Laboratory Instrumentation - Part One (1) PDF

LABORATORY INSTRUMENTATION & TECHNIQUES - PART ONE (1) DAVID NTIAMOAH OFOSU Department of Medical Laboratory Science LECTURER UNIVERSITY OF ENERGY AND NATURAL RESOURCES  Laboratory Instrumentation ❑ Laboratory instrumentation is...

LABORATORY INSTRUMENTATION & TECHNIQUES - PART ONE (1) DAVID NTIAMOAH OFOSU Department of Medical Laboratory Science LECTURER UNIVERSITY OF ENERGY AND NATURAL RESOURCES  Laboratory Instrumentation ❑ Laboratory instrumentation is the use or application of instruments for observation, measurement, or control. ❑ It involves the use of or operation with instruments; especially: the use of one or more instruments in carrying out laboratory tests. Instrumentation is the development or use of measuring instruments for observation, monitoring or control ❑ It could also include: "The design, construction, and provision of instruments for measurement, control, etc; the state of being equipped with or controlled by such instruments collectively." 2  Laboratory Instruments ❑ Laboratory Instrument is any implement, tool, or utensil used for laboratory test. ❑ An instrument is a device that measures a physical quantity, such as flow, concentration, temperature, level, distance, angle, or pressure. ❑ Medical instrument is a device used to diagnose or treat diseases. ❑ Instruments may be simple or complex 3  Laboratory Equipment ❑ Laboratory equipment refers to the various tools and equipment used by scientists working in a laboratory. It involves the measuring tools used in a scientific laboratory, often electronic in nature. ❑ It is generally used to either perform an experiment or to take measurements and gather data. ❑ Larger or more sophisticated equipment is generally called a scientific instrument ❑ The classical equipment includes tools such as Bunsen burners and microscopes as well as specialty equipment such as operant conditioning chambers, spectrophotometers and calorimeters. 4  Laboratory Techniques ❑ Laboratory techniques are the sum of procedures used on pure and applied sciences in order to conduct an experiment ❑ All of them follow scientific method; while some of them involves the use of complex laboratory equipment from laboratory glassware to electrical devices others require such specific or expensive supplies. 5  Laboratory Apparatus ❑ Laboratory apparatus is a set of equipment or tools or a machine that is used for a particular purpose ❑ It involves the individual instruments or pieces of equipment, or the entire set of equipment to conduct projects and experiments. ❑ The laboratory apparatus depends upon the type of laboratory you are in and the experiment you are going to perform. 6  Laboratory Tools ❑ Laboratory tool is any physical item that can be used to achieve a goal, especially if the item is not consumed in the process. ❑ Tools that are used in particular fields or activities may have different designations such as "instrument", "utensil", "implement", "machine", "device," or "apparatus". ❑ The set of tools needed to achieve a goal is "equipment". The knowledge of constructing, obtaining and using tools is technology. 7  THE AUTOCLAVE ❑An autoclave is essentially just a large steel vessel through which steam or another gas is circulated to sterilize things, perform scientific experiments, or carry out industrial processes. ❑Typically the chambers in autoclaves are cylindrical, because cylinders are better able to withstand extreme pressures than boxes, whose edges become points of weakness that can break. ❑The high-pressure makes them self-sealing (the words "auto" and "clave" mean automatic locking), though for safety reasons most are also sealed manually from outside. ❑Just like on a pressure cooker, a safety valve ensures that the steam pressure cannot build up to a dangerous level. 8 THE AUTOCLAVE 9  Cont... 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. Prions, such as those associated with Creutzfeldt- Jakob disease, may not be destroyed by autoclaving at the typical 134 °C for three minutes or 121°C for 15 minutes. Although archaea are known to withstand high temperature above 120 °C, no archaea is known to pose any health risk to man. This is because their biochemistry is so vastly different from our own and their multiplication rate is far too slow 10  Cont... ❑ Because damp heat is used, heat-labile products (such as some plastics) cannot be sterilized this way or they will melt ❑ Autoclaving is often used to sterilize medical waste prior to disposal in the standard municipal solid waste stream ❑ In dentistry, autoclaves provide sterilization of dental instruments according to health technical memorandum 01-05 (HTM01-05). ❑ According to HTM01-05, instruments can be kept, once sterilized using a vacuum autoclave for up to 12 months using sealed pouches 11  Working Principle of an autoclave ❑It is a large pressure cooker that operates by using steam under pressure as sterilizing agent ❑Most of the heating power of steam comes from its latent heat of vaporization which is the amount of heat required to convert boiling water to steam ❑Steam at 100ºC has almost seven times more heat than water at 100ºC 12  Cont… ❑ Steam is able to penetrate objects with cooler temperatures ❑ This is because, once the steam contacts a cooler surface, it immediately condenses to water, producing a concomitant 1,870 fold decrease in steam volume. ❑ This then creates negative pressure at the point of condensation and draws more steam to the area. ❑ Condensations continue so long as the temperature of the condensing surface is less than that of steam; once temperatures equilibrate, a saturated steam environment is formed. 13  How does killing occur? ❑ Moist heat is thought to kill microorganisms by causing coagulation of essential proteins. ❑ When heat is used as a sterilizing agent, the vibratory motion of every molecule of a microorganism is increased to levels that induce the cleavage of intramolecular hydrogen bonds between proteins. ❑ Death is therefore caused by an accumulation of irreversible damage to all metabolic functions of the organism. 14  Standard Temperature and Pressure for an autoclave Processes conducted at high temperatures for short time periods are preferred over lower temperatures for longer times. Some standard temperatures/pressures employed are 115 °C /10 p.s.i., 121 °C / 15 p.s.i., and 132 °C /27 p.s.i. (psi=pounds per square inch) In our university autoclave, autoclaving generally involves heating in saturated steam under a pressure of approximately 15 psi, to achieve a chamber temperature of a least 121°C (250 °F)but in other applications in industry, for example, other combinations of time and temperature are sometimes used. 15  How does the autoclave itself work? ❑ Steam enters the chamber jacket, passes through an operating valve and enters the rear of the chamber behind a baffle plate. ❑ It flows forward and down through the chamber and the load, exiting at the front bottom. ❑ A pressure regulator maintains jacket and chamber pressure at a minimum of 15 psi, the pressure required for steam to reach 121°C (250°F). ❑ Overpressure protection is provided by a safety valve. ❑ The conditions inside are thermostatically controlled so that heat (more steam) is applied until 121°C is achieved, at which time the timer starts, and the temperature is maintained for the selected time. 16 17 Test for the efficacy of an Autoclave(quality assurance) ❑ Chemical indicators on medical packaging on autoclave tape changes colour to signify appropriate processing of items inside. ❑ Autoclave tape is only a marker that steam and heat have activated the dye. The marker on the tape does not indicate complete sterility. ❑ A more difficult challenge device, named the Bowie-Dick device after its inventors, is also used to verify a full cycle. This contains a full sheet of chemical indicator placed in the center of a stack of paper ❑ To prove sterility, biological indicators which contains spores of bacterium Geobacillus stearothermophilus is used. If right temperature isn’t achieved, the spores will germinate and their metabolism will change the color of a pH- sensitive chemical 18  Application of an autoclave ❑ Autoclaves are widely used in microbiology, medicine, podiatry, tattooing, body piercing, veterinary science, mycology, funeral homes, dentistry, and prosthetics fabrication. ❑ A notable recent and increasingly popular application of autoclaves is the pre- disposal treatment and sterilization of waste material, such as pathogenic hospital waste ❑ Autoclaves are also widely used to cure composites and in the vulcanization of rubber. ❑ The aerospace industry and sparmakers (for sailboats in particular) have autoclaves well over 50 feet (15 m) long, some over 10 feet (3.0 m) wide. ❑ Synthetic quartz crystals used in the electronic industry are grown in autoclaves. ❑ Packing of parachutes for specialist applications may be performed under vacuum in an autoclave which allows the parachute to be warmed and inserted into the minimum volume. 19 CENTRIFUGES 20  INTRODUCTION ❑ A laboratory centrifuge is a piece of laboratory equipment, driven by a motor, which spins liquid samples at high speed. ❑ Like all other centrifuges, laboratory centrifuges work by the sedimentation principle, where the centripetal acceleration is used to separate substances of greater and lesser density. ❑ A centrifuge is a device for separating two or more substances from each other by using centrifugal force. ❑ Centrifugal force is the tendency of an object traveling around a central point to continue in a linear motion and fly away from that central point. ❑ Materials with different masses experience different centrifugal forces when traveling at the same velocity and at the same distance from the common center 21  TYPES OF CENTRIFUGES ❑Microcentrifuges (devices for small tubes from 0.2 ml to 2.0 ml (micro tubes), up to 96 well-plates, compact design, small footprint; up to 30,000 g) Clinical centrifuges (moderate-speed devices used for clinical applications like blood collection tubes) ❑Multipurpose high-speed centrifuges (devices for a broad range of tube sizes, high variability, big footprint) ❑Ultracentrifuges (analytical and preparative models) Because of the heat generated by air friction (even in ultracentrifuges, where the rotor operates in a good vacuum), and the frequent necessity of maintaining samples at a given temperature, many types of laboratory centrifuges are refrigerated and temperature regulated. 