WO1998051412A1 - Density gradient sedimentation and swim-up test tube - Google Patents

Density gradient sedimentation and swim-up test tube Download PDF

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Publication number
WO1998051412A1
WO1998051412A1 PCT/IT1997/000114 IT9700114W WO9851412A1 WO 1998051412 A1 WO1998051412 A1 WO 1998051412A1 IT 9700114 W IT9700114 W IT 9700114W WO 9851412 A1 WO9851412 A1 WO 9851412A1
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WO
WIPO (PCT)
Prior art keywords
test tube
sedimentation
swim
density gradient
small probe
Prior art date
Application number
PCT/IT1997/000114
Other languages
French (fr)
Inventor
Roberto Guglielmo
Original Assignee
Roberto Guglielmo
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Roberto Guglielmo filed Critical Roberto Guglielmo
Priority to AU30465/97A priority Critical patent/AU3046597A/en
Priority to PCT/IT1997/000114 priority patent/WO1998051412A1/en
Publication of WO1998051412A1 publication Critical patent/WO1998051412A1/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls

Definitions

  • the present invention relates to an improved test tube for the sedimentation by density gradient and for the selection of moving spermatozoa by swim-up techniques inhibiting the contamination of the already formed liquid layers and the infection due to the introduction of a probe.
  • the selection of the moving spermatozoa by the swim-up technique may be resorted to, wherein the spermatozoa move upwards and are collected in the upper culture medium.
  • the spermatozoa are picked by the operator manually by suction through a Pasteur pipette or a syringe connected to a probe of suitable length, with the danger of contaminating the already formed layers because of their mixing or due to the introduction of such pipette or probe.
  • the present invention seeks to solve such a problem by providing a test tube which is equipped with means inhibiting undesired mixing and contamination of the layers during the suction or deposition of the sample by the operator.
  • Fig. 1 is an exploded view of the test tube with relative screw cap
  • Fig. 2 is a section view according to the plane I-I of the test tube of Fig. 1.
  • an usual test tube consists of a plastic or glass tube 4 provided with a conical bottom 6 and at its upper mouth 8 with a screw thread 10 allowing a screw cap 12 to be screwed thereon .
  • such test tube is provided along its inner wall with a fixed small probe 14 extending from near the mouth 8 up to near the rounded bottom 6 where it is provided with a suitable goose bill 16, as can be seen from Fig. 2.
  • the diameter of such small probe is much lower than that of the test tube but large enough to receive a further flexible probe.
  • the diameter of the small probe is 1-2 mm while the test tube has an inner diameter of 15 mm.
  • such small probe 14 is provided at the upper side with a Luer cone for a syringe 18 so as to help the operator.
  • the small probe is made in one moulding piece with the test tube.
  • a flexible small probe having the above-mentioned features may, however, be secured to the test tube by adhesives or other known means.
  • the inner surface of the test tube is provided with a spiral protrusion 20 essentially extending for its whole length.
  • Such spiral protrusion provides a support for the Pasteur pipette or the syringe at the desired height and avoids any oscillation and causes a flow which is tangential to the formed layers and not perpendicular thereto with the risk of a not controlled contamination (vertical withdrawal) .
  • a test tube having the features mentioned above fully satisfies the requirements of the operator both in the sedimentation technique by density gradient and the swim-up technique.
  • the solution (Percoll, glucose or sucrose, etc.) having lower density is deposited by a syringe through the fixed small probe, and in succession the other solutions having higher density are deposited still through the same small probe.
  • the sample to be deposited as a layer above the formed gradients is then taken by a Pasteur pipette or a syringe.
  • the sample is then carefully deposited at the intersection between the outer wall of small probe 14 and spiral 20 within the test tube near the surface of the uppermost deposited layer.
  • the selected medium is deposited by small probe 14 and then the seminal liquid as such or a treated one is deposited by the same. After 1-2 hours the supernatant is sucked by laying the pipette on the spiral 20 at the desired height.
  • the fixed small probe extending to the bottom of the test tube allows the liquid layers having a biological density to be formed much more quickly without mixing the layers being formed with one another, thus avoiding the risk of inadvertent oscillations or sudden accelerations caused during the deposition.
  • the system prevents the already deposited liquid layers from being crossed to form underlying layers having higher density.
  • the particular spiral protrusion within the test tube allows the Pasteur pipette or the syringe to be firmly laid at the desired height without creating strong liquid flows.
  • the liquid to be either sucked or deposited will flow of course along the inclined spiral so as to avoid undesired mixing and contamination .

