Laboratory Centrifuge PDF

Laboratory Centrifuge Centrifuges are instruments that spin samples at high speeds, forcing the heavier particles to the bottom of the container (usually a tube). The part of the centrifuge that holds the tubes and rotates during operation is the rotor. Centrifuges are frequently used to separa...

Laboratory Centrifuge Centrifuges are instruments that spin samples at high speeds, forcing the heavier particles to the bottom of the container (usually a tube). The part of the centrifuge that holds the tubes and rotates during operation is the rotor. Centrifuges are frequently used to separate the cellular components of blood from serum or plasma and to centrifuge urine to obtain urine sediment. Centrifuges vary in size, capacity, and speed capability. Clinical centrifuge is the name given to models that can be used for urinalysis or serum separation. Clinical centrifuges can be large floor models or small enough to fit on a benchtop. These usually have a speed capacity of 0 to 3000 rpm (revolutions per minute), and hold tubes ranging in size from 5 to 50 mL, depending on the rotor or tube carriers. Correct way to balance tubes in a centrifuge rotor: Top, placing tubes in the rotor of a tabletop clinical centrifuge; Bottom, A-C, diagrams showing correct placement of tubes (small, dark blue circles) in rotor and, D, incorrect placement of tubes. Unquestionably the medical laboratory plays an important role in the detection, diagnosis, and ultimately the treatment of medical disorders. As emphasized earlier in this program, the meticulous handling of patient samples from collection, preparation, and all the way through analysis is essential for accurate test results. Centrifugation is one of the most important steps in the preparation of clinical specimens for analysis. Consequently, it is paramount that those working in the medical laboratory not only have a basic knowledge in the theory of centrifugation but also be proficient in how to safely use and maintain centrifuges in their laboratory. The next two sections will focus on the steps necessary to safely use a centrifuge and how to keep it functioning properly. Fortunately, centrifuge accidents are rare. When they do occur, human error is the most likely cause. Over the last several years, advancements in centrifugation technology have resulted in enhanced performance, ease of use, and built-in safety features. Nevertheless, one must always keep in mind that a well-trained, centrifuge-savvy laboratory staff will be of immense assistance in preventing centrifuge accidents and costly repairs due to misuse.  It is important that the laboratory staff whose work requires a centrifuge be familiar with the manufacturer’s operating instructions for each make and model of centrifuge located in the laboratory.  When using a centrifuge, proper protective equipment should be employed, such as eyewear, gloves, and a laboratory coat.  Make sure the centrifuge is on a flat, sturdy surface prior to operation.  Confirm that the rotor is properly seated on the drive shaft, that the chamber and tube holders/ buckets are clean and dry, and the spindle is clean.  Verify that all tube holders and buckets are in place and matched. (See Figures 1 & 2) Do not interchange tube holders and buckets with other centrifuges. Use only the tube holders and buckets that came with that model of centrifuge.  Before operating the centrifuge inspect all tube holders/buckets and rotor for any cracks or flaws.  When centrifuging possible hazardous material, make sure that biocontainment lids are in use and locked into place.  Sample tubes should be evenly filled, taking care not to overfill. Both swing-out and fixed rotors must be loaded symmetrically, with opposing tubes being of the same type and filled to the same mass. For example, if you have a tube filled with 7 mL of sample and another tube with 4 mL of sample you will need two additional tubes in order to load the rotor symmetrically. This is accomplished by filling another tube with 7 mL of water or a liquid of similar density and then a second tube to match the 4 mL sample.  When loading the rotor, the opposing tubes must be of the same weight in order to “balance” the centrifuge.  Capped tubes should always remain capped during centrifugation. This not only helps prevent possible contamination by infectious agents but also any dehydration of the fluid contained in the tubes resulting from the centrifugation.   As noted previously, for each tube placed into the rotor, a tube of equal weight must be placed into the rotor directly opposite it. By doing so, the operator is ensuring that the center of gravity remains at the center of the rotor. This is critical because balancing the centrifuge not only prevents damage but also better assures a safe operation.  Do not exceed the manufacturer’s recommended maximum speed. The operator should remain with the centrifuge until it reaches the set speed.  A minor vibration in the centrifuge is normal and to be expected, but excessive amounts of vibration can mean danger. If the centrifuge begins to shake or wobble, it more than likely means that it is not balanced properly and should be stopped immediately. 3-8. Microliter pipette Micropipettes come in different sizes. At Labster we use the following pipettes: To adjust the volume, turn the adjustable wheel or the plunger (depending on the pipette) until the desired volume is displayed on the readout. It is very important to stay within the volume range of the respective pipette to avoid contaminating the pipette or producing inaccurate measurements. Proper pipetting technique is important to ensure an accurate volume of liquid is transferred. Microliter pipette instructions After selecting a pipette with an appropriate volume, you are ready to transfer liquids using the following steps. Drawing up liquid Before drawing up liquid, make sure you add a sterile tip to the pipette. Then press the plunger from the rest position to the first stop. Hold the pipette vertically and dip the pipette tip 3-4 mm into the liquid and carefully release the plunger in a controlled manner (1). Dispensing liquid To dispense liquid, hold the pipette tip against the side of the tube at a 30-40 deg angle or dip the tip beneath the surface of the liquid already contained in the tube. Then press the plunger to the second stop to make sure no fluid remains inside the tip. Discard the tip into the trash after each pipetting event by using the ejector. By serial dilution Whereas if it was done by serial dilution, we first look at the final volume and concentrations needed, then determine the dilution factor. So if we need 10 ml of a 1 mM solution, our dilution factor is: Dilution factor = 1 / (final concentration / initial concentration) = 1 / (0.001M / 1M) = 1000X dilution factor equals one divided by the final concentration divided by the initial concentration, which equals one divided by 0.001 molar divided by 1 molar, which equals 1000 We can split up 1000X into multiple factors: 10 X 10 X 10. This means we can set up 3 tubes to perform the serial dilution. When it is a 10X dilution, it means that it is 1 part of the solution, 9 parts of the diluent (usually it is water). Similarly, for a 15X dilution, a thousand times dilution into multiple factors: 3 times a 10-fold dilution. This means we can set up 3 tubes to perform the serial dilution. When it is a 10-fold dilution, it means that it is 1 part of the solution, 9 parts of the diluent (usually it is water). Similarly, for a 15-fold dilution, it will be 1 part of the solution, 14 parts of the diluent. 1 mL of stock is transferred to the second tube that contains 9 mL of diluent (e.g water). Then 1 mL (aliquot) of the second tube is taken and transferred to the third tube. The process of transferring aliquot is repeated until the desired dilution concentration is achieved. Although serial dilution requires more preparation, it will give more accurate dilutions, provided that your technique is precise. terilization refers to the removal of all microorganisms in a material or on an object. Sterilization ensures that even fungal spores or highly resistant bacterial endospores are killed. Sterilizing media The medium and its container must be sterilized by autoclaving before use, to avoid contamination. The autoclave utilizes high-pressure saturated steam at 121o C for 15 to 20 minutes to kill the microorganisms in the medium and its container. To ensure that the autoclave procedure has been conducted correctly, the bottle that contains the medium is labeled with a piece of autoclave tape. The autoclave tape has a white stripe which will turn black if subjected to high-temperature steam. Therefore, we can identify whether a bottle of medium has been autoclaved/sterilized or not by looking at the stripe color of the autoclave tape. Autoclave tape. Before autoclave; white stripe, After autoclave: black stripe Sterilizing equipment Lab equipment can be sterilized by autoclaving, heating it up on a flame or using disinfectants such as ethanol to kill germs.