22  CENTRIFUGATION ❑A centrifuge is used to separate particles or macromolecules: -Cells -Sub- cellular components -Proteins -Nucleic acids Basis of separation: -Size - Shape -Density ❑Methodology: -Utilizes density difference between the particles/macromolecules and the medium in which these are dispersed - Dispersed systems are subjected to artificially induced gravitational fields PRINCIPLE OF CENTRIFUGATION ❑A centrifuge seperates particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. In a solution, particles whose density is higher than that of the solvent sink (sediment),and particles that are lighter than it float to the top. 23  CENTRIFUGE ROTORS 1. Fixed Angle Rotor Sedimenting particles have only short distance to travel before pelleting. Shorter run time. The most widely used rotor type. 24 2. Swinging Bucket Rotor Longer distance of travel may allow better separation, such as in density gradient centrifugation. Easier to withdraw supernatant without disturbing pellet. 25  CARE AND MAINTENANCE OF CENTRIFUGES MECHANICAL STRESS ❑ Always ensure that loads are evenly balanced before a run. ❑ Always observe the manufacturers maximum speed and sample density ratings. ❑ Always observe speed reductions when running high density solutions, plastic adapters, or stainless steel tubes. ❑ The combination of stress and corrosion causes the rotor to fail more quickly and at lower stress levels than an uncorroded rotor so CLEAN regularly. 26 ELECTRONIC BALANCE (WEIGHING SCALE) 27  INTRODUCTION ❑ Balances are designed to meet the specific weighing requirement in the laboratory working environment ❑ The range offered includes Analytical Balances, General Purpose Electronic Balance, Laboratory Balances and Precision Weighing Balances. These balances come in precision designs and operating characteristics that allows making quick and accurate measurements. ❑ The history of balances and scales dates back to Ancient Egypt. A simplistic equal- arm balance on a fulcrum that compared two masses was the standard. ❑ Today, scales are much more complicated and have a multitude of uses. Applications range from laboratory weighing of chemicals to weighing of packages for shipping purposes. 28  MASS AND WEIGHT ❑ Mass is a constant unit of the amount of matter an object possesses. It stays the same no matter where the measurement is taken. The most common units for mass are the kilogram OR gram. ❑ Weight is the heaviness of an item. It is dependent on the gravity on the item multiplied by the mass, which is constant. The weight of an object on the top of a mountain will be less than the weight of the same object at the bottom due to gravity variations. A unit of measurement for weight is the Newton. A newton takes into account the mass of an object and the relative gravity and gives the total force, which is weight. ❑ Although mass and weight are two different entities, the process of determining both weight and mass is called weighing. 29  BALANCE AND SCALE TERMS ❑ Accuracy: The ability of a scale to provide a result that is as close as possible to the actual value. The best modern balances have an accuracy of better than one part in 100 million when one-kilogram masses are compared. ❑ Calibration: The comparison between the output of a scale or balance against a standard value. Usually done with a standard known weight and adjusted so the instrument gives a reading in agreement. ❑ Capacity: The heaviest load that can be measured on the instrument. ❑ Precision: Amount of agreement between repeated measurements of the same quantity; also known as repeatability. Note: A scale can be extremely precise but not necessarily be accurate. ❑ Readability: This is the smallest division at which the scale can be read. It can vary as much as 0.1g to 0.0000001g. Readability designates the number of places after the decimal point that the scale can be read. ❑ Tare/Zero: The act of removing a known weight of an object, usually the weighing container, to zero a scale. This means that the final reading will be of the material to be weighed and will not reflect the weight of the container. Most balances allow taring to 100% of capacity. 30 BALANCE AND SCALE TYPES 1. Analytical Balance These are most often found in a laboratory or places where extreme sensitivity is needed for the weighing of items. Analytical balances measure mass ranges from 1 g to a few kilograms with precision and accuracy often exceeding one part in 106 at full capacity. Important parts of an analytical balance includes: A beam arrest which is a mechanical device that prevents damage to the delicate internal devices when objects are being placed or removed from the pan. The pan is the area on a balance where an object is placed to be weighed. Leveling feet are adjustable legs that allow the balance to be brought to the reference position. Analytical balances are so sensitive that even air currents can affect the measurement. To protect against this they must be covered by a draft shield. 31 2. EQUAL ARM BALANCE/TRIP BALANCE ❑This is the modern version of the ancient Egyptian scales. This scale incorporates two pans on opposite sides of a lever. It can be used in two different ways. ❑The object to be weighed can be placed on one side and standard weights are added to the other pan until the pans are balanced. The sum of the standard weights equals the mass of the object. ❑Another application for the scale is to place two items on each scale and adjust one side until both pans are leveled. ❑This is convenient in applications such as balancing tubes or centrifugation where two objects must be the exact same weight. 32 3.PLATFORM SCALE ❑This type of scale uses a system of multiplying levers. It allows a heavy object to be placed on a load bearing platform. ❑The weight is then transmitted to a beam that can be balanced by moving a counterpoise, which is an element of the scale that counterbalances the weight on the platform. ❑This form of scale is used for applications such as the weighing of drums or even the weighing of animals in a veterinary office. 33 4. SPRING BALANCE ❑This balance utilizes Hooke's Law which states that the stress in the spring is proportional to the strain. ❑Spring balances consist of a highly elastic helical spring of hard steel suspended from a fixed point. ❑The weighing pan is attached at the lowest point of the spring. ❑An indicator shows the weight measurement and no manual adjustment of weights is necessary. An example of this type of balance would be the scale used in a grocery store to weigh produce. 34 5. TOP-LOADING BALANCE ❑This is another balance used primarily in a laboratory setting. They usually can measure objects weighing around 150– 5000 g. ❑They offer less readability than an analytical balance, but allow measurements to be made quickly thus making it a more convenient choice when exact measurements are not needed. ❑Top-loaders are also more economical than analytical balances. Modern top-loading balances are electric and give a digital readout in seconds. 35 6. TORSION BALANCE ❑Measurements are based on the amount of twisting of a wire or fiber ❑Many microbalances and ultra- microbalances, that weigh fractional gram values, are torsion balances. ❑A common fiber type is quartz crystal. 36 7. TRIPLE-BEAM BALANCE ❑This type of balance is less sensitive than a top- loading balance. They are often used in a classroom situation because of ease of use, durability and cost. ❑They are called triple-beam balances because they have three decades of weights that slide along individually calibrated scales. ❑The three decades are usually in graduations of 100g, 10g and 1g. These scales offer much less readability but are adequate for many weighing applications. 37  8. PRECISION WEIGHING BALANCES ❑ They are laboratory standard high precision balances that are based on latest process technology and features best displayed increment of 0.001g (1mg) with maximum capacity available. ❑These perfectly match up the applications demanding more than a standard balance and assist in simplifying complex laboratory measurements including in determining difference between initial & residual weights. Here, the calculation of the density of solids & liquids also eliminates need for time consuming manual calculation and data logging. ❑The standard features include protective in-use cover and security bracket, working capacities from 0.1 mg to 230 gm, pan size of 90 mm, ACC of 0.1 mg, internal calibration, display using LCD with back light, standard RS-232 C interface and hanger for below balance weighing. 38  CARE AND USE OF BALANCES AND SCALES 1. Items to be measured should be at room temperature before weighing. A hot item will give a reading less than the actual weight due to convection currents that make the item more buoyant 2. Clean the balance regularly to prevent corrosion of metal surfaces 3. It is important to always check the previous usage of the balance to prevent contaminating yourself with harmful chemicals or prevent incompatible chemicals from reacting. 4. To avoid damaging the scale or putting others in danger, the balance should be kept extremely clean. A camel's hair brush can be used to remove any dust that can spill over during weighing. 5. Use the correct weight sets to calibrate the balance regularly at least once a year. 39 LABORATORY WATER BATHS 40  INTRODUCTION ❑ Water retains heat so well, using water baths was one of the very first means of incubation. ❑ Applications include sample thawing, bacteriological examinations, warming reagents, coliform determinations and microbiological assays. ❑ Boiling baths will boil water at 100oC (under normal conditions). Any baths that work above 100oC will need a liquid in them such as oil. Any baths that work below ambient will need an internal or external cooling system ❑ Laboratory water baths working below 4oC should be filled with a liquid which does not freeze. 