MXPA00008845A - Detection and characterization of microorganisms - Google Patents

Abstract

A method for separating microorganisms, especially infectious agents, from a mixture by two-dimensional centrifugation on the basis of sedimentation rate and isopycnic banding density, for sedimenting such microorganisms through zones of immobilized reagents to which they are resistant, for detecting banded particles by light scatter or fluorescence using nucleic acid specific dyes, and for recovering the banded particles in very small volumes for characterization by mass spectrometry of viral protein subunits and intact viral particles, and by fluorescence flow cytometric determination of both nucleic acid mass and the masses of fragments produced by restriction enzymes. The method is based on the discovery that individual microorganisms, such as bacterial and viral species, are each physically relatively homogeneous, and are distinguishable in their biophysical properties from other biological particles, and from non-biological particles found in nature. The method is useful for distinguishing infections, for identifying known microorganisms, and for discovering and characterizing new microorganisms. The method provides very rapid identification of microorganisms, and hence allows a rational choice of therapy for identified infectious agents. A particularly useful application is in clinical trials of new antibiotics and antivirals, where it is essential to identify at the outset individuals infected with the targeted infectious agent.

Description

DETECTION AND REPRESENTATION OF MICROORGANISMS BACKGROUND OF THE INVENTION The present invention relates to the field of separating and identifying microorganisms, particularly infectious agents, using two-dimensional centrifugation and exposure to enzymatic and chemical agents, combined with density gradient detection based on light scattering or fluorescence, counting by flow cytometry of fluorescence and representation of intact virions, bacteria, proteins and nucleic acids by mass spectrometry, flow cytometry and epifluorescence microscopy. The publications and other materials used herein are incorporated by reference to illustrate the background of the invention or provide additional details with respect to the practice, and for convenience, are respectively grouped in the attached reference list. The patents referred to herein are also incorporated by reference. In the prior art, the diagnosis of viral and bacterial infections has been made by cultivating causative agents in suitable media or in tissue cultures to obtain sufficient particles for analysis, followed by identification on the basis of which, conditions support growth , in reaction to specific antibodies, or based on nucleic acid hybridization (Gao and Moore, 1996). Biological growth can be omitted when the polymerase chain reaction (PCR) is used to amplify DNA, however, PCR requires sequence-specific primers and is thus limited to known or suspected agents (Bai et al., 1997). For all these methods, considerable time is required and the methods are useful for agents whose properties are known or presumed. Current methods do not provide means to isolate and rapidly represent new infectious agents. Hundreds of infectious agents are known and it is not feasible to have reagents available for a considerable fraction of them. Previously, techniques have been developed to recover infectious agents from blood, urine and tissues based on centrifugation or filtration, but they have not been widely used clinically (Anderson et al., 1996, Anderson et al., 1967). Higher resolution methods use zonal velocity centrifugation to separate fractions based on sedimentation rate (measured in Svedberg S units) and isopycnical banding density (measured in grams per mL or p). S-p separations have been used to isolate virus particles in a high state of purity from rat liver homogenates and have been used to isolate the equivalent of approximately 20 virions per cell (Anderson et al., 1996). In these studies, virus particles were detected by light scattering and visualized by electron microscopy. The separations required complex special equipment generally not available, one or more days of work and did not provide a definitive identification of the separated bacterial or viral species. It is important to show that candidate infectious particles isolated through centrifugal methods, in fact, contain nucleic acids. The DNA and RNA in both active and fixed bacterial particles and in viral particles were stained with fluorescent dyes specific to nucleic acids, and were observed and counted by fluorescent microscopy and flow cytometry. Many dyes are known which exhibit low fluorescence in the free state, but become highly fluorescent when bound to nucleic acids. Some bind differentially to DNA and RNA or to different specific regions and some show different emission spectra depending on whether they bound DNA or RNA. In this description, the colorants referred to are fluorescent dyes. Differential fluorescence spectroscopy can distinguish ssDNA, dsDNA and RNA. See Haugland, 1996; Mayor and Diwan, 1961; Major, 1961; Hobbie et al., 1977; Zimmerman, 1977; Peter and Feig, 1980; Paul, 1982; Suttle, 1993; Hirons et al., 1994; Hennes and Suttle, 1995; Hennes et al., 1995. Nucleic acid molecules isolated from the dimensions found in bacteria and viruses have been counted and their mass was estimated using fluorescence flow cytometry for molecules in solution and epifluorescence microscopy of immobilized molecules (Hennes and Suttle , 1995, Goodwin et al., 1993). In both cases, one can estimate the size of fragments produced by restriction enzymes and the molecules identified by reference to a database that lists the sizes of fragments of known DNA molecules produced by different restriction enzymes (Hammond et al., U.S. Patent No. 5,558,998; Jing et al., 1998). By using specific fluorescently labeled antibodies, specific identifications can also be made. These studies take time and require batteries of specific antibodies, along with epifluorescence microscopy or fluorimeters. Matrix-assisted laser ionization-desorption flight time mass spectrometry (MALDI-TOF-MS) currently allows accurate measurements of protein masses having molecular weights of more than 50,000 daltons. Previous individual virion proteins have been studied by mass spectrometry (Siuzdak, 1998); however, the resolution of complete sets of viral subunits from clinically relevant preparations of intact viruses and the demonstration that accurate measurements can be made from their individual masses has not been previously reported. Although simple protein mass measurements can reliably identify many proteins, when a series of proteins derived from a virus or bacterial cell cell is known, detection of such a series provides more definitive identification. In addition, methods are currently being developed which allow the partial sequencing of proteins or fragments of peptide enzymatically produced and in this way, the reliability of the identifications is further increased. For MALDI-TOF-MS, the currently used methods require a picomol or more protein, while mass spectrometry by electroaspersion currently requires 5-10 femtomoles. The limits of detection with mass spectrometry, especially MALDI, depend on obtaining a concentrated sample and a very small target area. The sensitivity will increase as ultramicro methods are developed to concentrate and transfer samples of a smaller volume. See, Claydon et al., 1996; Fenselau, 1994; Krishmanurthy et al., 1996; Loo et al., 1997; Lennon and Walsh, 1997; Shevchenko et al., 1996; Holland et al., 1996; Liang et al., 1996. Centrifugal methods for concentrating large volume particles in small volumes have been used for decades. By using microbanking centrifuge tubes which have a large cylindrical volume and cross section which is tapered in a downwardly centrifugal direction to a small tubular section, the particles can be concentrated or bound in a density gradient restricted to the tubular base narrow the tube, or they can be compressed. The basic design of these tubes is known to those skilled in the art. See, Tinkier and Challenger, 1917; Cross, 1928; ASTM Committe D-2, 1951; Davis and Outenreath; patent of E.U.A. No. 5,342,790; Sauders et al., Patent of E.U.A. No. 5,422,018; Sauders, patent of E.U.A. No. 5,489,396. The original tubes of this type were called Sutherland bulbs and were used to determine the water content of oil (The Chemistry of Petroleum and Its Substitutes, 1917, ASTM Tentative Method of Test for Water and Sediment by Means of Centrifuge, ASTM Designation: D 96-50T, 1947). In the patents of E.U.A. Nos. 4,106,907; 4,624,835; 4,861, 477; 5,422,018; 5,489,396 slight modifications of the basic design are described. Such tubes have been made of glass or plastic materials and the use of water or other fluids to support plastic or glass centrifuge tubes in metal centrifuge protectors has long been known in the art. However, the centrifuge tubes described in the prior art, which include a shape similar to that of the microbanking centrifuge tubes of the present invention could not withstand the centrifugal forces required to bind viral particles in gradients. The conventional centrifuge tubes, or tubes derived from the Sutherland design, have been used for gradient density separations and for separations in which plastic or wax barriers are used, which are placed between the regions of different density to allow the recovery of these fractions without mixing. There has been no previous discussion about barriers which avoid the mixing of gradient components of the stage at rest, but of barriers which are centrifuged away from the gradient during rotation. Also, tube housings for high speed thin wall oscillating blade centrifuge tubes, whose outer surfaces can be disinfected after the tubes are loaded, have not been described.

