 56°C for 5 days. No growth of Bacillus stearothermophilus indicates proper sterilization.

                      autoclave.gif
                        Horizontal & Vertical autoclave.

                        PRECAUTION:

                        Ø  Wearing appropriate Personal Protective Equipment (PPE) including a lab coat, heat resistant gloves, and eye protection, especially when unloading the autoclave.

                        Ø  Never sealing containers, under pressure they pose an explosion risk.

                        Ø  Never opening the door to the autoclave immediately turn off the power supply.

                        USES:

                        Ø  Sterilization autoclaves are widely used in microbiology, medicine, podiatry, veterinary, medicine, mycology at research work.

                        Ø  A medical autoclave is a device that uses steam to sterilize equipment and other objects. This means that all bacteria, viruses, fungi and spores are inactivated.

                        Ø  Research-grade autoclaves may be configured for "pass-through" operation. This makes it possible to maintain absolute isolation between "clean" and potentially contaminated work areas. Pass-through research autoclaves are especially important in biosafety level BSL-3 or BSL-4 facilities.

                        Ø  Research-grade autoclaves—which are not approved for use sterilizing instruments that will be directly used on humans—are primarily designed for efficiency, flexibility, and ease-of-use. They rely on efficient cylindrical pressure-chamber designs, are intended for intermittent use, and have highly customizable programmable controls

                        Protocol for autoclave:

                        Ø  To collect the measured media mix with D.H2O at the required volume.

                        Ø  Pour in a conical flask to close the flask using cotton plug.

                        Ø  Put the conical flask into the autoclave followed by locking the instrument properly

                        Ø  Check the water level instrument.

                        Ø  To set a certain time and Temp (121֯c for 15-20 mins) in liquid material for the sterilization process.

                        Ø  The same procedure is followed for contaminated sterilization process before use to discard the microbial culture, plant tissue culture, cell line study, medical wastage, etc.

                        Co2 Incubator

                        PRINCIPLE: 

                        The incubator is a device used to grow and maintain microbiological cultures or cell cultures. The incubator maintains optimal temperature, humidity and other conditions such as the CO (CO2) and oxygen content of the atmosphere inside.

                        FUNCTION OF INCUBATOR:

                        Ø  Laboratory incubators provide a controlled, contaminant-free environment for safe, reliable work with cell and tissue cultures by regulating conditions such as temperature, humidity, and CO2. Microbiological incubators are used for the growth and storage of bacterial cultures.

                        Ø  An incubator is a modern piece of laboratory equipment which is used to maintain the progressive development of microbiological cultures by regulating viable growth factors such as temperature, humidity, and ventilation. Incubators are available in different sizes.

                        Ø  Some superior qualities include being capable of controlling extreme low temperatures (microbiological incubator), humidity and carbon dioxide levels (cell culture incubator). A microbiological incubator focuses mainly on the growth and storage of bacterial cultures and control temperatures ranging from 5 to 7 degrees C.

                        Ø  Laboratory incubators are widely involved in a number of biological applications such as cell and tissue culture, pharmaceutical studies, haematological studies, biochemical studies, food processing, cell aeration, plant and animal studies, solubility studies, fermentation studies and bacterial culturing.

                        Ø  Incubators are extensively used to study tissue cultures that involve the extraction of tissue fragments from animals and plants, keep these "explants" (i.e., isolated cells from a piece of tissue) in a controlled environment and subsequently analyzing their growth. Study of these explants enables clinicians and scientists to understand the function of specific disease-causing cells such as cancer cells and help them in developing vaccines for diseases such as polio, mumps, and measles.

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                          BOD  Incubator

                          Gel Rocker

                          PRINCIPLE:

                          Ø  A gel rocker is a device used in the laboratory for molecular and biological mixing application. Rockers are used in a place of shakers when less aggressive mixing is required. Rockers are commonly used for staining and destaining of gels after electrophoresis, hybridization, washing, blotting, cell culture, and gentle mixing.

                          Ø  Two-dimensional rockers use a platform to remove in a seesaw motion to create waves in liquid at laboratory samples. Three-dimensional rockers have a platform in a three-dimensional gyratory motion to create a gentle swirling of sample.