41  TYPES OF WATER BATH 1. Unstirred water baths are the cheapest laboratory baths and have the least accurate temperature control because the water is only circulated by convection and so is not uniformly heated. 2. Stirred water baths have more accurate temperature control. They can either have an in-built pump/circulator or a removable immersion thermostat / circulator (some of which can pump the bath liquid externally into an instrument and back into the bath). 3. Circulating Water Baths (also called stirrers ) 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. 42  TYPES OF WATER BATH 4. Non-Circulating Water Baths This type of water bath relies primarily on convection instead of water being 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. 5. Shaking water baths have a speed controlled shaking platform tray (usually reciprocal motion i.e. back and forwards, although orbital motion is available with some brands) to which adaptors can be added to hold different vessels. 6. Cooled water baths are available as either an integrated system with the cooling system (compressor, condenser, etc.) built into the laboratory water baths or using a standard water bath as above using an immersion thermostat / circulator with a separate cooling system such as an immersion coil or liquid circulated from a circulating cooler. The immersion thermostat used must be capable of controlling at the below ambient temperature you require. 43  CONSTRUCTION AND DIMENSIONS: ❑Laboratory water baths usually have stainless steel interiors and either chemically resistant plastic or epoxy coated steel exteriors. Controllers are either analogue or digital. ❑Bath dimensions can be a bit misleading when litre capacity is quoted because it depends how high you measure (water baths are never filled to the top). To compare different bath volumes it is best to compare the internal tank dimensions. LABORATORY WATER BATH ACCESSORIES: ❑Lift-off or hinged plastic (depending on bath temperature) or stainless steel lids are available as well as different racks to hold tubes, etc. ❑Lids with holes with concentric rings are available for boiling water baths to hold different size flasks. 44  CARE AND MAINTENANCE 1. It is not recommended to use water bath with moisture sensitive or pyrophoric reactions. Do not heat a bath fluid above its flash point.\ 2. Water level should be regularly monitored, and filled with distilled water only. This is required to prevent salts from depositing on the heater. 3. Disinfectants can be added to prevent growth of organisms 4. Raise the temperature to 90 °C or higher to once a week for half an hour for the purpose of decontamination. 5. Markers tend to come off easily in water baths. Use water resistant ones 6. If application involves liquids that give off fumes, it is recommended to operate water bath in fume hood or in a well-ventilated area. 7. The cover is closed to prevent evaporation and to help reaching high temperatures. 8. Set up on a steady surface away from flammable materials. 45 46 ANAEROBIC JARS 47 48 49  METHOD OF USE ❑ The culture: The culture media are placed inside the jar, stacked up one on the other, and The Indicator system: Pseudomonas aeruginosa (aerobic bacteria), inoculated on to a nutrient agar plate is kept inside the jar along with the other plates. A growth free culture plate at the end of the process indicates a successful anaerobiosis. However, P. aeruginosa possesses a denitrification pathway. If nitrate is present in the media, P. aeruginosa may still grow under anaerobic conditions. ❑ 6/7ths of the air inside is pumped out and replaced with either unmixed Hydrogen or as a 10%CO2 + 90%H2 mixture. The catalyst (Palladium) acts and the oxygen is used up in forming water with the hydrogen. The manometer registers this as a fall in the internal pressure of the jar. ❑ Hydrogen is pumped in to fill up the jar so that the pressure inside equals atmospheric pressure. The jar is now incubated at desired temperature settings. 50  DESCRIPTION The jar (McIntosh and Filde's anaerobic jar), about 20×12.5″ is made up of a metal. Its parts are as follows: 1. The body made up of metal (airtight) 2. The lid, also metal can be placed in an airtight fashion 3. A screw going through a curved metal strip to secure and hold the lid in place 4. A thermometer to measuring the internal temperature 5. A pressure gauge to measuring the internal pressure (or a side tube is attached to a manometer) 6. Another side tube for evacuation and introduction of gases (to a gas cylinder or a vacuum pump) 7. A wire cage hanging from the lid to hold a catalyst that makes hydrogen react to oxygen without the need of any ignition source 51  GAS-PAK ❑ Gas-pak is a method used in the production of an anaerobic environment. It is used to culture bacteria which die or fail to grow in presence of oxygen (anaerobes). ❑ These are commercially available, disposable sachets containing a dry powder or pellets, which, when mixed with water and kept in an appropriately sized airtight jar, produce an atmosphere free of elemental oxygen gas (O2). ❑ They are used to produce an anaerobic culture in microbiology. It is a much simpler technique than the McIntosh and Filde's anaerobic jar where one needs to pump gases in and out. 52  CONSTITUENTS OF GAS-PAK SACHETS 1. Sodium borohydride - NaBH4 2. Sodium bicarbonate - NaHCO3 3. Citric acid - C3H5O(COOH)3 4. Cobalt chloride - CoCl2 (catalyst) 5. The addition of a Dicot Catalyst maybe required to initiate the reaction. Reactions 1. NaBH4 + 2 H2O = NaBO2 + 4 H2↑ 2. C3H5O(COOH)3 + 3 NaHCO3 + [CoCl2] → C3H5O(COONa)3 + 3 CO2 + 3 H2 + [CoCl2] 3. 2 H2 + O2 + [Catalyst] = 2 H2O + [Catalyst] 53  CONT. Consumption of oxygen These chemicals react with water to produce hydrogen and carbon dioxide along with sodium citrate and water (C3H5O(COONa)3) as byproducts. Again, hydrogen and oxygen reacting on a catalyst like Palladiumised alumina (supplied separately) combine to form water. Culture method The medium, the gas-pak sachet (opened and with water added) and an indicator are placed in an air-tight gas jar which is incubated at the desired temperature. The indicator tells whether the environment was indeed oxygen free or not. The chemical indicator generally used for this purpose is "chemical methylene blue solution" that since synthesis has never been exposed to elemental oxygen. It is colored deep blue on oxidation in presence of atmospheric oxygen in the jar, but will become colorless when oxygen is gone, and anaerobic conditions are achieved. 54 COLORIMETER 55  INTRODUCTION ❑A colorimeter is a device used for measuring colours, or colorimetry. It measures the absorbance of different wavelengths of light in a solution. It can be used to measure the concentration of a known solute. ❑It compares the amount of light getting through a solution with the amount that can get through a sample of pure solvent. ❑A colorimeter contains a photocell is able to detect the amount of light which passes through the solution under investigation. ❑A colorimeter measures that change so users can analyze the concentration of a particular substance in that medium. ❑The device works on the basis of Beer-Lambert's law, which states that the absorption of light transmitted through a medium is directly proportional to the concentration of the medium. 56  TYPES OF COLORIMETRY 1. Colour densitometers, which measure the density of primary colours. 2. Colour photometers, which measure the colour reflection and transmission. DESIGN OF A CALORIMETER ❑The three main components of a colorimeter are a light source, a cuvette containing the sample solution, and a photocell for detecting the light passed through the solution. WORKING PRINCIPLE ❑The colorimeter is based on Beer-Lambert's law, according to which the absorption of light transmitted through the medium is directly proportional to the medium concentration. 57  APPLICATION 1. Colorimeters are widely used to monitor the growth of a bacterial or yeast culture. 2. They provide reliable and highly accurate results when used for the assessment of color in bird plumage. 3. They are used to measure and monitor the color in various foods and beverages, including vegetable products and sugar. 4. Certain colorimeters can measure the colors that are used in copy machines, fax machines and printers. 5. They have many practical applications such as testing water quality by screening chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine. 6. They are also used to determine the concentrations of plant nutrients such as ammonia, nitrate and phosphorus in soil or hemoglobin in blood. 58  OPERATING INSTRUCTIONS FOR A TYPICAL COLORIMETER 1. Switch on the instrument at least 5 minutes before use to allow it to stabilize. 2. Select the most appropriate filter for the analysis and insert it in the light path (or dial it in with the selector) 3. Place the reagent blank solution (or water) in the cuvette and zero the instrument (consult your manufacturers instruction about how to do this.) Make sure the clear faces of the cuvette are in the light path 4. Place the sample in the colorimeter and read the absorbance of the solution. If the absorbance is "over range" (usually > 2.0) then the sample must be diluted to yield a value within the limits of the instrument. 5. At intervals, recheck the reagent blank to ensure that there is no drift in the zero value. 