JPH04503603A - Methods and gradient separation components for separating bacteria from bacteria-containing liquid samples - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Isolation of bacteria from liquid samples containing bacteria Methods and gradient separation components The present invention relates to a method for separating bacteria from a bacteria-containing liquid sample. For more details, The present invention allows the separation of bacteria from a bacteria-containing liquid according to the gradient separation theory known per se. Regarding how to

In many different cases, bacteria isolated from the sample after By simply counting the number of bacteria in an optical measuring device known per se. Microorganisms can be extracted from liquid samples containing bacteria in that the bacterial content of the sample can be determined by It is desirable to be able to isolate bacteria. The liquid sample may be of any biological origin; The sample may be a blood sample (see, e.g., U.S. Pat. No. 3,928,139) or a urine sample. , or suspensions or solutions of solid samples, e.g. organic components, e.g. tissue or It can be an aqueous solution or an alcoholic suspension of the food sample.

A very important example of a bacteria-containing liquid sample is a milk sample. As recognized, the original The measurement of the bacterial content of a liquid sample essentially depends on the accuracy of the isolation of bacteria from the liquid sample. Determined by Therefore, only bacteria are isolated from the liquid sample, while others Precise and precise particles, such as fat globules, blood cells, etc., are not separated from the liquid sample. And most importantly, it is possible to carry out very precise separation methods.

A method of the above type and an apparatus for carrying out the method are known; According to Bacteria are isolated from the body sample. Is the high-density component and low-density component a liquid sample containing bacteria? have higher and lower densities, respectively, than the average density of the bacteria to be isolated. Up According to the method known from the US and EP patents, the dense components of the gradient separation layer and the low Bacteria are retained at the interface defined between the density component and the density component.

The technique of separating and separating bacteria from a bacteria-containing liquid sample using a tube component gradient separation layer is Multicomponent gradient separation where multiple components are used to form a continuous density spectrum It was further developed into layer separation technology. Chemical Abstracts, Vo l, 101. No, 12. September 17, 1984 (Colus+bus+ 0hio, US), Maechtle W,: FjApi! Ldna s+ic water PERCOLL density radients f ormicro articles in the anal u ltracentrifue, 24 pages.

Summary 91793d i and Co11oid Po1ya+, Sci, 19 84.262(4).

270-82 (Get), as disclosed in that paper, for 20 minutes. A mechanical density gradient is built up within a period of . As understood, this technique is rather Multiple components of different densities are time-consuming and preferably limit the continuous density spectrum. need minutes.

The object of the present invention is to be simpler than the methods known from the above-mentioned US and EP patents, Additionally, bacteria can be extracted from bacteria-containing liquid samples without excluding other particles from the liquid sample. To provide a method of the above type that makes it possible to carry out accurate and highly precise separations. Is Rukoto.

Another object of the invention is to provide a method that allows separation to be carried out automatically and rapidly. It is to provide.

According to the invention, it is limited by a radially inwardly extending rim portion and an inner circumferential surface. Bacteria are separated from a bacteria-containing liquid sample using a dish-shaped centrifuge vessel with a defined top opening. A method comprising the following sequential steps: (a) rotating the centrifuge container at a high rotational speed; (b) approximately reaching an average density of the bacteria; A volume of gradient separation components of equal density is introduced into the centrifuge vessel and the inner circumferential surface is producing a separation layer of the gradient separation component on the surface; (c) While discharging the remaining components of the liquid sample from the upper opening of the centrifuge container, A continuous flow of the liquid sample is introduced into the centrifuge vessel at a discharge rate to remove bacteria through the separation layer. (d) introducing a flushing agent into the centrifuge container; and discharging the flushing agent from the top of the vessel; (e) slowing down the centrifuge vessel; and rotate it at low speed, (f) The liquid enters the centrifuge container without draining the liquid from the upper opening of the centrifuge container. a first position where the tip is away from said internal peripheral surface while introducing a volume of transfer liquid; the suction pipette from one position to a second position where the tip is placed close to said internal peripheral surface. actuating and moving a pump to aspirate liquid material from the centrifuge container; and (i) returning said suction pipette to its first position. .