The efficient stabilization of very low density gradients in centrifugal fields is well known and is used in ultra analytical centrifugation to cause a sample layer to flow rapidly to the centripetal surface of an unmixed gradient using a synthetic boundary cell (Anderson, patent of US No. 3,519,400). Consequently, the lightweight physical barrier disks between the stage gradient components can be moved away from the gradient by centrifugal force without significantly altering the gradient, since they are made of porous, woven or sintered materials having a physical density lower than the of the sample layer, such as polyethylene or polypropylene. Many authors have noticed that viruses and bacteria are often resistant to the actions of detergents and enzymes, which will digest or dissolve contaminating particles of biological origin, and efforts have been made to classify infectious agents based on their differential sensitivities. These differences have not been incorporated previously and advantageously into a method for detecting and quantifying infectious agents. See, Gessler et al, 1956; Theiler, 1957; Epstein and Hold, 1958; Kovacs, 1962; Planterose et al., 1962; Gard and Maaloe, 1959. Density differences between different species of viruses and bacteria are well known, but have not been used previously for identification purposes. Infectious particles show a broad scale of isopycnic banding densities ranging from about 117 g / ml to 1.55 g / ml, depending on the type of nucleic acid present and the radii between the amount of nucleic acid, protein, carbohydrate and lipid present. Although such differences in banding density are known, no attempt has previously been made to measure them systematically and use the data to classify infectious agents. The present invention is directed to an integrated system for concentrating, detecting and representing infectious agents using separations based on sedimentation rate and banding density, spectral analysis of fluorescent light emitted to distinguish DNA from RNA, differentiation of viral and bacterial particles from other particles by sedimentation through zones of enzymes or solubilizing reagents, determination of the isopycnic banding densities of infectious particles by means of reference to the positions of particles of synthetic density standardization, particle detection using fluorescent dyes for DNA or RNA, concentration additional particles grouped by compression, transfer of concentrated particles to mass spectrometer targets for determination and analysis of protein mass, counting of concentrated particles by epifluorescent microscopy and flow cytometry luorescence and identification of bacterial or viral nucleic acids by restriction fragment length polymorphism analysis, using either immobilized nucleic acid molecules or ultrasensitive fluorescence flow cytometry. These methods are especially useful for representing biological samples that have low virus titers and which contain viruses, which are not cultivable. In addition, all current methods used to detect and represent infectious agents, including the use of fluorescent antibodies, detection of agent-associated enzymes, culture to increase agent mass, PCR amplification, restriction fragment length polymorphism analysis , hybridization for immobilized probes in fragments, histochemical analysis and all forms of microscopy including electron microscopy, are greatly improved by preconcentration of the microorganisms using the methods of the present invention. These techniques have not been previously gathered in an operational system capable of use in routine field, hospital and clinical laboratory. The present application describes innovations and inventions which make said system feasible. To work with potentially lethal agents, the system will be assembled in containment and will be at least partially automated.

BRIEF DESCRIPTION OF THE INVENTION The overall objective of this invention is to develop a physical system for rapidly identifying agents of infectious diseases without cultivating them and for discovering new infectious agents. The procedure is based on the thesis that infectious agents constitute a single group of particles, which can be isolated through physical and chemical means from other natural particles and identified by their physical parameters using centrifugal means, fluorescence and spectrometry of dough. The system will allow a rapid clinical distinction between viral and bacterial infections, identification of specific agents with the aim of providing specific therapy and the rapid discovery of new infectious agents. In addition, the system will make it feasible to develop and test new antibiotics and antiviral agents in man, by measuring the effects of these agents on bacterial and viral loads. Currently, the development of new antiviral drugs is severely hampered by the inability to define populations of individuals in the early stages of infection that could benefit from treatment. In accordance with the present invention, an ultracentrifuge tube is provided which comprises upper, middle and lower regions of successively smaller diameters. In one embodiment, the tube has an upper region for receiving a sample, a funnel-shaped middle region and a lower region of narrow tubular microbending. The diameter of the lower region may be 0.635 cm or less, preferably 0.254 cm or less, preferably 0.254 to 0.2032 cm and more preferably 0.2032 to 0.099 cm. The smallest diameter microbanking regions are feasible and are within the scope of this description. The length of the lower region is usually between 5% to 25% of the length of the tube. In one aspect, the tube may also include a seal with a central opening, which can be connected and disconnected. In accordance with the present invention, a blade is provided to support an ultracentrifuge tube. The vane comprises upper and lower regions, which may be of successively smaller diameters, may have inserts for successively decreasing the internal diameter or may be of a uniform internal diameter and may further comprise a third region, which adds the vane to a rotor. In addition, according to the present invention, a method for concentrating microorganisms is provided. As used herein, the term "microorganisms" is intended to include viruses, myoplasmas, rickettsia, yeast and bacteria. The method comprises the ultracentrifugation of a sample containing the microorganism in an ultracentrifuge tube described herein. Ultracentrifugation can include the formation of density gradients and / or the staining of microorganisms. In one aspect, the staining can be used to distinguish the DNA or RNA content of a virus. The banding of the microorganisms in the ultracentrifugation can be used to identify the microorganisms. In a further aspect of the invention, the concentrated microorganisms are subsequently represented by conventional techniques such as mass spectrometry, flow cytometry, optical mapping, isopycnic banding densities, fluorescence, restriction enzyme analysis, genome size, resistance / enzymatic or chemical susceptibility, immunochemistry and the like. In another aspect of the invention, the amount or title of the microorganisms can be determined. In accordance with the present invention, a system is provided for measuring the fluorescence of a sample in a centrifuge tube. In one embodiment, the system includes a centrifuge tube, a light source, such as a laser, and a detector to detect light that passes through the sample or that is emitted from the sample at the time the light passes through. through this one. Optical filters select and separate the wavelengths of emitted and excitation light. The internal surfaces of the tubes and especially, the funnel portion must be very smooth in order to prevent the small virus particles from being retarded by surface irregularities and in addition, the surfaces must be treated in such a way that the infectious particles are not adsorbed. The polishing of plastic surfaces is done through a brief exposure to a solvent vapor. For example, polycarbonate is polished by brief exposure to heated gaseous methylene chloride. The plastic surfaces are modified to prevent the adsorption of infectious particles by exposure to diluted solutions of proteins such as bovine serum albumin or gelatin, or charged polymers, such as heparin or heparin derivatives. Both methods are well known to those skilled in the art. In addition, according to the present invention, a system for counting concentrated particles in a small volume is provided. The system includes a container in which the particles are concentrated, a capillary tube, two pumps, means for moving the container in relation to the capillary tube, a flow cell, a light source and detector. Alternatively, the fluid can be moved by gas pressure instead of pumps.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an S-rho plot for a typical tissue and for representative viruses. Figures 2A-2C are a schematic representation of one embodiment of a centrifuged microbanking tube and its use. Figures 3A-3G show alternative embodiments of micronized tubes and use in a rotor (3F). Figures 4A and 4B illustrate a centrifugal oscillating blade design that allows faster fractioning of large sample volumes. Figure 5 illustrates a complete system including vertical monochromatic laser illumination, goniometer and X-Y stage to support and position the microbanded tube, microbanded tube with pooled virus particles and camera system. Figure 6 illustrates a complete system that includes interference filter light sources, light conduit illumination, digital data acquisition and CRT data presentation.