59  SPECTROPHOTOMETRY ❑A spectrophotometer consists a spectrometer for producing light of any selected colour (wavelength), and a photometer for measuring the intensity of light ❑It involves the quantitative estimation of colours. ❑According to Beer’s law when monochromatic light passes through the coloured solution, the amount of light transmitted decreases exponentially with increase in concentration (thickness) of the coloured substance. Apparatus Parts and functions ✓ Light source ✓ Filter (the device that selects the desired wavelength) ✓ Cuvette chamber (where the sample to be tested is placed) ✓ Detector (photosensitive element that converts light into electrical signals) ✓ Galvanometer (measures electrical signal quantitatively) 60 61  CHOOSING THE WAVELENGTH OF ABSORPTION S/N COLOR OF SOLUTION COLOUR ABSORBED WAVELENGTH OF ABSORPTION 1 Yellow to Green Violet 400nm – 435nm 2 Yellow to Green Blue 435nm – 490nm 3 Red Blue to Green 490nm – 500nm 4 Purple Green 500nm – 560nm 5 Violet Yellow to Green 560nm – 580nm 6 Blue to Green Yellow to Orange 580nm – 650nm 7 Bluish Green Red 650nm – 700nm 62  FLAME PHOTOMETERS ❑In flame photometry, a branch of atomic spectroscopy also called “flame atomic emission spectrometry,” atoms are examined in the spectrometer. ❑This technique is suited to the quantitative and qualitative determination of a variety of cations—particularly for alkali and alkaline earth metals— since they are excited to higher levels of energy at low flame temperatures ❑Flame photometry is a process where in emission of radiation by neutral atoms is measured. ❑The neutral atoms are obtained by introduction of sample into flame. Hence the name flame photometry. Since radiation is emitted it is also called as flame emission spectroscopy. 63 64  WORKING PRINCIPLE OF FLAME PHOTOMETER ❑When a solution of metallic salt is sprayed as fine droplets into a flame. Due to heat of the flame, the droplets dry leaving a fine residue of salt. This fine residue converts into neutral atoms. ❑Due to the thermal energy of the flame, the atoms get excited and there after return to ground state. In this process of return to ground state, exited atoms emit radiation of specific wavelength. This wavelength of radiation emitted is specific for every element. ❑This specificity of wavelength of light emitted makes it a qualitative aspect. While the intensity of radiation, depends on the concentration of element. This makes it a quantitative aspect. ❑The process seems to be simple and applicable to all elements. But in practice only a few elements of Group IA and group IIA (like Li, Na, k & Ca, Mg) are only analyzed.4 ❑The radiation emitted in the process is of specific wavelength. Like for Sodium (Na) 589nm yellow radiation, Potassium 767nm range radiation. 65  FLAME PHOTOMETER INSTRUMENTATION 1. Burner: This is a part which produces excited atoms. 2. Fuel and oxidant are required to produce flame such that the sample converts to neutral atoms and get excited by heat energy. Examples include: Fuel + Oxidant Temperature of Flame ✓ Propane +Air 2100 ºC ✓ Propane +Oxygen 2800 ºC ✓ Hydrogen + Air 1900 ºC ✓ Hydrogen + Oxygen 2800 ºC ✓ Acetylene + Air 2200 ºC ✓ Acetylene + Oxygen 3000 ºc 3. Monochromators: Filters and monochromators are needed to isolate the light of specific wavelength from remaining light of the flame. 66  CONT… 1. Detector: Flame photometric detector is similar to that used in spectrophotometry. The emitted radiation is in the visible region i.e. 400nm to 700nm. Further the radiation is specific for each element so simple detectors are sufficient for the purpose like photo voltaic cells, photo tubes etc. 2. Recorders and display: These are the devices to read out the recording from detectors. 67  FLAME PHOTOMETRY LIMITATIONS Unlike other spectroscopy methods, flame photometry finds little use in research and analysis. This is due to 1. Limited number of elements that can be analyzed. 2. The sample requires to be introduced as solution into fine droplets. Many metallic salts, soil, plant and other compounds are insoluble in common solvents. Hence, they can’t be analyzed by this method. 3. Since sample is volatilized, if small amount of sample is present, it is tough to analyze by this method. As some of it gets wasted by vaporization. 4. Further during solubilisation with solvents, other impurities might mix up with sample and may lead to errors in the spectra observed. 68  ION SELECTIVE ELECTRODES AND POTENTIOMETRY 69 Equipment needed for Iron Selective electrode ❑One Ion-Selective electrode and one Reference electrode are inserted into a dual electrode head. ❑The head, one temperature sensor and one pH electrode are connected to a 2-channel electrode-computer interface. ❑The interface is connected to a serial or USB port of a computer running the 2- channel measurement software. ❑The computer screen shows the calibration graph for a potentiometric ammonium measurement, with sample results plotted on it. 70  APPLICATIONS OF ISE Ion-selective electrodes are used for determining the concentrations of various ions in aqueous solutions. The following is a list of some of the main areas in which ISEs have been used. 1. Pollution Monitoring: CN, F, S, Cl, NO3 etc., in effluents, and natural waters. 2. Agriculture: NO3, Cl, NH4, K, Ca, I, CN in soils, plant material, fertilisers and feedstuffs. 3. Salt content of meat, fish, dairy products, fruit juices, brewing solutions. F in drinking water and other drinks. 4. Ca in dairy products and beer. 5. K in fruit juices and wine making. Corrosive effect of NO3 in canned foods. 6. Detergent Manufacture: Ca, Ba, F for studying effects on water quality. Paper 7. Manufacture: S and Cl in pulping and recovery-cycle liquors. 8. Explosives: F, Cl, NO3 in explosive materials and combustion products. 9. Biomedical Laboratories: Ca, K, Cl in body fluids (blood, plasma, serum, sweat). 10. F in skeletal and dental studies. 71 Simple laboratory set-up for Ion Selective Electrode calibration and measurement 72 73 HOT AIR OVEN 74  INTRODUCTION ❑Hot air oven is used for sterilization by providing the dry heat. They were originally developed by Pasteur. ❑The oven uses dry heat to sterilize articles. Generally, they can be operated from 50 to 300ºC (122 to 572ºF). ❑There is a thermostat controlling the temperature. These are digitally controlled to maintain the temperature. ❑Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outer layer being metallic. ❑It is used in the sterilization of pharmaceutical products and other materials. It is double walled chamber made of steel. 75  TYPES OF OVEN 1. Laboratory Oven. 2. High Temperature Lab Oven. 3. Industrial Oven. 4. Top Loading Annealing Oven. 5. Pharmaceutical Oven. 6. Vacuum Oven. 7. Bench Oven. Hot air ovens are also referred to as forced air thermal convection ovens 76  LABORATORY OVENS ❑ These hot air ovens are used to attain constant temperatures inside every corner of the oven chamber. ❑ The most common size of Laboratory oven being used has (24 x 24 x 24) inches Inner Chamber dimensions, although they start from (12 x 12 x 12) inches Inner Chamber Dimension. ❑ The Maximum temperature for Laboratory Ovens can vary from 100ºC to over 350°C, However 250ºC is the most preferred range as it falls midway and is suitable for most lab applications. ❑ These can also be termed as Clean Room Ovens. 77  HIGH TEMPERATURE LAB OVENS ❑This is an advanced version for Laboratory oven as discussed above having the same principle of operation i.e through forced air thermal convection. ❑The only difference in High Temperature Lab Oven is the temperature range, these ovens are generally classified as ovens having temperatures from 300ºC to 550ºC control unit ensure a homogenous temperature profile in the chamber, precise course of processes and short recovery time (return to the preset temperature) after door opening. ❑This line of drying ovens is characterized by its economical operation. It is suitable for simple process of drying and heating of common materials. The units work noiseless. 78  WORKING PRINCIPLE The working of the hot air oven is based on the hot air inside the chamber of oven by the forced circulation. As it is a universal scientific fact that in any chamber hot air rises above, So by utilizing this principle when the hot air reaches the top of chamber it is circulated back to bottom by a fan installed inside the chamber and hence optimum amount of heat is achieved gradually inside the hot air oven. After heating the content of the oven for two hours at 160 @c, the articles are allowed to remain there, till the temperature comes down to 40 @c. then the sterilized materials is then removed from the oven. 79  APPLICATIONS ❑It is mainly used for the sterilization of glasswares such as pestle and motar, petridishes, flasks, pipettes, bottles, test tubes etc. ❑It is used for the sterilization of powders such as sulphacetamides, sulphadiazenes, kaolin, zinc oxide, starch etc. ❑Injections where fixedoils is used as the vehicle are sterilised by the dry heat method. Example: injections of progestrone, injection of testosterone propionate and injections of oestradiols dipropionate. ❑It is also used for sterilisation of scalpels, scissors, spatula, blades and glass syringes. ❑The chemicals, glassware in laboratories, research institutions, industries, hospitals use hot air ovens are suitable for temperature up to 250degC. ❑Hot air ovens suits to various applications like heating, drying, sterilizing & baking. 80  ADVANTAGES 1. It is used for the sterilization of those substances which gets spolied during moist heat sterilization. Eg: oily materials and powders. 2. The method is suitable for sterilization of assembled equipment such as all glas syringes due to expose to high temperature for a long time. 3. It is not so damaging to glass and metals equipment as moist heat. 4. Dry heat will not corrode or rust instruments or needles. 5. Dry heat will sterilize instruments containing many parts that can not be disassembled 81  DISADVANTAGES 1. This method is not suitable for the surgical dressings. 2. This method is not suitable for the most of the medicaments, rubber and plastic good because the articles are exposed to a very high temperature for a long period. 3. Dry heat penetrates slowly and unevenly. 4. Dry heat requires long exposure times to effectively achieve sterility. 5. Dry heat requires higher temperatures that many items cannot be safely exposed to. 6. Dry heat requires specialized packaging materials that can sustain integrity under high heat conditions. 