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                            Gel rocker

                            ADVANTAGE:

                            Ø  Provides regulated gentle rocking motion of the platform and is ideal for mini gel destaining after electrophoresis, conducting Northern, Southern and Western blotting.

                            Ø  Shaker can be used in cold rooms or incubators, operating at ambient temperature range +4°C to +40°C.

                            Ø  The device when installed inside a bio-incubator it is ideal for growing cells and cell cultures in disposable plastic reactor-bags (working volumes up to 10 litres, media volumes up to 5litres.

                            Orbital Shaker

                            "Orbital shaker Invented by Jack A. Kraft and Harold D. Kraft in 1962."

                            WORKING MECHANISM:

                            An orbital shaker works by generating a circular shaking motion at a slow speed of 25-500 rpm. The shaker contains an oscillating board that holds the vessels as the device shakes to blend, agitate, or mix the substances in the vessels.

                            USAGE IN ORBITAL SHAKER:

                            Ø To use the orbital shaker for agitating the microbial culture, chemical mixing, various sample solution at continuously at certain rpm and time.

                            Ø It is mainly used in the fields of chemistry and biology. A shaker contains an oscillating board that is used to place the flasks, beakers, or test tubes.

                            Ø A shaker contains an oscillating board that is used to place the flasks, beakers and test tubes. Although the magnetic stirrer has lately come to replace the shaker, it is still the preferred choice of equipment when dealing with large volume substances or when simultaneous agitation is required.

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                               Orbital shaker.

                              UV-vis Spectrophotometer

                              "In July 1941, Arnold Beckman introduced the UV-vis spectrophotometer".

                              PRINCIPLE:

                              Ø The Beer-Lambert law states that the quantity of light absorbed by a substance dissolved in a fully transmitting solvent is directly proportional to the concentration of the substance and the path length of the light through the solution

                              Ø Molecules containing bonding and non-bonding electrons (n-electrons) can absorb energy in the form of ultraviolet or visible light to excite these electrons to higher.

                              APPLICATION OF UV VIS SPECTROSCOPY

                              1.      UV/Vis spectroscopy is routinely used in analytical chemistry, the quantitative determination of different analytes.

                              2.      Detection of functional groups: UV spectroscopy is used to detect the presence or absence of chromophore in the compound. The absence of a band at a particular band can be seen as evidence for the absence of a particular group. If the spectrum of a compound comes out to be transparent above 200 nm than it confirms the absence of

                              a) Conjugation

                              b) A carbonyl group

                              c) Benzene or aromatic compound.

                              3.      Detection of the extent of conjugation: The extent of conjugation in the polyenes can be detected with the help of UV spectroscopy. With the increase in double bonds the absorption shifts towards the longer wavelength. If the double bond is increased by 8 in the polyenes then that polyene appears visible to the human eye as the absorption comes in the visible region.

                              4.      Identification of an unknown compound: An unknown compound can be identified with the help of UV spectroscopy. The spectrum of an unknown compound is compared with the spectrum of a reference compound and if both the spectrums coincide then it confirms the identification of the unknown substance.

                              5.       Determination of configurations of geometrical isomers: It is observed that cis-alkenes absorb at a different wavelength than the trans-alkenes. The two isomers can be distinguished with each other when one of the isomers has non-coplanar structure due to steric hindrances. The cis-isomer suffers distortion and absorbs at a lower wavelength as compared to trans-isomer.

                              6.      Determination of the purity of a substance: Purity of a substance can also be determined with the help of UV spectroscopy. The absorption of the sample solution is compared with the absorption of the reference solution. The intensity of the absorption can be used for the relative calculation of the purity of the sample substance.

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                                UV-VIS specteroscopy.
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                                   UV-Vis reading

                                  SAMPLES RUN IN UV-VIS SPECTROSCOPY:

                                  SAMPLE: A Gold nanoparticle in sphere shape.

                                  SAMPLE: B Gold nanoparticle in a rod shape.

                                  RESULT:

                                  Through the UV-VIS spectroscopy to know about the concentration and excitation range of Gold Nanoparticle is maximum absorption is 250nm.

                                  Agarose Gel Electrophoresis

                                  "The development of gel electrophoresis began with the pioneering work of Arne Tiselius"

                                  PRINCIPLES OF ELECTROPHORESIS:

                                  Ø  Electrophoresis is a method used to separate charged particles from one another based on differences in their migration speed. In the course of electrophoresis, two electrodes (typically made of an inert metal, e.g. platinum) are immersed in two separate buffer chambers.

