Laboratory Centrifuges A device that can rapidly spin & yoe L boc ce ny fod a % Why do we use centrifuge? oS ° A centrifuge is a laboratory device that is used for the Separation of fluids, as or liquid, based om density. Separation is achieve: by spinning a_vessel containing material at high speed, the centrifugal force pushes heavier materials to the outside of the vessel. ml wel. awhat is centrifugation? \“ t = The proces: Ss where a mixture is separated through spinning. = Cen trifugation is a separation process which uses the action of centrifugal force to promote accelerated settling oO i ‘i a mixture and is used in laboratory settings. = Two distinct major phases are formed in. the vessel during centrifugation : = sediment and = supernatant. « The performance of a centrifuge is characterised by: “lV. =4,?. (4,- 4). Wer] 180 « where Vc= eee rte (gs), « dp= particle diameter, ds = density of solid, dl = density of liquid, = + w= angular speed, F = distance of the particle to the axis of rotation « and n= viscosity of medium.> 4 SEDIMENT AND SUPERNATANT, On centrifugation, effective gravitational force cay and complete deposition of suspended in the bottom of the tube as a pellet. 5 Partic * hy 4 rapid gather The remaining solution above the pellet js cai supernate or supernatant. led — The supernatant liquid is then either + decanted from the tube without disturbing the pellet, 2 * withdrawn with a Pasteur pipette. the ¥ Application of centrifugation = General applications of centrifugation include: = Separating fat from milk to produce skimmed milk. = Separating particles from an air flow using cycloni: separation. 1 = The clarification and stabilization of wine. icesor In the field of laboratory medicine: ¥ Centrifugation separates particulate materials from a solution in which they are suspended. For example: 1 S in forensic and research laborat 2 nee in separation of portions using purification techniques such a .[Link] sulfate! to provide cell- 8.. from an analytical specimen, e.g. protein free filtrate. 9. Separating protein-bound or antibody-bound legenc from free legend in immunochemical or other assay: 10. To separate two liquid phases of different densities.Wt How does a centrifuge work? * A centrifuge is a device that spins liquid samples at high speeds and thus creates a strong centrifugal force causing the denser materials to travel towards the bottom of the centrifuge tube more rapidly than they would under the force of normal gravity. How do you calculate centrifugal force? ° F=mv/r * where F.= centrifugal force, m=mass, v = speed, and r= radius. * Online centrifugal force calculator ‘Easy [Link]’. = Online Hettich RCF / RPM Calculator¥ Revolutions Per Minute (RPM) + Revolutions Per Minute (RPM) in regards to centrifugation is simply a measurement of how fast the centrifuge rotor does a full rotation in one minute. — Basically, it tell us how fast the rotor is spinning (centrifugation speed) and does not express the centrifugal force. See eae ee ea sores Relative Centrifugal Force RCF) * Relative Centrifugal Force (RCF) or g-force (both are the same, RCF is expressed as units of gravity) is a at a sample is subject to. The force is generated from the spinning of the rotor which, in turn, exerts this force outward on the centrifuge tube. — Not only does RCF * ke into account the speed of rotation, it us a.g-force measurement. * RCF is the preferred method of measurement as it will remain thé same even if you are using a different centrifuge with a different rotor size.& eae: Speed of Centrifuge * Centrifuges will have a speed range that they are capable of achieving and will vary depending on the centrifuge. 1.6A ge might spin at speed below 10,000 rpm. 2. Whilst alhigh Speed / Super speed centrifuge could spin between 10,000 and 20,000 rpm. 3. einen also available and are the most powerful typ entrifuge, they can spin in excess of 150,000 rpm. s 4 \ Difference Between RPM and Ror? SSF = RPM and RCF/g-force can be measured in centrifugation but , both RPM and RCF are very different. — RPM simply measures how fast the rotor is spinning and this information is helpful, but it lacks the ability to truly measure theg’sthe sample is'subject fo. * For example a centrifuge spinning at 5000 RPM, will spin at 5000 RPM regardless of the centrifuge being used or the size of the rotor. LA — RCF accommodates the rotor size into its measyrement so we can determine the grav nal force being applied. This is why we should always document our procedures as 'g’s rather than RPM. x “ =ymmutRotor or Centrifuge head * It holds the tubes Or other containers of the sample . The rotor can be of three types. Ee i. Fixed angle rotor ii. Vertical tube rotor ili, Swinging bucket rotor