7. Dry heat may require different temperature and exposure times, depending on the type of item being sterilized. 82  PRECAUTIONS ❑Glass apparatus must be wrapped with the clean cloth or filter paper and containers must be plugged with non absorbents cotton wool. ❑The article and substances which are to be sterilised should not be placed at the floor of the oven as it receives direct heat and becomes much hotter. ❑The oven should not be over loaded with the materials meant for sterilisation. ❑There should be sufficient space in between the articles, so that there is uniform distribution of heat. 83  LABORATORY REFRIGERATOR 84  HOW DOES A REFRIGERATOR WORK? ❑ In the refrigeration cycle, there are five basic components: fluid refrigerant; a compressor, which controls the flow of refrigerant; the condenser coils (on the outside of the fridge); the evaporator coils (on the inside of the fridge); and something called an expansion device. Here’s how they interact to cool your food. ❑ Refrigeration is the removal and relocation of heat. ❑ The compressor constricts the refrigerant vapor, raising its pressure, and pushes it into the coils on the outside of the refrigerator. ❑ When the hot gas in the coils meets the cooler air temperature of the kitchen, it becomes a liquid. ❑ Now in liquid form at high pressure, the refrigerant cools down as it flows into the coils inside the freezer and the fridge. ❑ The refrigerant absorbs the heat inside the fridge, cooling down the air and the refrigerant evaporates to a gas, then flows back to the compressor, where the cycle starts all over 85  MAIN COMPONENTS There are 4 main components in a mechanical refrigeration system. Any components beyond these basic 4 are called accessories. 86  PURPOSE OF REFRIGERATION UNITS Storage of ❑ Reagents ❑ Stock cultures ❑ Media ❑ Patient specimens ❑ Blood and its derivatives ❑ Biological fluids ❑Never store food or drink for human consumption in a laboratory refrigerator ❑Always label and warning signs to potentially hazardous substances. 87 LABORATORY MIXER 88  INTRODUCTION ❑A laboratory mixer allows for mixing of smaller quantities of material, typically up to 100 gallons (380 liters). ❑Lab mixers are capable of handling solutions with viscosities up to 150,000 cps, which is dependent on the torque, horsepower, and speed of the mixer. ❑Laboratory mixers are also available with the following options: 1. Speeds up to 10,000 rpm 2. Through-shaft design for easy shaft and propeller adjustment 3. Digital display of speed, torque, and timer functions 4. Sample light for easy viewing during low light conditions 5. Remote controller for easy adjustment from up to six feet away 6. RS232 connectivity for easy data collection 89  CONT. NB: When considering your laboratory mixing needs, consideration has to be given to a wide range of factors including: 1. Container capacity 2. Liquid viscosity 3. Torque 4. Horsepower (hp) 5. Rotational speed (rpm) and diameter of mixing propeller 6. Duty cycle 7. Power supply 90 BLOOD MIXERS/ROLLERS 91 LABORATORY INCUBATOR 92  INTRODUCTION ❑An incubator comprises a transparent chamber and the equipment that regulates its temperature, humidity, and ventilation. ❑The first incubators were used in ancient China and Egypt, where they consisted of fire- heated rooms in which fertilized chicken eggs were placed to hatch, thereby freeing the hens to continue laying eggs. Later, wood stoves and alcohol lamps were used to heat incubators. ❑Today, poultry incubators are large rooms, electrically heated to maintain temperatures between 99.5°F and 100°F (37.5°C and 37.8°C). ❑During the late nineteenth century, physicians began to use incubators to help save the lives of babies born after a gestation period of less than 37 weeks (an optimal human pregnancy lasts 280 days, or 40 weeks). The first infant incubator, heated by kerosene lamps, appeared in 1884 at a Paris women's hospital. ❑In 1933, American Julius H. Hess designed an electrically heated infant incubator (most are still electrically heated today). 93  USES OF THE LABORATORY INCUBATOR ❑After a sample has been obtained, it is transferred to a Petri dish, flask, or some other sterile container and placed in a rack inside the incubator. ❑To promote pathogenic growth, the air inside the chamber is humidified and heated to body temperature (98.6°F or 37°C). ❑Incubators provide the amount of atmospheric carbon dioxide or nitrogen necessary for the cell's growth. As this carefully conditioned air circulates around it, the microorganism multiplies, enabling easier and more certain identification. ❑A related use of incubators is tissue culture, a research technique in which clinicians extract tissue fragments from plants or animals, place these explants in an incubator, and monitor their subsequent growth. ❑Incubators are also used in genetic engineering, an extension of tissue culturing in which scientists manipulate the genetic materials in explants, sometimes combining DNA from discrete sources to create new organisms. 94  DESIGN ❑Like standard refrigerators, incubators are measured in terms of the chamber's volume, which ranges from 5 to 10 cubic feet (1.5 to 3 cubic meters) for countertop models and from 18 to 33 cubic feet (5.5 to 10 cubic meters) for free-standing models. ❑Humidity is generated by heating a small copper bowl that contains limited amounts of purified water; the resulting steam can be introduced into the chamber by means of a control valve. ❑Two types of heat sources are used: electrical heaters that use fans to circulate the warmth they generate, and hot water jackets. ❑Fluorescent and UV (ultra-violet) lamps can be installed separately or in combination. 95  LABORATORY INSTRUMENTATION & TECHNIQUES - PART TWO (2) DAVID NTIAMOAH OFOSU Department of Medical Laboratory Science LECTURER UNIVERSITY OF ENERGY AND NATURAL RESOURCES  MICROTOMES 97  MICROTOMES ❑A microtome is a tool used to cut extremely thin slices of tissues, knownas sections. TYPES: Sliding microtomes Rotary microtomes Cryostat Freezing microtomes Rocking microtomes Ultra-thin section microtomes ❑Most commonly used microtome for routine histopathology is the rotary microtome. 98 99  APPLICATIONS OF MICROTOMES ❑Traditional Histology Technique: Tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 50 µm. ❑Cryosectioning Technique: Water-rich tissues are hardened by freezing and cut in the frozen state with a freezing microtome or microtome- cryostat; sections are stained and examined with a light microscope. This technique is much faster than traditional histology (15 minutes vs 16 hours) ❑Electron Microscopy Technique: After embedding tissues in epoxy resin, a microtome equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically 60 to 100 nanometer). ❑Botanical Microtomy Technique: Hard materials like wood, bone and leather require a sledge microtome. These microtomes have heavier blades and cannot cut as thin as a regular microtome. 100  ROTARY MICROTOME ❑It is most commonly used microtome. This device operates with a staged rotary action such that the actual cutting is part of the rotary motion. ❑The knife is typically fixed in a horizontal position. The block holder or block (depends upon the type of cassette) is mounted on the steel carriage that moves up and down and is advanced by a micrometer screw. ADVANTAGES ❑An advantage over the rocking type is that it is heavier and there by more stable ❑Hard tissues can be cut without vibration. ❑Serial sections or ribbons of sections can easily be obtained. 101  USE OF A ROTARY MICROTOME 10 2  AUTO-CUT MICROTOME It has a built-in motor drive with foot and hand control. With suitable accessories the machine can cut thin sections of paraffin wax blocks and 0.5 to 2.0 micrometer thin resin sections. ADVANTAGES 1. The machine is heavy, so it is stable and does not vibrate during cutting. 2. Serial sections can be obtained. 3. Cutting angle and knife angle can be adjusted. 4. It may also be used for cutting celloidin embedded sections with the helpof special holder to set the knife. 103 Sledge Microtome It is a microtome where the sample is placed into a fixedholder (shuttle), the sledge placed upon a linear bearing, a design that allows for the microtome to readily cut many coarse sections. Applications for this design of microtome are of the preparation of large samples, such as those embedded in paraffin for biological preparations. Typical cut thickness achievable on a sledge microtome is between is 10 and 60 micron. 104  MICROTOME TYPES AND FUNCTIONS Type of Microtome Functions Most common microtome. Used for sectioning of Paraffin embedded blocks. Also can be used for Frozen sections in cryostat and also for resin embeded cases, for example Renal Rotary Microtome biopsies LR white resin embedded tissue and Bone marrows embeddded in methyl methacrylate (MMA). Sectioning occurs by movement of microtome head containing block across blade. Heavy duty microtome that is able to cut celloidin embedded tissue from brain, gelatine Base SledgeMicrotome embedded whole organs using Gough wentworth method and for sectioning undecalcified teeth and bone embedded in resin. Rocking microtome Small microtome that has 2 rocking arms one to cut sections other to feed through tissue block. Limited to sectioning small soft blocks as it uses spring action to cut. Sliding Microtome Unusual design microtome with blade moving over block, rather than block moving. Good for celloidin sectioning, although can produce good paraffin sections. Ultramicrotome Microtome used mainly for Electron microscopy, for example for cutting Epon resin embedded sections. Hand Microtome Very early microtome, with scalpel blade placed above micrometer screw. It is not useful in histology main use for botanical specimens. 