Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to a method and an apparatus for mixing of
liquids, especially in laboratories for serial dilutions of microbiological
sam~les.
Mixing and dilution of liquids belong to the routine work in most
chemical and microbiological laboratories. A large number of such working
moments are especially carried out in test laboratories and often in a
similar and standardized way. For example, a microbiological sample is often
analysed in respect of several different microorganisms, several nutrients,
and aerobically as well as anaerobically. It is also typical of microbiologi-
cal analyses that test cultivation is carried out at a number of different
dilution degrees of the sample to obtain at some dilution of the unknown
sample a suitable concentration for quantitative determination of the number
of cultures arisen or to establish the presence or absence of growth. In
these cases the sample is repeatedly subjected to serial dilution with a
nutrient or another diluent for a series of samples with decreasing concen-
tration, e.g. with tenfold dilution between each test.
The more the extent of routine testing of this kind increases, the
greater the need of mixing, dilution and analysis methods will be that are
rationalJ require not much equipment and can be carried out by relatively
untrained personnel with little risk arising due to uncontrolled contact with
dangerous chemicals or organisms and without increasing risks of erroneous
analysis results.
A usual procedure of carrying out a microbiological analysis has
previously been to add to a test dish a measured amount of samples by means
of a pipette, to add to the dish a measured amount of culture medium, manually
stirring the mixture and then to incubate the sample and to examine it in
respect of growth.
This procedure involves several disadvantages. The method is
time-consuming due to the great number of steps involved relatively numerous
pieces of equipment are required for preparation of each sample, which is
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expensive both in view of procuring and cleaning, involves a need of training
and a risk of confusion. The whole procedure takes place quite openly, which
means risks of exposure to the personnel and risks of contamination of the
samples. In anaerobic cultivation additional moments are added to prevent
air admission. The handling steps take place independently of each otherJ
which results in that some steps, even the important mixing, can be forgotten
or be carried out insufficiently at a high working pace.
In the Swedish patent specification 377,811 it is suggested that
mixing is to be carried out in a syringe in such a way that liquid containing
a sample and a diluent or nutrient respectively are sucked each by themselves
into the syringe and mixed there with each other. This is a considerable
simplification of previous technique, especially as cultivation and examina-
tion can also be made in the syringe. As the diluent is normally the major
liquid amount it is most advantageous, from a mixing point of view first to
suck in the sample and then the dilution liquid. When doing this the tip of
the syringe must be moved from the vessel containing the sample to a vessel
or a hose containing the dilution liquid, and then it cannot be avoided that
the kit containing the diluent is contaminated with sample, analysis errors
being risked in a subsequent test where the same diluent is utili~ed. Of
course the problems can be avoided if the tip is cleaned between the different
stages or special intermediate pieces are used between syringe tip and the
liquids, which are disposed of after use. However, these procedures compli-
cate the mixing process to a large extent, as they must be performed after
each moment. Another disadvantage is that the normally placed spout sometimes
does not meet the highest demands on mixing effect as minor ranges of rather
stationary liquid can form during the suction moments, which results in
dilution errors. In serial dilution this method also provides an unnecessary
number of handling steps.
