Molecular Biology (Laboratory) Instruments for Measurement PDF

Molecular Biology (Laboratory) 1st Sem 3rd Year Topic: Instruments for Volumetric flask Measurements Large amounts of Types of Glasswares standard s...

Molecular Biology (Laboratory) 1st Sem 3rd Year Topic: Instruments for Volumetric flask Measurements Large amounts of Types of Glasswares standard solutions and reagents that require highly Flasks, beakers, and graduated accurate concentrations cylinders are used to hold solutions. Flat-bottomed bulb Volumetric and Erlenmeyer flasks with elongated neck are two types of containers in calibrated to hold a set general use in the clinical laboratory volume at a mark on the neck. Aka, graduated flask or measuring Class A and Class B flask Glassware Ehrlenmeyer flask Class A Named for has the highest level of accuracy out Emil Erlenmeyer of all the different types of The flask is glassware. often used for stirring manufactured from borosilicate or heating solutions material. and is purposefully ass A borosilicate glassware has designed to be useful for those superior thermal and chemical tasks. resistance properties, which are Used in solution preparation but are helpful when working with less accurate than volumetric flasks. chemicals common in laboratory Used in preparing a solution of experiments. known volume rather than determining the volume Class B Beakers made for more general-purpose use They are also used around the laboratory. to catch liquids manufactured from soda-lime glass, from titrations and filtrates which is suitable for most materials from but not for long-term chemical filtering operations. holding or exposure They would not be accurate in ⚬ manufactured from soda-lime determining the total volume of an unknown glass, which is suitable for most fluid because the markings are not materials but not for long-term calibrated to a level of chemical holding or exposure accuracy for that purpose. Used to hold stock solutions or diluents for short-term storage or during laboratory procedures Molecular Biology (Laboratory) 1st Sem 3rd Year To read the level of the liquid properly Graduated cylinder If the meniscus is Tall flask with a concave meniscus, multiple volume read at the lowest gradations level of the curve. The purpose of If the meniscus is this flask is to aliquot convex, take your volumes of a fluid in measurement at the highest point of making up a reagent the curve dilution or to determine the volume of an To read the level of the liquid properly unknown fluid. Has a higher degree of accuracy Water, or clear than an Ehrlenmeyer flask since the liquids – read the volume markings are graduated to measurement at the at least 5% of the total volume. bottom of the The sizes routinely used are 10, 25, meniscus. 50, 100, 500, 1,000, and 2,000 mL For mercury, or liquids with color - read the Reading a meniscus measurement at the top of the meniscus The meniscus is the curve seen at the top of a liquid in response to its PIPETTE CLASSIFICATION container. The meniscus can be either concave or convex, depending on the surface tension of the liquid and its adhesion to the wall of the container To read the level of the liquid properly Eye should be at the same level as the bottom of the meniscus Sometimes a white card or a white card with a black mark on it will help a person to see the meniscus clearly. Molecular Biology (Laboratory) 1st Sem 3rd Year To Deliver A TC pipette holds or contains a particular volume but does not While in ‘TD’ marked pipette, the dispense that exact volume, delivered quantity of liquid whereas a TD pipette will dispense corresponds to the capacity printed the volume indicated on the pipette Blowout and self-draining Pipettes that are calibrated “to deliver” A blowout pipette has a continuous (TD) but with no etched etched ring or two small, close, rings on the top should continuous rings located near the be allowed to drain out top of the pipette. This means that the fluid since the volume has been the last drop of liquid should be calibrated in this manner. expelled into the receiving vessel. Pipettes that are calibrated TD but Without these markings, a pipette is with etched rings on the top should self-draining, and the user allows have the last drop blown out if the the contents of the pipette to drain full volume is to be delivered by gravity To Contain Measuring pipettes In a ‘TC’ marked pipette, Measuring pipettes the contained quantity of the deliver multiple volumes and liquid corresponds to the capacity are used when less accuracy printed on the pipette of measurement is required, Not used often in clinical such as for delivering chemistry. reagents or small amounts of This type of pipette is solvent. used when diluting a highly This type of pipette viscous fluid such as whole blood should not to be used for A TC pipette is calibrated to contain making standard solutions or the designated volume so, in order quality control materials. to accurately dispense the full Mohr and Serological pipets volume, the pipette must be rinsed out in the diluting fluid several time Transfer pipettes TD vs. TC Transfer pipettes deliver one volume and are used when a higher degree The major difference is the amount of accuracy is required. of liquid needed to wet the interior This type of pipette is used for surface of the pipette and the preparation of standard solutions to amount of any residual liquid left in be used in calibration. the pipette tip Transfer pipettes resemble measuring pipettes except that only Molecular Biology (Laboratory) 1st Sem 3rd Year one measurement mark appears on the neck and there is a round or cylindrical bulb toward the middle or base of the pipette. Volumetric transfer pipettes vs. Ostwald-Folin pipettes, and Ostwald-folin vs. Sahli vs. Micropipettes automated micropipettes Ostwald-Folin pipettes Serological pipet vs. Mohr have a bulb closer to the delivery tip unlike volumetric Serological measuring pipette which has at center. pipettes are graduated so that These (OF) are used for multiple volumes can be accurate measurement of delivered?ered. Typical serological viscous fluids, such as blood or pipette volumes range from a total serum. In contrast to volumetric volume of 0.2 ml to 10.0 mL and pipette, an Ostwald-Folin have gradations extending into the pipette has an etched ring near tip of the pipette. the mount piece, indicating