According to the invention, the gradient separation components are separated into a centrifugal volume at high rotational speed by a two-speed motor. While rotating the container, it is introduced into the centrifuge container. U.S. Patent No. 4,541,445 Compared to the method of EP 0.128°509 and EP 0.128°509, the method of the present invention is even more It is a simple and accurate separation method. According to the teachings of the present invention, , since it uses a single gradient separation component. Surprisingly, the teachings of the present invention According to the study, bacteria can be separated from a sample using a separation layer composed of a single gradient separation component. and can be left in place. Various densities, i.e. low and high densities, e.g. ．． Bacteria with a density within the range of 04 to 1.18 g/cell are approximately the average density of bacteria. with a single gradient separated component of approximately equal density, e.g. 1.13 g/c11 Although there is no technical explanation for this unexpected effect, there are some possible technical explanations. The theory is that a bacteria-containing liquid layer and a separation layer are separated by high-speed introduction of a liquid sample into a centrifugal container. An interface is created between the two, at which the liquid sample enters the gradient separation component of the separation layer. This results in suspension. The above technical theory should not be construed as limiting the present invention. It's not possible.

Since the average density of bacteria is approximately equal to the average density of the gradient separated components, the liquid sample is Once introduced into the centrifuge vessel, the bacteria are retained by the gradient separation components while the liquid sample The remaining components of the material are discharged from the centrifuge vessel through the top.

Therefore, the method for isolating bacteria from a liquid sample containing bacteria should theoretically be a continuous separation process. It can be carried out at any time. Flushing into the centrifuge vessel after the separation process itself By introducing the singe agent, substances adhering to the separation layer, such as fat globules, are washed away. and is discharged from the top of the centrifuge container. Contains bacteria isolated from the original liquid sample To remove the separated layer, place it in the centrifuge container while rinsing the container with the transfer liquid. by means of a suction pipette movable from a first position to a second position with an extending tip. It is done.

According to a preferred embodiment of the invention, the method of the invention includes steps (a) to (g) followed by (h) draining the liquid from the top of the centrifuge vessel; Introduce a small amount of migration liquid into the centrifuge container, (i) accelerating the centrifuge vessel and rotating it at a high rotational speed; and (j) Repeat steps (e) to (g).

A further amount of said liquid is introduced into the centrifuge vessel and the centrifuge vessel is accelerated to a high rotational speed. Any material, i.e. the gradient separation component or bacteria contained therein, is The heart is washed out of the container, and as a result, repeating steps (e) to (g) results in the be transferred.

In order to completely clean the centrifuge vessel, steps (a) to (j) are followed by the following further sequential steps. (k) Accelerate the centrifuge vessel to rotate it at high rotational speed. (1) Immediately direct a stream of flushing agent and heated air far into the spray solution. Introduce it into the heart container, wash out the substances attached to the centrifuge container, (m) decelerate the centrifuge container to rotate it at low rotational speed; (n) remove the aspiration pipette; Actuate and move from a first position to a second position to aspirate liquid material from the centrifuge container and (o) return the suction pipette to its first position.

According to the invention, it is advantageous to cause a delay in the discharge of liquid from the centrifuge container. The notch that limits the minimum width of the radially inwardly extending rim of the centrifuge container is After that, the liquid can be discharged from the upper opening of the centrifuge container. As a result, the centrifuge container Due to the delay in draining the liquid, the liquid is retained in the centrifuge container for a long period of time and can increase the rate of liquid delivery to the centrifuge vessel and obtain higher separation rates. Wear.

To perform the isolation of bacteria from bacteria-containing liquid samples under controlled conditions, this book The method of the invention provides the Preferably, the air further comprises thermostating the centrifuge vessel to a temperature of Any suitable method may be used, such as maintaining the outer surface of the centrifuge vessel at a predetermined temperature. may be supplied from below so that the Air at a predetermined temperature can be supplied from above to the centrifuge container. Air can be introduced into the centrifuge container through the mouth.

The liquid discharged from the top of the centrifuge container rotates the centrifuge container at high rotational speeds. to ensure that it is not reintroduced into the centrifuge vessel due to the suction effect that occurs when , a flow of pressurized air is introduced into the centrifuge vessel while the centrifuge vessel is rotated at high rotational speed. Preferably, the flow of pressurized air is used for the purpose of temperature conditioning the centrifuge vessel. Advantageously, the pressurized air is temperature conditioned to said predetermined temperature.