105  THE MICROTOME KNIVES ❑Microtome knives are usually developed to fit a particular microtome and to cope with different degrees of hardness of tissue and embedding media. ❑In modern laboratories, the most widely used knife is the disposable steel blade. ❑Resin embedded tissues are sectioned using glass knives and the old steel knives are used for cryostat subjected tissues in histology 106  ENZYME LINKED IMMUNOSORBENT ASSAYS (ELISAs)  PRINCIPLE OF ELISA ❑ELISA is a rapid test used for detecting or quantifying antibody or antigen against viruses, bacteria and other materials. It is so named because the test technique involves the use of an enzyme system and immunosorbent. It is as sensitive as radioimmunoassay (RIA) and requires only microlitre quantities of test reagents. ❑ELISA makes use of basic antigen-antibody interactions to determine the presence or absence of a corresponding antibody or antigen in question in biological samples. The enzyme catalyses (usually hydrolyses) the substrate to give a colour end point. The intensity of the colour gives an indication of the amount of bound antibody or antigen. 108  TYPES OF ELISA There are four major types of ELISA ❑ Direct Elisa ❑ Indirect ELISA ❑ Sandwich ELISA ❑ Competitive ELISA 10  EQUIPMENTS USED IN ELISA Microwell Plate (Flat bottom polystyrene plate, contain 8x12 wells holding 350µL Multichannel Micro-pipette each) (8-channel 100µL) Microplate Washer 110 REAGENTS USED IN ELISA 111 ELISA READER 112  ELISA READER PRINCIPLE ❑The basic principle in Elisa readers are the special filters for only 5-6 standard wavelengths for all Elisa kits (which depends from substrate type). ❑Always check your kit’s instructions with the reader filters (or the substrate electronic absorbance spectrum). For instance, you can measure the maximum to reach the highest sensitivity of your elisa photometer by putting your colored substrate in the plate reader for the absorbance spectra. ❑The Elisa photometers have these filters which fit to almost all substrates commonly used. ELISA READER VS. SPECTROPHOTOMETER ❑The major difference between the Elisa plate reader and the spectrophotometer is that the Elisa readers are commonly used for intensity measurements on a large number of samples where you can also use a very small volume of sample. 113 MICROSCOPY  INTRODUCTION ❑Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye). ❑There are three well- known branches of microscopy: ❑Optical ❑Electron ❑Scanning probe microscopy. ❑Optical and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/electron beams interacting with the specimen, and the collection of the scattered radiation or another signal in order to create an image 115  STEREO MICROSCOPE ❑Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample. ❑The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally. ❑Limitation: This technique can only image dark or strongly refracting objects effectively. The stereo microscope and its parts 116  FLUORESCENCE MICROSCOPE ❑When certain compounds are illuminated with high energy light, they emit light of a lower frequency. This effect is known as fluorescence. Often specimens show their characteristic autofluorescence image, based on their chemical makeup. ❑Many different fluorescent dyes can be used to stain different structures or chemical compounds. One particularly powerful method is the combination of antibodies coupled to a fluorophore as in immunostaining. Examples of commonly used fluorophores are fluorescein or rhodamine. 117  CONFOCAL MICROSCOPE ❑Confocal microscopy uses a scanning point of light and a pinhole to prevent out of focus light from reaching the detector. ❑Compared to full sample illumination, confocal microscopy gives slightly higher resolution, and significantly improves optical sectioning. Confocal microscopy is, therefore, commonly used where 3D structure is important. 118  X-RAY MICROSCOPE ❑As resolution depends on the wavelength of the light. Electron microscopy has been developed since the 1930s that use electron beams instead of light. Because of the much smaller wavelength of the electron beam, resolution is far higher. ❑Though less common, X-ray microscopy has also been developed since the late 1940s. The resolution of X-ray microscopy lies between that of light microscopy and electron microscopy. 119  ELECTRON MICROSCOPE ❑Until the invention of sub-diffraction microscopy, the wavelength of the light limited the resolution of traditional microscopy to around 0.2 micrometers. In order to gain higher resolution, the use of an electron beam with a far smaller wavelength is used in electron microscopes. ❑Transmission electron microscopy (TEM) is quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. The resolution limit in 2005 was around 0.05 nanometer and has not increased appreciably since that time. ❑Scanning electron microscopy (SEM) visualizes details on the surfaces of specimens and gives a very nice 3D view. It gives results much like those of the stereo light microscope. The best resolution for SEM in 2011 was 0.4 nanometer. ❑Electron microscopes equipped for X-ray spectroscopy can provide qualitative and quantitative elemental analysis. 120 THE ELECTRON MICROSCOPE 121 Functional parts of the electron Microscope  Scanning probe microscopy ❑This is a sub-diffraction technique. ❑Examples of scanning probe microscopes are the atomic force microscope (AFM), the Scanning tunnelling microscope, the photonic force microscope and the recurrence tracking microscope. ❑ All such methods use the physical contact of a solid probe tip to scan the surface of an object, which is supposed to be almost flat. 122  Bright field microscopy ❑Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light, i.e. illuminated from below and observed from above. ❑Limitations include low contrast of most biological samples and low apparent resolution due to the blur of out of focus material. ❑The simplicity of the technique and the minimal sample preparation required are significant advantages. 123 12 4  ELECTROPHORESIS ❑The term Electrophoresis means “Electro” = electric field + “Phoresis” =migration. ❑Electrophoresis is a method of separation where charged molecules migrate in differential speeds in an applied electric field. ❑Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. ❑This electrokinetic phenomenon was observed for the first time in 1807 by Ferdinand Frederic Reuss (Moscow State University) 125  PRINCIPLE ❑ The charged molecules under the influence of electric field migrate towards oppositely charged electrodes. ❑ Molecules with +ve charge move towards cathode and -ve molecules move towards Anode. 126  EQUIPMENTS USED IN ELECTROPHORESIS a. Electrophoresis Chamber a. Electrophoresis Chamber 127  TYPES OF ELECTROPHORESIS & THEIR TECHNIQUES Electrophoresis can be broadly divided into 2 types as 1. Capillary electrophoresis 2. Slab electrophoresis: it is the sole method available for separation of proteins like enzymes, hormones, antibodies and nucleotides like DNA and RNA. Slab electrophoresis is further divided into 3 types based on the principle used for separation. a) Zone electrophoresis (Paper and Gel electrophoresis) b) Isoelectric-focusing c) Immune-electrophoresis d) Capillary electrophoresis 128  APPLICATIONS OF ELECTROPHORESIS 1. To separate complex molecules: Many complex biological molecules like vitamins B12, antibiotics, and proteins can be separated efficiently by electrophoresis. This is possible due to charge difference among the mixtures. 2. For analysis of nucleic acid molecules like RNA and DNA studies. These long chain molecules can be analyzed only after separation by electrophoresis. This helps to determine the size or breaks in the DNA or b. Separation of blood into various haemoglobin types using electrophoresis RNA molecule. 129  POLYMERASE CHAIN REACTION (PCR) ❑The polymerase chain reaction (PCR) can be used for the selective amplification of a specific segment (target sequence or amplicon) of a DNA molecule. ❑The size of the DNA region amplified during the PCR reaction typically falls within the range of 100 bp to 10 kbp. ❑PRINCIPLE: The double stranded DNA of interest is denatured to separate into two individual strands. Each of the strands is renatured using primers. The primer-template duplex is used for DNA synthesis by a DNA polymerase. This process is repeated several times to generate multiple forms of the target DNA if present. 130  THE PCR SCHEME (PROCESSES) 1. A The basic function of this machine is copying the sections of DNA and this is performed though a heating cycle. 2. This is performed when the temperature rises to 95 degree Celsius which in turn melts the DNA strands. This melting of DNA strands causes the backbones of sugar phosphate to split apart. 3. Then as the temperature lowers, the primers bind them 3 inch end of each sequence of target. Primers are able to perform this task as the DNA polymerase taq and free nucleotides aid it in the process. 4. This process goes on so that there are two strands of double partially stranded molecules of DNA at the end of first cycle. 5. The same process continues to be repeated again and again causing thousands of copies of the particular target sequence. 131 CYCLES OF THE POLYMERASE CHAIN REACTION (PCR) 132  EQUIPMENTS USED IN PCR PCR thermal recycler 133  THE PCR MACHINE ❑Introduction PCR stands for Polymerase Chain Reaction, which is often used in biological and chemical labs. ❑It is also called a DNA amplifier or A thermal cycler, or PCR machine, has the ability to produce DNA copies of a specific segment that can range from thousands to millions in numbers. ❑Also, it’s used in the field of forensic sciences in arriving at the results based on fingerprints patterns and for testing paternity. ❑Kary B. Mullis invented PCR technique in 1985. 134  TYPES OF PCR MACHINES 1. Quantitative and Real Time PCR Machine: To quantify and detect DNA sample, this type of machine is widely used for amplification. This thermal cycler uses DNA dyes and fluorescent reporter for its method of probing. 2. Inverse PCR Machine: This is used to carry out amplification method that helps you identify the flanking sequence of different genomic inserts. This is DND amplification from known sequence to unknown sequence. 3. Anchored PCR Machine: When small sequence of nucleotides needs to be tagged to a particular DNA of target, the anchor is frequented by poly G by using poly C primer. 