It has been suggested that in mixing by means of a syringe the tip
thereof is provided with a three-way valve, to which a hose with dilution
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liquid can also be connected while the third aperture is intended as outlet
of the finished mixture. A device for a very simple serial dilution of
samples is obtained because the three-way valve is provided with suitable
non-return valves in such a way that diluent flows into the syringe at suction
with the syringe while the outlet aperture is closed whereas the outlet to-
wards the diluent supply is closed at ejection of the finished mixture while
the outlet is opened. A serial dilution of the concentration ratio of 1:10
and the sample amount 9 ml can e.g. be made so that 1 ml of sample is intro-
duced into the syringe, after which the three-way valve is applied. By with-
drawing the piston 9 ml of diluent will flow into the syringe, a dilution of
1:10 of the sample being obtained. By an inward movement of the piston 9 ml
of this mixture can be sprayed out through the valve and be supplied to a test
dish or another vessel. In the syringe l ml of the diluted sample will then
remain, to which at a repeated extraction of the piston 9 ml diluent are
supplied until a mixture of the concentration 1:100 is obtained, of which 9 ml
can be sprayed into a test dish by means of an inward movement of the piston
in the same wa~ as before. An arbitrary number of dilutions can be obtained
with a concentration decreasing with the factor 1:10 only by means of recip-
rocating movements of the piston. However, this method does not solve the
problem with contamination of the dilution means unless the three-way valve
is replaced when continuing with the next sample. Nor is the mixing effect
in the syringe improved but the dilution errors have rather a tendency to
increase because ranges with stationary liquid will also arise in the three-
way valve. The 10w space in the three-way valve restricted by the main
volume of the syringe and the non-return valves on the three-way valve is
moreover not subjected to the mixing in the syringe, which adds another
dilution error to this procedure. That is to say the space in the three-way
valve closest to the syringe forms a channel common for both the liquids to
be sucked into the syringe, and hence this space will be filled with a volume
of the liquid last sucked into the syringe, which volume will add to the
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mixture when this is ejected. After ejection of the mixture,
the same volume of the three-way valve will be filled with the
mixture, which will add to the liquid next sucked into the
syringe. Both additions cause measuring faults.
According to the invention there is provided an
apparatus for laboratory mixing of two liquids comprising a
mixing chamber designed as a syringe, with a syringe body, a
front wall, a rear opening, a piston extending through the
rear opening and on the front wall two apertures, separated
from each other and each connecting the interior of the chamber
with the exterior of the chamber, characterized in that the
mixing chamber is flat with two substantially parallel walls.
Preferably at least one aperture may be provided with a station-
ary or detachable non-return valve and conveniently both aper-
tures may be provided with non-return valves, which non-return
valves may be arranged for flow in opposite directions.
According to a feature of the invention the syringe
body may be provided with means for limiting the movement of the
piston between predetermined volume values.
By the arrangement of a separate aperture for one
liquid the connection means thereof can be kept practically com-
pletely free from contamination with the other liquid or the
mixture. This means that the connection means of the mentioned
one liquid can be detached from the syringe and attached to
another syringe to which the same mentioned one liquid is to be
added without any cleaning operation or another measure being
necessary to secure the purity of the liquid and to avoid analy-
sis errors due to this. This is of course a great advantage
e.g. when the same liquid is used as diluent Eor a great number
of samples or for many different types of analysis, such as dis-
tilled water at chemical analysis, or culture medium at microb-
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iological analysis.
As is preferred, arranging two apertures separated
from each other on the syringe at least one aperture will
always be displaced from the middle of the syringe front towards
the edge of the syringe envelope. This will cause a circulation
flow in the main volume of the syringe at suction as well as
ejection, which considerably improves mixing in the syringe and
reduces the risks of stationary liquid volumes in the same.
Both apertures
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are pxeferably placed eccentrically relatiYe to the centre of
the syringe front and as close to the edge and as far from each
other as possible. This improved mixing is an advantage as no
additional mixing in this way is requlred. It will also be
impossible in this manner to forget the mixing as it is carried
out as a direct consequence of the necessary suction and
ejection operations.
By arranging separate apertures no ranges in the syringe
tip with countercurrent flow will arise, where the mixing effects
cannot be anticipated and are subjected to random variations.
Nor to any great volumes, which are not subjected to the mixing
in the main volume of the syringe and where ranges of stationary
liquid may result, arise as at three-way valves. The improvements
in these respects contribute to reduced dilution and mixing
errors.
The arrangement of two separate apertures for different
purposes provides the possibility of designing these in a
different way without respect tocompromisesin order that they
should fulfil their different functions better. To minimize the
risks of contamination the suction nozzle may e.g. be designed
with such a length or cross-section that the mixing in the main
body of the syringe does not involve the liquid contents in the
outer portion of the aperture, to which the supply means for the
dilution liquid is connected. In contrast to this the outlet
aperture can be formed so that as great a portion as possible of
its volume is comprised by the mixing in the main volume of the
dilution chamber. This possibility of selective design of the
apertures is not available in syringes with only one aperture
that has to serve several purposes.