In a first embodiment of the method of the invention, the gradient separation components have an average of about 1.13 g/cj. It has a density and constitutes an aqueous solution. I! ! .

The gradient separation components were 300 m of 85% glycerol; 60. Og no Shi w II ;30. Og of NaHCO3; 15.9 g of Na, COz; 0.7 Consisting of 5g NazEDTA; and 0.02d Br1j96@.

In a second embodiment of the method of the invention, the gradient separation component has an average concentration of about 1.07 g/cd. It has a homogeneous density and constitutes an aqueous solution. 1i! .

The gradient separation components were 94m of 85% glycerol; 1B, 8g(7)'/ Yose sugar; 30. OA of NaHCOs; 15.9 g of NatCOs; 0. Consisting of 75g Na, EDTA; and 0.006d Br1j96@ Useful for cleaning purposes when transferring the zero separation layer and the bacteria it contains from the centrifuge container. , and also serves the purpose of washing out any material in the separation layer and any bacteria contained therein. The transition liquid to be used can advantageously be an enzyme solution. This is further explained by the above optical coefficient It also serves the purpose of pre-treating bacteria for manipulation.

In a preferred embodiment of the method of the invention, the high rotational speed of the centrifuge vessel is 45.00O rpm, and the low rotational speed of the centrifuge vessel is 480 rpm. The net capacity of the centrifuge vessel is limited when the centrifuge vessel is rotated at high rotational speed. The volume is on the order of 2d. The centrifuge container was rotated at a rotation speed of 45,000 rpm. When rotated, 50.0 A virtual extreme gravity field of the order of 00 G is provided.

By providing such an extremely strong gravitational field, a resolution of 1.33 d/s can be achieved. obtained, i.e. 10- samples separated within 10 seconds or 20-d samples separated from 15 Separated within seconds.

Although the method of the invention can be carried out using any suitable centrifuge vessel, No. 4.591.445 (which is incorporated herein by reference) and Furthermore, using a centrifuge vessel of the type disclosed in European Patent No. 0.128,509, It is preferable to carry out the

The invention further relates to gradient separation components for use in accordance with the above method. The slope The separation component has an average density approximately equal to the average density of the bacteria to be separated. .

According to the first type B of the gradient separation components of the present invention, the two gradient separation components are approximately 1.13 It has an average density of g/cil and constitutes an aqueous solution. This gradient separated component 1 2 is 300 d of 85% glycerol; 60. Og's Sea! Sugar; 30. Og of NaHCOi; 15.9 g of NaHCO3; 0.75 g of NatED TA; and 0.02m1! Br1j 9 (consists of i @.

According to another embodiment of the gradient separation component of the present invention, the gradient separation component contains about 1.07 g/ ctil, and constitutes an aqueous solution. This gradient separation component 11 is , 94d of 85% glycerol; 18゜゛ 8g of Si; 30. O g NaHCOz; 15.9 g NatCOs i 0.75 g Naz EDT^; and 0.006 d of Br1j96''.

The invention will be further explained with reference to the drawings.

FIG. 1 shows an apparatus for carrying out the method of the present invention, in which bacteria are extracted from a liquid sample containing bacteria. 1 is a vertical cross-sectional view of a preferred embodiment of an apparatus for separating FIG. 2 is a schematic partial cross-sectional view illustrating the general separation method of the present invention.

In Figure 1, an apparatus for separating bacteria from a bacteria-containing liquid sample, e.g. a milk sample. is shown. The device is designated as 10 as a whole and is provided with a bottom plate 11 and above the bottom plate. A cylindrical casing 12 is placed on it. The lower end portion of the cylindrical casing 12 is It is fixed to the bottom plate 11 and embedded in a cylindrical circumferential recess 13. outside At the peripheral surface of the cylindrical casing 12 is provided a fin 28 constituting a heat sink. It is. That is, this fin 28 is designed to provide a large heat dissipation or heat transfer surface. the residual heat is radiated through its surface or a coolant, e.g. or transferred to the coolant. A floor 14 is placed on top of the cylindrical casing 12 . The floor 14 has a cylindrical case with a cylindrical rim projecting from the lower surface of the floor 14. A motor 16 is enclosed within a cylindrical casing 12 which is fixed relative to the housing 12. entered. The motor 16 has high speed rotation on the order of 45.00 Orpm and 48 It is a two-speed motor adapted to produce low speed rotation on the order of orpm. Ru. The motor 16 has an upper bearing 18 and a lower bearing placed on the floor 14 and the bottom plate 11, respectively. It has a shaft 17 journal-bearing in a bearing 19. The shaft 17 is an upper bearing 18 and is provided with an internally tapered cone 20 at its upper end.

