MLS 11Aa Clinical Chemistry Pipetting Techniques PDF

MLS 11Aa CLINICAL CHEMISTRY MLS 3A MODULE 3: Pipetting Techniques PIPET...

MLS 11Aa CLINICAL CHEMISTRY MLS 3A MODULE 3: Pipetting Techniques PIPETTE CLASSIFICATION I. Calibration Marks/ Design: WHAT ARE PIPETTES? A. To Contain (TC) B. To Deliver (TD) A pipette, (sometimes spelled as pipet), is a laboratory tool that functions to II. Drainage Characteristics transfer specific or measured amounts of liquid from one container to another; they A. Blowout may be reusable or disposable. Although pipettes may transfer any volume, they are B. Self-draining usually used for volumes of 20mL or less. Larger volumes are usually transferred III. Types or Function A. Transfer Pipettes or dispensed using automated pipettes devices or jar-style pipetting apparatus. 1. Volumetric Pipette 2. Ostwald-Folin Pipette Pipettes are made with glass or plastic and are usually open at both ends. 3. Pasteur Pipette Pipettes come in various classifications and can be designed as a single piece glass 4. Automatic macropipet and micropipette to a more complex adjustable or electronic pipette called, automatic pipettes, which B. Measuring or graduated Pipettes is the most routinely used pipette in today’s clinical chemistry laboratory. 1. Serologic Pipette 2. Mohr Pipette Note: - Glass pipettes are reusable but they can be disposed if they are broken. 3. Bacteriologic Pipette -Automatic pipettes like the micropipettes are the most routinely used pipette in 4. Ball, Kolmer, Kahn Pipette 5. Graduated Micropipette the CC laboratory because of their convenience, they are easy to use, accurate, and easy to clean. I. CALIBRATION MARKS/ DESIGN -Micropipettes usually have a capacity from 10 µL to 1 mL/5 mL Most manufacturers stamp TC or TD near the top of the pipette to alert the user as to the type of pipette. Like other TC-designed labware, a TC pipette holds or Fixed volume contains a particular volume but does not dispense that exact volume, whereas a TD  automatic pipette associated with only one volume pipette will dispense the volume indicated. Variable  models able to select different volumes A. To Contain (TC)  Also known as “rinse-out” pipettes (Rinse out: in order to get rid of the Note: Only one volume may be used at a time. The widest volume range usually residue that adheres to the glass) seen in a single pipette is 0 to 1 mL.  Holds the particular volume but does not dispense the exact volume. Micropipette  Must be filled and rinsed-out prior to refilling again and delivery of final volume.  a pipette with a pipetting capability of less than 1mL Macropipette  Contains exact amount of liquid which must be completely transferred for accurate measurement.  a pipette that dispenses greater than 1mL Examples of TC pipets: All laboratory utensils including pipettes can have specifications such as Class A, Class B or student grade. But in critical measurement, Class A should be used Micro-folin, dual purpose whenever possible to maximize accuracy and precision and thus decrease calibration Sahli hemoglobin, Kirk Micro White-Black lambda time. Transfer micropipette, measuring micropipette. 1 MLS 11Aa CLINICAL CHEMISTRY MLS 3A B. To Deliver (TD) B. Self-draining (Non-blown-out)  Delivers the exact amount it holds into a container.  Content of pipet drained by gravity.  Has 2 types: 1. Blown out Note: - We use the rubber aspirator to blow out (NOT THE MOUTH). 2. Non-blown-out III. PIPETTE CLASSIFICATION ACCORDING TO FUNCTION/ TYPE (TRANSFER or GRADUATED PIPETTE) A. Transfer Pipettes  Transfer pipettes can either be “TD” or “TC” 1. TD: Blown-out pipets  Calibrated for one specified volume measurement  Two etched rings near the top.  More accurate than the measuring pipet  Transfer or deliver exact amount of liquid.  Indicated in class A pipettes.  These are not rinsed out.  Small amount of liquid remaining at the tip is blown out as it is included EXAMPLES OF TRANSFER OF PIPETTES: to the initial volume a. Volumetric pipette  TD (non-blown pipette) Example:  For non-viscous fluid. Serological pipets, serological large tip, and serological long tip,  Self-draining; small amount of fluid left in the tip should not be blown Ostwald-Folin out.  Contains a mark to which the pipette is to be filled. 2. TD: Non-blown-out-pipets  Holds and deliver fluid specific volumes indicated at the upper end of  Filled and allowed to self-drain. the pipette.  The most accurate pipette and it is used for diluting standards, II. DRAINAGE CHARACTERISTICS calibrators or QC materials.  The bulb is located at the middle of the pipette A. Blown-out  A blowout pipet has continuous etched ring or two small, close continuous rings located near the top of the pipet.  A pipette calibrated to deliver its nominal volume by permitting it first to drain and then blowing out the last drop of liquid in the tip. 2 MLS 11Aa CLINICAL CHEMISTRY MLS 3A Specifications on a Volumetric pipette Note: -Compared to the volumetric pipette, this one needs to be aspirated. -We also have the limit indicator, where the fluid must not exceed that line because when it goes over it, excess amount of fluid is aspirated on the pipette. c. Pasteur Pipet  no calibration marks  used to transfer biologic fluids without consideration of a specific Note: - Nominal volume: the volume capacity of the volumetric pipette volume -The bulb is the distinguishing feature of the volumetric pipette. It is found at the middle of the pipette. -The liquid at the tip of the pipette should not be blown out. -We have the specifications of a volumetric pipette (based on the picture):  Brand  Nominal Volume – always read the volume it can carry.  Reference Temperature – sometimes we transfer fluids from water bath or boiling specimens. Note: -Sometimes when we aspirate samples, we use Pasteur pipettes.  