Even if these advantages are obtained in mixing on a
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laboratory scale ~t Is prefexred that the invention is utilized
for analyses includin~ m~crobiolo~lcal culti~ation stages. At
such analyses organisms dan~erous to the health are often handled,
and the closed mixing procedure in a mixing vessel formed as a
syringe as well as the slight risk of contamination will here
bring special advantages of security. It is a specific problem
when analysing living organisms that their number is increased
under suitable conditions, and therefore the risk of a non-
desired contamination may increase by time and a contamination
unimportant per se of e.g. a nutrient solution over a night or a
week-end may turn into a serious contamination. The reduced
contamination risks of the invention will thus provide special
advantages here.
It is also preferred that the invention be utilized
in serial dilutions. As two apertures are present each with
their function, no shifting or transfer of various vessels will
be required between each dilution moment, which b~ings especially
great advantages at a great number of dilutions with the same
starting liquids.
As has been indicated, it is further preferred that the
device be given a non-circular cross-section with two substant- ~ ~-
ially parallel walls. With a syringe of a flat design the
apertures can be arranged further away from each other and from
the centre of the syringe front than what is possible at a
circular syringe with a corresponding volume, which improves the
circulating mixture described above in the syringe. Mixing will
also be improved because the syringe form in this case is better
adapted to the form of the circulation flow arisen, whereas in
a syringe of a circular cross-section zones of relatively
stationary liquid may arise more easily in the spaces existing
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here above and below the level of the ci~culation flow. The
flat syringe wtll also make it easier to o~serve the contents
of the syringe without the distortions of the picture arising
when looking through heavily curved surfaces. The flat syringe
form with two apertures is especially preferred for micro-
biological analyses due to the possibility of a direct examin-
ation of a cultivation result in the syringe. Moreover, its
good mixing effect in this connection is especially important
as the only mixing obtained in such a syringe culturing is at
the suction of the dilution liquid, whereas in other mixing
situations ejection and possible collection of the mixture in
a vessel also contribute to the mixing effect.
Again, another preferred embodiment of the device is
characterized in that at least one aperture is provided with
a stationary or detachable non-return valve. Both apertures
are preferably provided with non-return valves, which are then
mounted for flow in different directions through the two
apertures. This design will simplify the mixing procedure in
about the same way as the use of the three-way valve described
above, however without the same disadvantages. Thanks to the
non-return valves the liquids will always flow in a correct
direction without any closing measure or the like being
necessary after the initial changing of the syringe. Therefore
the embodiment is especially suitable at serial dilution, as
only reciprocating movements of the piston are required for the
number of times corresponding to the desired
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number of dilutions of the sample. If only one of the apertures is equipped
with a non-return valve, the simplification is only gained at this aperture
whereas the other aperture must be closed when no flow is desired through it.
Closing can be carried out for example by flattening a supply or spray hose,
by a special stop valve or by application of a plug over the aperture.
The following is a description by way of example of an embodiment
of the present invention with reference to the drawings which show a syringe
with a flattened cross-section, as seen from above, in figure 1 and in a front
view in figure 2.
The dilution chamber designed as a syringe consists of a syringe
body 1 of a substantially rectangular cross-section, the biggest walls of
which are parallel to each other. 2 is a piston with sealing against the
inside of the syringe body 1 and a handle in conventional manner, however with
its forms adapted to the rectangular cross-section of the syringe body 1.