Contd... = Fixed-angle rotor — The rotor (mainly made of aluminium) is very compact. — There are boreholes with a specific angle (like 45°) within the rotor. - — These boreholes are used for the sample tubes.Centrifuge Rotors (mems.3,2) BFixed Angle Rotor =f BSwinging Seas Bucket Rotor 1% || Contd... " Vertical tube rotor — They are considered as zero angle fixed angle rotors in which the tubes are aligned vertically in the body of the rotors at all times.Contd... » Swing-out rotor (= horizontal rotor) — The rotor looks like across with bucket. — Within these buckets, different tubes can be centrifuged. - For a safe centrifugation, a specific adadpter for every tube shape is mandatory. : Rotor shaft * The rotor shaft is Usually driven by a gyro system ( reduces noise and vibration), and the bearings are Usually sealed, tinimizing vibration and the need for lubtcation: = igeiamaee sees :Tachometer * The tachometer measures rotor speed in rpm * Centrifuge speed is controlled by a potentiometer that modulates the voltage supplied to the motor. Speed is also determined by the mass of the load in the rotor. * The tachometer measures rotor speed if rpm. ) = aS Alarm * Some centrifuges are equipped with an alarm that sounds when a malfunction, such as a tube imbalance occurs. “= Some. centrifuges automatically shut down _under these conditions, preventing tube breakage and the potential for exposure to bio-hazardous agents. — Newer centrifuges automatically decelerate OY weproaee : rriers are improperly balancedS__ Improper _ ae Improper halancing an cause the centrifuge to vibrate, disrupting the formed pellet.Safety lock All modern centrifuges have a required safety latch that Prevents the operator from opening the instrument befoi o instrument the rotor has sto} a Timer * The timer permits the rotor to reach a preprogrammed speed: the rotor then decelerates without braking after a set time has elapsed.Brake * The brake decel erates the rotor by r ; the current to the motor. — ¥Teversing the polarity g — Refrigeration Unit °* When necessary, refrigeration units are «included. — Refrigerated centrifuges are used when th generated during centrifugation could on evaporation or denaturation of protein or lea a of cellular components in the a. a temperature can be controlled between tear. f. allowing centrifugation at higher SP and for prolonged periods. e heat gutProtective shield + Whenever possible, tubes containing bio-hazardous materials should be centrifuged with the caps or stoppers in place to minimize aerosols. iy \ ee tubes . Be comtituse tubes can be used with most solvents, but ec, paiclexexpensive) They can be cleaned like other Ty glassware, and can be sterilized by autoclaving. * Plasti i reuse tubes, especially micro-centrifuge tubes less expensive. They are more difficult to clean thoroughly, andare Y, and are usu i i be considered ually inexpensive enough toCentrifuge tubes ontrifuge tubes or centrifuge tips are tapered tubes of srious sizes made of glass or plastic. os hey may vary in capacity from tens of mm, to much smaller ru, S\ myrv apacities used in micro-centrifuges used extensively in—) m» notecular biology laboratories. orn £ fhe most commonly encountered tubes are of about the size and shape of a normal test tube (~ 10 cm long). ~~ \o we Micro-centrifuges typically accommodate micro-centrifuge Capacities from 250 ul to 2.0 mi ~—isowl te These are exclusively made of plastic. om) ee) Centrifuge tubes Four screw-top micro-centrifuge tubes., Ore tae mation © completed, let the maching stp ng Chen the Rd removes the centrifuge tube, use supertatane Geposit as required for examination. - Types of Centrifuges on The Basis Of'Speed_ . There are four major types of centrifuges. They are: A. Small Bench Centrifuges: i + They are used2. Large Capacity Refrigerated Centrifuges: have They ‘have Bifigefatédirotor chamber and varying size. of 6500 g and use to * They can go up to maximum sediment or collect the substances that sediment rapidly like erythrocytes, yeast ¢! cell, nuclei and 4. Ultra Centrifuges: chloroplast @ @ fe) . as tive centrifugation objective is to isolate specific) ich can be reused. = There are many type of preparative centrifugation such as differential,(Genisty gradient {rate zonal and isopycnic) centrifugation. of force of Srige oaronar eieneemem on co of shot + tt is employed for separation bes inde suots mpm of a ee - ~~ fractions from plasma and deprotonisation of p Se3. High Speed Refrigerated Centrifuges: * They can generate speed of about 60000g ahd are used to collect micro-organism, cellular debris, larger cellular organelles _and_ proteins Precipitated by ammonium sulphate. What is differential centrifugation? + Differential centrifugation is a