it is a They are termed serological blow out pipette. With the use of because they are designed to easily pipetting bulb, liquid is blown out of make dilutions or titers of patient the pipette only after blood or serum serum for serological pro?cedures. has drained to the last drop in They are termed serological delivery because they are designed to easily The volumetric make dilutions or titers of patient transfer pipettes is serum for serological procedures calibrated to deliver accurately a fixed volume of Mohr pipettes a dilute aqueous solution Graduated, but the markings Sahli pipettes do not extend into the tip. Commonly are small transfer these pipettes are available in total pipettes similar to volumes of 2.0, 5.0, or 10.0 mL, but Ostwald-Folin pipettes since there is no frosted ring at the but are used for making mouthpiece, they are not blown out dilutions of whole blood, to deliver the full volume. typically for hematology It is a self-draining pipette, but procedures the tip should not be allowed to Micropipettes touch the vessel while the pipette is pipettes to measure draining microliters of volume. Inner wall of pipette can be coated with liquid so most micropipettes are calibrated to contain (TC) that stated volume rather than to deliver it. a pipette with a total holding volume of less Molecular Biology (Laboratory) 1st Sem 3rd Year than 1 mL; it may be designed as A pipette with a pipetting capability either a Mohr or a serological of less than 1 mL is considered a pipette micropipette, and a pipette that dispenses greater than 1 mL is called an automatic micropipette Disposable pipettes Multichannel Pipettes Disposable transfer Multichannel pipettes may or may not pipettes generally have calibration marks and come with either 8 or are used to transfer solutions 12 pipette heads, or biologic fluids without easily allowing for a consideration of a specific single device to fill volume. multiple wells at a These pipettes should not be used in single time. This any quantitative analytic techniques enables the user to quickly and easily fill Automatic Pipettes multi-well plates used Advantages: in tissue culture, Safety drug screening, or enzyme assays. Stability Multichannel pipettes are able to Ease of use attach multiple pipette tips to a Increased precision single handle and can then be used Save time to dispense a fixed volume of fluid Less cleaning (tips are to multiple wells, such as in delivery disposables to a multiwell microtiter plate Most routinely used pipette in today's clinical chemistry laboratory. Automatic pipettes can also be categorized Automatic pipettes come in a variety according to their mechanism of types including fixed volume, variable volume, and multichannel Air-displacement It may be a fully Relies on a piston for automated/self-operating, semi creating suction to draw the automatic, or completely manually sample into a disposable tip operated device that must be changed after Pipette associated with only one each use. volume is termed a fixed volume; The piston does not models able to select different come in contact with the volumes are termed variables. liquid The available range of volumes is 1 Positive displacement μL to 5,000 mL. The widest volume Positive-displacement the range usually seen in a single pipettes work like a syringe. The pipette is 0.5 μL to 1,000 μL. samples come into direct contact with the tip and piston, increasing Molecular Biology (Laboratory) 1st Sem 3rd Year precision and accuracy and Liquid examples:concentrated eliminating the air cushion. The sulfuric acid, concentrated direct contact with samples results in hydrochloric acid, biological the aspiration force remaining samples, Iodine-125 constant. It does not require a different tip for Detergent containing/ foaming liquids each use Sealing of piston with the tip wall reduces foaming and Sealing of Dispenser pipettes piston with the tip wall reduces automatic pipettes that foaming and bubble formation. obtain the liquid from common Liquid examples: serum, SDS, reservoir and dispense it Mucasol repeatedly Calibration of pipettes Positive displacement pipettes are The most accurate method of recommended for: validation is gravimetric. A gravimetric method can Viscous and dense liquids accomplish this task by delivering Samples are aspirated and and weighing a solution of known dispensed more slowly than water in specific gravity, such as water. A a traditional air cushion pipette, currently calibrated analytic balance which can lead to inaccurate and at least Class 2 weights should volumes and residue attached to the be used. A pipette should be used tip wall. Syringe/ Piston tips wipe the only if it is within ±1.0% of the tip walls clean. expected value following calibration Liquid examples: Tween 20, sulfuric acid, concentrated DNA / RNA, Other pipette accuracy testing glycerol Photometric methods: Volatile liquids Spectrophotometry Evaporation can lead to inaccuracy Uses Potassium dichromate, or and the pipette dripping In traditional another colored liquid with air-cushion pipettes, positive appropriate absorbance spectra displacement pipettes prevent These calibration techniques are time vapors from moving into air space. consuming and, therefore, impractical for Liquid examples: acetone, ethanol, use in daily checks. It is recommended that methanol pipettes be checked initially and subsequently three or four times per year, or Radioactive, infectious, and corrosive as dictated by the laboratory's liquids accrediting agency Positive displacement pipettes prevent aerosolization of solutions into an open-air space. Molecular Biology (Laboratory) 1st Sem 3rd Year Few reminders: Pipette calibration results should be recorded for long-term monitoring and to track changes over time with a particular pipette Class A pipettes Conversion of volumes like all other Class A labware, do not need to be recalibrated by the laboratory. Automatic pipetting devices, as well as non–Class A materials, do need Conversion of masses recalibration A class A transfer pipette is accurate to within 0.1%. Tips for positive-displacement pipettes are made of straight columns of glass or plastic. These tips must fit snugly to avoid carryover and can be used repeatedly without being changed after each use. As previously mentioned, these devices may need to be rinsed and dried between samples to minimize carryover