The front wall of the chamber is provided with two apertures 3 in the form of
spouts, which are placed close to the sides of the syringe body 1 located ,- -
~urthest away from each other. According to the above one or two non-return
valves can be mounted on the spouts 3 of the apertures in a detachable manner,
and at two non-return valves so that they permit a flow in different direction
through the two apertures 3. In order to simplify the determination of the
sucked and ejected amounts at serial dilutions the syringe can be provided
with means limiting the motion of the piston 2. In the figure such a form of
limiting means is shown consisting of a clamp shown above the syringe in .
figure 1 that can be attached over the syringe body 1 and is provided with a
stop means in the form of a pin intended to be inserted into a hole through
the syringe body 1 so that it penetrates the cross-section of the syringe
body a little and ends between two ribs located on the piston, which ribs are
arranged in right angle to the movement direction of the piston~ In this way
the movements of the piston are limited so that the syringe can only contain
a volume in a preselected range, e~g. 1 - 10 ml. ~hus reading of the volume
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need not be carried out at serial dilution, but the piston is
alternatingly brought to the one or the other of the two end
positions.
As mentioned above a flattened-out cross-section
form of the syringe body is preferred to facilitate the examin-
ation of the contents of the chamber, preferably with two sub-
stantially parallel sides. If the chamber is to be used for
cultivation and examination, the height of the cross-section
should not exceed 20 mm and should preferably be 5 - 12 mm.
The greatest length of the section can vary within a wide range.
Usually it is within the range 20 - 70 mm, preferably 25 - 50
mm. Preferably the greatest length is more than twice the
length of the shortest dimension. If examination is not impor-
tant the measures can of course be selected in another way. The
syringe body is preferably made from a transparent material, such
as glass or a transparent plastic, e.g. polystyrene plastic, and
can in that case be prepared by means of injection moulding.
The design of the piston 2 is not critical. However,
its front portion must seal against the inside of the syringe
body 1 and be made from a material capable of withstanding the
treated liquids. The form is preferably adapted to the front
wall of the syringe and can be provided with protrusions dimen-
sioned and shaped to completely enter and fill the spouts 3 of
the apertures when the piston is completely pushed in. It can
also be provided with recesses to form an air-cushion for aero-
bic cultivation tests or be designed for modification of the flow -;
in the chamber in order to obtain the best mixing effect. That
is to say the front of the piston can be provided with a groove
or notch, so that with the piston fully advanced and before
suction of liquid into the syringe, this contains air suitable
for aerobic cultivation. Alternatively, the front of the piston
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can be provided with tracks or guiding means for causing tur-
bulence or additional circulation movements to the liquid stream
coming into the syringe during suction.
The apertures 3 are preferably placed as far from ~ :
each other as is
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practically possible, but other positions are also possible which need not be
symmetrical. According to the invention two apertures must be present, but
of course more apertures for special purposes are not excluded, e.g. when
mixing more liquids than two. As indicated above the design of the apertures
can vary and they can possibly be different. They are conveniently made so
that hoses, nozzles or the like can be easily attached to the aperture, e.g.
as spouts integral with the syringe body 1.
The non-return valves can be formed as a part of the syringe or
be detachably fastened to the apertures as a special unit or as a part of an
inlet or outlet hose.
A method of using the device according to the figure will be
described. 1 ml of a sample containing microorganisms is sucked into the
device through one aperture. This aperture is then provided with a non-return
valve only permitting outflow. The other aperture is provided with a non-
return valve only permitting inflow and with a supply hose for culture medium.
The piston is moved back so far that 9 ml culture medium enter and are mixed
with the sample. The piston is thereafter moved forwards so that 9 ml mixture
flow out through the outlet aperture and are supplied to a collecting vessel,
while 1 ml of the mixture remains in the syringe. The piston is now moved
back again and forwards once more, whereby two dilution degrees, 1/10 and
1/100, have been obtained. The procedure is repeated until a desired number
of dilutions has been obtained, the last dilution, however, not being ejected
but left in the device and subjected to incubation and examination of the
cultivation result. The other dilution degrees of the sample can be incubated
in their collecting vessels or rather be supplied to a syringe according to
the invention or a syringe according ~o the Swedish patent specification
377,811 with only one spout and there be subjectcd to cultivation and examina-
tion. ~hen ejecting the mixtures according to the above they can be directly
supplied to such another syringe via a connection without first being sup-
plied to a collecting vessel.
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Of course the invention is not limited to the embodiments shown
above but can be varied in several manners within the scope of the following
claims.
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