4. Reverse Transcriptase PCR Machine: This is used of RNA molecules amplification. This type of machine is highly used for profiling expression, finding gene expression and identifying RNA sequence in a transcript. 135  TYPES OF PCR MACHINES 5. Asymmetric PCR Machine: When there is a requirement for single strand molecules synthesis of DNA, which is essential for DNA sequencing, this PCR machine is widely used. It can perform 20 to 25 cycles of amplification using two primers. When one cycle is completed, one primer gets exhausted and in another 5 to 10 cycles, single strand DNA is produced. 6. Allele Specific PCR Machine: When there is a need to identify and detect a selective single nucleotide polymorphism, this thermal cycler is widely used. It uses a special design primer, which will match or not match the alleles at the primer end of 3′. 7. Colony PCR Machine: This machine is used to detect the formation of bacterial colonies after a plasmid transformation. 8. Conventional PCR Machine: This makes use of standard Polymerase Chain Reaction process that helps you produce billion copies of DNA and RNA strand. 9. Nested PCR Machine: After the initial 30 to 35 Polymerase Chain Reaction cycles, the new primers are nested along with the old primers to aid in a sensitive process as it reduces the risk involved in it. 136  FLUOROMETRY /SPECTROFLUOROMETRY ❑A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. ❑The phenomenon of fluorescence was discovered and published by Sir John Fredrick William Herschel in the mid-1800s. ❑These parameters are used to identify the presence and the amount of specific molecules in a medium on the basis of their unique fluorescent nature. ❑Although some information about molecular structure may be derived from excitation and emission spectra, qualitative application of spectrofluorimetry are rare and vast majority of applications in pharmaceutical analysis concern the quantitative assay of drugs, decomposition products and metabolites. 137  CONT. Fluorescence: When an illuminating system emits light of wavelength different from the incident light High sensitivity results from a difference in wavelength between the exciting and fluorescence radiation. These results in a signal contrasted with essentially zero background. High specificity results from dependence on two spectra: the excitation and the emission spectrum. 138 PRINCIPLE 139 INSTRUMENTATION Portable Fluorometer 140  LYOPHILISATION (FREEZE-DRYING) ❑ Lyophilization is also known as cryodesiccation. It is a low temperature dehydration process that involves freezing a product and lowering temperature, removing the ice by sublimation. ❑ Protein (or any other non-volatile molecule) samples can be concentrated by evaporating water and other volatile compounds from the sample. In principle, this could be achieved by simply heating the sample. However, most proteins would unfold during such a simple evaporation process. ❑ This method is frequently used not only to concentrate but to preserve proteins or other sensitive biomolecules for long-term storage. Such samples can usually be stored for years without a significant deterioration of quality. ❑ However, before lyophilisation it is very important to carefully consider all non-volatile compounds of the initial sample as these will concentrate along with the proteins. Non-volatile acids or bases can cause extreme pH, and the presence of salts can result in very high ionic strength when the sample is resolubilised. 141  PRINCIPLE In order to prevent the denaturation of proteins, 1. The sample is transferred into a glass container and is frozen as quickly as possible, usually by immersing the outside of the container into liquid nitrogen. 2. The container is rotated in order to spread and freeze the sample on a large surface area. 3. The glass container with the sample is then placed into an extremely low-pressure space (vacuum) that contains a cooling coil (condenser), usually lower than -50°C. 4. Volatile compounds of the frozen sample will evaporate (sublimate) in the vacuum. The process of evaporation (in this case, sublimation) absorbs heat. 5. This effect keeps the sample frozen. Evaporated molecules are captured from the gas phase by the cooling coil, forming a frozen layer on it. At the end of the process, proteins and other non-volatile compounds of the sample remain in the container in a solid form. This process does not cause irreversible denaturation of proteins. 142 THE LYOPHILISER 143  APPLICATIONS OF LYOPHILISATION ❑Pharmaceutical companies use free-drying to increase the self-life of drugs and vaccines such as vaccines, biological specimen and other injectables. ❑Food industries use freeze-drying to increase shelf-life of food and to maintain food quality ❑Astronauts use lyophilized food substances due to its light weight and long term preservation. 144  OSMOMETRY ❑Osmometry is the measurement of the osmotic strength of a substance. This is often used by chemists for the determination of average molecular weight. ❑Osmometers are useful for determining the concentration of dissolved salts or sugars in blood or urine samples. ❑Osmometry is also useful in determining the molecular weight of unknown compounds and polymers. TYPES: Membrane Osmometry Vapour Pressure Osmometry Freezing point depression osmometer 145  TYPES OF OSMOMETRY ❑An osmometer is a device for measuring the osmotic strength of a solution, loid, or compound. ❑Vapor pressure osmometers determine the concentration of osmotically active particles to reduce the vapor pressure of a solution. ❑Membrane osmometers measure the osmotic pressure of a solution separated from pure solvent by a semipermeable membrane. ❑Freezing point depression osmometer may also be used to determine the osmotic strength of a solution, as osmotically active compounds depress the freezing point of a solution. 146  WORKING PRINCIPLE 14 7 EQUIPMENT (OSMOMETER) 148  TURBIDIMETRY AND NEPHELOMETRY ❑Turbidimetry and nephelometry are two techniques based on the elastic scattering of radiation by a suspension of colloidal particles: (a) In turbidimetry the detector is placed in line with the source and the decrease in the radiation’s transmitted power is measured. (b) In nephelometry scattered radiation is measured at an angle of 90° to the source. ❑The similarity of turbidimetry to the measurement of absorbance and of nephelometry to the measurement of fluorescence is evident in these instrumental designs. ❑Turbidity can be measured using a UV/ Visible spectrophotometer and a spectrofluorometer is suitable for nephelometry. 149  WORKING PRINCIPLE OF THE TURBIDIMETER & NEPHELOMETER 150 TURBIDIMETER & NEPHELOMETER 151 Turbidimeter Nephelometer  CONDUCTOMETRY, COULOMETRY AND POLAROGRAPHY ❑Conductometry is a measurement of electrolytic conductivity to monitor a progress of chemical reaction. Conductometry has notable application in analytical chemistry, where conductometric titration is a standard technique. ❑In usual analytical chemistry practice, the term conductometry is used as a synonym of conductometric titration, while the term conductimetry is used to describe non-titrative applications. ❑Conductometry is often applied to determine the total conductance of a solution or to analyse the end point of titrations that include ions. ❑The method can be used for titrating coloured solutions or homogeneous suspension (e.g.: wood pulp suspension), which cannot be used with normal indicators. 152  POLAROGRAPHY ❑Polarography is a subclass of voltammetry where the working electrode is a dropping mercury electrode (DME) or a static mercury drop electrode (SMDE), which are useful for their wide cathodic ranges and renewable surfaces. ❑Principle: Polarography is a voltametric measurement whose response is determined by combined diffusion/convection mass transport. The simple principle of polarography is the study of solutions or of electrode processes by means of electrolysis with two electrodes, one polarizable and one unpolarizable, the former formed by mercury regularly dropping from a capillary tube. 153  COULOMETRY ❑Coulometry is the name given to a group of techniques in analytical chemistry that determine the amount of matter transformed during an electrolysis reaction by measuring the amount of electricity (in coulombs) consumed or produced. ❑It is named after Charles-Augustin de Coulomb. ❑There are two basic categories of coulometric techniques. ❖ Potentiostatic coulometry involves holding the electric potential constant during the reaction using a potentiostat. ❖ coulometric titration or amperostatic coulometry, keeps the current (measured in amperes) constant using an amperostat. 154  WORKING PRINCIPLE ❑ Coulometer is a device for determining the amount of a substance released during electrolysis by measuring the electrical charge that results from the electrolysis. Coulometers can be used to detect and measure trace amounts of substances such as water. 155  RADIOIMMUNOASSAY (RIA) ❑Radioimmunoassay (RIA) is a sensitive method for measuring very small amounts of a substance in the blood. Radioactive versions of a substance, or isotopes of the substance, are mixed with antibodies and inserted in a sample of the patient's blood. The same non- radioactive substance in the blood takes the place of the isotope in the antibodies, thus leaving the radioactive substance free. ❑The amount of free isotope is then measured to see how much of the original substance was in the blood. This isotopic measuring method was developed in 1959 by two Americans, biophysicist Rosalyn Yalow (1921-) and physician Solomon A. Berson (1918-1972). ❑Yalow and Berson developed the first radio-isotopic technique to study blood volume and iodine metabolism. They later adapted the method to study how the body uses hormones, particularly insulin, which regulates sugar levels in the blood. 