Figure 7 illustrates a method for recovering virus particles pooled using a micropipette and counting them by flow cytometry. Figures 8A-8C illustrate details of band recovery. Figures 9A-9F illustrate one embodiment of a housing for oscillating rotor rotor centrifuge tubes and detection of a sample with respect to the tubes.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to methods for identifying and measuring the presence of microbial agents, such as bacteria and viruses in biological samples. The methods include centrifugation steps to purify the microbial agents in a very small volume. The agents are then analyzed by means such as isopycnical banding density, fluorescence or mass spectrometry. It is an object of this invention to develop integrated systems and methods in which suspensions containing microorganisms, including infectious agents, are stained with one or more fluorescent dyes, in which a continuous step or gradient is automatically formed during centrifugation, in the which microorganisms are centrifuged away from the dispersed medium containing stain and washes free from external staining, concentrated in a gradient of very small cross section, separated according to their isopycnical banding densities, with their determined banding densities and the microorganisms detected by fluorescence. It is a further object of this invention to concentrate microorganisms, which include infectious agents, in microstrips by a factor of 1-5,000. It is a further object of this invention to expose microorganisms, such as infectious agents, to reagents that include detergents, surfactants, enzymes or organic solvents contained in different zones in a density gradient, to dissolve or separate contaminating particles to prevent them from group with microorganisms and to separate stained particles from free staining of the initial sample volume. It is a further object of this invention to use one or more dyes, which bind differentially to RNA, double stranded DNA or DNA to allow them to be distinguished by their fluorescent spectra. It is a further object of this invention to provide the concentration of pooled microorganisms, for example infectious agents, by re-dispersing the banding gradient, which is normally 0.04 mL, to about 4 mL in water or a very dilute pH regulator and compressing the microorganisms one or more times to provide a concentrated tablet free of gradient materials for mass spectrometric analysis, for epifluorescent microscopy counting or by flow cytometry. It is a further object of this invention to provide means for the diagnosis of infectious diseases which minimize the exposure of laboratory personnel to infectious agents. It is a further object of this invention to provide means for preparing nucleic acids from small amounts of microorganisms, which include infectious agents, to determine the masses of intact nucleic acid molecules and to represent fragments produced by restriction enzymes using either flow cytometry or epifluorescence microscopy. It is a further object of this invention to precisely determine the baiting densities of the microorganisms, such as infectious agents, by reference to the positions of calibrated particles added to the gradients. For ease of description, the invention will be described with reference to viruses as microorganisms. It will be understood that the invention is also applicable to other microorganisms, including mycoplasmas, yeast and bacteria. The invention is particularly suitable for the identification of infectious agents and will be described in this context. Figure 1 is a graph that describes the sedimentation coefficients and densities of isopycnic banding of subcellular organelles and viruses to illustrate the concept of "Virus Window" (Anderson, 1966). It is evident that viruses have a relatively narrow scale of sedimentation coefficients and banding densities and can be isolated from a homogenate of tissue or blood in a high state of purity using high resolution S-p separation systems. For a complete description of centrifugal methods of high resolution Sp and centrifuge development for virus isolation, refer to National Cancer Institute Monograph 21, The Development of Zonal Centrifuges and Ancillary Systems for Tissue Fractionation and Analysis, Department of Health, Education and Welfare of the USA, Public Health Service, 1966. This paper describes the theory and separation systems Sp and the use of colored plastic beads of graduated densities as density indicators. In practice, a sample of blood or tissue homogenate is centrifuged to pellet all particles that have settling velocities higher than that of the particle or particles to be analyzed. For viruses, that means particles of around 10,000 S and more are discarded. After such separation, the supernatant is used as the sample for second dimensional isopycnic banding separations made in microbanking tubes as described herein, using centrifugation conditions which will pellet and cluster all known infectious particles. A picomol of virus would contain 6,022x1011 of viral particles, while 6x109 of virions would contain a picomol of a viral coat protein present in 100 copies per virion. The quantitative polymerase chain reaction (PCR) has been used to show that in many infectious diseases >108 of virus particles are present per mL of plasma or serum. Consequently, if the virions of a biological sample of 5-10 mL containing 108 virions / mL are concentrated to one microliter or two, and then applied to a very small target area, the individual viral proteins can be detected using MALDI-TOF -MS (Krishmanurthy et al., 1996; Holland et al., 1966). By using electrospray techniques, samples containing 106 virus particles / mL can be detected, while with flow cytometry and immobilized DNA epifluorescence microscopy, fewer particles are required (Hará et al., 1991; Hennes and Suttle nineteen ninety five). The application of these methods to bacteria may require a preseparation of proteins to reduce the complexity of the sample. In mass spectrometry, the detection has been by charged ion detection and the limitations of such detection have established the upper limits on the size of proteins and nucleic acids that can be detected. We have now described mass spectrometric methods which allow the measurement of biological particle masses greater than 100,000 daltons. In order to work with such virus levels, the virus must be concentrated in a very small volume. This concentration is achieved during the second dimension of centrifugation (the isopycnic banding stage) by pooling the viruses using a specially designed centrifuge tube to concentrate the virus in a microstrip after passing through gradient layers that wash the particles and the expose selected reagents. An example of such a microbanking centrifuge tube is shown in Figures 2A-2C. Figure 2A schematically illustrates a hollow, transparent centrifuge tube 1 with an upper sample volume 2, which gradually becomes a denture funnel region 3 having sections of parallel and successively tapered walls 4-6, which taper toward down in a narrow region of tubular microbending 7. The denture funnel region 3 is an improvement in the centrifuge tubes, which simply tapers from the top to the bottom without including a denture region. By denture it refers, for example, to concentric rings or edges or edges. These rings, edges or edges preferably are continuous around the inner diameter of the centrifuge tube, but this is not mandatory. For example, three projections can be used from the inner wall of the centrifuge tube equally spaced around the diameter to support a disc in place. The term dentures includes those possibilities but does not include a straight taper with no ring, edge, edge or projection on the inner surface of the centrifuge tube. Dentures can be used as supports in which discs can be placed to separate two or more layers of fluid. Although the discs can be placed in tubes which simply taper without dentures, the discs in such tapered tubes can be easily tilted on one edge by pressing on the opposite edge. This would cause premature mixing of the layers which will be separated by the discs. The denture region allows the discs to be extended and prevents the discs from accidentally tilting. As an example, the centrifuge tube may be 8,763 cm from the top to the bottom, have an outer diameter at the top of 1427 cm and have an internal diameter at the bottom microbanking region 7 of 0J625 cm. Said tube is suitable for use in a SW41 Ti rotor (Beckman). The internal surface of the tube is preferably polished using conventional techniques, including steam polishing, so that the virus particles do not stick to the wall of the tube. In addition, the internal surfaces of the tubes can be coated with a protein or polymer to prevent particle adhesion, as is known in the art. Figure 2B illustrates how the standing tube is loaded with a series of fluids of descending physical density. The tube shown comprises a series of dentures in which disks can be extended to separate a fluid layer from the next fluid layer. The liquid 8 is denser than any particle to be recovered, and it is used to partially fill the microbanded region 7. When a less dense fluid 9 is introduced with a micropipette, an air bubble 10 can be left (whereby air is collected). refers to atmospheric air or other gas) to keep fluids 8 and 9 separated. Similarly, when the first layer 11 fluid is introduced, an air bubble 12 can be left in place, thus keeping the three liquids separated until that centrifugation starts. A tube with an internal diameter of 0J625 cm in the microbanking region 7 is suitable to allow an air bubble to remain in place to separate two layers of liquid. Alternatively, air bubbles can be omitted and the fluids spread together to create a density gradient. The fluid 11 is covered with a light porous plastic disc 13, preferably of sintered polyethylene or polypropylene, which fits in place in the first denture. A fluid 14, less dense than the fluid 11, which may contain one or more reagents, is introduced and covered with the disk 15, followed by an even less dense liquid 16 which is covered with the disk 17. The complete system It is stable until it is centrifuged. Before use, the sample layer 18, which has a density lower than that of the fluid 16 is added to level 19. The tubes are then centrifuged at high speed in metal shields, usually with water or other liquid added to the shields. In addition, the tubes can be supported by fitting adapters which will fill the space between the tubes and the protectors and water can be added to fill any space between the tubes, adapters and protectors to provide additional support. Optionally, the tubes can be capped (as shown in Figures 9A-9F, described in greater detail below), to minimize the chances of operator infection. Figure 2C shows schematically a tube after centrifugation. The porous separation discs 13, 15 and 17 have been raised to the top of the tube and the sample layer 18 is cleared of virus and the original cap gradient has changed, by diffusion, into one of a series of low gradients. In addition, gas bubbles 10 and 12 have also moved centripetally and fluids 8 and 9 have been contacted to form a stage gradient by diffusion. As the centrifugation progresses, the slope of this gradient decreases, producing a gradient of banding of a suitable width to group the infectious agents. For cesium chloride gradients, the densities are usually in the range of 1 J8 to 1.55 g / ml. These gradient steps can not only contain reagents to dissolve non-viral particles, but also serve to wash the excess fluorescent dye from the particles. For example, various detergents or enzymes such as proteases may be added, either to the sample layer 18 or to other layers such as 14 or 16. Fluorescent dyes may also be present in these regions. The free dye will not enter the lower and denser regions in which the virus is grouped and therefore, the centrifugation will purify the viruses from all the reagents that may be present in the upper and less dense layers. After centrifugation, the microbanking region of the tube contains the upper portion of the banding gradient 27, pooled viruses 28 (including any dye attached to the virus or viral nucleic acid) and the lower dense portion of the banding gradient. 29 and the gradient formed between them by diffusion. Figures 3A-3G illustrate alternative embodiments of tubes useful for microbanking of viruses and bacteria and all have an internal construction in dentition that allows to place and maintain at rest one or more light barriers. The tubes shown in Figures 3A-3D and 3G are designed to be centrifuged in oscillating blade rotors so that the tubes are horizontal during centrifugation and vertical at rest. The tube shown in Figure 3A is the most conventional design with a sample reserve 31, a denture funnel region 32 and a microstrip section 33. The tube shown in Figure 3B is similar to that of Figure 3A, but is supported in a centrifuge shield by a support insert 34 which may be plastic or metal. The tubes shown in Figures 3B-3E completely fill a rotor chamber. The tube of figure 3C has an opaque lower section 35 which absorbs scattered light, while that shown in figure 3D, has a bulbous section 36 in the lower part of the microbanking tube 37 to contain an excess volume of the fluid that forms the dense end of the gradient, thus stabilizing the gradient. The tube shown in Figure 3D is designed to be centrifuged in an angular head rotor as shown in Figure 3F and has a linearly continuous wall 38 along a side placed in the rotor, so that the particles can be slide down easily to the micro-band region 40. The tube shown in Figure 3G illustrates how a very long micro-band tube can be made. Figures 4A-4B illustrate the manner in which the tube of the figure 3G can be centrifuged at a higher speed than tubes that have a constant radius from the top to the bottom curve. This is done using a carbon fiber, plastic or metal shield 45 which matches the dimensions of the tube 46. The shield has a cap 47 and the shield or flange oscillates at the integral joint 48, as is conventionally done on rotors of high speed oscillating blade. The nozzle 49 of the protector is of a diameter much smaller than the upper section of the protector, has much less mass that oscillates at its maximum radius and consequently, can reach much higher speeds than with protectors of uniform internal diameter. This makes it possible to isolate fatty amounts of virus from much larger volumes than would otherwise be the case. During centrifugation using the rotor 50 driven by the motor 51, the shield and the tube 52 assume a horizontal position as shown. The microbanded viruses can be analyzed in this stage or they can be collected, diluted and processed later. In order to analyze the microbanded viruses in this step, they can be detected by a system as shown in Figure 5. For example, the banding step or a previous step may have included a fluorescent dye or fluorescent dyes within the solution with which the virus was mixed or through which the virus was centrifuged. Dyes are known with which intact viruses can be stained and which can distinguish between RNA, DNA, single-stranded nucleic acid and double-stranded nucleic acid allowing the presence or absence of an infectious agent to be detected and subsequently determined what type of virus has been purified. The apparatus of Figure 5 can be used to analyze these stained particles.