                                  Ø  A method used in biochemistry and molecular biology to separate DNA or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electronic field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones.

                                  APPLICATION

                                  Ø  Estimation of the size of DNA molecules.

                                  Ø  Genetic disease diagnosis or DNA fingerprinting and forensic crime investigation.

                                  Ø  Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to Northern analysis.

                                  PROTOCOL:

                                  Ø  Gels for DNA separation are often made out of a polysaccharide called agarose, which comes as dry, powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores 1.0% of agarose enhance resolution (100-500 nm) size pore.

                                  Ø  At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed.

                                  Ø  Before the DNA samples are added, the gel must be placed in a gel box. One end of the box is hooked to a positive electrode, while the other end is hooked to a negative electrode. The main body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct current. The buffer fills the gel box to a level where it just barely covers the gel.

                                  Ø  The end of the gel with the wells is positioned towards the negative electrode. The end without wells (towards which the DNA fragments will migrate) is positioned towards the positive electrode.

                                  Ø  As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than longer ones. After the gel has run for a while, the shortest pieces of DNA will be close to the positive end of the gel, while the longest pieces of DNA will remain near the wells. Very short pieces of DNA may have run right off the end of the gel.

                                  Ø  EtBr is used to visualize the DNA.

                                  Ø  BPB is used as a sample loading dye.

                                  Ø  To give the power supply foe 1-1.1/2 hrs. The DNA sample are separate based on the length of the band size.

                                  Ø  Finally, place the gel carefully on the Transilluminator platform and close the lead of the Transilluminator.

                                  Ø  Turn on the UV light and the result will be observed. EtBr intercalates between DNA bases and emits fluorescent light. Orange coloured DNA bands are observed under the UV Transillumination.

                                  Well no: 1-6 contain the various type of DNA sample is used from the various plant.

                                  Well .no: 01 Polyalthia longifolia

                                  Well .no: 02 Albizia amara

                                  Well .no: 03 Mimusops elengi

                                  Well .no: 04 Tectona grandis

                                  Well .no: 05 Peltophorum pterocarpum

                                  Well .no: 06 Azadirachta indica

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                                     Agarose  gel electrophoresis.

                                    Result:

                                    Through the agarose gel electrophoresis to separate the DNA molecules based on the size of the length of the DNA.

                                    SDS PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)

                                    "SDS-PAGE was first known as the Laemmli method, after its inventor by U.K. Laemmli."

                                    PRINCIPLE:

                                    Ø  SDS-PAGE is working upon the principle in which, the charged molecule will migrate towards the oppositive charged electrode through a highly cross-linked matrix. Separation occurs due to different rates of migration occurs by the magnitude of charge and frictional resistance related to the size.

                                    Ø  Such as proteins and nucleic acids. In general, macromolecules may be run in their native state or in denatured forms. 

                                    Ø  To separate molecules based on their lengths, samples are run in denaturing conditions. For proteins, sodium dodecyl sulfate (SDS) is used to linearize proteins and to negatively charge the proteins.

                                    Ø  The binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass.

                                    Ø  As a result, negatively charged proteins will migrate towards the positive electrode and will be fractionated by approximate size during electrophoresis.

                                    Ø  Western blotting is the combination of SDS-PAGE and antibody-based detection protein immunoblot application to detect proteins from complex biological mixtures.

                                    MATERIALS:

                                    Ø  To Pour Gels: 30% acrylamide, 10% SDS, 10% APS (make fresh each time), TEMED, 1.5 M Tris, pH 8.8 (resolving gel), 1.0 M Tris, pH 6.8 (stacking gel)

                                    Ø  5x SDS Running Buffer (1 L): Tris 15 g, Glycine 72 g, SDS 5 g

                                    Ø  Coomassie Blue Stain: 10% (v/v) acetic acid, 0.006% (w/v) Coomassie Blue dye, 90% ddH2O

                                    Ø  Isopropanol Fixing Solution: 10% (v/v) acetic acid, 25% (v/v) isopropanol, 65% ddH2O

