Microtome, Water Bath, PCR and Hot Air Oven (PDF)

Microtome Lecture 01 Marwa Mahmood 2023/2024 Tuesday, April 23, 2024  Introduction At the beginning of light microscope development, sections from plants and animals were manually prepared using razor blades. It was found that to observe the structure of the specimen under observa...

Microtome Lecture 01 Marwa Mahmood 2023/2024 Tuesday, April 23, 2024  Introduction At the beginning of light microscope development, sections from plants and animals were manually prepared using razor blades. It was found that to observe the structure of the specimen under observation it was important to make clean reproducible cuts on the order of 100 μm, through which light can be transmitted. This allowed for the observation of samples using light microscopes in a transmission mode  Microtome Machines that cut extremely thin sections from a sample for applications in histology or pathology use special metal, glass, or diamond blades, depending on the type of specimen and the desired thickness. consists of a blade holding unit with a blade holder and clamped blade, the clamp, an advancing mechanism, and a mechanism for adjusting section thickness. A microtome (from the Greek mikros, meaning "small", and temnein, meaning "to cut") is a cutting tool used to produce extremely thin slices of material known as sections, with the process being termed micro sectioning. Important in science, microtomes are used in microscopy for the preparation of samples for observation under transmitted light or electron radiation.  Types of microtomes Based on the mechanism: Rotary Rocking Base-sledge Sliding Freezing Vibrating Saw Cryostat Ultra-Microtome  Rotary microtome Most widely used, also called Minot microtome, after its inventor Professor Minot. The knife is stationary and the block is moved up and down in a vertical plane by the rotary action of the hand wheel Suitable for paraffin-embedded sections What is the importance of rotary microtome (or any microtome)? Microtomes are used in microscopy, allowing for the preparation of samples for observation under transmitted light or electron radiation. Microtomes help us in giving desirable sections of any μm we desire also provide section trimming and cutting features of any μm and we get our thin section which helps us in identifying the tissue and its parts. Advantages: Ideal for cutting serial sections Heavier, so more stable The heavier knife is used, so less vibration The cutting angle(Tilt) of the knife is adjustable Ability to cope with harder tissue Can cut Celloidin-embedded sections by using a special holder to set the knife obliquely. Manual or electrically driven rotary microtomes are successfully used in cryostats. Disadvantage: Not suitable for hard blocks of hard tissue.  Parts of microtome:  Block Holder Holds the paraffin block in place. Typically, the block moves up and down with each revolution while the blade is stationary. The block holder may have knobs that allow the user to manipulate the block face in various directions to bring the tissue in alignment with the blade. Microtome base plate or stage: A plate form that has rails that secure the knife holder base Knife holder base: A part that anchors the knife holder to the microtome stage. The knife holder base can be moved toward or away from the block, but MUST be stationary and locked during microtomy. Knife holder: This part is comprised of several components including the blade clamp that holds the blade, the knife tilt for adjusting the knife angle, and the face plate that guides the ribbons away from the blade and towards the operator. Coarse handwheel: Moves the block holder either toward the knife or away from the knife. Micron adjustment: Micron settings for section thickness can range from 1 to 60 microns on most microtomes. Advancement hand wheel: Turns in one direction and advances the block toward the knife at the specified microns. Most hand wheels are equipped with a safety lock to prevent the wheel from releasing and having the block holder come down towards the blade while a block is inserted or removed. The safety lock should be used anytime the microtomist is not actively sectioning paraffin blocks Thank you… Water bath Lecture 02 Marwa Mahmood 2023/2024 Friday, May 10, 2024  A water bath is a laboratory instrument. It is a container or vessel filled with heated water. The temperature of the water is maintained at a constant level. It is used to incubate samples over a period of time at a constant temperature. The instrument used in the laboratory for carrying out serological, biomedical, and pharmaceutical tests at specific temperature ranges. Parts of water bath The main parts of the water bath: 1-Container or tank bath 2-Heater. 3-Thermometer. 