Figure 5 schematically illustrates a scanning and detection system wherein the microbanking tube 60 is held in a vertical position on the assembly 61 supported by the goniometers 62 and 63 which in turn are supported by XY movements 64 and 65 so as to align and center the microstrip section of the tube 60 with respect to the laser beam 66. The laser beam 66 is generated by a laser 67, which may be an argon ion laser that produces coherent light at 458, 488, 496, 502, and 515 nm. The beam passes through an interference or other filter 68 to isolate a wavelength and is reflected downwardly in the microbanking tube by a dichroic mirror 69. The fluorescent clustered particle zone 70 is photographed or scanned electronically by the camera 71. through the emission filter 72. The complete system may be enclosed to eliminate deviated light, and the filters 68 and 72 may be replaced by filtering wheels (not shown) to optimize detection, using fluorescent dyes which absorb and emit in different wavelengths, or to distinguish ssDNA, dsDNA and RNA by differences in the spectra of fluorescent light emitted. Electronic shutters can be added to the laser to minimize exposure of the sample to light and to the camera to control exposure. The goniometers and X-Y movements can also be motor-driven and remotely controlled and the entire system can be controlled by a computer (not shown). Figure 6 illustrates a different version of the scanning system which can encompass the entire visible spectrum and the near ultraviolet. The microbanking tube 80 is aligned in a fixed support between the transparent intensity balancers 81 and 82 attached to the light conduits 83 and 84 which in turn are attached to the intensity balancer 85 illuminated through the filter 86 by the lens of condensation 87 and light source 88. The filter 86 is one of a series attached to the filtering wheel 89 adjusted by the motor 90. The result is uniform illumination of two sides of one or more bands 91., 92 and 93. The image is captured through the emission filter 94 by digital camera 95 and the image stored, processed and displayed by the computer 96 in CRT 97. The filter 94 can be replaced by a filter wheel identical to the 89 and 90 so that, with both an excitation filter wheel and an emission filter wheel and a broad spectrum light source, such as a xenon lamp or a halogen lamp, a wide variety of combination of excitation light and emission, which in turn makes possible the use of a wide variety of fluorescent dyes. Both fluorescent light and light scattering at a selected wavelength can be used for particle detection. This arrangement facilitates the distinction between ssDNA, dsDNA and RNA. The processed image 98 can be displayed to show a photograph of the tube and contained bands 99, 100 and 101. The amount of light from each band can be integrated and displayed as peaks 102, 103, and 104, and in addition the integrated values can be unfold digitally (not shown). The complete system including shutters in the light and camera source (not shown), filter movement and positioning, and camera focus, can be digitally controlled by the computer 96. The 99 and 101 display bands, which represent bands of centrifuge tubes 91 and 93 can be fluorescent or non-fluorescent density indicator beads of known density and the band of virus 92 represented by the deployment band 103. The banding density of the virus can be determined by interpolation of the positions of the density indicators. When non-fluorescent density indicators are used, they are detected by scattered light using identical filters at positions 86 and 94. A second image using suitable and different filters is then captured, which comprises only fluorescent light. The two images are compared electronically with each other and the physical density of the infectious agent is determined by interpolation. In this stage, the virus can be identified as a DNA virus or an RNA virus and if it is a DNA virus, it can be determined whether it is of a chain or double chain. In addition, the density of the virus can be determined. These data can be used to help identify the type of virus that has been purified. However, it may be convenient or necessary to gather more data to fully determine which virus is exactly and also to determine the original viral titre. Figure 7 schematically illustrates the counting of individual fluorescent particles recovered from a tube 110 containing pooled virus zones 111 and 112 after it has been removed and all the fluid replaced above the banding gradient. The tube is placed in a tube rack 113 and a layer of deionized water or a very dilute pH regulator 114 is introduced over the gradient supplied through the tube 115 to replace the volume held in the waves 117. The tube can be closed in the upper part by means of a plastic closure 116. The capillary probe 117 is held in a fixed manner and the microbending tube 110 slowly rises below it. The tube rack 113 is part of a precision drive mechanism 108 and the associated stepper motor 119 which moves the tube holder vertically at a very low and controllable speed. A slow uniform stream of fluid is brought to the contraction 120 which is centered in the stream of shells 121 provided by the pump 122. The result is a constant flow of fluid through the flow cell 123 with a fine stream containing virus in the center, prolonged and extended by the envelope of the flow. A second pump 124 removes the fluid upward at a constant speed from the flow cell, whose speed is higher than the speed at which the piston pump 122 injects fluid into the casing 121. The difference in the speeds of pumps 124 and 122 is adjusted by the fluid that passes through capillary probe 117. The fluid that passes through capillary probe 117 is a mixture of virus plus fluid from the layer, which is introduced through the tube 115. The flow cell 123 is illuminated by the laser beam 125 produced by the laser 126, which passes through the excitation filter 127. The emitted light is isolated by the emission filter 128. and is detected by a photomultiplier 129. The output of photomultiplier 129 is integrated in intervals by computer 130 and the integrated signal against time is displayed in CRT 131. When two viral bands are present, two peaks such as 132 and 133 are displayed. Depending on the number of fluorescent particles present, the signal generated from one band can be integrated to a peak, or if the solution is sufficiently diluted, the particles can be counted individually, the values are stored and the integrated results are displayed. In order to account for the particles as just described, it is necessary that the virus particles are greatly diluted as they pass through the flow cell 123. Figure 8 schematically illustrates how the problem of making a Initial dilution of a virus band of very small volume to count individual particles. Figure 8A shows a tube 110 as in Figure 7, with a section indicated which is shown enlarged in Figure 8B, which in turn shows the enlarged section of this board in Figure 8C. As the upward movement of the microbanking tube causes the capillary tube to move to the bottom of the tube, the difference in pumping rates of the two pistons attached to the flow cell causes the fluid to flow up the capillary where it is diluted as described by the combined action of the pumps 122 and 124 of figure 7. However, the amount of fluid carried to the capillary 117 is much greater than the volume of the fluid effectively displaced from the band gradient by the relative movements of the capillary and the capillary. microbanded tube. This volume is replaced by fluid flowing to the tube 115 through the layer 116 which is initially allowed to flow until the tube 110 is full. This fluid is much less dense than the density of the fluid in the upper part of the gradient in the microbanking region and causes minimal alteration in the gradient. As shown in Figure 8B, the capillary 117 slowly approaches the virus band 114 and, as shown in Figure 8C, a small amount of gradient liquid 145 is diluted by a larger amount of fluid from the supernatant 114. as it flows into the capillary. In this way, a thin band of virus particles 144 is diluted and moved through the flow cell as a larger band volumetrically, but with a small effective loss of resolution. This technique provides the necessary solution to make feasible and accurate the counting of individual virus particles. The amount of solution can be controlled so that the concentration of microorganisms in the capillary tube is less than one-half or one-tenth, or one hundredth, or one thousandth, or one ten thousandth, or one millionth, or one billionth of the concentration in the band in the lower region of said tube. In addition to counting the particles or determining the titration of a virus, the amount of DNA in the virus or other microbe for individual particles can be determined. In this aspect of the invention, the amount of DNA in the particles is measured through flow fluorescence analysis (Goodwin et al., 1993) or epifluorescence analysis (Jing et al., 1998). In this way, yeast, bacteria, mycoplasma and viruses can be distinguished as groups. For example, it is known that viruses contain 5-200 x 103 bases or base pairs, E. coli, a typical bacterium, contains 4 x 104 base pairs, while a typical yeast cell contains 1.3 x 107 of base pairs. In this way, an estimate of the amount of DNA or RNA present allows the class of an infectious agent to be determined. A) Yes, the size of a genome can be determined. In this embodiment of the invention, the genome is extracted from the band of microorganisms and immobilized on a solid support. The immobilized DNA is stained and electronically imaged using an epifluorescence microscope (Jing et al., 1998). The length of the individual nucleic acid molecules can then be measured. The microbanking technique is useful not only to dye the virus with dyes and to be able to count the virus particles. Once viruses from a biological sample have been highly purified and concentrated by the two-dimensional centrifugation technique as described above using microwell centrifuge tubes, the viruses are ready for use in many other assays. When an infectious agent is grouped in a microbanded tube, the band can also be removed prudently using a capillary needle in a volume of a few microliters, diluted to 5 mL or more with very dilute pH regulator or deionized water to dilute Gradient materials by a factor as high as 1,000, and then compressed into a fresh microbanded tube. The supernatant can then be carefully removed by a suction capillary and the virus or other agent is redispersed in approximately 1 microliter using a syringe made, for example, of a thin material for Teflon® tubes fitted with a steel wire plunger very small stainless for adjustment. The sample can then be transferred to a mass spectrometer target, mixed with a matrix dye and used for matrix-assisted laser ionization-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) to directly determine the masses of viral coat protein or bacterial cell proteins. The technology described for the sample concentration, without a matrix dye, can also be applied to mass spectroscopic or electro-sputtering analysis systems, which includes an intact viral mass detection. A system similar to that shown in Figure 7 can also be used to produce the equivalent of molecular restriction fragment length maps of DNA molecules using restriction enzymes. For this work, bacterial virus or particle bands can be diluted and pelleted as described, after which, the DNA can be extracted using detergents or other reagents known in the art, treated with a restriction enzyme and a dye Fluorescent and fragment sizes can be determined by flow cytometry (Goodwin et al., 1993; Hammond et al., U.S. Patent No. 5,558,998). The extracted DNA can also be immobilized on a solid support, stained with a fluorescent dye and photographed using an epifluorescence microscope to determine the length of DNA molecules. The preparation can then be treated with a restriction endonuclease and the number and lengths of the oligobucleotide fragments can be determined (Jing et al., 1998). These data are compared to a database that lists the expected fragment lengths for different bacterial or viral species to identify each agent. DNA fragment lengths can also be determined by gel electrophoresis. Fluorescence-labeled antibodies can also be added to the particle suspension studied and the presence or absence of the label in isopycnically pooled particles can be determined. This method is useful for specific identifications and the use of a series of antibodies labeled with dyes having different and unique spectral characteristics allows the presence or absence of a series of agents to be determined. Alternatively, antibodies labeled with quelators for rare earths such as Europium and Terbium can be used, in which case the delayed fluorescence is measured. Whey or plasma typically have a physical density between 1. 026 and 1.031. Normally, viruses have banding densities between 1.17 and 1.55 in cesium chloride and much lower densities in iodinated gradient materials such as lodixanol or sucrose (Graham et al., 1994).