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Devices For Use In Laboratory Experiments (AREA)

Abstract

A test tube (4) for sedimentation by density gradient and swim-up techniques provided in combination with: a fixed small probe (14) extending along the inner wall of the test tube (4) up to near the bottom (6) of the same in order to allow the sedimentation medium to be either deposited or sucked without causing any contamination or mixing of the deposited layers by a Pasteur pipette and/or a syringe or the like connected or not to another small probe of lower diameter; a spiral protrusion (20) on the inner wall of the test tube (4) which allows said Pasteur pipette or a syringe to be firmly supported at the desired height without causing a strong liquid flow.

Description

Density gradient sedimentation and swim-up test tube
The present invention relates to an improved test tube for the sedimentation by density gradient and for the selection of moving spermatozoa by swim-up techniques inhibiting the contamination of the already formed liquid layers and the infection due to the introduction of a probe.
In the biology of the reproduction and, more specifically, in the practice of the artificial insemination, techniques of seminal liquid sedimentation by density gradient are commonly used in order to gather the spermatozoa below liquid phases of different densities.
Alternately, the selection of the moving spermatozoa by the swim-up technique may be resorted to, wherein the spermatozoa move upwards and are collected in the upper culture medium.
In both cases the spermatozoa are picked by the operator manually by suction through a Pasteur pipette or a syringe connected to a probe of suitable length, with the danger of contaminating the already formed layers because of their mixing or due to the introduction of such pipette or probe.
The present invention seeks to solve such a problem by providing a test tube which is equipped with means inhibiting undesired mixing and contamination of the layers during the suction or deposition of the sample by the operator.
This is achieved according to the invention by providing a test tube equipped with:
a) a fixed small probe extending along the inner wall of the test tube up to the bottom of the same in order to allow the desired layers to be either deposited or sucked by a Pasteur pipette or a syringe or the like connected or not to another small probe of lower diameter;
b) a spiral protrusion on the inner wall of the test tube which allows said Pasteur pipette or a syringe to be firmly supported at the desired height without causing a strong liquid flow. Actually, the liquid to be sucked or deposited will flow downwards along the inclined spiral so as to avoid undesired mixing and contamination.
A better understanding of the invention will ensue from the following detailed description with reference to the accompanying drawings which show by way of a not limiting example a preferred embodiment of the invention. In the drawings:
Fig. 1 is an exploded view of the test tube with relative screw cap;
Fig. 2 is a section view according to the plane I-I of the test tube of Fig. 1.
With reference to the figures, an usual test tube consists of a plastic or glass tube 4 provided with a conical bottom 6 and at its upper mouth 8 with a screw thread 10 allowing a screw cap 12 to be screwed thereon .
According to a first feature of the invention such test tube is provided along its inner wall with a fixed small probe 14 extending from near the mouth 8 up to near the rounded bottom 6 where it is provided with a suitable goose bill 16, as can be seen from Fig. 2. The diameter of such small probe is much lower than that of the test tube but large enough to receive a further flexible probe. By way of example, the diameter of the small probe is 1-2 mm while the test tube has an inner diameter of 15 mm.
Advantageously, such small probe 14 is provided at the upper side with a Luer cone for a syringe 18 so as to help the operator. Preferably, the small probe is made in one moulding piece with the test tube. Alternately, a flexible small probe having the above-mentioned features may, however, be secured to the test tube by adhesives or other known means. According to another feature of the invention, the inner surface of the test tube is provided with a spiral protrusion 20 essentially extending for its whole length. Such spiral protrusion provides a support for the Pasteur pipette or the syringe at the desired height and avoids any oscillation and causes a flow which is tangential to the formed layers and not perpendicular thereto with the risk of a not controlled contamination (vertical withdrawal) . A test tube having the features mentioned above fully satisfies the requirements of the operator both in the sedimentation technique by density gradient and the swim-up technique.
The way for use is very easy. By the first technique, the solution (Percoll, glucose or sucrose, etc.) having lower density is deposited by a syringe through the fixed small probe, and in succession the other solutions having higher density are deposited still through the same small probe. The sample to be deposited as a layer above the formed gradients is then taken by a Pasteur pipette or a syringe. The sample is then carefully deposited at the intersection between the outer wall of small probe 14 and spiral 20 within the test tube near the surface of the uppermost deposited layer.
After centrifugation, a suitable amount of pellet is sucked by small probe 14 or, if Percoll is used, the whole supernatant is sucked and the desired amount of pellet is preserved. In the other cases, the desired layers are sucked 'by laying the Pasteur pipette or a syringe on spiral 20.
In the selection of the moving spermatozoa by the swim-up technique, the selected medium is deposited by small probe 14 and then the seminal liquid as such or a treated one is deposited by the same. After 1-2 hours the supernatant is sucked by laying the pipette on the spiral 20 at the desired height.
From the foregoing the advantages of using the test tube according to the invention are self-evident.
The fixed small probe extending to the bottom of the test tube allows the liquid layers having a biological density to be formed much more quickly without mixing the layers being formed with one another, thus avoiding the risk of inadvertent oscillations or sudden accelerations caused during the deposition. In addition, the system prevents the already deposited liquid layers from being crossed to form underlying layers having higher density. During the suction of any supernatant and the careful deposition of upper layers of medium or biological liquids the particular spiral protrusion within the test tube allows the Pasteur pipette or the syringe to be firmly laid at the desired height without creating strong liquid flows. The liquid to be either sucked or deposited will flow of course along the inclined spiral so as to avoid undesired mixing and contamination . The present invention is described and illustrated according to a preferred embodiment thereof, however, it should be understood that anyone skilled in the art can make modifications and/or equivalent replacements without departing from the scope of the present industrial invention.