                                    Ø  SDS sample prepare loading buffer (40 ml): ddH2O 16 ml, 0.5 M Tris, pH 6.8 5 ml, 50% Glycerol 8 ml, 10% SDS 8 ml, β mercaptoethanol 2 ml (add immediately before use), bromophenol blue, 10% (v/v) acetic acid

                                    PROTOCOL:

                                    1. Prepare polyacrylamide gel according to standard protocol.

                                    2. Load samples and run gel 100 mA to150 mA in 1x SDS Running Buffer.

                                    3. At this point, the gel can either be transferred to a membrane stained with Coomassie brilliant blue.

                                    4. Place gel in a plastic container. Cover with isopropanol fixing solution and shake at room temperature. For 0.75 mm-thick gels, shake 10 to 15 min; for 1.5 mm thick gels, shake 30 to 60 min.

                                    5. Pour off fixing solution. Cover with Coomassie blue staining solution and shake at RT for 2 hr.

                                    6. Pour off staining solution. Wash gel with 10% acetic acid to destaining, shaking at RT ON.

                                    Laminar Air Flow Chamber

                                    "Willis Whitfield invented the laminar air flow chamber".

                                    PRINCIPLE:

                                    In a laminar flow hood, the air is passed through a HEPA (High-Efficiency Particulates Air) filter which removes all airborne contamination to maintain sterile conditions. ... Now the sterile air flows into the working area where you can do all your work without risk of contamination.

                                    WORKING MECHANISM:

                                    Ø  In a laminar flow hood, the air is passed through a HEPA (High-Efficiency Particulates Air) filter which removes all airborne contamination to maintain sterile conditions. A laminar flow hood consists of a filter pad, a fan, and a HEPA (High-Efficiency Particulates Air) filter.

                                    Ø  A laminar flow cabinet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive materials. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user.

                                    Ø  Mostly to use the laminar airflow chamber in the Microbiology, Biotechnology, Botany, Biochemistry, clinical laboratory, and research unit.

                                    Ø  Laminar air flow chamber provide the sterile and aseptic condition for the chamber.

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                                      Laminar air flow chamber

                                      CLEANING OF CHAMBER:

                                      Ø  Use isopropyl alcohol 70% to clean the compounding area prior to the beginning of each shift.

                                      Ø  Allow the alcohol to remain for at least 30 seconds.

                                      Ø  Clean from back to front and top to bottom and avoid contact and contamination of the HEPA filter.

                                      Ø  Ultraviolet C light with wavelengths between 200 – 280 nanometer (nm).

                                      Ø  Light in the UV C wavelength can be used for disinfecting water, sterilizing surfaces, destroying harmful micro-organisms in food products and in air. UV for 20-30 mins to kill the micro-organism.

                                      Biosafety Cabinet

                                      "In 1943, Van den Ende published the first formal description of a dedicated biological safety cabinet."

                                      PRINCIPLE:

                                      The air enters the cabinet via the front aperture passing through a built-in exhaust fan, HEPA or Carbon filter, thus providing operator and environmental protection. The air then exits the cabinet at the rear of the work surface.

                                      WORKING MECHANISM:

                                      Ø  The velocity of air flowing through the front opening into a BSC is about 0.45 m/s. At this velocity, the integrity of the directional air inflow is fragile and can be easily disrupted by air currents generated by people walking close to the BSC.

                                      Ø  Operators need to be careful to maintain the integrity of the front opening air inflow when moving their arms into and out of cabinets. Arms should be moved in and out slowly, perpendicular to the front opening.

                                      Ø  Materials to be placed inside the cabinet should be surface-decontaminated with 70% alcohol. Work may be performed on disinfectant-soaked absorbent towels to capture splatters and splashes.

                                      Ø  Cabinets should be turned on at least 5 min before beginning work and after completion of work to allow the cabinet to “purge”, i.e. to allow time for contaminated air to be removed from the cabinet environment.

                                      Ø  Ultraviolet light intensity should be checked when the cabinet is recertified to ensure that light emission is appropriate. Ultraviolet lights must be turned off while the room is occupied, to protect eyes and skin from inadvertent exposure.

                                      Ø  Open flames should be avoided in the near microbe-free environment created inside the BSC.

                                      Ø  To sterilize bacteriological loops, micro-burners or electric “furnaces” are available and are preferable to open flames.

