CA2050325A1 - Method of separating bacteria from a bacteria containing liquid sample and a gradient separation component - Google Patents
Method of separating bacteria from a bacteria containing liquid sample and a gradient separation componentInfo
- Publication number
- CA2050325A1 CA2050325A1 CA002050325A CA2050325A CA2050325A1 CA 2050325 A1 CA2050325 A1 CA 2050325A1 CA 002050325 A CA002050325 A CA 002050325A CA 2050325 A CA2050325 A CA 2050325A CA 2050325 A1 CA2050325 A1 CA 2050325A1
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- Prior art keywords
- centrifuge container
- bacteria
- centrifuge
- liquid
- rotational speed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/10—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by centrifugation ; Cyclones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Centrifugal Separators (AREA)
Abstract
In a method of separating bacteria from a bacteria containing liquid sample, a dish-like centrifuge container (21) having an upper opening (25) defined by a radially inwardly extending rim portion (24) is employed. A volume of a gradient separation component of a density approximately identical to the average density of the bacteria is introduced into the centrifuge container, which the centrifuge container is rotating, so that a separation layer (60) is formed within the centrifuge container (21). The liquid sample is introduced into the centre of the centrifuge container at a fairly high discharge rate. The liquid is thrown outwardly from the centre of the centrifuge container and into contact with the separation layer (60) so that a liquid film layer (62) is generated. The bacteria are detained by the separation component, while the constituents of the liquid sample are discharged (66) from the centrifuge container.
Description
2C1~32~
W O 90tl0057 PCT/DK90/00052 L
A METHOD OF SEPARATIN~ BACTERIA FROM A BACTERIA CONTAI~ING LIQ~ID
SAMPLE AND A GRADIE~T SEPARATION COMPONENT
The presene invention relaees to a method of separating bacteria fron, a bacteria containing liquid sample. More specifically, the present invention relates eo a method of separating bacteria from a bacteria containing liquid in accordance with the 8radient separation prin-ciples known pe~ se.
In many different cases, it is desirable to be able to separate bacteria from a bacteria containing liquid sample in that the bacteria content of the sample may be determined after having separated the bacteria from the sample by simply counting the number of bacteria separated from the sample in an optical measuring apparatus known per se. The liquid sample may have any organic origin. Thus, the sample may be a blood sample (vide e.g. US patent No. 3,928,139) or urine sample, or a suspension or solution of a solid sample, e.g. an aqueous solution or an alcoholic suspension of an organic component, e.g. a tissue or food-stuff sample. A very i~-portant example of a bacteria containing liquid sample is a milk .
sample. As will be appreciated, the measurement of the bacteria - 20 content of the original liquid sample is basically determined by the exactitude of the separation of the bacteria from the liquid sample.
Consequently, it is of the utmost importance to be able to carry out an exact and highly accurate separation process in which bacteria exclusively are separated from the liquid sample while other particles, e.g. fat globules, blood cells or the like are not separated from the liquid sample.
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From U.S. patent No. 4,541,445 and European patent No. 0,128,509 a method of the above kind and an apparatus for carrying out the method are known, in accordance with which method the bacteria are separated f~om the bacteria containing liquid sample by means of a two compo-nent gradient separation layer comprising a high density component and a low density component. The high density component and the low density component have densities which are higher and lower, respec-tively, than the average density of the bacteria to be separated from the bacteria containing liquid sample. In accordance with the meehod ... j- .
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known from the above US and EP patents, the bacteria are detained in an interface defined between the high density and the low density components of a gradient separation layer.
The technique of separating bacteria from a bacteria containing liquid sample by means of a tube component gradient separation layer has been further developed into a multi-component gradient separation layer separation technique, in which a plurality of components together constituting a continuous density spectrum is employed, vide Chemical Abstracts, Vol. 101, No. 12, 17 September 1984 (Columbus, Ohio, US), Maechtle W.: Rapid dyna~ic water/PE~COLL densicy gradients for microparticles in the analy~ical ultracen~rifuge, page 24, abstract 91793d; and Colloid Polym. Sci. 1984, 262(4), 270-82 (Ger). As is disclosed in the article, a dynamical density gradient is built up within a period of time of 20 minutes. As will be - 15 understood, this technique is rather time consuming and requires a plurality of components of different densities, preferably defining a continuous spectrum of densities.
It is an object of the present invention to provide a method of the above kind which is more simple than the method known from the above ~S and EP patents and which further makes it possible to carry ou~ an exact and highly accurate separation of bacteria from the bacteria containing liquid sample, excluding other particles from being separated from the liquid sample.
A further object of the present invention is to provide a method which renders it possible to carry out the separation automatically and at a hi8h speed.
In accordance with the present invention a method of separating bacteria from a bacteria containing liquid sample by means of a dish-like centrifuge container having an upper opening defined by a radially inwardly extending rim portion and an inner peripheral surface, comprising the following sequential steps:
(a) rotating said centrifuge container at a high rotational speed, (b) introducing a volume of a gradient separation component of a density approximately identical to the average density of said 2 ~
W O 9D/l0057 PCT/DK90/00052 bacteria into the centrifu~e container so as to generate a separa~ion layer of said gradient separation component at said inner peripheral surface~
(c) introducing a continuous flow of said liquid sample into said centrifuge container at a discharge rate so as to detain the bacteria by said separation layer, while the remaining constituents of said liquid sample are discharged through said upper opening of said centrifuge container, (d) introducing a flushing agent into said centrifuge container and discharging said flushing agent from said upper opening of said centrifuge container, (e) decelerating said centrifuge~container and rotating it at a low rotational speed, (f~ activating and moving a suction pipette from a first posicion having its tip remote from said inner peripheral surface to a second position having its tip arranged adjacent to said inner peripheral surface, so as to suck liquid substance from said centrifuge con-tainer, while introducing a volume of a transfer liquid into said centrifuge container without discharging any liquid through said upper opening of said centrifuge container, and (g) returning said suction pipette to its first position.
In accordance with the present invention, the gradient separation component is introduced into the centrifuge container while rotating the centrifuge container by means of a two speed motor at its high rotational speed. As compared to the method known from U.S. patent No. 4,541,445 and European patent No. 0128509, the method according - to the present invention is a more simple yet accurate separation method as, in accordance with the teaching of the present invention, a single gradient separation component is employed. Surprisingly, th~
bacteria may be separated from the sample and be detained by the separation layer constituted by a single gradient separation compo-nent in accordance with the teaching of the present invention.
Although there is no technical explanation of the surprising effect that bacteria of varying density i.e. of low and high densities such 35 as densities within the range of 1.04-1.18 g/cm3, may be detained by a single gradient separation component of a density approximately identical to the average density of the bacteria. such as a density ' - ' : ' :'' :
.:.: -wo 90/10057 2 ~ ~ O ~ PCTtDK90/00052 of 1.13 g/cm3, a possible technical theory is the one that by the high rate introduction of the liquid sample into the centrifu~e con~ainer, an interface is generated between a bacteria containing liquid layer and the separation layer, in which interface a suspen-sion of the liquid sample in the gradient separation component of theseparation layer is generated. The above technical theory is not to be construed as limiting the invention in any way.
Since the average density of the bacteria is approximately identical to the average density of the gradient separation component, the bacteria are detained by the gradient separation component while the remaining constituents of the liquid sample are discharged from the centrifuge container through the upper opening thereof as the liquid sample is introduced into the centrifuge container in a continuous flow.
Consequently, the method of separating bacteria from the bacteria containing liquid sample may in principle be performed in a conti-nuous separation process. By the introduction of the flushing agent into the centrifuge container after the carrying out of the separa-tion process itself, any material adhering to the separation layer, e.g. fat globules or the like, are flushed off and discharged from the upper opening of the centrifuge container. The removal of the separation layer including the bacteria separated from the original liquid sample is carried out by means of a suction pipette which is movable from a first position to a second position having its tip extending into the centrifuge container while rinsing the centrifuge container by means of the transfer liquid.
In accordance with a preferred embodiment of the method according to the present invention, the method further comprises the following sequential steps succeeding the steps (a)-(g):
(h) introducing a small volume of the transfer liquid into the centrifuge container without discharging any liquid through the upper opening of the centrifuge coneainer, ~i) accelerating the centrifuge container and rotating it at its higl rotational speed, and (j) repeating the steps (e)-(g).
, .. .
.,:, ' :
2a~032a By introducing a further volume of said liquid into the centrifuge container and accelerating the centrifuge container to its high rota-tional speed, any material, i.e. any gradient separation component or any bacteria included therein is rinsed off the centrifuge container, and consequently when repeating the steps (e)-(g) transferred to the measuring container.
In order to completely rinse the cen~rifuge container it is preferred to carry out the following further sequential steps succeeding the steps (a)~
(k) accelerating the centrifuge container and rotating it at its high rotational speed, (l) intermittently introducing a flow of a flushing agent and heated air into the centrifuge container in a spray, so as to rinse off any material adhering to the centrifuge container, (m) decelerating the centrifuge container and rotating it at its low rotational speed, .
(n) activating and moving the suction pipette from its first position to its second position, so as to suck any liquid substance from the centrifuge container, and (o) returning the suction pipette to its first position.
In accordance-with the present invention, the liquid may advantage-ously be discharged from the upper opening of the centrifuge con-tainer through a minimum width defining notch of the radially inward-ly extending rim portion thereof, so as to cause a delay of the dis-charge of the liquid from the centrifuge container. Consequently,caused by the delay of the discharge of liquid from the centrifuge container, the liquid is maintained in the centrifuge container for a longer period of time, and consequently, the rate of supply of liquid to the centrifuge container may be increased so that a higher separa-tion rate may be obtained.
In order to carry out the separation of bacteria from the bacteriacontaining liquid sample under controlled conditions, it is preferred that the method according to the invention further comprises thermo-stating the centrifuge container tO a predetermined ~emperature by - , . - - . : : :
- , , , - . .
;, , ' , ' " ' ' ; ' ' ' W090/10057 2 ~ J ~ 3 /. i PCT/DK90/00052 supplying air of said temperature to the centrifuge con~ainer. The air may be s~pplied in any appropriate manner, e.~. from below, so that the exterior surface of the centrifuge container is maintained at the predetermined temperature, and furthermore or alternatively, the air of the predetermined temperature may be supp~ied to the centrifuge container from above so that the air is introduced into the centrifuge container through the upper opening thereof.
In order to guarantee that the liquid which is discharged from the upper opening of the centrifuge container is not reintroduced into the centrifuge container caused by the suction effect generated when rotating the centrifuge container at its high rotational speed, it is preferred that a flow of pressurized air is introduced into the cen-trifuge container while rotating it at its high rotational speed.
Preferably, the pressurized air is thermostated to the said pre-determined temperature so that the flow of pressurized air further serves the purpose of thermostating the centrifuge container.
In a first embodiment of the method according to the present invention, the gradient separation component has an average density of approximately 1.13 g/cm3 and constitutes an aqueous solution. 1 litre of the gradient separation component is composed of 300 ml 85%
glycerol; 60.0 g sucrose; 30.0 g NaHC03; 15.9 g Na2CO3; 0.75 g Na2 EDTA; and 0.02 ml Brii 96~. .
In a second embodiment of the method according to the present invention, the gradient separation component has an average density of approximately 1.07 g/cm3 and constitutes an aqueous solution. 1 litre thereof is composed of 94 ml 85Z glycerol; 18.8 g Sucrose; 30.0 g NaHC03; 15.9 g Na2C03: 0.75 g Na2 EDTA; and 0.006 ml Brij 96~ The transfer liquid serving rinsing purpose when transferring the separation layer and the bacteria included therein from the centrifuge container and also serving the purpose of rinsing off anymaterial of the separation layer and any bacteria included therein, may advantageously be an enzyme solution which fur~her serves the purpose of pretreating the bacteria for the above optical counting procedure.
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W O 90/100~ PCT/DK90~00052 ln the preferred e~bodi~ent of the method according to the presen~
invention, the high rotational speed of the centrifuge container is in the order of 45,000 rpm, and the low rotational speed of the centrifuge container is in the order of 480 rpm, the net volume of the centrifuge container defined therein when rotating the centrifuge container at its high rotational speed is in the order of 2 ml. When rotating the centrifuge container at a rotational speed of 45,000 rpm, an extreme virtual gravitational field in the order of 50,000 G is provided in the inner space of the centrifuge container having an inner diameter in the order of 47 mm. By providing a gravitational field of such extreme intensity, a separation capacity of 1.33 ml/s is obtained, i.e. a 10 ml sample is separated within 10 s, or, alternatively, a 20 ml sample is separated within 15 s.
The method according to the present invention may be carried out by means of any appropriate centrifuge container, however, it is preferably carried out by means of a centrifuge container of the type disclosed in ~S patent No. 4,591,445, to which reference is made, and which is herewith incorporated in the present specification by reference, and further in European patent No 20 0,128,509.
The present invention further relates to a gradient separation component to be used in accordance with the above method, which gradient separation component has an average density approximately identical to the average density of the bacteria to be separated.
In accordance with a first embodiment of the gradient separation component according to the present invention, the gradient separation component has an average density of approx. 1.13 g/cm3 and constitutes an aqueous solution. 1 litre thereof is composed of 300 ml 85-~ glycerol; 60.0 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2C03;
30 0.75 g Na2 EDTA; and 0.02 ml Brij 96~.
In an alternative embodiment of the gradient separation component according to the present invention, the gradient separation componen, has an average density of approx. 1.07 g/cm3 and constitutes an aqueous solution. 1 litre thereof is composed of 94 ml 85% glycerol;
' : ~ ., ..
2 0i~ ;3 18.8 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2CO3; 0.75 ~ Na2 EDTA; and 0.006 ml Brij 96~
The invention will now be further described wieh reference to the drawing, wherein Fig. 1 is a vertical sectional view of a preferred embodiment of an apparatus for carrying out the method according to the present invention and of separating bacteria from a bacteria containing liquid sample, and Fig. 2 a partly sectional, diagrammatical view illustrating the general separation method according to the invention.
In Fig. L an apparatus for separating bacteria from a bacteria con-taining liquid sample, e.g. a milk sample is shown. The apparatus is designated 10 in its entirety and provided with a base 11 on which a cylindrical casing 12 is mounted. The lower end portion of the lS cylindrical casing 12 is secured to the base 11 and received in a cylindrical, circumferential recess 13 thereof. At its outer periphe-ral surface the cylindrical casing 12 is provided with fins 28 which constitute heat sinks, i.e. the fins serve the purpose of providing a large heat radiating or heat transmitting surface through which excess heat is radiated or transmitted to a cooling medium such as a cooling gas or a cooling liquid. On top of the cylindrical casing 12 a floor 14 is mounted. The floor 14 is secured in relation to the cylindrical casing 12 by means of a cylindrical rim projecting from the lower side surfac~e of the floor 14. In the cylindrical casing 12 a motor 16 is encapsulated. The motor 16 is a two speed motor which is adapted to generate a high speed ro.ation in the order of 45,000 rpm and a low speed rotation in the order of 480 rpm. The motor 16 has its shaft 17 journalled in a top bearing 18 and a bottom bearing l9 arranged in the floor 14 and the base 11, respectively.
The shaft 17 extends beyond the top bearing 18 and is provided with an inwardly tapering cone 20 at its upper end.
A dish-like centrifuge container 21 is mounted on the shaft 17 and ar its lower end provided with an outwardly tapering conical recess of â
tapering, rate identical to the tapering rate of the inwardly tapering 35 cone 20 of the shaft 17. Consequently, the cones of the shaft 17 and : ' ~ ' ::
': ' ' ' ' ~- . :
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`2~32.`i ~0 90/10057 PCT/DK90/00052 of the dish-like centrifuge container 21 secure ehe cen~rifuge con-tainer 21 in relation to the mo~or shaft 17. The centrifu~e con~ainer 21 which is shown in greater detail in Fig. 2 and is to be described below, comprises a conical bottom portion 22, a vertical cylindrical S portion 23, and an inwardly extending rim portion 24 defining an upper opening 25 of the centrifuge container 21. At the upper opsning 25 of the centrifuge container 21, the inwardly extending rim portion 24 is provided with a notch 26 defining the minimum radial width of the rim portion. Furthermore, the centrifuge container 21 is provided with a downwardly projecting separating skirt 27 which serves the purpose of preventing liquid from getting into contact with the ~op bearing 18. In order to further pre~ent liquid or dr~plets from getting into contact with the top bearing 18, a flow of air of a predetermined temperature, further serving the purpose of thermo-stating the centrifuge to the predetermined temperature, i.s suppliedto the centrifuge container from below through a supply tube, not shown on the drawings, so as to generate a separating air curtain surrounding the lower part of the centrifuge container.
On top of the floor 14, a housing 30 is arranged encapsulating the centrifuge container 21. The housing 30 is at its vertical cylindri-cal side wall provided with outlets 31 serving the purpose of letting - out liquid from the housing 30. A supply tube 32 for supply of a liquid sample extends into the interior of the housing 30 through a bore of the housing 30 and through the upper opening 25 of the centrifuge container 21 and into the interior thereof. The supply tube 32 is adapted to be connected to an external liquid sample container through an appropriate tubing, not shown on the drawings.
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In another cylindrical bore at the top of the housing 30, a suction pipette, generally designated 40, is arranged. The suction pipette 40 comprises a pipette tube 41 which extends into the interior of the housing 30 and through the opening 25 of the centrifuge container 21 into the interior thereof. The pipette tube 41 is to be connected to external containers, not shown on the drawings, through appropriate tubing and valves, not shown on the drawings. The suction pipette 40 further comprises a pneumatic motor 43. A piston of the pneumatic motor 43 acts on an upper side surface of a plate member 44 which is - , - . , . ~ . , : ~:
', '. ' . ':
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~ J~ `3.~j W O 90/10057 PCT/DK90/000~2 fixedly connected to the pipette tube 41 so that a~y motion of ehe plate member 44 results in a similar motion of the pipet~e tube 41.
On the lower side surface of the plate member 44, a coil 45 acts so as to maintain the plate member in a first position, shown in Fig. l, provided the pneumatic motor 43 is not activated. By the activation of the pneumatic motor 43, the plate member 44 is moved from its first position to a second position in which the coil 45 is com-pressed. The positions of the pipette tube 41 corresponding to the above first and second positions are a position shown in Fig. 1 and an extended position in which the outer end of the pipette tube 41 is moved into a position in proximity with the vertical cylindrical portion 23 of the centrifuge container 21, respectively. The pres-surized air is supplied to the pneumatic motor 43 through a fitting 46, and the pneumatic motor 43 is also provided with a throttle valve 47 which serves the purpose of reducing the rate by which the pipette tube 41 is moved from its retracted position shown in Figs. 1 and 2 to its above extended position corresponding to the above first and second positions of the plate member 44, respectively.
At the centre of the top of the housing 30, a body 50 is arranged. In a through-going bore of the body 50, a tube 51 is arranged and sealed in relation to the body 50 by means of a sealing ring or gasket 52.
The upper end of the tube 51 is provided with a connecting fitting 53 allowing connection to an external fluid source, not shown on the drawings, through an appropriate tubing, not shown on the drawings.
Perpendicularly to the through-going bore of the body 50, a bore 54 provides communication thereto from a connecting fitting 55. The connecting fitting 55 is adapted to be connected to an external air pressurizing source, not shown on the drawing, through an appropriate tubing, not shown on the drawings, e.g. the above mentioned pressur-izing source connected to the connecting fitting 46 of the suctionpipette 40. The supply of pressurized air through the connection fitting 55 and further through the through-going bore of the body SO
serves two purposes. Firstly, the air, which is maintained at a predetermined temperature, serves the purpose of thermostating the centrifuge container to the predetermined temperaeure. Secondly, the pressurized air serves the purpose of preventing liquid from being collected at the interior surface of the housing 30 and from being 2 ~ 2 ~
W O 901tO057 PCT/DK90/00052 sucked into the centrifuge container when rotating the centrif~ge container at its high rotational speed of ~S,000 rpm.
In Fig. 2 ehe centrifuge container 21 is shown in greater detail.
While the centrifuge container 21 is rotating at its high rotational speed, driven by the motor 16 as indicated by an arrow 64, a gradient separation layer 60 is arranged in a circumferential inner space of the centrifuge container 21 defined by the minimum width defining notch 26 of the inwardly extending rim portion 24. The separation layer 60 comprises a gradient separation component having a density approximately identical to the average density of the bacteria to be separated. The liquid sample is supplied to the centre of the centrifuge container 21 through the supply tube 32 and discharged therefrom. The liquid sample is discharged from the supply tube 32 at a fairly high discharge rate and due to the centrifugal force generated by the rotation of the centrifuge container 21 at its high rotational speed it is thrown outwardly from the centre of the centrifuge container and forced into contact with the separation layer 60. The liquid supplied from the supply tube 32 in a continuous flow generates a liquid film layer 62.
It is believed that in the interface between the liquid film layer 62 and the separation layer 60, the liquid sample is suspended in the gradient separation component of the separation layer 60, and as the bacteria to be separated from the liquid sample have an average density approximately identical to the der,sity of the gradient separation component, the bacteria are detained by the separation layer 60. Those constituents of the liquid sample having densities lower than the density of the gradient separation component and, consequently, lower than the average density of the bacteria, are forced upwardly and are discharged from the centrifuge container through the notch 26 as indicated by the reference numeral 66. It is to be realized that since the bacteria constitute the components of the liquid sample having the highest densities, due eo the high centrifugal gradient field generated by the high rotational speed of the centrifuge container the bacteria are inevitably forced from the liquid film layer 62 into the separation layer 60. As the liquid is only discharged through the notch 26, the liquid is retained in the .
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W O Q0/l0057 PCTiDK90100052 centrifuge container for a longer period of time when compared ~o a dish^like centri~uge container not having the minimum width defining notch 26 in which the liquid is discharged from the ~o~al upper opening of the centrifuge container and, consequently, the rate of supply of liquid to the cen~rifuge con~ainer may be increased further increasing the rate of separation of bacteria when compared to an apparatus not having the notch 26.
The operation of the apparatus 10 is described in the example below.
In a practical embodiment of the kind described above with reference to the drawings, the dish-like centrifuge container 21 was made of titanium and provided with an interior Teflon~-PFA (Perfluoroalkoxy) surface coating. The inner diameter of .he centrifuge container 21 was 47 mm. The motor 16 was a thyristor controlled three phase AC
motor adapted to be driven at a low rotational speed of 480 rpm and a high rotational speed of 45,000 rpm providing a virtual gravitational field in the centrifuge container in the order of 50,000 G. The circumferential inner space of the centrifuge container 21, the height thereof being defined by the minimum width defining notch 26 of the inwardly extending rim portion 24, was in the order of 2 ml.
In this embodiment, a 10 ml liquid sample was separated within 10 s.
Alternatively, a 20 ml liquid sample was separated within 15 s. The 10 ml and 20 ml liquid samples were aqueous dilutions of 2.5 ml and 5 ml milk samples, respectively. Through the connecting fitting 55 and through the through-going bore of the body 50 a flow of pressuri-zed air heated to a temperature of 40C was continuously supplied from a l.S Bar pressurizing source through a tubing of an interior diameter of l.S mm and of a length of 250 mm. As mentioned above a flow of air of a temperature of 40C was also supplied to the centrifuge container from below.
In carrying out the separation method according to the invention, the above apparatus was driven in an automatized sequence comprising the following steps~
, . . : - .
, ~ 13 ~
~0 90/10057 PCT/DK90/00052 (a) the motor 16 was accelerated to its high rotational speed for rotating the centrifuge container 21 at its high rotational speed of 45,000 rpm, (b) a 2.2 ml gradient separation component, 1 litre of which was composed of 300 ml 85X glycerol; 60.0 g Sucrose; 30.0 g NaHC03;
15.9 g Na2C03; 0.75 g Na2 EDTA; and 0.02 ml Brij 96~, of an average density of 1.13 g/cm3 was supplied from a separate supply tube, not shown on the drawings, to the centre of the centrifuge container 21, whereupon the gradient separation component was thrown outwardly from the centre of the centrifuge container 21 and was arranged in the separation layer 60 shown in Fig. 2, and approximately 0.2 ml excessive gradient separation component was discharged through the notch 26 of the centrifuge container 21.
(c) the liquid sample was supplied from the supply tube 32 in a continuous flow at a rate of 1 ml/sec. so that the interface 62 was generated and so that the bacteria having densities of the order of 1.04-1.18 g/cm3 were deposited in the separation layer 60, and the liquid was discharged through the upper opening 25 of the centrifuge container as indicated by the reference numeral 66 of Fig 2, (d) thus having concluded the separation process itself, a flushing agent constituted by a detergent solution was supplied from the supply tube 32 to the centre of the centrifuge container so that any particles, e.g. fat globules which might influence the bacteria counting process to bs carried out later, were rinsed off and discharged from the centrifuge container 21, (e) the motor 16 was decelerated to its low rotational speed for rotating the centrifuge container 21 at its low rotational speed of 480 rpm, (f) the suction pipette 40 was activated and moved from a first position having its tip remote from the inner surface of the periphe-ral wall of the centrifuge container to a second position having its tip arranged adjacent to said inner surface as evident from Fig. 2, while a 1.5 ml volume of the enzyme solution was supplied from a separate supply tube, not shown on the drawings, to the periphery of the centrifuge container 21 so that the material including the separation layer 60 was transferred in an enzyme solution through the pipette tube 41 and further through the connecting fitting 42 and an . .
.
~ ' ' . ' W O 90/l00~7 2 ~ O ~ ` `i PCT/DK90/00052 external tubin~, not shown on the drawings, to an external measurin~
container or measuring equipment, noc shown on the drawings, (g) the suction pipette was returned to its first position, (h) a 1.5 ml enzyme solution volume was supplied through the above supply tube, not shown on the drawings, to the periphery of the cen-trifuge container 21, (i) the motor 16 was accelerated for rotating the centrifuge con-tainer 21 at its high rotational speed of 45,000 rpm so that any material arranged within the circumferential inner space of the centrifuge container 21 was rinsed off by means of the enzyme solution volume, and (j) thereupon the steps (e)-(g) were repeated for transferring the rinsing 1.5 ml enzyme solution volume together with any material dissolved therein to the above measuring container or measuring equipment, not shown on the drawings.
For rinsing the entire apparatus, the following sequential steps were carried out:
~k) the motor 16 was accelerated for rotating the centrifuge con-tainer 21 at its high rotational speed of 45,000 rpm, (1) a flushing agent supplied through the supply tube 51 and air supplied through the bore 54 from a preheater heating the air to a temperature of approximately 40C were intermittently introduced into the interior space of the housing 30, the interval being in the order of 0.5 s, in a spray serving the purpose of rinsing the interior surfaces of the hous,ing 30 and the outer surfaces of the centrifuge container 21, (m) the motor 16 was decelerated for rotating the centrifuge con-tainer at its low rotational speed of 480 rpm, (n) the suction pipette was activated and was moved from its first to its second position so that any liquid and any material present in the centrifuge container were suc~ed off and transferred through the pipette tube 41 and further through the connecting fitting 42 and through an external tubing, not shown on the drawings, to a waste container, and (o) finally the suction pipette was returned to its initial position, and the motor 16 was turned off.
~09Ofl00~7 2 0 ~ ~ 3 ,~ ~ PCT/DK90/00052 Alternatively, in carrying out ehe separation method according ~o ~he invention, in (b) in example l above, a 2.2 ml gradient separation component was used, l litre of which was composed of 94 ml 85/.
glycerol; 18.8 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2C03, 0.75 g Na2 EDTA; and 0.006 ml Brij 96~, of an average density of 1.07 g/cm3 instead of the gradient separation component described above in (b) in example l.
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W O 90tl0057 PCT/DK90/00052 L
A METHOD OF SEPARATIN~ BACTERIA FROM A BACTERIA CONTAI~ING LIQ~ID
SAMPLE AND A GRADIE~T SEPARATION COMPONENT
The presene invention relaees to a method of separating bacteria fron, a bacteria containing liquid sample. More specifically, the present invention relates eo a method of separating bacteria from a bacteria containing liquid in accordance with the 8radient separation prin-ciples known pe~ se.
In many different cases, it is desirable to be able to separate bacteria from a bacteria containing liquid sample in that the bacteria content of the sample may be determined after having separated the bacteria from the sample by simply counting the number of bacteria separated from the sample in an optical measuring apparatus known per se. The liquid sample may have any organic origin. Thus, the sample may be a blood sample (vide e.g. US patent No. 3,928,139) or urine sample, or a suspension or solution of a solid sample, e.g. an aqueous solution or an alcoholic suspension of an organic component, e.g. a tissue or food-stuff sample. A very i~-portant example of a bacteria containing liquid sample is a milk .
sample. As will be appreciated, the measurement of the bacteria - 20 content of the original liquid sample is basically determined by the exactitude of the separation of the bacteria from the liquid sample.
Consequently, it is of the utmost importance to be able to carry out an exact and highly accurate separation process in which bacteria exclusively are separated from the liquid sample while other particles, e.g. fat globules, blood cells or the like are not separated from the liquid sample.
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From U.S. patent No. 4,541,445 and European patent No. 0,128,509 a method of the above kind and an apparatus for carrying out the method are known, in accordance with which method the bacteria are separated f~om the bacteria containing liquid sample by means of a two compo-nent gradient separation layer comprising a high density component and a low density component. The high density component and the low density component have densities which are higher and lower, respec-tively, than the average density of the bacteria to be separated from the bacteria containing liquid sample. In accordance with the meehod ... j- .
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known from the above US and EP patents, the bacteria are detained in an interface defined between the high density and the low density components of a gradient separation layer.
The technique of separating bacteria from a bacteria containing liquid sample by means of a tube component gradient separation layer has been further developed into a multi-component gradient separation layer separation technique, in which a plurality of components together constituting a continuous density spectrum is employed, vide Chemical Abstracts, Vol. 101, No. 12, 17 September 1984 (Columbus, Ohio, US), Maechtle W.: Rapid dyna~ic water/PE~COLL densicy gradients for microparticles in the analy~ical ultracen~rifuge, page 24, abstract 91793d; and Colloid Polym. Sci. 1984, 262(4), 270-82 (Ger). As is disclosed in the article, a dynamical density gradient is built up within a period of time of 20 minutes. As will be - 15 understood, this technique is rather time consuming and requires a plurality of components of different densities, preferably defining a continuous spectrum of densities.
It is an object of the present invention to provide a method of the above kind which is more simple than the method known from the above ~S and EP patents and which further makes it possible to carry ou~ an exact and highly accurate separation of bacteria from the bacteria containing liquid sample, excluding other particles from being separated from the liquid sample.
A further object of the present invention is to provide a method which renders it possible to carry out the separation automatically and at a hi8h speed.
In accordance with the present invention a method of separating bacteria from a bacteria containing liquid sample by means of a dish-like centrifuge container having an upper opening defined by a radially inwardly extending rim portion and an inner peripheral surface, comprising the following sequential steps:
(a) rotating said centrifuge container at a high rotational speed, (b) introducing a volume of a gradient separation component of a density approximately identical to the average density of said 2 ~
W O 9D/l0057 PCT/DK90/00052 bacteria into the centrifu~e container so as to generate a separa~ion layer of said gradient separation component at said inner peripheral surface~
(c) introducing a continuous flow of said liquid sample into said centrifuge container at a discharge rate so as to detain the bacteria by said separation layer, while the remaining constituents of said liquid sample are discharged through said upper opening of said centrifuge container, (d) introducing a flushing agent into said centrifuge container and discharging said flushing agent from said upper opening of said centrifuge container, (e) decelerating said centrifuge~container and rotating it at a low rotational speed, (f~ activating and moving a suction pipette from a first posicion having its tip remote from said inner peripheral surface to a second position having its tip arranged adjacent to said inner peripheral surface, so as to suck liquid substance from said centrifuge con-tainer, while introducing a volume of a transfer liquid into said centrifuge container without discharging any liquid through said upper opening of said centrifuge container, and (g) returning said suction pipette to its first position.
In accordance with the present invention, the gradient separation component is introduced into the centrifuge container while rotating the centrifuge container by means of a two speed motor at its high rotational speed. As compared to the method known from U.S. patent No. 4,541,445 and European patent No. 0128509, the method according - to the present invention is a more simple yet accurate separation method as, in accordance with the teaching of the present invention, a single gradient separation component is employed. Surprisingly, th~
bacteria may be separated from the sample and be detained by the separation layer constituted by a single gradient separation compo-nent in accordance with the teaching of the present invention.
Although there is no technical explanation of the surprising effect that bacteria of varying density i.e. of low and high densities such 35 as densities within the range of 1.04-1.18 g/cm3, may be detained by a single gradient separation component of a density approximately identical to the average density of the bacteria. such as a density ' - ' : ' :'' :
.:.: -wo 90/10057 2 ~ ~ O ~ PCTtDK90/00052 of 1.13 g/cm3, a possible technical theory is the one that by the high rate introduction of the liquid sample into the centrifu~e con~ainer, an interface is generated between a bacteria containing liquid layer and the separation layer, in which interface a suspen-sion of the liquid sample in the gradient separation component of theseparation layer is generated. The above technical theory is not to be construed as limiting the invention in any way.
Since the average density of the bacteria is approximately identical to the average density of the gradient separation component, the bacteria are detained by the gradient separation component while the remaining constituents of the liquid sample are discharged from the centrifuge container through the upper opening thereof as the liquid sample is introduced into the centrifuge container in a continuous flow.
Consequently, the method of separating bacteria from the bacteria containing liquid sample may in principle be performed in a conti-nuous separation process. By the introduction of the flushing agent into the centrifuge container after the carrying out of the separa-tion process itself, any material adhering to the separation layer, e.g. fat globules or the like, are flushed off and discharged from the upper opening of the centrifuge container. The removal of the separation layer including the bacteria separated from the original liquid sample is carried out by means of a suction pipette which is movable from a first position to a second position having its tip extending into the centrifuge container while rinsing the centrifuge container by means of the transfer liquid.
In accordance with a preferred embodiment of the method according to the present invention, the method further comprises the following sequential steps succeeding the steps (a)-(g):
(h) introducing a small volume of the transfer liquid into the centrifuge container without discharging any liquid through the upper opening of the centrifuge coneainer, ~i) accelerating the centrifuge container and rotating it at its higl rotational speed, and (j) repeating the steps (e)-(g).
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2a~032a By introducing a further volume of said liquid into the centrifuge container and accelerating the centrifuge container to its high rota-tional speed, any material, i.e. any gradient separation component or any bacteria included therein is rinsed off the centrifuge container, and consequently when repeating the steps (e)-(g) transferred to the measuring container.
In order to completely rinse the cen~rifuge container it is preferred to carry out the following further sequential steps succeeding the steps (a)~
(k) accelerating the centrifuge container and rotating it at its high rotational speed, (l) intermittently introducing a flow of a flushing agent and heated air into the centrifuge container in a spray, so as to rinse off any material adhering to the centrifuge container, (m) decelerating the centrifuge container and rotating it at its low rotational speed, .
(n) activating and moving the suction pipette from its first position to its second position, so as to suck any liquid substance from the centrifuge container, and (o) returning the suction pipette to its first position.
In accordance-with the present invention, the liquid may advantage-ously be discharged from the upper opening of the centrifuge con-tainer through a minimum width defining notch of the radially inward-ly extending rim portion thereof, so as to cause a delay of the dis-charge of the liquid from the centrifuge container. Consequently,caused by the delay of the discharge of liquid from the centrifuge container, the liquid is maintained in the centrifuge container for a longer period of time, and consequently, the rate of supply of liquid to the centrifuge container may be increased so that a higher separa-tion rate may be obtained.
In order to carry out the separation of bacteria from the bacteriacontaining liquid sample under controlled conditions, it is preferred that the method according to the invention further comprises thermo-stating the centrifuge container tO a predetermined ~emperature by - , . - - . : : :
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;, , ' , ' " ' ' ; ' ' ' W090/10057 2 ~ J ~ 3 /. i PCT/DK90/00052 supplying air of said temperature to the centrifuge con~ainer. The air may be s~pplied in any appropriate manner, e.~. from below, so that the exterior surface of the centrifuge container is maintained at the predetermined temperature, and furthermore or alternatively, the air of the predetermined temperature may be supp~ied to the centrifuge container from above so that the air is introduced into the centrifuge container through the upper opening thereof.
In order to guarantee that the liquid which is discharged from the upper opening of the centrifuge container is not reintroduced into the centrifuge container caused by the suction effect generated when rotating the centrifuge container at its high rotational speed, it is preferred that a flow of pressurized air is introduced into the cen-trifuge container while rotating it at its high rotational speed.
Preferably, the pressurized air is thermostated to the said pre-determined temperature so that the flow of pressurized air further serves the purpose of thermostating the centrifuge container.
In a first embodiment of the method according to the present invention, the gradient separation component has an average density of approximately 1.13 g/cm3 and constitutes an aqueous solution. 1 litre of the gradient separation component is composed of 300 ml 85%
glycerol; 60.0 g sucrose; 30.0 g NaHC03; 15.9 g Na2CO3; 0.75 g Na2 EDTA; and 0.02 ml Brii 96~. .
In a second embodiment of the method according to the present invention, the gradient separation component has an average density of approximately 1.07 g/cm3 and constitutes an aqueous solution. 1 litre thereof is composed of 94 ml 85Z glycerol; 18.8 g Sucrose; 30.0 g NaHC03; 15.9 g Na2C03: 0.75 g Na2 EDTA; and 0.006 ml Brij 96~ The transfer liquid serving rinsing purpose when transferring the separation layer and the bacteria included therein from the centrifuge container and also serving the purpose of rinsing off anymaterial of the separation layer and any bacteria included therein, may advantageously be an enzyme solution which fur~her serves the purpose of pretreating the bacteria for the above optical counting procedure.
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W O 90/100~ PCT/DK90~00052 ln the preferred e~bodi~ent of the method according to the presen~
invention, the high rotational speed of the centrifuge container is in the order of 45,000 rpm, and the low rotational speed of the centrifuge container is in the order of 480 rpm, the net volume of the centrifuge container defined therein when rotating the centrifuge container at its high rotational speed is in the order of 2 ml. When rotating the centrifuge container at a rotational speed of 45,000 rpm, an extreme virtual gravitational field in the order of 50,000 G is provided in the inner space of the centrifuge container having an inner diameter in the order of 47 mm. By providing a gravitational field of such extreme intensity, a separation capacity of 1.33 ml/s is obtained, i.e. a 10 ml sample is separated within 10 s, or, alternatively, a 20 ml sample is separated within 15 s.
The method according to the present invention may be carried out by means of any appropriate centrifuge container, however, it is preferably carried out by means of a centrifuge container of the type disclosed in ~S patent No. 4,591,445, to which reference is made, and which is herewith incorporated in the present specification by reference, and further in European patent No 20 0,128,509.
The present invention further relates to a gradient separation component to be used in accordance with the above method, which gradient separation component has an average density approximately identical to the average density of the bacteria to be separated.
In accordance with a first embodiment of the gradient separation component according to the present invention, the gradient separation component has an average density of approx. 1.13 g/cm3 and constitutes an aqueous solution. 1 litre thereof is composed of 300 ml 85-~ glycerol; 60.0 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2C03;
30 0.75 g Na2 EDTA; and 0.02 ml Brij 96~.
In an alternative embodiment of the gradient separation component according to the present invention, the gradient separation componen, has an average density of approx. 1.07 g/cm3 and constitutes an aqueous solution. 1 litre thereof is composed of 94 ml 85% glycerol;
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2 0i~ ;3 18.8 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2CO3; 0.75 ~ Na2 EDTA; and 0.006 ml Brij 96~
The invention will now be further described wieh reference to the drawing, wherein Fig. 1 is a vertical sectional view of a preferred embodiment of an apparatus for carrying out the method according to the present invention and of separating bacteria from a bacteria containing liquid sample, and Fig. 2 a partly sectional, diagrammatical view illustrating the general separation method according to the invention.
In Fig. L an apparatus for separating bacteria from a bacteria con-taining liquid sample, e.g. a milk sample is shown. The apparatus is designated 10 in its entirety and provided with a base 11 on which a cylindrical casing 12 is mounted. The lower end portion of the lS cylindrical casing 12 is secured to the base 11 and received in a cylindrical, circumferential recess 13 thereof. At its outer periphe-ral surface the cylindrical casing 12 is provided with fins 28 which constitute heat sinks, i.e. the fins serve the purpose of providing a large heat radiating or heat transmitting surface through which excess heat is radiated or transmitted to a cooling medium such as a cooling gas or a cooling liquid. On top of the cylindrical casing 12 a floor 14 is mounted. The floor 14 is secured in relation to the cylindrical casing 12 by means of a cylindrical rim projecting from the lower side surfac~e of the floor 14. In the cylindrical casing 12 a motor 16 is encapsulated. The motor 16 is a two speed motor which is adapted to generate a high speed ro.ation in the order of 45,000 rpm and a low speed rotation in the order of 480 rpm. The motor 16 has its shaft 17 journalled in a top bearing 18 and a bottom bearing l9 arranged in the floor 14 and the base 11, respectively.
The shaft 17 extends beyond the top bearing 18 and is provided with an inwardly tapering cone 20 at its upper end.
A dish-like centrifuge container 21 is mounted on the shaft 17 and ar its lower end provided with an outwardly tapering conical recess of â
tapering, rate identical to the tapering rate of the inwardly tapering 35 cone 20 of the shaft 17. Consequently, the cones of the shaft 17 and : ' ~ ' ::
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`2~32.`i ~0 90/10057 PCT/DK90/00052 of the dish-like centrifuge container 21 secure ehe cen~rifuge con-tainer 21 in relation to the mo~or shaft 17. The centrifu~e con~ainer 21 which is shown in greater detail in Fig. 2 and is to be described below, comprises a conical bottom portion 22, a vertical cylindrical S portion 23, and an inwardly extending rim portion 24 defining an upper opening 25 of the centrifuge container 21. At the upper opsning 25 of the centrifuge container 21, the inwardly extending rim portion 24 is provided with a notch 26 defining the minimum radial width of the rim portion. Furthermore, the centrifuge container 21 is provided with a downwardly projecting separating skirt 27 which serves the purpose of preventing liquid from getting into contact with the ~op bearing 18. In order to further pre~ent liquid or dr~plets from getting into contact with the top bearing 18, a flow of air of a predetermined temperature, further serving the purpose of thermo-stating the centrifuge to the predetermined temperature, i.s suppliedto the centrifuge container from below through a supply tube, not shown on the drawings, so as to generate a separating air curtain surrounding the lower part of the centrifuge container.
On top of the floor 14, a housing 30 is arranged encapsulating the centrifuge container 21. The housing 30 is at its vertical cylindri-cal side wall provided with outlets 31 serving the purpose of letting - out liquid from the housing 30. A supply tube 32 for supply of a liquid sample extends into the interior of the housing 30 through a bore of the housing 30 and through the upper opening 25 of the centrifuge container 21 and into the interior thereof. The supply tube 32 is adapted to be connected to an external liquid sample container through an appropriate tubing, not shown on the drawings.
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In another cylindrical bore at the top of the housing 30, a suction pipette, generally designated 40, is arranged. The suction pipette 40 comprises a pipette tube 41 which extends into the interior of the housing 30 and through the opening 25 of the centrifuge container 21 into the interior thereof. The pipette tube 41 is to be connected to external containers, not shown on the drawings, through appropriate tubing and valves, not shown on the drawings. The suction pipette 40 further comprises a pneumatic motor 43. A piston of the pneumatic motor 43 acts on an upper side surface of a plate member 44 which is - , - . , . ~ . , : ~:
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~ J~ `3.~j W O 90/10057 PCT/DK90/000~2 fixedly connected to the pipette tube 41 so that a~y motion of ehe plate member 44 results in a similar motion of the pipet~e tube 41.
On the lower side surface of the plate member 44, a coil 45 acts so as to maintain the plate member in a first position, shown in Fig. l, provided the pneumatic motor 43 is not activated. By the activation of the pneumatic motor 43, the plate member 44 is moved from its first position to a second position in which the coil 45 is com-pressed. The positions of the pipette tube 41 corresponding to the above first and second positions are a position shown in Fig. 1 and an extended position in which the outer end of the pipette tube 41 is moved into a position in proximity with the vertical cylindrical portion 23 of the centrifuge container 21, respectively. The pres-surized air is supplied to the pneumatic motor 43 through a fitting 46, and the pneumatic motor 43 is also provided with a throttle valve 47 which serves the purpose of reducing the rate by which the pipette tube 41 is moved from its retracted position shown in Figs. 1 and 2 to its above extended position corresponding to the above first and second positions of the plate member 44, respectively.
At the centre of the top of the housing 30, a body 50 is arranged. In a through-going bore of the body 50, a tube 51 is arranged and sealed in relation to the body 50 by means of a sealing ring or gasket 52.
The upper end of the tube 51 is provided with a connecting fitting 53 allowing connection to an external fluid source, not shown on the drawings, through an appropriate tubing, not shown on the drawings.
Perpendicularly to the through-going bore of the body 50, a bore 54 provides communication thereto from a connecting fitting 55. The connecting fitting 55 is adapted to be connected to an external air pressurizing source, not shown on the drawing, through an appropriate tubing, not shown on the drawings, e.g. the above mentioned pressur-izing source connected to the connecting fitting 46 of the suctionpipette 40. The supply of pressurized air through the connection fitting 55 and further through the through-going bore of the body SO
serves two purposes. Firstly, the air, which is maintained at a predetermined temperature, serves the purpose of thermostating the centrifuge container to the predetermined temperaeure. Secondly, the pressurized air serves the purpose of preventing liquid from being collected at the interior surface of the housing 30 and from being 2 ~ 2 ~
W O 901tO057 PCT/DK90/00052 sucked into the centrifuge container when rotating the centrif~ge container at its high rotational speed of ~S,000 rpm.
In Fig. 2 ehe centrifuge container 21 is shown in greater detail.
While the centrifuge container 21 is rotating at its high rotational speed, driven by the motor 16 as indicated by an arrow 64, a gradient separation layer 60 is arranged in a circumferential inner space of the centrifuge container 21 defined by the minimum width defining notch 26 of the inwardly extending rim portion 24. The separation layer 60 comprises a gradient separation component having a density approximately identical to the average density of the bacteria to be separated. The liquid sample is supplied to the centre of the centrifuge container 21 through the supply tube 32 and discharged therefrom. The liquid sample is discharged from the supply tube 32 at a fairly high discharge rate and due to the centrifugal force generated by the rotation of the centrifuge container 21 at its high rotational speed it is thrown outwardly from the centre of the centrifuge container and forced into contact with the separation layer 60. The liquid supplied from the supply tube 32 in a continuous flow generates a liquid film layer 62.
It is believed that in the interface between the liquid film layer 62 and the separation layer 60, the liquid sample is suspended in the gradient separation component of the separation layer 60, and as the bacteria to be separated from the liquid sample have an average density approximately identical to the der,sity of the gradient separation component, the bacteria are detained by the separation layer 60. Those constituents of the liquid sample having densities lower than the density of the gradient separation component and, consequently, lower than the average density of the bacteria, are forced upwardly and are discharged from the centrifuge container through the notch 26 as indicated by the reference numeral 66. It is to be realized that since the bacteria constitute the components of the liquid sample having the highest densities, due eo the high centrifugal gradient field generated by the high rotational speed of the centrifuge container the bacteria are inevitably forced from the liquid film layer 62 into the separation layer 60. As the liquid is only discharged through the notch 26, the liquid is retained in the .
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W O Q0/l0057 PCTiDK90100052 centrifuge container for a longer period of time when compared ~o a dish^like centri~uge container not having the minimum width defining notch 26 in which the liquid is discharged from the ~o~al upper opening of the centrifuge container and, consequently, the rate of supply of liquid to the cen~rifuge con~ainer may be increased further increasing the rate of separation of bacteria when compared to an apparatus not having the notch 26.
The operation of the apparatus 10 is described in the example below.
In a practical embodiment of the kind described above with reference to the drawings, the dish-like centrifuge container 21 was made of titanium and provided with an interior Teflon~-PFA (Perfluoroalkoxy) surface coating. The inner diameter of .he centrifuge container 21 was 47 mm. The motor 16 was a thyristor controlled three phase AC
motor adapted to be driven at a low rotational speed of 480 rpm and a high rotational speed of 45,000 rpm providing a virtual gravitational field in the centrifuge container in the order of 50,000 G. The circumferential inner space of the centrifuge container 21, the height thereof being defined by the minimum width defining notch 26 of the inwardly extending rim portion 24, was in the order of 2 ml.
In this embodiment, a 10 ml liquid sample was separated within 10 s.
Alternatively, a 20 ml liquid sample was separated within 15 s. The 10 ml and 20 ml liquid samples were aqueous dilutions of 2.5 ml and 5 ml milk samples, respectively. Through the connecting fitting 55 and through the through-going bore of the body 50 a flow of pressuri-zed air heated to a temperature of 40C was continuously supplied from a l.S Bar pressurizing source through a tubing of an interior diameter of l.S mm and of a length of 250 mm. As mentioned above a flow of air of a temperature of 40C was also supplied to the centrifuge container from below.
In carrying out the separation method according to the invention, the above apparatus was driven in an automatized sequence comprising the following steps~
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~0 90/10057 PCT/DK90/00052 (a) the motor 16 was accelerated to its high rotational speed for rotating the centrifuge container 21 at its high rotational speed of 45,000 rpm, (b) a 2.2 ml gradient separation component, 1 litre of which was composed of 300 ml 85X glycerol; 60.0 g Sucrose; 30.0 g NaHC03;
15.9 g Na2C03; 0.75 g Na2 EDTA; and 0.02 ml Brij 96~, of an average density of 1.13 g/cm3 was supplied from a separate supply tube, not shown on the drawings, to the centre of the centrifuge container 21, whereupon the gradient separation component was thrown outwardly from the centre of the centrifuge container 21 and was arranged in the separation layer 60 shown in Fig. 2, and approximately 0.2 ml excessive gradient separation component was discharged through the notch 26 of the centrifuge container 21.
(c) the liquid sample was supplied from the supply tube 32 in a continuous flow at a rate of 1 ml/sec. so that the interface 62 was generated and so that the bacteria having densities of the order of 1.04-1.18 g/cm3 were deposited in the separation layer 60, and the liquid was discharged through the upper opening 25 of the centrifuge container as indicated by the reference numeral 66 of Fig 2, (d) thus having concluded the separation process itself, a flushing agent constituted by a detergent solution was supplied from the supply tube 32 to the centre of the centrifuge container so that any particles, e.g. fat globules which might influence the bacteria counting process to bs carried out later, were rinsed off and discharged from the centrifuge container 21, (e) the motor 16 was decelerated to its low rotational speed for rotating the centrifuge container 21 at its low rotational speed of 480 rpm, (f) the suction pipette 40 was activated and moved from a first position having its tip remote from the inner surface of the periphe-ral wall of the centrifuge container to a second position having its tip arranged adjacent to said inner surface as evident from Fig. 2, while a 1.5 ml volume of the enzyme solution was supplied from a separate supply tube, not shown on the drawings, to the periphery of the centrifuge container 21 so that the material including the separation layer 60 was transferred in an enzyme solution through the pipette tube 41 and further through the connecting fitting 42 and an . .
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~ ' ' . ' W O 90/l00~7 2 ~ O ~ ` `i PCT/DK90/00052 external tubin~, not shown on the drawings, to an external measurin~
container or measuring equipment, noc shown on the drawings, (g) the suction pipette was returned to its first position, (h) a 1.5 ml enzyme solution volume was supplied through the above supply tube, not shown on the drawings, to the periphery of the cen-trifuge container 21, (i) the motor 16 was accelerated for rotating the centrifuge con-tainer 21 at its high rotational speed of 45,000 rpm so that any material arranged within the circumferential inner space of the centrifuge container 21 was rinsed off by means of the enzyme solution volume, and (j) thereupon the steps (e)-(g) were repeated for transferring the rinsing 1.5 ml enzyme solution volume together with any material dissolved therein to the above measuring container or measuring equipment, not shown on the drawings.
For rinsing the entire apparatus, the following sequential steps were carried out:
~k) the motor 16 was accelerated for rotating the centrifuge con-tainer 21 at its high rotational speed of 45,000 rpm, (1) a flushing agent supplied through the supply tube 51 and air supplied through the bore 54 from a preheater heating the air to a temperature of approximately 40C were intermittently introduced into the interior space of the housing 30, the interval being in the order of 0.5 s, in a spray serving the purpose of rinsing the interior surfaces of the hous,ing 30 and the outer surfaces of the centrifuge container 21, (m) the motor 16 was decelerated for rotating the centrifuge con-tainer at its low rotational speed of 480 rpm, (n) the suction pipette was activated and was moved from its first to its second position so that any liquid and any material present in the centrifuge container were suc~ed off and transferred through the pipette tube 41 and further through the connecting fitting 42 and through an external tubing, not shown on the drawings, to a waste container, and (o) finally the suction pipette was returned to its initial position, and the motor 16 was turned off.
~09Ofl00~7 2 0 ~ ~ 3 ,~ ~ PCT/DK90/00052 Alternatively, in carrying out ehe separation method according ~o ~he invention, in (b) in example l above, a 2.2 ml gradient separation component was used, l litre of which was composed of 94 ml 85/.
glycerol; 18.8 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2C03, 0.75 g Na2 EDTA; and 0.006 ml Brij 96~, of an average density of 1.07 g/cm3 instead of the gradient separation component described above in (b) in example l.
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Claims (13)
1. A method of separating bacteria from a bacteria containing liquid sample by means of a dish-like centrifuge container having an upper opening defined by a radially inwardly extending rim portion and an inner peripheral surface, comprising the following sequential steps:
(a) rotating said centrifuge container at a high rotational speed, (b) introducing a volume of a gradient separation component of a density approximately identical to the average density of said bacteria into said centrifuge container so as to generate a separa-tion layer of said gradient separation component at said inner peripheral surface, (c) introducing a continuous flow of said liquid sample into said centrifuge container at a discharge rate so as to detain the bacteria by said separation layer, while the remaining constituents of said liquid sample are discharged through said upper opening of said centrifuge container, (d) introducing a flushing agent into said centrifuge container and discharging said flushing agent from said upper opening of said centrifuge container, (e) decelerating said centrifuge container and rotating it at a low rotational speed, (f) activating and moving a suction pipette from a first position having its tip remote from said inner peripheral surface to a second position having its tip arranged adjacent to said inner peripheral surface, so as to suck liquid substance from said centrifuge con-tainer, while introducing a volume of a transfer liquid into said centrifuge container without discharging any liquid through said upper opening of said centrifuge container, and (g) returning said suction pipette to its first position.
(a) rotating said centrifuge container at a high rotational speed, (b) introducing a volume of a gradient separation component of a density approximately identical to the average density of said bacteria into said centrifuge container so as to generate a separa-tion layer of said gradient separation component at said inner peripheral surface, (c) introducing a continuous flow of said liquid sample into said centrifuge container at a discharge rate so as to detain the bacteria by said separation layer, while the remaining constituents of said liquid sample are discharged through said upper opening of said centrifuge container, (d) introducing a flushing agent into said centrifuge container and discharging said flushing agent from said upper opening of said centrifuge container, (e) decelerating said centrifuge container and rotating it at a low rotational speed, (f) activating and moving a suction pipette from a first position having its tip remote from said inner peripheral surface to a second position having its tip arranged adjacent to said inner peripheral surface, so as to suck liquid substance from said centrifuge con-tainer, while introducing a volume of a transfer liquid into said centrifuge container without discharging any liquid through said upper opening of said centrifuge container, and (g) returning said suction pipette to its first position.
2. A method according to claim 1, further comprising the following sequential steps succeeding the steps (a)-(g):
(h) introducing a small volume of said transfer liquid into said centrifuge container without discharging any liquid through said upper opening of said centrifuge container.
(i) accelerating said centrifuge container and rotating it at its high rotational speed, and (j) repeating the steps (e)-(i).
(h) introducing a small volume of said transfer liquid into said centrifuge container without discharging any liquid through said upper opening of said centrifuge container.
(i) accelerating said centrifuge container and rotating it at its high rotational speed, and (j) repeating the steps (e)-(i).
3. A method according to claim 2, further comprising the following sequential steps succeeding the steps (a)-(j):
(k) accelerating said centrifuge container and rotating it at its high rotational speed, (l) intermittently introducing a flow of a flushing agent and heated air into said centrifuge container in a spray, so as to rinse off any material adhering to said centrifuge container, (m) decelerating said centrifuge container and rotating it at its low rotational speed, (n) activating and moving said suction pipette from its first position to its second position, so as to suck any liquid substance from said centrifuge container, and (o) returning said suction pipette to its first position.
(k) accelerating said centrifuge container and rotating it at its high rotational speed, (l) intermittently introducing a flow of a flushing agent and heated air into said centrifuge container in a spray, so as to rinse off any material adhering to said centrifuge container, (m) decelerating said centrifuge container and rotating it at its low rotational speed, (n) activating and moving said suction pipette from its first position to its second position, so as to suck any liquid substance from said centrifuge container, and (o) returning said suction pipette to its first position.
4. A method according to any of the preceding claims, the liquid being discharged from said upper opening of said centrifuge container through a minimum width defining notch of said radially inwardly extending rim portion thereof so as to cause a delay of the discharge of the liquid from said centrifuge container.
5. A method according to any of the preceding claims, further com-prising thermostating said centrifuge container to a predetermined temperature by supplying air of said temperature to said centrifuge container.
6. A method according to any of the preceding claims, a flow of pressurized air being introduced into said centrifuge container while rotating it at its high rotational speed.
7. A method according to any of the preceding claims, said gradient separation component having an average density of approximately 1.13 g/cm3 and constituting an aqueous solution, 1 1 thereof being composed of 300 ml 85% glycerol; 60.0 g Sucrose; 30.0 g NaHCO3: 15.9 g Na2CO3; 0.75 g Na2 EDTA; and 0.02 ml Brij 96?.
8. A method according to any of the preceding claims, said gradient component having a density of approx. 1.07 g/cm3 and constituting an aqueous solution, 1 litre thereof being composed of 94 ml 85%
glycerol; 18.8 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2CO3; 0.75 g Na2 EDTA; and 0.006 ml Brij 96?.
glycerol; 18.8 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2CO3; 0.75 g Na2 EDTA; and 0.006 ml Brij 96?.
9. A method according to any of the preceding claims, said high rotational speed of said centrifuge container being of the order of 45,000 rpm, and said low rotational speed of said centrifuge con-tainer being of the order of 480 rpm.
10. A method according to any of the preceding claims, the net volume of said centrifuge container defined therein when rotating said centrifuge container at its high rotational speed being of the order of 2 ml.
11. A gradient separation component to be used in carrying out the method according to any of the claims 1-10, having an average density approximately identical to the average density of the said bacteria.
12. A gradient separation component according to claim 11, the gradient separation component having an average density of approx.
1.13 g/cm3 and constituting an aqueous solution, 1 litre thereof being composed of 300 ml 85% glycerol; 60.0 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2C03; 0.75 g Na2 EDTA; and 0.02 ml Brij 96?.
1.13 g/cm3 and constituting an aqueous solution, 1 litre thereof being composed of 300 ml 85% glycerol; 60.0 g Sucrose; 30.0 g NaHCO3; 15.9 g Na2C03; 0.75 g Na2 EDTA; and 0.02 ml Brij 96?.
13. A gradient separation component according to claim 11, the gradient separation component having an average density of approx.
1.07 g/cm3 and constituting an aqueous solution, 1 litre thereof being composed of 94 ml 85% glycerol; 18.8 g Sucrose; 30.0 g NaHCO3;
15.9 g Na2CO3; 0.75 g Na2 EDTA; and 0.006 ml Brij 96?.
1.07 g/cm3 and constituting an aqueous solution, 1 litre thereof being composed of 94 ml 85% glycerol; 18.8 g Sucrose; 30.0 g NaHCO3;
15.9 g Na2CO3; 0.75 g Na2 EDTA; and 0.006 ml Brij 96?.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK081389A DK81389D0 (en) | 1989-02-22 | 1989-02-22 | PROCEDURE AND APPARATUS FOR SEPARATING BACTERIES |
| DK0813/89 | 1989-02-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2050325A1 true CA2050325A1 (en) | 1990-08-23 |
Family
ID=8097861
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002050325A Abandoned CA2050325A1 (en) | 1989-02-22 | 1990-02-22 | Method of separating bacteria from a bacteria containing liquid sample and a gradient separation component |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0460033A1 (en) |
| JP (1) | JPH04503603A (en) |
| AU (1) | AU5198190A (en) |
| CA (1) | CA2050325A1 (en) |
| DK (1) | DK81389D0 (en) |
| WO (1) | WO1990010057A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5846439A (en) * | 1996-02-28 | 1998-12-08 | Marshfield Medical Research & Education Foundation, A Division Of Marshfield Clinic | Method of concentrating waterborne protozoan parasites |
| US6500107B2 (en) | 2001-06-05 | 2002-12-31 | Baxter International, Inc. | Method for the concentration of fluid-borne pathogens |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB983760A (en) * | 1962-02-20 | 1965-02-17 | Ici Ltd | Apparatus and method for separating particles in liquids |
| FR1597877A (en) * | 1968-12-04 | 1970-06-29 | ||
| US3928139A (en) * | 1973-02-12 | 1975-12-23 | Wadley Res Inst & Blood Bank | Detection of microbial pathogens |
| DK366778A (en) * | 1978-08-18 | 1980-02-19 | Foss Electric As | METHOD OF COUNTING BACTERIES |
| DK268283D0 (en) * | 1983-06-10 | 1983-06-10 | Metrofoss Ks | METHOD AND APPARATUS FOR BACTERIAL SEPARATION |
-
1989
- 1989-02-22 DK DK081389A patent/DK81389D0/en not_active Application Discontinuation
-
1990
- 1990-02-22 AU AU51981/90A patent/AU5198190A/en not_active Abandoned
- 1990-02-22 CA CA002050325A patent/CA2050325A1/en not_active Abandoned
- 1990-02-22 JP JP2504130A patent/JPH04503603A/en active Pending
- 1990-02-22 WO PCT/DK1990/000052 patent/WO1990010057A1/en not_active Application Discontinuation
- 1990-02-22 EP EP90903794A patent/EP0460033A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| WO1990010057A1 (en) | 1990-09-07 |
| JPH04503603A (en) | 1992-07-02 |
| DK81389D0 (en) | 1989-02-22 |
| AU5198190A (en) | 1990-09-26 |
| EP0460033A1 (en) | 1991-12-11 |
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