Therefore, the intermediate wash and the reagent layers between the sample and the banding gradient should have densities less than the density of the lightest virus to be grouped. The pH regulators used to dissolve gradient material for virus isolation include 0.05 M sodium borate, and 0.02 M sodium cyanide, which prevent bacterial growth. With human serum or plasma, sufficient centrifugation is used to remove platelets and other particles that have sedimentation coefficients of approximately 104 S before virus particle banding. The density of virus particle banding depends on the nucleic acid / protein ratio, and the presence or absence of lipids and lipoproteins. Accordingly, the binding of specific identification antibodies labeled with fluorescent dyes must not only allow identification through fluorescence, but through a change in banding density. To aid in the identification of particles by density, fluorescent particles of known density can be included in the sample as presented in Figure 6. These particles can include bacterial or fluorescently stained virus particles and fixed a banding density known, or very small fluorescent or non-fluorescently labeled plastic beads. When the polystyrene latex particles are coated with antibodies, their band densities increase markedly and the density can be further increased by the reaction of the particles coated with antibody to the antigen for which the antibodies are specific (Anderson and Breillatt, 1971). ). The fluorescent polystyrene beads coated with antibody can therefore be used, not only to locate virus particles, but to identify them. The dyes that are currently most useful are described in the Handbook of Fluorescent Probes and Research Chemicals, R.P. Haugland, ed., Molecular Probes, Inc., Eugene, Oregon (1996), which mentions the names of abbreviated dyes, their chemical names, the absorption and emission maxima and the most commonly used filter combinations. To work with human pathogens, safe operation and containment is important (Cho et al., 1996). The use of oscillating rotor rotors, optimal from a physical-chemical point of view, require extensive manipulation and have more parts than an angular head rotor. The tubes described in Figure 3E are designed for use on angle head rotors and allow the settling particles to travel along a wall at an invariable angle. Such rotors are easier to use and handle in contention than oscillating blade rotors, however, sedimentation in an angular head rotor is far from ideal. Consequently, it is important to develop methods and devices to work safely with oscillating blade rotors. High-speed centrifuge tubes are remarkably difficult to seal effectively and are a potential source of infection for laboratory personnel. In practice, almost all the high-speed oscillating rotor rotor tubes do not close on their own, but are enclosed in a metal blade which is sealed with a metal cap, which does not seal the tube. Therefore, the centrifuge tubes are opened when they are loaded, moved to the centrifuge rotor, inserted and removed. It is very difficult to disinfect the outer part of an open tube that contains a density gradient without altering the gradient. In the present application, it is convenient to be able to effectively seal the plastic tubes in such a way that the external surfaces can be cleaned with a suitable disinfectant before the tubes are inserted into centrifuge guards and can be handled safely until they are scanned. The seal has been made, as shown in Fig. 9A, by inserting an annular ring seal 151, having a physical density lower than that of water, into the tube 152. The ring 151 is slightly tapered so that it fits closely into the tube 152 and has a central hole, which can be connected and disconnected. In one embodiment, the central hole is screwed in to accept a short plastic flat head screw. Initially two gradient components, including a lighter solution 153 and a denser solution 155, are introduced to the lower microbanking region with a small bubble of air 154 in between, as described above. As shown in Figure 9B, the solution 156 containing the infectious agent or other particles is then introduced through the tube 157, leaving the bubble 158 to separate the sample from the higher gradient solution. The volume of sample introduced does not completely fill the centrifuge tube, leaving the space 159 empty. The tube is then sealed, as shown in Figure 9C with, for example, a plastic flat head screw 160, leaving the air bubble 160 in place. The outside of the tube is then sterilized by immersing it in a disinfectant such as a sodium hypochlorite solution or a hydrogen peroxide solution, followed by a water wash and gentle drying - all with the tube upright. After centrifugation, as shown in Figure 9D, the upper plastic seal has been driven downward by the centrifugal force a small distance since trapped air rises around the plastic seal. However, because the seal has a lower density than water, it is retained in the upper part of the liquid sample, leaving a small flange 160 that can be fastened with a hemostat to remove the tube from the centrifuge protector. During centrifugation, the infectious particles settle out of the liquid 163 and produce the band 164 in the gradient. The screw is then removed from the ring seal, and, as shown in FIG. 9E, supernatant liquid 165, which may contain a fluorescent dye, is withdrawn through tube 166, leaving meniscus 167. As shown in Figure 9F, a laser beam 168, which enters the tube from above, is then aligned with the tube, causing the pooled infectious agent to emit fluorescent light for detection as described above.

When a step gradient containing several reagents is used in addition to those used for technical sampling, as illustrated in Figure 2, the disks used to separate the various solutions are raised to the top and would not allow the use of illumination with vertical laser as shown in Figure 9F without removing the seal and discs. In this case, lateral illumination would be used, as illustrated in Figure 6. The laser or delayed fluorescence systems can be fully included, the mechanical operations are performed remotely through small stepper motors, and the tubes they move in and out of the included system under remote control. These techniques can be combined with mass spectrometry and restriction fragment mapping based on fluorescence to allow rapid diagnosis and identification of infectious agents. However, estimates of individual protein masses are generally taken from published sequence data, and do not include numerous post-translational modifications. Mass spectrometric databases should be created to include real mass measurements of different microorganisms. In addition, mass measurements of virion protein will allow the detection of many genetic variants. However, for many studies of microorganisms, including the development of databases, the key problem has been the development of methods to systematically provide microsamples of highly concentrated and purified microorganisms from patient samples, natural waters, and from fluids. of tissue culture. This problem is solved by means of the present invention. The present invention is described by reference to the following example, which is offered by way of illustration and is not intended to limit the invention in any way. Standard techniques well known in the art or the techniques specifically described below were used.