4-Thermostat or regulator. Types of water bath 1-Circulating water bath. 2-Non-circulating water bath. 3-Shaking water bath. Circulating water bath: is also called a stirrer water bath. It is perfect for uses when temperature uniformity and regularity is critical; water is thoroughly distributed throughout the bath resulting in a uniform temperature: It is used for some specific substances or chemical reactions and reagents for example, enzymatic and serologic experimentations. To ensure optimum temperature uniformity throughout the whole bath. An electric motor with a rotary magnet is flanged to the bath bottom Non-circulating water bath: It is less accurate in terms of temperature control. This type of water bath depends mainly on convection instead of water being uniformly heated Shaking water bath: This type of water bath has additional control for shaking, which moves liquids from one place to another place. The shaking feature can be turned on or turned off. Constant shaking allows the incubated liquid sample to be mixed at a constant level and the temperature is maintained at a constant point. Principle of water bath: the principle of water bath depends on the heat applied to the sample using the heater.  Uses of water bath: 1-used to improve the solubility of poorly soluble substances. 2-It is used for melting some substances. 3-It is used for the warming of chemical reagents. 4- It is used for facilitating some chemical reactions. 5. For incubation of cell cultures. 6. It is used as a heat source for some substances such as flammable chemicals.  Applications 1-If the equipment has been stored in cold or humid conditions, condensation may form inside it. Therefore, allow time (at least 2 hours) for the condensation to evaporate before using the equipment. 2-It is not recommended to use a water bath with moisture sensitive reactions. 3-Water levels should be regularly monitored and filled with distilled water or deionized water. This is required to prevent salts from depositing on the heater. 4-Disinfectants or bactericidal agents can be added to prevent the growth of organisms. 5-For the purpose of decontamination, the temperature of the water bath may be raised to 90°C or higher once a week for half an hour 6-If application involves liquids that give out vapors (gases), It is recommended to operate the water bath in the gas hood or in a well-ventilated area 7-The cover is closed to prevent evaporation and to help to reach high temperatures. 8-Set up on a steady surface away from flammable materials. 9-Change the water regularly and empty it when not in use for prolonged periods. 10- Before emptying a bath, allow the water temperature to fall to a safe level 11- Do not use the equipment in an area where there are aggressive or explosive chemical mixtures. 12- Do not use the bath to heat any material that could cause a fire or any other kind of hazard. Hot air oven  Hot air oven An oven provides a temperature higher than that of the atmosphere. The temperature range covered by ovens is between 50- 250°C. These are used for rapid evaporation of materials, rapid drying, and for sterilization of articles that can be sterilized by dry heat. The dry heat sterilization technique requires longer exposure time (1.5 to 3 hours) and higher temperatures than moist heat sterilization.  Principle Sterilizing 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. 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.  Application 1. It is mainly used for the sterilization of glass wares like pipettes, bottles test tubes, Petri dishes, etc. 2. It is used for the sterilization of powders as well as oils which is not possible by moist heat sterilization. 3. Injectables can be sterilized by a hot air oven. 4. Hot air oven is used for the sterilization of scalpels, scissors, spatula, blades, and glass syringes Advantages A dry heat cabinet is easy to install and has relatively low operating costs It penetrates materials It is non-toxic and does not harm the environment. it is noncorrosive for metal and sharp instruments. Disadvantages Time-consuming method because of the slow rate of heat penetration and microbial killing. High temperatures are not suitable for most materials. Thank you…  PCR Polymerase Chain Reaction Lecture 03 Marwa Mahmood 2023/2024 Saturday, May 4, 2024 PCR PCR Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.  PCR is a technique that takes specific sequence of DNA of small amount and amplifies it to be used for further testing. In vitro technique The process cycle can be repeated up to 30x. Developed in 1983 by Kary Mullis, PCR is now a common technique used in clinical and research laboratories for a broad variety of applications. In 1993, Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.  Components of PCR DNA template: DNA template is DNA target sequence. The DNA template is the DNA molecule that contains the DNA region (segment) to be amplified, the segment we are concerned with which is the target sequence. DNA polymerase: DNA polymerase sequentially adds nucleotides complimentary to the template strand at 3’-OH of the bound primers and synthesizes new strands of DNA complementary to the target sequence. The most commonly used DNA polymerase is Taq DNA polymerase (from Thermus aquatics, a thermophilic bacterium) because of high-temperature stability. Primers: Primers are synthetic DNA strands of about 18 to 25 nucleotides complementary to the 3’ end of the template strand. DNA polymerase starts synthesizing new DNA from the 3’ end of the primer.  