156  Radioimmunoassay (RIA) method ❑The target antigen is labeled radioactively and bound to its specific antibodies (a limited and known amount of the specific antibody has to be added). ❑A sample, for example a blood-serum, is then added in order to initiate a competitive reaction of the labelled antigens from the preparation, and the unlabelled antigens from the serum- sample, with the specific antibodies. The competition for the antibodies will release a certain amount of labelled antigen. This amount is proportional to the ratio of labelled to unlabelled antigen. A binding curve can then be generated which allows the amount of antigen in the patient's serum to be derived. ❑That means that as the concentration of unlabeled antigen is increased, more of it binds to the antibody, displacing the labeled variant. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigens remaining in the supernatant is measured. A binding curve can be generated using a known standard, which allows the amount of antigens in the patient's serum to be derived. 157 RIA PROCEDURE 158  NEEDED SUBSTANCES AND EQUIPMENT ❑Specific antiserum to the antigen to be measured ❑Availability of a radioactive labelled form of the antigen ❑A method in which the antibody-bound tracer can be separated from the unbound tracer ❑An instrument to count radioactivity NB: 125-I labels are usually applied although other isotopes such as C14 and H3 have also been used. Usually, high specific activity radio-labelled (125-I) antigen is prepared by iodination of the pure antigen on its tyrosine residue(s) by chloramine-T or peroxidase methods and then separating the radio-labelled antigen from free-isotope by gel-filtration or HPLC. Other important components of RIA are the specific antibody against the antigen and pure antigen for use as the standard or calibrator. 159  AUTOANALYZERS/ AUTOMATED ANALYSER ❑An automated analyser is a medical laboratory instrument designed to measure different parameters and other characteristics in a number of biological samples quickly, with minimal human assistance. ❑Some analysers require samples to be transferred to sample cups. However, the effort to protect the health and safety of laboratory staff has prompted many manufacturers to develop analysers that feature closed tube sampling, preventing workers from direct exposure to samples. ❑The automation of laboratory testing does not remove the need for human expertise (results must still be evaluated by medical technologists and other qualified clinical laboratory professionals), but it does ease concerns about error reduction, staffing concerns, and safety. 160  ROUTINE BIOCHEMISTRY ANALYSERS ❑ The history of discrete sample analysis for the clinical laboratory began with the introduction of the "Robot Chemist" invented by Hans Baruch and introduced commercially in 1959. ❑ Perform tests on whole blood, serum, plasma, or urine samples to determine concentrations of analytes (e.g., cholesterol, electrolytes, glucose, calcium) ❑ To provide certain haematology values (e.g., haemoglobin concentrations, prothrombin times), and to assay certain therapeutic drugs (e.g., theophylline), which helps diagnose and treat numerous diseases, including diabetes, cancer, HIV, STD, hepatitis, kidney conditions, fertility, and thyroid problems. ❑ The types of tests required include enzyme levels (such as many of the liver function tests), ion levels (e.g. sodium and potassium, and other tell-tale chemicals (such as glucose, serum albumin, or creatinine).Simple ions are often measured with ion selective electrodes, which let one type of ion through, and measure voltage differences. ❑ Enzymes may be measured by the rate they change one coloured substance to another; in these tests, the results for enzymes are given as an activity, not as a concentration of the enzyme. Other tests use colorimetric changes to determine the concentration of the chemical in question. Turbidity may also be measured. 16 FULLY AUTOMATED BIOCHEMISTRY ANALYZERS 162  PRINCIPLES OF OPERATION ❑After the tray is loaded with samples, a pipette aspirates a precisely measured aliquot of sample and discharges it into the reaction vessel; a measured volume of diluent rinses the pipette. Reagents are dispensed into the reaction vessel. After the solution is mixed (and incubated, if necessary), it is either passed through a colorimeter, which measures its absorbance while it is still in its reaction vessel, or aspirated into a flow cell, where its absorbance is measured by a flow-through colorimeter. The analyzer then calculates the analyte’s chemical concentrations. 163  USE AND MAINTENANCE ❑Maintenance: Laboratory Scientist; biomedical or clinical engineer ❑Training: Initial training by manufacturer and manuals ❑Environment of use (Setting): Clinical laboratory ❑Requirements: Adequate benchtop or floor space, water supply, line power, biohazard disposal. 164  Immuno-based analysers ❑Antibodies are used by some analysers to detect many substances by immunoassay and other reactions that employ the use of antibody-antigen reactions. ❑When concentration of these compounds is too low to cause a measurable increase in turbidity when bound to antibody, more specialised methods must be used. ❑Recent developments include automation for the immunohaematology lab, also known as transfusion medicine 165  Hematology analysers ❑These are used to perform complete blood counts, erythrocyte sedimentation rates (ESRs), or coagulation tests. CELL COUNTERS ❑Automated cell counters sample the blood, and quantify, classify, and describe cell populations using both electrical and optical techniques. ❑Electrical analysis involves passing a dilute solution of the blood through an aperture across which an electrical current is flowing. The passage of cells through the current changes the impedance between the terminals (the Coulter principle). A lytic reagent is added to the blood solution to selectively lyse the red cells (RBCs), leaving only white cells (WBCs), and platelets intact. Then the solution is passed through a second detector. This allows the counts of RBCs, WBCs, and platelets to be obtained. The platelet count is easily separated from the WBC count by the smaller impedance spikes they produce in the detector due to their lower cell volumes. 166 HAEMATOLOGY ANALYZER (FBC Machines) 167  COAGULOMETERS ❑ Automated coagulation machines or Coagulometers measure the ability of blood to clot by performing any of several types of tests including Partial thromboplastin times, Prothrombin times (and the calculated INRs commonly used for therapeutic evaluation), Lupus anticoagulant screens, D dimer assays, and factor assays. ❑ Coagulometers require blood samples that have been drawn in tubes containing sodium citrate as an anticoagulant. These are used because the mechanism behind the anticoagulant effect of sodium citrate is reversible. 168  Automated Westergren-based ESR 'analyzer' ❑the only reference method, being Westergren, explicitly indicating the use of diluted blood (with sodium citrate), in 200 mm pipettes, bore 2.55 mm. After 30 or 60 minutes being in a vertical position, with no draughts and vibration or direct sunlight allowed, an optical reader determines how far the red cells have fallen by detecting the level. Automated Westergren-based ESR 'analyzer' 169  Miscellaneous analysers ❑Some tests and test categories are unique in their mechanism or scope, and require a separate analyser for only a few tests, or even for only one test. Other tests are esoteric in nature—they are performed less frequently than other tests, and are generally more expensive and time-consuming to perform. Even so, the current shortage of qualified clinical laboratory professionals has spurred manufacturers to develop automated systems for even these rarely performed tests. ✓ Analysers that fall into this category include instruments that perform: ✓ DNA labeling and detection ✓ Osmolarity and osmolality measurement ✓ Measurement of glycosylated haemoglobin (haemoglobin A1C), and ✓ Aliquotting and routing of samples throughout the laboratory 170  SOLVENT EXTRACTION ❑A technique, also called liquid extraction, for separating the components of a liquid solution. This technique depends upon the selective dissolving of one or more constituents of the solution into a suitable immiscible liquid solvent. ❑It is particularly useful industrially for separation of the constituents of a mixture according to chemical type, especially when methods that depend upon different physical propertie. ❑Extraction takes advantage of the relative solubilities of solutes in immiscible solvents. If the solutes are in an aqueous solution, an organic solvent that is immiscible with water is added. The solutes will dissolve either in the water or in the organic solvent. If the relative solubilities of the solutes differ in the two solvents, a partial separation occurs. The upper, less dense solvent. 171  SOXHLET EXTRACTION ❑Solvent extraction is carried out regularly in the laboratory as a commonplace purification procedure in organic synthesis, and in analytical separations in which the extraordinary ability of certain solvents preferentially to remove one or more constituents from a solution quantitatively is exploited. Batch extractions of this sort, on a small scale, are usually done in separatory funnels, where the mechanical agitation is supplied by handshaking of the funnel. ❑A Soxhlet Extractor has three main sections: A percolator (boiler and reflux) which circulates the solvent, a thimble (usually made of thick filter paper) which retains the solid to be laved, and a siphon mechanism, which periodically empties the thimble. 172  CHROMATOGRAPHY ❑A technique for analysis of chemical substances. The term chromatography literally means colour writing, and denotes a method by which the substance to be analysed is poured into a vertical glass tube containing an adsorbent, the various components of the substance moving through the adsorbent at different rates of speed, according to their degree of attraction to it, and producing bands of colour at different levels of the adsorption column. 173  FLOW CYTOMETRY ❑Flow cytometry is a technology that is used to analyse the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths.