Note: Descriptions are shown in the official language in which they were submitted.
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.
..
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
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W O 90/10057 PCT/DK90/00052
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
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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
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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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W O 90110057 PCT/DK90/00052
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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W O 90/10057 PCT/DK90/00052
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.
.
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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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
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W O 901tO057 PCT/DK90/00052
11
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
12
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.
EXAMPLE 1
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
13
(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
EXAMPLE 2
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.
: :
: . . : : . , : , :
,
- , . . .
.
- : ' '' ' .':