EXAMPLE To illustrate the use of microbanking tubes, experimental studies using a small non-pathogenic single-stranded DNA virus FX 174. have been carried out. Approximately 1010 virus particles that had been purified by isopycnical banding in CsCl in a tube of microbanded as shown in Figure 2A were suspended in 5 mL of 0.05 M borate pH regulator, compressed in a microbanking tube at 35,000 rpm in an oscillating blade rotor, and resuspended in approximately 3 μL For analysis. The analyzes were performed on a PerSeptive Biosystems DESTR instrument with an extraction delay time adjusted to 150 nseconds using a sinapinic acid matrix. Bovine insulin (MW = 5,734.59) and horse heart apomioglobin (MW = 16,952) were used as patterns 1 and 2 pmolar. The results are shown in table 1. The FX 174 masses for virion capsid proteins F, G, H and J are calculated from published sequence data. The differences between the calculated and experimental values for F and H are probably due to post-translational modifications. The probability that a non-related virus could have subunits of the same masses listed is very small. However, even more definitive protein identifications can be made by treating virus proteins with proteolytic enzymes such as trypsin and determining the masses of the produced peptide fragments. Computer programs are available that calculate the sizes of protein fragments of known sequence by well-represented enzymes. Such programs include Protein Prospector (available from the University of California, San Francisco) and ProFound (available from Rockefeller University).

TABLE 1 Mass spectrometric analysis of virion proteins FX 174 Mass Mass Difference% Calculated Protein Experimental Mass Difference F 48,351.53 48,407.4 +55.9 0.12% G 19,046.73 19,046.7 0.0 0.00% H 34,419.25 34,446.1 +46.9 0.14% J 4,095.78 4,097.03 +1.2 0.03% This example demonstrates that suspensions of highly purified and concentrated microorganisms can be isolated from biological samples such as, but not limited to, patient samples such as plasma, urine, feces and tissues, natural water and tissue culture fluids. This example also demonstrates that said purified and concentrated microorganisms can then be identified, for example, using mass spectrometry to identify viruses. Although the invention has been described in this patent application by reference to the details or preferred embodiments of the invention, it should be understood that the description is intended to illustrate and not to limit, since it is contemplated that those skilled in the art modifications will easily occur to them, within the spirit of the invention and the scope of the appended claims.

LIST OF REFERENCES Anderson, N.G., ed. (1966). "The Development of Zonal Centrifuges." Nati Cancer Inst. Monogr. 2J. Anderson, N.G., et al. (1966). "Separation of subcellular components and viruses by combined rate- and isopycnic-zonal centrifugation." In The Development of Zonal Centrifuges. N.G. Anderson, ed. National Cancer Institute Monograph 21 / 253-284. Anderson, N.G., et al. (1967). "Isolation of viral particles from large fluid volumes." In Transmission of Viruses bv the Water Route, G. Berg ed., Interscience Publishers, NY, pp. 75-88 Anderson, N.G. (1968). "Analytical techniques for cell fractions, VIII, Analytical differential centrifugation in angle-head rotors." Anal. Biochem. 23: 72-83. Anderson, N.G. and Breillatt, J.P. (1971). "Isopycnometric serology: A new technique based on buoyant density changes latex beads." Nature 231: 112-114. Bergh, O. et al. (1989). "High abundance of viruses found in aquatic environments." Nature 340: 467-468. Borsheim, K.Y. et al. (1990). "Enumeration and biomass estimation of planktonic bacteria and viruses by electron microscopy transmission." Applied Environ. Microbiol. 56: 352-356.

Chang, Y. et al. (1994). "Identification of herpes virus-like DNA sequences in AIDS-associated Kaposi's sarcoma." Science 265: 1865-1869. Cho, N. et al. (1966). "Problems in biocontainment." Nati Cancer Inst. Monogr. 21 .: 485-502. Claydon, M.A. et al. (nineteen ninety six). "The rapid identification of intact microorganisms using mass spectrometry." Nature Biotechnology 14: 1584-1586. Fenselau, C. (ed.) (1994). "Mass spectrometry for the characterization of micro-organisms." ACS Symposium Seríes, Vol. 541, ACS, Washington DC. Fredricks, D.M. and Relman, D.A. (nineteen ninety six). "Sequence-based evidence of microbial disease causation: When Koch's postulates do not fit." J. NIH Res. 8: 39-44. Gao, S.-J. and Moore, P.S. (nineteen ninety six). "Molecular approaches to the identification of unculturable infectious agents." Emerging Infectious Diseases 2: 159-167. Goodwin, P.M. et al. (1993). "Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry." Nucí Acids Res. 21: 803-806. Graham, J. et al. (1994). "The preparation of subcellular organelles from mouse liver in self-generated gradients of lodixonol." Anal. Biochem. 220: 367-373.

Hara, S. et al. (1991). "Abundance of viruses in marine waters: Assessment by epifluorescence and transmission electron microscopy." Applied Environ. Microbiol. 57: 2731-1734. Harris, W.W. et al. (1966). "Unusual particles in human plasma from leukemia and lymphosarcoma." In The Development of Zonal Centrifuges. National Cancer Institute Monograph 21 .: 289-394. Haugland, R.P. (1996) Handbook of Fluorescent Probes and Research Chemicals, 6th ed. Molecular Probes Inc., Eugene OR, p. 679. Hennes, K.P. and Suttle, C.A. (nineteen ninety five). "Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy." Limno Oceanogr. 40: 1050-1055. Hennes, K.P. et al. (nineteen ninety five). "Fluorescently labeled virus probes show that natural virus populations can control the structure of marine microbial communities." Applied Environ. Microbiol. 61 .: 3623-3627. Hirons, G.T. et al. (1994). "TOTO and YOYO: New very bright fluorochromes for DNA content analysis by flow cytometry." Cvtom.15: 129-140. Holland, R.D. et al. (nineteen ninety six). "Rapid dentition of intact whole bacteria based on spectral patterns using matrix assisted laser desorption ionization with time of flight mass spectrometry." Rapid Commun. Mass Spectrom. 10: 1227-1232.

Jing, J. et al. (1998). Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules. Proc. Nati Acad. Sci. USA 95: 8046-51. Kovacs, E. (1962). Chemical dissection of mammalian cells with liberation of biologically intact viruses. Z. Naturforschg. 17: 234-240. Krishmanurthy. T. et al. (nineteen ninety six). "Detection of pathogenic and non-pathogenic bacteria by matrix assisted laser desorption ionization time of flight mass spectrometry." Rapid Commun. Mass Spectrom. 10: 883-888. Liang, X. et al. (nineteen ninety six). "Determination of bacterial protein profiles by matrix assisted laser desorption ionization mass spectrometry with high performance liquid chromatography." Rapid Commun. Mass Spectrom. 10: 1219-1226. Linnen, J. et al. (nineteen ninety six). "Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent." Science 271: 505-508. Mazzone, H.M. (1998). CRC Handbook of Viruses. CRC Press, Boca Ratón, p. 206. Samad, A.H. et al. (nineteen ninety five). "Mapping the genome one molecule at a time - optical mapping." Nature 378: 516-517. Simons, J.N. et al. (nineteen ninety five). "Identification of two flavi-virus-like genomes in the GB hepatitis agent." Proc. Nati Acad. Sci. USA 92: 3401-5. Siuzdak, G. (1998). Probing viruses with mass spectrometry. J. Mass Spec. 33: 203-211.

Suttle, C.A. (1993). "Enumeration and isolation of viruses." In Handbook of Methods in Aguatic Microbial Ecologv. Chapter 15. Kemp, P.F., Sherr, B.F., Sherr, E.B. and Cole, J.J., eds. Lewis Publishers, Boca Ratón, Fia., Pp 121-134. Suttle, C.A. and Chen, F. (1992). "Mechanisms and rates of decay of marine viruses in seawater." Appl. Environ. Microbiol. 58: 3721-3729. Wommack, K.E. et al. (1992). "Distribution of viruses in the Chesapeake Bay." Appl. Environ. Microbiol. 58: 2965-2970. Yolken, R.H. and Torrey, E.F. (nineteen ninety six). "Viruses, schizophrenia and bipolar disorder." Clinical Microbiology Reviews 8: 131-145. Zeuzem, S. et al. (nineteen ninety six). "Phylogenetic analysis of hepatitis C virus isolates and their correlation to viremia, liver function tests, and histology." Hepatology 24: 1003-1009.