Purpose To amplify a lot of double-stranded DNA molecules (fragments) with the same (identical) size and sequence by enzymatic method and cycling condition.  Steps 1. Denaturation of ds DNA template 2. Annealing of primers 3. Extension of ds DNA molecules Denaturation Temperature: 92-94C, high temperature to break down the hydrogen bonds between the bases. Double-stranded DNA melts to single-stranded DNA. Annealing Temperature: ~50-70C (dependent on the melting temperature of the expected duplex and the temperature for the primers) Primers bind to their complementary sequences 5’ 3’ Forward primer Reverse primer 3’ 5’  Extension Temperature: ~72C DNA polymerase binds to the annealed primers and extends DNA at the 3’ end of the chain. Application: Amplification and quantification of DNA Medical and diagnostic applications Infectious disease applications Forensic applications Research applications Gel Electrophoresis  Introduction Electrophoresis is a process of migration of charged particles through a solution under the influence of an external electric field.  Electrophoresis of positively charged particles is sometimes called cataphoresis, while electrophoresis of negatively charged particles is sometimes called anaphoresis. Electrophoresis is used in laboratories to separate macromolecules based on size. Electrophoresis is used extensively in DNA, RNA, and Protein analysis.  Gel Electrophoresis Gel electrophoresis is separation technique which uses the gel as a separating pocket. Molecules are separated in aqueous buffer supported within a polymeric gel matrix. Based on the molecular size of the substance molecular sieving technique is employed to facilitate the separation. Molecular sieving technique is the one in which electrophoretic mobility and migration of solute are purely dependent on viscosity and pore size. Hence the molecular weight decides the migration of macromolecules in the system.  Agarose gel electrophoresis Commonly used support medium Less expensive Equally good separation Agar is a complex acidic polysaccharide containing monomers of sulfated galactose The electrophoretic run takes about 1-1.5 hours.  Principle: This porous gel matrix is used which consists of the cross-linked polymer network. Through this network, molecules of different size, charges, and shape pass-through This relies upon the fact that negatively charged molecules will attract toward the positive end and vice versa. After the migration, bands will appear on the gel matrix at different levels those which lag behind will be the heavy molecules and those which moves faster are lighter molecules through the pores of the gel matrix.  Visualization After the electrophoresis is complete, the molecules in the gel can be stained to make them visible.  Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyzed molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.  Application  It is a tool for macromolecular separation  Many biological complex samples can be separated by using various methods of electrophoresis.  In some cases, the identification of molecules is also possible  The gel method is more commonly used for routine laboratory experiments as well as research-oriented separations and identification  Used for estimation of molecular weight of protein and nucleic acid Chromatography Lecture 04 Marwa Mahmood Asst. lecturer 2023/2024 Tuesday, May 14, 2024  Introduction Chromatography is a physical process where the components (solutes) of a sample mixture are separated as a result of their differential distribution between stationary and mobile phases. Greek chroma meaning ‘color’ and graphein meaning ‘writing’ Stationary Phase History Tswet, a Russian botanist (referred to as the Father of chromatography) is credited for the development of chromatography.  Principle Chromatography is usually based on the principle of partition of solute between two phases. It usually consists of a Mobile Phase and a Stationary Phase. The Mobile phase is a compound used to separate components in a mixture, this phase can move along with the components. The mobile phase is either a liquid or a gas. The Stationary Phase is a porous solid matrix through which the sample contained in the mobile phase moves. This phase does not move with the sample. Different affinities of the various components towards the stationary and mobile phase results in the separation of the components. Affinity, is affected by Adsorption and Solubility. Adsorption means how well a component of the mixture sticks to the stationary phase.  Solubility is how well a component of the mixture dissolves in the mobile phase. Higher the adsorption to the stationary phase, the slower the molecule will move through the column.  Applications of chromatography  The chromatographic technique is used for the separation of amino acids, proteins & carbohydrates.  It is also used for the analysis of drugs, hormones, vitamins  Helpful for the qualitative & quantitative analysis of complex mixtures.  