CENTRIFUGATION

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CENTRIFUGATION:

CENTRIFUGATION

Introduction :

Introduction Centrifugation is one of the most important and widely applied research techniques in biochemistry, cellular and molecular biology, and in medicine. A centrifuge is one of the best devices for separation of microorganisms from a suspending fluid using centrifugal force (g-force)

Rate of settling (r[cm/sec]) Stroke’s law:

Rate of settling (r[cm/sec]) Stroke’s law r= 2a²g( dp -dm) 9 η dp =density of spherical particle a=radius of spherical particle(cm) dm=density of medium η = viscosity of medium( cgs ) g=acceleration due to gravity(981cm/s²)

Factors increasing Rate of settling :

Factors increasing Rate of settling An increase in size of the particle. (Fungi > Bacteria> Viruses) increase of radius by factor 2 will increase the Rate of settling by 4. An increase in the difference between the density of the particle dp & that of the medium dm. A decrease in the viscosity of the medium. An increase in the force due to gravity.

RCF(RELATIVE CENTRIFUGAL FORCE):

RCF(RELATIVE CENTRIFUGAL FORCE) RCF (in g )= 1.118X10̄⁻⁵XRXN² R=radius of centrifuge rotor in cm, being the distance from the centre of the centrifuge shaft to the tip of the centrifuge tube. N= revolutions/min(rev/min)

Types of centrifuge:

Types of centrifuge Small bench centrifuge: For bacteriology or virology lab. 10 – 40 specimen containers of 5 – 30ml capacity RCF of 5000g Refrigerated centrifuge: For research purpose 6L with max. 50000 – 70000 g. Ultra centrifuge: Speed ranging from 40000 – 75000rev/min RCF 100000 – 500000 g. Viruses and rickettsias , for subcellular fractions.

Types of rotor:

Types of rotor Horizontal swing – out rotors. Fixed – angle rotors. Vertical rotors. Continuous - flow rotors.

Swinging bucket rotors:

Swinging bucket rotors Sample tubes are loaded into individual buckets that hang vertically while the rotor is at rest. When the rotor begins to rotate the buckets swing out to a horizontal position. Useful when samples are to be resolved in density gradients. The longer pathlength permits better separation of individual particle types from a mixture. However, this rotor is relatively inefficient for pelleting .

Fixed-angle rotors:

Fixed-angle rotors Sample tubes are held fixed at the angle of 20 – 45⁰ in the rotor cavity. When the rotor begins to rotate, the solution in the tubes reorients. This rotor type is most commonly used for pelleting applications. Examples include pelleting bacteria, yeast, and other mammalian cells. It is also useful for isopycnic separations of macromolecules such as nucleic acids.

Vertical rotors:

Vertical rotors Sample tubes are held in vertical position during rotation. This type of rotor is not suitable for pelleting applications but is most efficient for isopycnic (density) separations due to the short pathlength . Applications include plasmid DNA, RNA, and lipoprotein isolations.

Continuous - flow rotors:

Continuous - flow rotors Not used in medical microbiology bcoz of aerosol problems.

Materials of choice:

Materials of choice Glass Plastics Cellulose nitrate Stainless steel

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Glass: Limited to low RCF. Not recommended for bacteriological work. Stainless steel Very inert. Can be heat sterilized. Can Withstand very high RCF. Plastics Can Withstand very high RCF.

Selection of Centrifuge Tubes:

Selection of Centrifuge Tubes Selection of the appropriate centrifuge tube: Prevents sample leakage or loss Ensures chemical compatibility Allows easy sample recovery Major factor in selection of a tube (plastic) material: Clarity Strength ,Chemical & heat resistance Sealing mechanism

Types of Centrifugal Separations :

Types of Centrifugal Separations Differential centrifugation. Separation is achieved primarily based on the size of the particles in differential centrifugation. It is commonly used in simple pelleting and in obtaining partially-pure preparation of subcellular organelles and macromolecules. For the study of subcellular organelles, tissue or cells are first disrupted to release their internal contents. This crude disrupted cell mixture is referred to as a homogenate. During centrifugation of a cell homogenate, larger particles sediment faster than smaller ones and this provides the basis for obtaining crude organelle fractions by differential centrifugation. A cell homogenate can be centrifuged at a series of progressively higher g-forces and times to generate pellets of partially-purified organelles.