                                      Ø  All items within BSCs, including equipment, should be surface-decontaminated and removed from the cabinet when work is completed.

                                      Ø  A solution of bleach or 70% alcohol should be used were effective for target organisms. A second wiping with sterile water is needed when a corrosive disinfectant, such as bleach, is used.

                                      Ø  The most common decontamination method is by fumigation with formaldehyde gas or chlorine dioxide or hydrogen peroxide

                                      Ø  BSCs can be equipped with one of two kinds of alarm. Sash alarms are found only on cabinets with sliding sashes. The alarm signifies that the operator has moved the sash to an improper position. Corrective action for this type of alarm is returning the sash to the proper position.

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                                        Biosafety cabinet.

                                        USES:

                                        Ø  Mainly cell line study, microbiology, cell biology, biochemistry, genetic engineering, research unit.

                                        Liquid Nitrogen

                                        LIQUID NITROGEN FREEZING PRINCIPLE AND PROCESS

                                        Liquid nitrogen is a relatively ideal refrigerant, coloured transparent, slightly lighter than water, inert, no corrosion, vibration, the spark is stable, an atmospheric pressure, liquid nitrogen vaporization and the temperature is -195.81֯c.

                                        CHARACTER OF LIQUID NITROGEN:

                                        Ø  Liquid nitrogen is a diatomic liquid, which means that the diatomic character of the covalent N bonding in N2 gas is retained after liquefaction.

                                        Ø  Liquid nitrogen is a cryogenic fluid that can cause rapid freezing on contact with living tissue.

                                        Ø  The temperature of liquid nitrogen can readily be reduced to its freezing point 63 K (−210 °C; −346 °F) by placing it in a vacuum chamber pumped by a vacuum pump.

                                        Ø  Any liquid in contact with an object significantly hotter than its boiling point. Faster cooling may be obtained by plunging an object into a slush of liquid and solid nitrogen rather than liquid nitrogen alone.

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                                          Liquid nitrogen

                                          USES OF LIQUID NITROGEN:

                                          Ø  To store cells a tissue at low temperature for laboratory work.

                                          Ø  The cryopreservation of blood, reproductive cells (sperm and egg), and other biological samples and materials.

                                          Ø  To preserve tissue samples from surgical excisions for future studies.

                                          Ø  To facilitate cry conservation of animal genetic resources.

                                          Ø  For vacuum pump traps and in controlled-evaporation processes in chemistry.

                                          Ø  As a component of cooling baths used for very low-temperature reactions in chemistry.

                                          Ø  Cryogenic preservation in hopes of future reanimation. 

                                          DISADVANTAGE:

                                          Ø  Special containment vessels and Dewar flasks are required to contain it.

                                          Ø  You have to work in a confined space as the nitrogen gas build up can render the air unbreathable.

                                          Ø  It is simpler to transport liquid nitrogen compared to gaseous nitrogen. Liquid nitrogen is pure elemental nitrogen in liquid form.

                                          PRECAUTION:

                                          Ø  Safety goggles (unvented) – Required at all times.

                                          Ø  Face shield – Required when pouring or filling. Insulating gloves, lab coat or long sleeve

                                          CONCLUSION

                                          • In this work to done myself to learn about the various instrumentation & nanotechnology techniques

                                          ACKNOWLEDGEMENTS

                                          I would like to acknowledge all those people who have made this research work possible, an experience that I will cherish forever.

                                          i. First and foremost, I bow my head, as a mark of thanks for giving to our Almighty God, without him nothing is possible.

                                          ii. Then, I am indebted to the Indian Academy of Sciences, Bangalore for giving me this opportunity to work at SRM University, Amaravathi-(AP).

                                          iii. I would like to express my sincere thanks and gratefulness to my supervisor Dr.Anil K. Suresh for his constant inspiration, most valuable guidance & supervision helpful suggestion in carrying out this the tenure of my summer research fellowship programme.

                                          iv. I would like to extend my thanks to all our faculties in the Department of Botany, Bharathidasan University, at Tiruchirappalli.

                                          v. My special thanks to my family and friends who have supported in all possible ways throughout the course of this project.

                                          vi. I thank all the supporting staff of AuthorCafe for providing such an excellent space to write my report and collaborate with my guide.

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