The technique is also useful for the determination of the molecular weight of proteins. Types of Chromatography Paper Chromatography  Thin Layer Chromatography(TLC)  Gel Chromatography Column Chromatography Ion Exchange Chromatography Gel Filtration Chromatography Gas Liquid Chromatography Affinity Chromatography  Paper chromatography Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample mixture, which starts to travel up the paper with the solvent. Sometimes, it is rather difficult to separate a complex mixture of substances by a single run with one solvent system. In such a case, a second run is carried out by a different solvent system, in a direction perpendicular to the first run. This is referred to as two-dimensional chromatography.  Thin layer chromatography Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbents like silica gel, alumina, or cellulose. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents.  Column Chromatography The Stationary bed is within the tube. In column Chromatography, the stationary Phase may be pure silica or polymer or may be coated onto, chemically bonded to, support particles. Depending on whether the mobile phase is a gas or a liquid it is divided into gas Chromatography or liquid Chromatography. When the Stationary phase in LC consists of small-diameter particles, the technique is High-Performance Liquid Chromatography (HPLC). Automated analyzer Lecture 05 Marwa Mahmood Asst. lecturer 2023/2024 Wednesday, May 15, 2024 Automated analyzer Definition: “ The process whereby an analytical instrument performs many tests with only minimal involvement of an analyst” “ Controlled operation of an apparatus, process, or system by mechanical or electronic devices without human intervention”  The International Union of Pure and Applied Chemistry (IUPAC) defines automation as: "The replacement of human manipulative effort and facilities in the performance of a given process by mechanical and instrumental devices that are regulated by feedback of information so that an apparatus is self-monitoring or self- adjusting”.  Phases in the analytical process 1. Pre-analytical phase: sample processing 2. Analytical phase: chemical reaction 3. Post-analytical phase: Data management, processing, and report dispatch Substantial improvements have occurred in all three areas. The analytical phase is the most automated. Most research and developments are focusing on the increasing automation of pre and post-analytical processes.  Types of Auto analyzer Basically, there are two types of automatic analysis techniques/instruments:- Discrete sampling instrument: In discrete sampling, each sample undergoes reaction and measurement in a separate cuvette or chamber. These samples may be analyzed sequentially or in parallel.  Continuous–flow sampling instrument: In continuous-flow sampling, the samples flow sequentially and continuously in a tube perhaps being separated by an air bubble. They are each sequentially mixed with the reagent in the same tube at the sample point downstream and then flow sequentially into a detector.  Advantages of automation instruments enable laboratories to:  Process much larger workloads  Reduce the number of staff Reduction in the variability of results and errors of analysis Significant improvement in the quality of lab tests  Cost reduction Filters Filtration: It may be defined as a process of separation of solids from a fluid by passing the sample through a porous medium that retains the solids but allows the fluid to pass through. Applications of filtration  Production of sterile products: HEPA filters or laminar air bench Membrane filters  Productions of bulk drugs  Production of liquid dosage  Effluents and waste water treatment  Filtration of Liquids in the Lab In laboratories, liquids are filtered through microbial filters to remove any microbes present. It is an effective method of sterilization for heat-sensitive liquids. There are four types of filters: Membrane filters are thin filters that are made of cellulose. They can be used for sterilization during injection by placing the membrane between the syringe and the needle.  Seitz filters are usually made of asbestos. They are pad-like and thicker than membrane filters. Sintered glass filters are an alternative type of filter that is made of glass and hence does not absorb liquids during filtration. Candle filters are made of clay-like mud. This special mud has tiny pores made of algae. The microbes get stuck during their travel through the pores. Advantages of Filtration Relatively inexpensive, except for those with the smallest pore sizes Filters do not clog easily Suitable for heat sensitive liquids as filters do not use heat They can filter large volumes of fluid reasonably fast  Disadvantages of Filtration Filters can only work on liquids and gasses Autoclaving is usually cheaper than filtration since filters are expensive to replace, especially Nano-filters Glass filters are very brittle and can break easily Membrane filters rupture easily The solution in Sietz Filters might get absorbed by the filter pad itself Clogging may occur Thank you..