Claims

Cl a ims
1. A test tube for sedimentation by density gradient and swim-up techniques, wherein it is provided with a fixed small probe extending along the inner wall of the test tube up to near the bottom of the same in order to allow the desired sedimentation medium to be either deposited or sucked without causing contamination or mixing of the deposited layers by a Pasteur pipette and/or a syringe or the like connected or not to another small probe of lower diameter.
2. A test tube for sedimentation by density gradient and swim-up techniques, wherein it is provided with a spiral protrusion on its inner wall which allows said Pasteur pipette and/or a syringe or the like connected or not to another small probe to be firmly supported at the desired height without causing a strong liquid flow.
3. A test tube for sedimentation by density gradient and swim-up techniques, wherein it is provided in combination with: a fixed small probe extending along the inner wall of the test tube up 'to near the bottom of the same in order to allow the sedimentation medium to be either deposited or sucked without causing any contamination or mixing of the deposited layers by a Pasteur pipette and/or a syringe or the like connected or not to another small probe of lower diameter; a spiral protrusion on the inner wall of the test tube which allows said Pasteur pipette or a syringe to be firmly supported at the desired height without causing a strong liquid flow.
4. The test tube for sedimentation by density gradient and swim-up techniques of claims 1 and 3, characterized in that said fixed small probe is provided at the upper side with a Luer cone for a syringe .
5. The test tube for sedimentation by density gradient and swim-up techniques of claims 1, 3 and 4, characterized in that the fixed small probe is made in one moulding piece with the test tube.
6. The test tube for sedimentation by density gradient and swim-up techniques of claims 1, 3 and 4, characterized in that said small probe is inserted and fixed by adhesives or the like.
7. The test tube for sedimentation by density gradient and swim-up techniques of claims 2 and 3 , characterized in that said spiral is made in one moulded piece with' the test tube.
PCT/IT1997/000114 1997-05-16 1997-05-16 Density gradient sedimentation and swim-up test tube WO1998051412A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU30465/97A AU3046597A (en) 1997-05-16 1997-05-16 Density gradient sedimentation and swim-up test tube
PCT/IT1997/000114 WO1998051412A1 (en) 1997-05-16 1997-05-16 Density gradient sedimentation and swim-up test tube

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IT1997/000114 WO1998051412A1 (en) 1997-05-16 1997-05-16 Density gradient sedimentation and swim-up test tube

Publications (1)

Publication Number Publication Date
WO1998051412A1 true WO1998051412A1 (en) 1998-11-19

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2827196A1 (en) * 2001-07-13 2003-01-17 Bio Merieux Vessel for biological samples, to be transferred by suction to another vessel, has an inner container with an angled base to concentrate the sample in a small zone for total extraction without residue
WO2007079936A1 (en) * 2005-12-22 2007-07-19 Sartorius Stedim Biotech Gmbh Disposable bioreactor for culturing cells in a nutrient medium
WO2007139652A2 (en) * 2006-05-26 2007-12-06 Prospect Biosystems, Llc A method for the fractionation and separation of particles by step-wise gradient density extraction
JP2009119457A (en) * 2007-10-24 2009-06-04 Jms Co Ltd Separation chamber, attachment, and separation method
US8278118B2 (en) 2006-05-26 2012-10-02 Prospect Biosystems, Inc. Method for the fractionation and separation of particles by step-wise gradient density extraction
CN103411095A (en) * 2013-07-18 2013-11-27 吴江市黎里建兴铸件厂 Anti-scaling water pipe
WO2015154115A3 (en) * 2014-04-09 2016-02-04 Greiner Bio-One Gmbh Receiver container and receiver unit for receiving body fluid
CN106000204A (en) * 2016-07-31 2016-10-12 浙江大学 Uniform mixing tube for vortex oscillator
CN106076174A (en) * 2016-07-31 2016-11-09 浙江大学 Coordinate the shearing mixing pipe that vortex oscillator uses
ITUB20156317A1 (en) * 2015-12-04 2017-06-04 Univ Degli Studi Di Catania DEVICE, KIT AND METHOD FOR SEPARATION IN GRADIENT OF DENSITY
WO2019138433A1 (en) * 2018-01-12 2019-07-18 Roberto Guglielmo Method of manufacturing a test tube for use with suction or sedimentation pipettes and test tube so obtained