Claims (91)

1. NOVELTY OF THE INVENTION CLAIMS 1. - An ultracentrifuge tube 1 comprising an upper centripetal region 2, an intermediate region 3 and a lower centrifugal region 7 characterized in that an internal diameter of said upper region 2 is larger than an internal diameter of said intermediate region 3 and characterized in that a The internal diameter of said intermediate region 3 is larger than an internal diameter of said lower region 7.
2. 2. The ultracentrifuge tube 1 according to claim 1, further characterized in that said intermediate region 3 comprises one or more serrations.
3. 3. The ultracentrifuge tube 1 according to claim 1, further characterized in that a wall 38 of said centrifuge tube is linearly continuous.
4. 4. The ultracentrifuge tube 1 according to claim 1, further characterized in that said internal diameter of said lower region 7 is less than 0.63 cm.
5. 5. The ultracentrifuge tube 1 according to claim 1, further characterized in that said lower region 7 is at least 5% of the total length of said tube. 6. - The ultracentrifuge tube 1 according to claim 1, further characterized in that the internal surfaces are polished by steam polishing. 7. The ultracentrifuge tube 1 according to claim 1, wherein the internal surfaces are coated with adhesion polymer to avoid adsorption of biological particles. 8. The ultracentrifuge tube 1 according to claim 1, further characterized in that said lower region 7 has an internal diameter small enough to trap an air bubble between two layers of liquid in such a way that the air bubble maintains said two separate liquid layers as long as said centrifuge tube 1 is at rest. 9. A vane for holding a centrifuge tube of claim 1, characterized in that said vane comprises an upper region and a lower region, and characterized in that said lower region has a smaller external diameter than said upper region. 10. The blade according to claim 9, further characterized in that said blade comprises a third region wherein said third region joins said blade to a rotor. 11. A method for analyzing a microorganism in a biological sample, characterized in that said method comprises the steps of: a) adding said biological sample containing microorganisms to the ultracentrifuge tube of claim 1 and b) centrifuging said sample in said tube to concentrate the microorganisms. 12. The method according to claim 11, further characterized in that density gradients are formed in said lower region 7 of said tube. 13. The method according to claim 11, further comprising placing two or more layers of fluid in said lower region 7 of said tube prior to the addition of the sample, the layers being separated by air bubbles. 14. The method according to claim 11, further comprising placing two or more layers of fluid in said intermediate region 3 of said tube before the addition of the sample, the layers of the intermediate region separated by one or more disks inserted in said tube, the disks are able to keep the fluid layers separated before centrifugation and to float upwards during centrifugation. 15. The method according to claim 14, wherein all fluid layers are reduced in physical density from a centrifugal direction to a centripetal direction. 16. The method according to claim 14, in which enzymes, reagents, dyes, or fixatives are present in individual layers and do not sediment appreciably under the centrifugation conditions employed. 17. - The method according to claim 14, in which gradient solutes are chosen so that microorganisms preferably survive passage through reagent layers while contaminating particles are degraded or dissolved. 18. The method according to claim 14, wherein the reagent layers are chosen to selectively degrade known classes of microorganisms while allowing others to settle to the gradient of centripetal synpcnic banding. 19. The method according to claim 14, in which bacteria of viruses are easily distinguished. 20. The method according to claim 14, further characterized in that said discs are porous. 21. The method according to claim 11, further comprising adding fluorescent staining to said sample. 22. The method according to claim 11, further comprising: (c) analyzing said microorganisms for DNA and RNA by flow fluorescence analysis or epifluorescence analysis. 23. The method according to claim 12, further characterized in that said biological sample contains a virus, further comprising (c) contacting said sample with dyes that distinguish between single-stranded DNA viruses, double-stranded DNA RNA virus and chain; and (d) detecting dyes bound in a band of virus, by which a type of virus is determined in said band. 24. - The method according to claim 23, further characterized in that said dyes are added to said sample. 25. The method according to claim 23, further characterized in that said dyes are added to a layer of fluid added to the tube before the addition of said sample and said viruses are in contact with said dyes during centrifugation. 26. The method according to claim 23, further characterized in that said dyes are fluorescent and said attached fluorescent dyes are detected by passing a fluorescent excitation light through said band of virus and determining a wavelength of peak intensity of light fluorescent emitted from said virus band. 27. The method according to claim 26, further comprising removing unbonded dyes from said tube before determining said peak intensity wavelength. 28. The method according to claim 23, further characterized in that said bound dyes are detected by passing an excitation light through said band of virus and determining the spectral distribution of the emitted light. 29. The method according to claim 26, further characterized in that an intensity of said fluorescent light emitted is measured to determine a titer of an infectious agent in said biological sample. 30. The method according to claim 11, further comprising the steps of: (c) removing fluid from above said lower banding region 7; (d) covering the remaining liquid with water or pH regulator less dense than the fluid in said lower region 7; (e) inserting a capillary tube with an open lower end into said centrifuge tube so that said open lower end is above one or more bands of microorganisms; (f) extracting fluid through said open lower end of said capillary tube so that said fluid that is drawn through said capillary tube forms a fluid stream that passes through a flow cell where it is analyzed; (g) adding water or pH regulator to said upper region of said centrifuge tube as the fluid is removed in step (e) or as needed to maintain the water or pH regulator above any band of virus; (h) moving said centrifuge tube relative to said capillary tube so that the capillary tube moves in said lower region 7 of the centrifuge tube and through any band of microorganism viruses; (i) analyzing for microorganisms in said fluid stream flowing through the flow cell to determine a number of microorganisms present; and (j) calculating a titer from the determined number of microorganisms and known volume of said biological sample. 5 31. - The method according to claim 30, further comprising pumping fluid in a wrap around said fluid stream leaving the capillary tube thus diluting said stream before passing through the flow cell. 32. The method according to claim 31, further characterized in that said fluid envelope is pumped at a rate lower than the speed at which the fluid passes through said flow cell. 33. The method according to claim 32, further characterized in that the flow of each liquid is controlled by gas pressure instead of pumps. 34. The method according to claim 30, further characterized in that said microorganisms are at a concentration in said capillary tube less than a medium of its concentration in a band of microorganisms in said lower region 7 of the centrifuge tube. The method according to claim 11, further comprising the steps of: (c) recovering the microorganism in a concentrated form; (d) submitting said microorganism to mass spectroscopy to measure the masses of individual proteins; (e) determining a mass spectrum of said protein sizes; (f) comparing said protein mass spectrum with mass spectra obtained using known microorganisms; and (g) determining that the microorganism in said biological sample is the same as a known microorganism that produces an identical mass spectrum with the mass spectrum obtained for the microorganism from said biological sample. 36. The method according to claim 35, further characterized in that said mass spectrometry is time-of-flight mass spectrometry by matrix-assisted laser ionization-desorption. 37. The method according to claim 35, further characterized in that said mass spectrometry is mass spectrometry by electrospray. 38. The method according to claim 35, further characterized in that said proteins are digested enzymatically before obtaining a mass spectrum. 39. The method according to claim 11, further comprising the steps of: (c) extracting nucleic acid from said concentrated microorganism; (d) incubating said nucleic acid with restriction enzymes to produce nucleic acid fragments; (e) dyeing said nucleic acid or nucleic acid fragments; (f) determining a size pattern of said nucleic acid fragments; (g) comparing said size pattern with nucleic acid size standards, digested with said restriction enzymes, obtained from known microorganisms; and (h) identifying said microorganism in said biological sample as a microorganism having an identical restriction fragment pattern. 40. - The method according to claim 39, further characterized in that said sizes of nucleic acid molecules or fragments thereof are determined using flow cytometry. 41. The method according to claim 39, further characterized in that said sizes of nucleic acid molecules or fragments thereof are determined by gel electrophoresis. 42. The method according to claim 39, further characterized in that said sizes of nucleic acid molecules or fragments thereof are determined by mass spectrometry. 43. The method according to claim 39, further characterized in that said sizes of nucleic acid molecules or fragments thereof are determined by optical cartography. 44. The method according to claim 11, further comprising the steps of: (c) dyeing said microorganism genome; (d) purifying said microorganism genome; (e) subjecting said microorganism genome to fluorescence flow cytometry to obtain data; and (f) determining the mass of a genome of a microorganism in said biological sample from the data of step (e). 45. The method according to claim 44, further characterized in that said microorganism genome is digested with restriction enzymes before step (d). 46. The method according to claim 11, further comprising the step of: (c) incubating with fluorescent antibodies specific for known microorganisms, further characterized in that if said antibodies bind to said concentrated microorganism then said microorganism is identified as the microorganism to which antibodies are known to bind, by their fluorescence. 47. The method according to claim 46, further characterized in that said fluorescent antibodies are present in said upper region of the centrifuge tube during the centrifugation of the biological sample, they are bound to the microorganism for which they are specific during the incubation, they sediment and group together with said microorganism, and are detected by the fluorescence of said joint antibody-microorganism grouping. 