A dish-shaped centrifuge container 21 is placed on the shaft 17, and its lower end is connected to the internal taper of the shaft 17. An external taper cone recess with a taper amount equal to the taper amount of the cone 20 is provided. has been done. As a result, the cone of the shaft 17 and the cone of the dish-shaped centrifuge vessel 21 are - Fixing the centrifuge container 21 with respect to the axis 17, as shown in more detail in FIG. 2 and described below. The centrifuge container 21 has a conical bottom portion 22, a vertical cylindrical portion 23, and an upper portion of the centrifuge container 21. It comprises an inwardly extending rim portion 24 defining a mouth 25. of centrifugal container 21 At the upper opening 25, an inwardly extending rim portion 24 is provided which limits the minimum radial width of the rim portion. A notch 26 is provided. Furthermore, the liquid in the centrifugal container 21 is in the upper bearing 18. A downwardly protruding isolation skirt 27 is provided to help prevent contact with It will be done. To further prevent liquids or droplets from coming into contact with the upper bearing 18, a predetermined airflow at a predetermined temperature (this is used to thermostat the centrifuge to a predetermined temperature) ) is supplied from below to the centrifuge vessel via a supply pipe (not shown), and the centrifuge vessel forming an isolation air curtain surrounding the lower part of the

A housing 30 is placed on the floor 14 so as to enclose the centrifuge container 21. C The housing 30 has a vertical cylindrical side wall that serves to expel liquid from the housing 30. (Exit 31 is provided.

A supply tube 32 for supplying a liquid sample extends from the lumen of the housing 30 to the housing 3 0 and extends into the interior of the centrifuge container 21 through the upper opening 25 thereof. supply pipe 32 can be connected to an external liquid sample container via a suitable tube extrusion (not shown). Adapted to suit.

In a separate cylindrical lumen above the housing 30 is a suction pipette generally designated 40. is placed. A suction pipette 40 extends inside the housing 30 and into the centrifugal volume. pipette tube 4, including a pipette tube 41 extending from the mouth 25 of the vessel 21 to its interior; 1 is connected to an external container (not shown) via a suitable tube extrusion and valve (not shown). can be connected to. The suction pipette 40 further includes an air motor 43. Ru. The piston of the air motor 43 is connected to the pipette tube 41 by any movement of the plate part 44. plate part firmly connected to the pipette tube 41 so as to cause the same action of 44 on the upper side. On the lower side of the plate part 44, an air motor 43 is operated. If not moving, maintain the plate part in the first position as shown in Figure 1. Coil 45 is activated. Operation of the air motor 43 causes the plate part 44 to move into its first position. from the position to a second position where the coil 45 is compressed. The first position above and the position of the pipette tube 41 corresponding to the second position are respectively shown in FIG. location, and the outer end of the pipette tube 41 is close to the vertical cylindrical portion 23 of the centrifuge vessel 21 This is the extended position when moved to the position. Pressurized air is supplied from the joint 46 to the air motor 4 3 and the air motor 43 is provided with a throttle valve 47, which throttle valve 47, the pipette tubes 4 are respectively aligned with the above-mentioned first and second positions of the plate part 44. reducing the speed of movement from the retracted position to the extended position shown in Figures 1 and 2 corresponding to Work in vain.

A main body 50 is centrally located at the top of the housing 30. Inside the main body 500 through-hole A tube 51 is placed and secured to the body 50 by a sealing ring or gasket 52. is sealed. The upper end of the tube 51 is provided with an external tube via a suitable tube extrusion (not shown). A connection fitting 53 (not shown) is provided which allows connection of the unit to a liquid source.