common Proce microbiology and cytology used t dure in © Sehaate certain organelles from whole cells for further analysis of specifi in the process, a tissue sample IC Parts of Cells. Js first homogeniseg t the cell membranes and mix yp th ‘0 _brea| i cell contents, breakProtective shield + Whenever possible, tubes containing bio-hazardous materials should be centrifuged with the Caps or stoppers in place to minimize aerosols. oy ‘ cee tubes * Glass centrifuge tubes can be used with most solvents, but tend to be more expensive. They can be cleaned like other laboratory glassware, and can be sterilized by autoclaving. ~ a * Plastic centrifuge tubes, especially micro-centrifuge tubes tend to be less expensive. They are more difficult to clean thoroughly, and are usually inexpensive enough to be *| Calibrated glass t that fit into centrifuge slots for theCentrifuge tubes Z Centrifuge tubes and bottles are available in different range of peel thickness and rigidity from different variety of materials. — including glass, Cellulose, esters, polyallomer, ppolvcarponat Polyethylene, Polypropylene, kynar, nylon and stainless 2+ The type of container used will depend upon nature and volume of sample to be centrifuged along with cent ve forces to be withstood. n of centrifuge On The Basis of There are at least five types of centrifuge: WY — Preparative centrifuge 1 50 S = Analytical centrifuge ¢ ae — Angle fixed centrifuge NY — Swing head centrifuge = Microhaematocrit centrifuge = Microfuge refrigerated in Molecular biology (PCR) : ee la 43. High Speed Refrigerated Centrifuges: * They can generate speed of about 60000g and are used to collect micro: organism, cellular debris, larger cellular and proteins precipitated by ammonium organelles sulphate, What is differential centrifugation? a= * Differential centrifugation is a common procedure in microbiology and cytology used to separate ¢ rganelles from whole cells for further analysis of specific parts of cells. In the process, a tissue sample is first homogenised to break the cell membranes and mix up the cell contents.eee Lsop' : cen ri} ({ f ? A” what isdensity gradient centrifugation, + density gradient centrifugation is a procedure for such as er i articles, seperate which the enti is placed ona molecules such such as sucrase or Cesium chloride, (b) Analytical ultracentrifuge: Involves measuring the physical properties of the sedimenting particles such as sedimentation coefficient or molecular weight. = It is capable of operating at 500000 g. = Three kinds of optical systems are available in analytical ultracentrifuges: |. Light absorption system. ee ll Schlieren system and. Re Ill. Rayleigh interferometric system, both of which detect changes in the refractive index of the solution.CPrecautionary Measured with Centrifuges: . Centrifuges are extremely dangerous in * = am fang strumi ii properly maintained and correctly used. sas — It is, therefore, always advisable that i always aavis that one mi understand the operating manual for at ey s + Manufacturers of centrifuges should en: effective li ks. — Access to the rotor chamber of centri : ¢ tora ges should avoided when spin is in progress. ee + Centrifuges should have imbalance detectors, Ih. wee * It is important when centrifuging hazardous materials like pathogenic microorganisms, infectious viruses, carcinogenic, rr or toxic chemicals, radioactive materials), especially in low speed non-refrigerated centrifuges in which rotor temperature is controlled by air-flow through the rotor bowl. — Samples should be kept in air-tight, leak-proof containers. — This is to prevent aerosol_ formations arising from accidental spillage of sample which would contaminate the rotor, centrifuge and possibly the whole laboratory.+ To prevent possible physical injury when rotors are filled and emptied, : — Care must be taken to ensure that the moving rotor is not rari that long hair and loose clothing (e.g., Ties) do not get caught in any, y rotating part. = This is especially important with older centrifuges where lid can be opened before the rotor has stopped rotating. oe Maintenance and Quality Assurance in contact with the blood or other jotentially infectious agent must be imm lecontami y wi should be cleaned with dalocCentrifuge ge speeds that are routinely used should be checked perlodtcaly: using a reliable photoelectric or strobe achometer in accordance with CAP inspection guidelines. — © The accuracy of thé centrifuge timershould also be checked and verified according to CAP inspection guidelines. © The pee eet refrigerated centrifuges should be checked at least monthly under standardized conditions. The agreement between the measured and expected (or programmed) temperature should be within 2 0 C.