Error Limit  Class A – Highest quality grade d. Automatic Macropipette or Micropipette  S – Swift Grade  These are programmable pipets that can automatically take up the -As much as possible, be familiarized with these various specifications. There specified volume and can dispense in multiple wells at same time. can be errors if you are not familiar with the information on the pipette,  Three types: air-displacement, positive- especially the volume. displacement and dispenser pipets. b. Ostwald-Folin Pipette  Used for measurement of viscous solution such as blood  Liquid is delivered drop by drop  Blown out  The bulb is located near the tip of the pipette 3 MLS 11Aa CLINICAL CHEMISTRY MLS 3A Note: -These are programmable pipettes that can automatically take up the specified  Shaft – found at the bottom. volume and can dispense multiple wells at the same time.  Tip ejector arm – we have disposable tips where we can get rid of the -We have this usually at the serology department, where we use multiple wells tips once used. It also has tip ejector button. No need to touch the especially for volatile samples. disposable tip, just push the button and it will detach to the micropipette.  Sealing end of Shaft – this carries the disposable tip. Take note that the tip ejector button and arm is absent in P5000 and P10ML types of micropipettes. Some of them are present in other types. 1. Air-displacement pipette  relies on a piston creating suction to draw a sample into a disposable tip that must be changed after each use. Note: -If you remember it has a spring (piston), so if you push the plunger, it will spring up, suctioning the fluid. When we first push the plunger, it will first blow the air. Then after that we present it to the sample to which the pipette tip must only touch the sample and not the shaft as it may contaminate the micropipette. As -What are the parts of the Automatic/ Micropipette (based on the picture)? much as possible, we limit the contamination. Once we pushed the plunger, we  Plunger Button present it to the fluid. When we released our thumb on the plunger, the fluid will  Plunger be suctioned in.  Volume Adjustment Knob – we can adjust the volume to be aspirated and sometimes it has labels on what volume it can carry (from 50 2. Positive-displacement pipette microliters to 1 mL). There are windows on the body of the  operates by moving the piston in the pipette tip or a barrel, much like micropipette and this can show the volume that it can hold. It can be a syringe. It does not require a different tip after each use. Because adjusted based on your preferred volume to be drawn. of carryover concerns, rinsing and blotting between samples maybe  Digital Volume Indicator – some of the micropipettes are digital, while required. some are mechanically changed using upward and downward buttons to change the volume.  Micrometer  Branding  Shaft (Coupling)  Piston – it is found inside. It’s a spring that supplies the force for aspirating the fluids. 4 MLS 11Aa CLINICAL CHEMISTRY MLS 3A Note: -It works like a syringe, where we pull the lever to suction the fluid. The problem is that as our positive-displacement pipette has disposable tip, so meaning it can have carry-over concerns, where the sample you aspirated first can contaminate the next sample you will aspirate. To solve this, we need to rinse and blot between samples. Thus, we prefer to use air-displacement pipette because of its convenience and accuracy because the positive displacement Note: -It has graduations up to the tip (from the lowest to the highest). It has two can be prone to contamination. etched rings so it has to be blown out. -It has the same parts as the micropipette but differs in interior parts. It has the shaft, disposable piston where it suctions the fluid, disposable capillary and the 2. Mohr Pipette piston seal.  TD (non-blown out pipette)  Without graduations to the tip. 3. Dispenser pipette- Dispenser/ Dilutors  Calibrated between 2 marks or calibrated to deliver in between  Automatic pipettes that obtain the liquid from a common reservoir and  Self-draining pipette dispense it repeatedly.  Graduations with uniform interval but well away from the delivery tip  Accuracy is for the full volume delivery  The smaller the volume used, the less accurate is the volume delivered. Note: -It has the same structure with the juicer, but it can deliver a specified amount of fluid. It has dispenser on top where it will suction the fluid and it will deliver to the receiving vessel. It also has reservoir where it can have maximum capability to which it can repetitively dispense the fluid. B. Graduated or measuring Pipettes  Long, cylindrical tubes drawn out to a tip Note: -In the blown out pipettes, liquid that are still inside the pipette are blown out  Calibrated in fractional uniform volume because it is still part of the graduation. Unlike the liquid in the non-blown out  Example of Graduated or measuring pipettes: Serologic Pipette, pipettes, in which excess liquids left in the pipette are not part of the graduation. Mohr Pipette, Bacteriologic Pipette, Ball, Kolmer and Kahn Pipettes, Graduated Micropipettes Examine pipettes A and B. Which is the Serological and which is the Mohr Pipette? 1. Serologic pipette  With graduation up to the tip  Blown out pipette 5 MLS 11Aa CLINICAL CHEMISTRY MLS 3A 3. Bacteriologic Pipette 2. Disposable Capillary micropipettes  With the ability to deliver samples containing particulates, calibrated  With a capillary tubing marked with a specified volume bacteriological pipettes are used routinely in the dairy field for  Blown-out undiluted milk.  Calibrated TC, requires rinsing  The single use device has wide tips to transfer thick liquids easily for routine quality assessment studies. Note: -Undiluted milk has bacteria so a bacteriologic pipette is used when handling it. 4. Ball, Kolmer and Kahn Pipette Note: -Same as capillary tubes but with marks. -You use the tubing to aspirate. This is used as a substitute of our aspirators. 5. Graduated Micropipette  TC pipettes designed as either Mohr or serologic Specifications on a Graduated pipette  Used when small amount of blood or specimen is needed (