Perpendicular to the throughbore of the body 50, the lumen 54 provides a communication passage therefrom from the connecting fitting 55. provide The connecting fitting 55 is connected to external air via a suitable tube extrusion (not shown). a compression source (not shown), e.g. connected to the connection fitting 46 of the suction pipette 40; Adapted for connection to a pressurized source. From the connecting joint 55 and further to the main body The supply of pressurized air from the 50 throughbore serves two purposes. First, decide in advance The air maintained at a predetermined temperature thermostats the centrifuge vessel to a predetermined temperature. useful for.

Second, pressurized air rotated the centrifuge vessel at a high rotational speed of 45.00 Orpm. At times, liquid collects on the inner surface of housing 30 and is sucked into the centrifuge container. This will help prevent you from getting caught.

In FIG. 2 the centrifuge container 21 is shown in more detail.

As indicated by arrow 64, centrifuge container 21 is actuated by motor 64 and raised to a high temperature. Bounded by a minimum width limiting notch in the inwardly extending rim portion while being rotated at rotational speed. A gradient separation layer 60 is arranged in the circumferential interior space of the centrifugal vessel 21, which is defined by the centrifuge vessel 21. separation Layer 60 is a gradient separation layer having a density approximately equal to the average density of the bacteria to be separated. It consists of ingredients. The liquid sample is supplied from the supply tube 32 to the center of the centrifuge container 21, and is ejected from there. The liquid sample is drained from the supply tube 32 at a fairly high drain rate. Due to the centrifugal force generated by the rotation of the centrifuge container 21 at high rotational speed, the liquid sample The material is sprinkled outward from the center of the centrifuge container and brought into contact with the separation layer 60. Ru. The liquid supplied in a continuous flow from the supply pipe 32 forms a liquid film layer 62 .

At the interface between the liquid film layer 62 and the separation layer 60, the liquid sample passes through the separation layer 60. The bacteria to be separated from the liquid sample are suspended in the gradient separation components of the gradient and the bacteria to be separated from the liquid sample are When the average density is approximately equal to the density of the distributed components, bacteria are retained by the separation layer 60. It seems that it will be placed. density lower than that of the gradient separated components, resulting in an average bacterial density Components of the liquid sample with a density lower than the density are forced upwards and It exits the centrifuge vessel through the notch 26, as indicated by reference numeral 66.

The high rotational speed of the centrifuge vessel because bacteria constitute the component of the liquid sample with the highest density Due to the centrifugal gradient created by It can be understood that it is necessarily forced into. Liquid drains only from notch 26 Since the liquid is discharged from all the upper ports of the centrifuge container, the minimum width limiting groove 26 is formed. is not retained in the centrifuge container for a longer period of time compared to a dish-shaped centrifuge container, and therefore The rate of supply of liquid to the centrifuge container can be further increased, and the notch 26 is not included. The rate of bacterial isolation can be further increased compared to older devices.

The operation of apparatus 10 is described in the Examples below.

Example 1 In an embodiment of the type described above with reference to the drawings, the dish-shaped centrifuge vessel 21 is made of titanium. and Tef　Ion■-PFA (perfluoroalkoxy) inner surface It was covered. The inner diameter of the centrifuge container 21 was 47 mm. The motor 16 is 48, providing a virtual gravitational field to the centrifuge vessel on the order of 50,0 OOG. To be driven at a low rotation speed of 0 rpm and a high rotation speed of 45,000 rpm. It was an adapted thyristor controlled three phase AC motor. Inside the circumference of the centrifugal container 21 The height of the space is limited by a minimum width limiting notch 26 in the inwardly extending rim portion 24. Its interior space was of the order of 2 relatives. In this embodiment, a liquid sample of lO− were separated within 10 seconds. Alternatively, 20 liquid samples can be separated within 15 seconds. It was. The 10ml and 20d liquid samples are 2.5ml and 5ml of the aqueous milk samples, respectively. It was a diluted solution. Through the connection joint 55 and the through hole of the main body 50, the temperature reaches 40°C. A heated pressurized air stream is applied from a pressurized source of 1.5 bar to an internal diameter of 1.5 rtm and It was fed continuously through a 250-long tube. As mentioned above, at a temperature of 40°C Air flow was also supplied to the centrifuge vessel from below.

When carrying out the method of the invention, the above-mentioned in an automatic operating sequence comprising the following steps: The device was activated.