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When a cell homogenate is centrifuged at 1000 x g for 10 minutes, unbroken cells and heavy nuclei pellet to the bottom of the tube. The supernatant can be further centrifuged at 10,000 x g for 20 minutes to pellet subcellular organelles of intermediate velocities such as mitochondria, lysosomes , and microbodies . Some of these sedimenting organelles can obtained in partial purity and are typically contaminated with other particles. Repeated washing of the pellets by resuspending in isotonic solvents and re- pelleting may result in removal of contaminants that are smaller in size. Obtaining partially-purified organelles by differential centrifugation serves as the preliminary step for further purification using other types of centrifugal separation (density gradient separation).

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2. Density gradient centrifugation. Density gradient centrifugation is the preferred method to purify subcellular organelles and macromolecules. Density gradients can be generated by placing layer after layer of gradient media such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode. The cell fraction to be separated is placed on top of the layer and centrifuged. Density gradient separation can be classified into two categories. 2a. Rate-zonal (size) separation. 2b. Isopycnic (density) separation.

2a. Rate zonal (size) separation :

2a. Rate zonal (size) separation Rate-zonal separation takes advantage of particle size and mass instead of particle density for sedimentation. Examples of common applications include separation of cellular organelles such as endosomes or separation of proteins, such as antibodies. For instance, Antibody classes all have very similar densities, but different masses. Thus, separation based on mass will separate the different classes, whereas separation based on density will not be able to resolve these antibody classes.

Criteria for successful rate-zonal centrifugation: :

Criteria for successful rate-zonal centrifugation: Density of the sample solution must be less than that of the lowest density portion of the gradient. Density of the sample particle must be greater than that of the highest density portion of the gradient. The pathlength of the gradient must be sufficient for the separation to occur. Time is important. If you perform too long runs, particles may all pellet at the bottom of the tube.

2b. Isopycnic separation :

2b. Isopycnic separation In this type of separation, a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. Once this quasi-equilibrium is reached, the length of centrifugation does not have any influence on the migration of the particle. A common example for this method is separation of nucleic acids in a CsCl gradient.

Criteria for successful isopycnic separation: :

Criteria for successful isopycnic separation: Density of the sample particle must fall within the limits of the gradient densities. Any gradient length is acceptable. The run time must be sufficient for the particles to band at their isopycnic point. Excessive run times have no adverse effect.

PRESERVATION OF CULTURES:

PRESERVATION OF CULTURES Regular subculturing Cold storage Drying methods Freeze – drying in vacuo (LYOPHILIZATION) culture is dried rapidly in vacuo from frozen state

LYOPHILIZATION:

LYOPHILIZATION Lyophilization (freeze drying) is the removal of water from frozen material. It is an excellent method for preserving microbes and heat-sensitive materials such as proteins, plasma, etc. Three steps in lyophilization Freezing. The sample is placed in a freezing vial/flask. The purpose is to completely freeze the sample. Primary drying . Approximately 90% of the total water in the sample (essentially all of the free water and some of the bound water) is removed by sublimation(Vaporization or evaporation wholly from a solid phase without melting). Secondary drying . Bound water is removed by desorption(The release of liquids and gas trapped within a substance), resulting in a product that has <1-3% residual water. This step requires 1/3 - 1/2 the time needed for primary drying.

Slide 36:

Two methods of freezing: Prefreezing Evaporative freezing Greaves Centrifugal method is used during initial stage of evacuation and drying to prevent frothing.(primary drying)

Slide 38:

Used: stock strains of bacteria,guinea pig complement serum,antisera for reference or std purpose,therapeutic antisera,human plasma,antibiotics & vaccines.

2 methods of centrifugation can be used to rapidly detect S.aureus from blood culture broth:

2 methods of centrifugation can be used to rapidly detect S.aureus from blood culture broth DIRECT CENTRIFUGATION Quicker process 10ml bl broth + 20ml st DW(to lyse RBC) centrifuge at 1000g for 10min, discard Supernatant, Use pellet for aurease test in RAPIDEC staph kit. DIFFERENTIAL CENTRIFUGATION cleaner preparation 10ml of bl broth Centrifuge at low speed to sediment blood cells Remove supernatant Centrifuge at 1000g for 15min to pellet bacteria Remove & discard Supernatant, Resuspend the pelleted bt in .5-1ml st DW used for testing

Applications for centrifugation:

Applications for centrifugation sedimentation of cells and viruses, separation of subcellular organelles, and isolation of macromolecules such as DNA, RNA, proteins, or lipids.

Slide 42:

Real-time PCR Rapid procedure for isolation of plasmid DNA Bacterial magnetosomes : molecular detection and differentiation of Malassezia yeast species on human skin Purification of plasma membranes of rat liver, application of zonal centrifugation to isolation of cell  membranes flow cytometry

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Thank you

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