48. The method according to claim 47, further characterized in that a plurality of antibody species is present in said upper region 2 of said centrifuge tube during the centrifugation of the biological sample and further characterized in that each species of antibody is labeled with a marker distinct from any marker in any other species of antibody present in said upper region 2. 49. The method according to claim 46, wherein the antibody microorganism complex has a different banding density than the antibody. of the free microorganism, thus allowing the presence of the complex to be detected. 50. - A method for separating layers in the centrifuge tube of claim 1 prior to centrifugation characterized in that the fluid in said centrifuge tube comprises a first dense layer and a second less dense layer, characterized in that said method comprises the steps of: a) inserting said first dense layer in said tube; (b) providing a means for separating the first and second layers; and (c) inserting said second less dense layer in said tube. 51. The method according to claim 50, further characterized in that said means for separating said layers is an air bubble. 52. The method according to claim 50, further characterized in that said means for separating said layers is a porous disk and said porous disk is inserted in the upper part of the first layer. 53. The method according to claim 52, further characterized in that said disk floats during centrifugation to a region above said second less dense layer, thus allowing said dense first layer to come into contact with said second less dense layer. 54. The method according to claim 52, further characterized in that said disc is made of sintered polyethylene or polypropylene. 55. - The centrifuge tube according to claim 1, further characterized in that said centrifuge tube is prepared from materials so that said tube can be centrifuged at sufficiently high speeds to group microorganisms into CsCl gradients without said tube centrifuge breaks. 56.- The centrifuge tube according to claim 55, further characterized in that said tube is made of polycarbonate. 57.- The centrifuge tube according to claim 1, further characterized in that said upper region 2, intermediate region 3, and said lower region 7 have external diameters equal to each other. 58.- The centrifuge tube according to claim 1, further characterized in that said upper region 2 has an external diameter larger than an external diameter of said lower region 7. 59.- The method according to claim 11, characterized in addition because said centrifuge tube is supported by an adapter with internal dimensions contoured to match the external dimensions of said centrifuge tube. 60. - The method according to claim 59, further characterized in that said adapter is made of polycarbonate or Delrin®. 61.- A system for measuring the fluorescence of a sample in a centrifuge tube characterized in that said system comprises: a fastener for said centrifuge tube; a light source to produce light that will pass through said sample; and a detector for detecting light that is emitted from said sample as light passes through it. 62.- The system according to claim 61, further comprising: a condensing lens through which said light passes from said light source; a filter through which said light passes from said light source; an intensity balancer through which said light passes; light tubes through which said light passes; and two intensity balancers through which the light exiting said light tubes passes. 63.- The system according to claim 62, further comprising: an emission filter through which the light emitted from said sample passes. 64.- The system according to claim 62, further characterized in that said filter is replaced by a filter wheel comprising more than one filter. The system according to claim 62, further comprising: a filtering wheel comprising more than one emission filter characterized in that any emission filter of said filtering wheel can be placed between the light emitted from said sample and said detector. 66.- The system according to claim 61, which also comprises a computer. 67. The system according to claim 66, further comprising a monitor. 68.- The system according to claim 67, further characterized in that said monitor is a cathode ray tube. 69.- The system according to claim 61, further characterized in that said fastener holds a centrifuge tube in a vertical position, said light source is a laser that produces a laser beam, and further comprises i) a first filter for isolating light of a wavelength; and ii) a second filter through which the light emitted by the excited dye attached to said sample passes which has been grouped in a centrifuge tube when said centrifuge tube is placed in said centrifuge holder. centrifuge tube; and further characterized in that said detector detects the light passing through said second filter. 70.- The system according to claim 69, further comprising goniometers on which said centrifuge tube holder is mounted thereby allowing a centrifuge tube that is in said centrifuge tube holder to be oriented to coincide with the angle vertical laser beam; an X-Y movement to align the tubes with the laser beam in any X-Y direction; a filter through which said laser beam passes; and a mirror for deflecting said laser beam through a centrifuge tube attached to said centrifuge tube holder. 71.- The system according to claim 69, which also comprises a computer. 72.- The system according to claim 71, further comprising a monitor. 73.- The system according to claim 72, further characterized in that said monitor is a cathode ray tube. 74.- A system for counting particles that are concentrated in a small volume, characterized in that said system comprises: a container in which said particles are concentrated; a capillary tube; a first pump and a second pump; means for moving said container relative to said capillary tube; a flow cell; a light source; and a detector. The system according to claim 74, further characterized in that said first pump pumps fluid in a shell around an upper end of said capillary. The system according to claim 75, further characterized in that said second pump pumps fluid out of said flow cell. 77. - The system according to claim 76, further characterized in that said second pump pumps more fluid in a given period than the first pump. 78.- The system according to claim 77, further characterized in that the fluid is extracted through a lower end of said capillary tube, said fluid leaves said upper end of the capillary tube where it is surrounded by fluid pumped from the first pump , and further characterized in that said pump pumps the fluid mixture leaving the upper end of the capillary tube and said fluid from the first pump through the flow cell. 79. The system according to claim 78, further characterized in that said light source produces a beam of light that passes through said flow cell. 80.- The system according to claim 79, further comprising: a filter between said light source and said flow cell. 81.- The system according to claim 80, further comprising: a filter between said flow cell and said detector. 82. The method according to claim 11 further comprising the steps of: (c) extracting a genome from said concentrated microorganisms in step (b) to produce extracted nucleic acid; (d) immobilizing said extracted nucleic acid on a solid support; (e) dyeing said extracted nucleic acid; and (f) electronically imageing said nucleic acid extracted and stained on said solid support using an epifluorescence microscope; and (g) measuring the length of individual nucleic acid molecules to determine the size of said genome. 83. The method according to claim 11 further comprising the steps of: (c) extracting a genome of said concentrated microorganism in step (b) to produce extracted nucleic acid; (b) dyeing said extracted nucleic acid; (e) immobilizing said extracted nucleic acid on a solid support to produce immobilized nucleic acid; (f) treating said immobilized nucleic acid with one or more restriction enzymes; (g) determining the number of nucleic acid fragments and the lengths of nucleic acid fragments produced; and (h) determining a restriction enzyme map of said microorganism. 84. The method according to claim 83 further comprising (i) comparing said restriction map with restriction maps of known microorganisms, wherein a concordance of restriction maps of said microorganism in said biological sample with a restriction map of a known microorganism identifies the microorganism of said biological sample as that of said known microorganism with a restriction map identical to that of the microorganism of said biological sample; and G) determining the identity of said microorganism. The method according to claim 11, further comprising: (c) dyeing the microorganism with a fluorescent dye; (d) measuring the amount of nucleic acid in the concentrated dye microorganism in step (b); (e) comparing the amount of said nucleic acid with the known amount of nucleic acid for viruses, mycoplasmas, yeast and bacteria; and (f) distinguish the type of infection between viruses, mycoplasma, yeast and bacteria. 86.- The method according to claim 85, further characterized in that the microorganism is dyed during centrifugation. 87. The method according to claim 85, further characterized in that the microorganism is stained in the biological sample. 88. The method according to claim 85, further characterized in that the amount of nucleic acid is measured by flow cytometry. 89. The method according to claim 85, further characterized in that the amount of nucleic acid is measured by optical cartography. The method according to claim 29, in which changes in the titer of an infectious agent are used to determine which known pharmacological agents are therapeutic, to evaluate the efficacy of new drugs in animal and human tests, and choose between analogues of medicines in development. 91. The method according to claim 29, wherein changes in the titer of an infectious agent are used to discover new antibiotics and other therapeutic agents. SUMMARY OF THE INVENTION A method for separating microorganisms, especially infectious agents, from a mixture by means of two-dimensional centrifugation based on the rate of sedimentation and isopycnical banding density, to sediment said microorganisms through areas of immobilized reagents to which they are resistant, for detecting clustered particles by light scattering or fluorescence using specific nucleic acid dyes, and to recover the clustered particles into very small volumes for mass spectrometry representation of protein subunits of viruses and intact virus particles, and by flow cytometric determination fluorescence of both the nucleic acid mass and the masses of fragments produced by restriction mes; the method is based on the discovery that individual microorganisms, such as bacterial species and viruses, are each physically relatively homogeneous, and can be distinguished in their biophysical properties from other biological particles, and from non-biological particles found in nature; the method is useful to distinguish infections, to identify known microorganisms, and to discover and represent new microorganisms; the method provides very rapid identification of microorganisms, and therefore allows a rational choice of therapy for identified infectious agents; A particularly useful application is in clinical trials of new antibiotics and antivirals, where it is essential to initially identify individuals infected with the target infectious agent. MC / PV / agm * mmr * rcp * jtr * asg * eos * kra * yac * P00 / 1143F