(a) To rotate the centrifuge container 21 at a high rotational speed of 45,000 rpmO The motor 16 is accelerated to a high rotational speed, and (b) the average density of 1.13 g/cffl is 2.2 degrees of gradient separation component (1! is 85% glycerol of 300; 60 ．． Og no Shi! F sugar; 30. Og of NaHCOs; 15.9 g of NazCO s; 0.75 g NazEDTA; and 0.02 d Br1j 96■ ) into the center of the centrifuge vessel 21 from another supply tube (not shown) and Immediately, the gradient separated components are sprinkled outward from the center of the centrifuge container 2, and as shown in Figure 2. and approximately 0.2 volumes of extra gradient separation component. (c) the liquid sample is discharged from the notch 26 of the centrifuge vessel 21 at a rate of ld/s. from the supply tube 32 in continuous flow, thereby creating an interface 62, and Bacteria with a density on the order of 1.04 to 1.188/- are deposited in the separation layer 60 and the liquid flows into the upper opening 2 of the centrifuge vessel, as indicated by reference numeral 66 in FIG. Exhausted from 5, (d) Flushing constituted by a washing solution after the separation process itself has been completed. The agent is fed into the center of the centrifuge vessel through the feed tube 32 and any particles obtained, e.g. fat globules, are washed out and discharged from the centrifugal container 21, (e) Motor 1 to rotate centrifuge container 21 at a low rotational speed of 48 Orpm. 6 to a low rotational speed, and (f) as can be seen from FIG. 2, the suction pipette 40 and the first pipette tip is away from the inner surface of the peripheral wall of the centrifuge vessel. from the position to a second position in which the tip of the pipette is disposed near the inner surface. 1.5 male enzyme solution is added to the outer periphery of the centrifuge container 21 from another supply pipe (not shown) while pipette tube 4 to supply the liquid, thereby dissolving the substance containing the separation layer 60 as an enzyme solution. 1. Also from the connecting fitting 42 and the external pipe (not shown) to the external measuring vessel or measuring (g) return the aspiration pipette to its first position; (h) 1.5 d of enzyme solution is supplied to the outer periphery of the centrifuge container 21 from the supply pipe (not shown). supply, (i) A motor for rotating the centrifuge container 21 at a high rotation speed of 45.00 Orpm. Accelerate the motor 16, thereby placing the centrifuge vessel 21 in the circumferential inner space. the substance is washed out with said amount of enzyme solution, and (j) 1.3rd volume of enzyme solution as above along with any substances dissolved therein. Steps (e) to (g) for flushing into a measuring vessel or measuring device (not shown). repeat.

The following sequential steps were performed to thoroughly clean the entire apparatus.

(k) A motor for rotating the centrifuge container 21 at a high rotation speed of 45.00 Orpm. Accelerate Tar 16, (1) Flushing agent supplied from the supply pipe 51 and heated to a temperature of approximately 4°C in advance. Air supplied through the lumen 54 from a preheater that heats the air is supplied to the housing 30. In the spray that serves to clean the inner surface and the outer surface of the centrifuge container 21, 0 ．． (a+ ) Reduce the motor 16 to rotate the centrifuge container at a low rotational speed of 480 rpm. Speed it up, (n) activating and moving the suction pipette from a first position to a second position; Aspirate any liquids and substances present in the centrifuge container and pipette. from the outer pipe 41, furthermore from the connecting fitting 42 and from the external pipe (not shown) to a waste container, and (o) finally returning the suction pipette to its first position and motor 16 was stopped.

Example 2 'Alternatively, when carrying out the separation method of the present invention, in (b) of Example 1 above, In place of the gradient separation component described in Example 1 (b), a flat rate of 1.07 g/cn was added. Gradient separation components of 2.2d of homogeneous density (11 of which is 85% glycerol of the 94 relative; 18.8 g sucrose; 30. Og of NaHCOs; 15.9 g of Naz CO+; 0.75 g NatEDTA * and 0°006- Br1j (consisting of 96 ■) was used.

international search report 1st floor pottery i-^oan-mMIl & P mouth A cumulative 90100052 international investigation report

Claims (1)

[Claims] 1. an upper mouth defined by a radially inwardly extending rim portion and an inner circumferential surface; A method of separating bacteria from a liquid sample containing bacteria using a dish-shaped centrifugal container with Then, the following sequential steps; (a) rotating the centrifuge container at a high rotational speed; (b) approximately reaching an average density of the bacteria; A volume of gradient separation components of equal density is introduced into the centrifuge vessel and the inner circumferential surface is producing a separation layer of the gradient separation component on the surface; (c) While discharging the remaining components of the liquid sample from the upper opening of the centrifuge container, A continuous stream of the liquid sample is introduced into the centrifuge vessel at a discharge rate, and the separation layer isolates the bacteria. (d) introducing a brushing agent into the centrifuge container; and (e) decelerating the centrifuge container; and and rotate it at low speed, (f) in the centrifuge container without draining the liquid from the upper opening of the centrifuge container; a first position where the tip is away from said internal peripheral surface while introducing a volume of transfer liquid; the suction pipette from one position to a second position where the tip is placed close to said internal peripheral surface. actuating and moving a pump to aspirate liquid material from the centrifuge container; and (8) returning the suction pipette to its first position. 2. Steps The next sequential step following steps (a) to (g); (h) the top of the centrifuge vessel; Introducing a small amount of transition liquid into the centrifuge container without draining the liquid from the (i) accelerating the centrifuge vessel, causing it to rotate at a high rotational speed, and (j) Repeat steps (e) to (g). 2. The method of claim 1, further comprising: 3. Steps After steps (a) to (j) the following sequential steps; (k) accelerating the centrifuge vessel; (l) Immediately flush the stream of brushing agent and heated air. Introduce the spray solution into the centrifuge container, wash out the substances adhering to the centrifuge container, (m) decelerate the centrifuge container to rotate it at low rotational speed; (n) remove the aspiration pipette; Actuate and move from a first position to a second position to aspirate liquid material from the centrifuge container and (o) return the suction pipette to its first position. 3. The method of claim 2, further comprising: 4. The liquid is a minimum width limiting notch in the rim portion extending radially inward from the upper opening of the centrifuge container; 6. A method according to any one of the preceding claims, wherein: 5. The centrifuge is heated by supplying air at a predetermined temperature to the centrifuge container. Any one of the preceding claims further comprising thermostating the container to said temperature. The method described in section. 6. A flow of pressurized air is applied to the centrifuge vessel while the centrifuge vessel is rotated at a high rotational speed. A method according to any one of the preceding claims, wherein the method is introduced. 7. The gradient separation components have an average density of about 1.13 g/cm2 and the aqueous solution is 300 ml of 85% glycerol; 60.0 g of sucrose; 3 0.0g NaHCO3; 15.9g Ha2CO3; 0.75g Na2ED TA; and 0.02 ml of Brij96. The method described in section. 8. The gradient separation components have an average density of about 1.07 g/cm2 and the aqueous solution is 1 liter of 94 ml of 85% glycerol; 18.8 g of sucrose; 30 ．． 0g NaHCO3; 15.9g Na2CO3; 0.75g Na2EDT A; and 0.006 ml of Brij96. The method described in section. 9. a high rotational speed of the centrifuge vessel is on the order of 45,000 rpm, and Any of the preceding claims, wherein the low rotational speed of the centrifuge vessel is of the order of 480 rpm. The method described in item 1. 10. The accuracy of the centrifuge container is limited when the centrifuge container is rotated at high rotational speed. A method according to any one of the preceding claims, wherein the taste volume is of the order of 2 ml. 11. A gradient used in carrying out the method according to any one of claims 1 to 10. a gradient component that is a distributed component and has an average density approximately equal to the average density of the bacteria; separation component. 12. It has an average density of about 1.13 g/cm2 and constitutes an aqueous solution, of which 1 liter is 300 ml of 85% glycerol; 60.0 g of sucrose; 30. Og of NaH CO3; 15.9 g Na2CO3; 0.75 g Na2EDTA; and 0. 12. The gradient separation component of claim 11, consisting of 0.2 ml of Brij96. 13. having an average density of about 1.07 g/cm2 and constituting an aqueous solution, the 12 is 94 ml of 85% glycerol; 18.8 g of sucrose; 30.0 g of NaHC. O3; 15.9 g Na2CO3; 0.75 g Na2EDTA; and 0.0 12. The gradient separation component of claim 11, consisting of 0.6 ml of Brij96.