Citations (7)

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Publication number Priority date Publication date Assignee Title
DE1171178B (en) * 1960-02-06 1964-05-27 Martin Christ Fa Centrifuge cell
GB1064901A (en) * 1965-01-19 1967-04-12 Alfred George Wright Improvements relating to centrifuges
DE2024618A1 (en) * 1969-05-21 1970-11-26 Beckman Instruments Ltd., Glenrothes, Fife, Schottland (Großbritannien) Device for introducing or removing a fluid, in particular a liquid, into or from a container
US3977598A (en) * 1973-05-24 1976-08-31 Mcdonald Bernard Centrifuge tube
US4397954A (en) * 1980-06-02 1983-08-09 The Regents Of The University Of California Flowcell fractionator
FR2539628A1 (en) * 1983-01-25 1984-07-27 Fondation Rech Hormonologie Method for separating motile cells, especially motile spermatozoa, as well as a device for implementing this method
EP0247340A1 (en) * 1986-05-17 1987-12-02 Gesellschaft für Strahlen- und Umweltforschung mbH (GSF) Method and apparatus for isolating fractions of macromolecules in a density gradient

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1171178B (en) * 1960-02-06 1964-05-27 Martin Christ Fa Centrifuge cell
GB1064901A (en) * 1965-01-19 1967-04-12 Alfred George Wright Improvements relating to centrifuges
DE2024618A1 (en) * 1969-05-21 1970-11-26 Beckman Instruments Ltd., Glenrothes, Fife, Schottland (Großbritannien) Device for introducing or removing a fluid, in particular a liquid, into or from a container
US3977598A (en) * 1973-05-24 1976-08-31 Mcdonald Bernard Centrifuge tube
US4397954A (en) * 1980-06-02 1983-08-09 The Regents Of The University Of California Flowcell fractionator
FR2539628A1 (en) * 1983-01-25 1984-07-27 Fondation Rech Hormonologie Method for separating motile cells, especially motile spermatozoa, as well as a device for implementing this method
EP0247340A1 (en) * 1986-05-17 1987-12-02 Gesellschaft für Strahlen- und Umweltforschung mbH (GSF) Method and apparatus for isolating fractions of macromolecules in a density gradient

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2827196A1 (en) * 2001-07-13 2003-01-17 Bio Merieux Vessel for biological samples, to be transferred by suction to another vessel, has an inner container with an angled base to concentrate the sample in a small zone for total extraction without residue
WO2003011463A1 (en) * 2001-07-13 2003-02-13 Biomerieux S.A. Improved container for easier complete emptying
WO2007079936A1 (en) * 2005-12-22 2007-07-19 Sartorius Stedim Biotech Gmbh Disposable bioreactor for culturing cells in a nutrient medium
US9174181B2 (en) 2005-12-22 2015-11-03 Sartorius Stedim Biotech Gmbh Disposable bioreactor for culturing cells in a nutrient medium
WO2007139652A3 (en) * 2006-05-26 2008-12-11 Prospect Biosystems Llc A method for the fractionation and separation of particles by step-wise gradient density extraction
WO2007139652A2 (en) * 2006-05-26 2007-12-06 Prospect Biosystems, Llc A method for the fractionation and separation of particles by step-wise gradient density extraction
US8278118B2 (en) 2006-05-26 2012-10-02 Prospect Biosystems, Inc. Method for the fractionation and separation of particles by step-wise gradient density extraction
US8318507B2 (en) 2006-05-26 2012-11-27 Marcus Joseph Horn Method for the fractionation and separation of particles by step-wise gradient density extraction
EP2208540A1 (en) * 2007-10-24 2010-07-21 JMS Co., Ltd. Separation container, attachment and separation method
EP2208540A4 (en) * 2007-10-24 2011-09-21 Jms Co Ltd Separation container, attachment and separation method
KR101201005B1 (en) 2007-10-24 2012-11-13 가부시끼가이샤 제이엠에스 Separation container, attachment and separation method
JP2009119457A (en) * 2007-10-24 2009-06-04 Jms Co Ltd Separation chamber, attachment, and separation method
CN103411095A (en) * 2013-07-18 2013-11-27 吴江市黎里建兴铸件厂 Anti-scaling water pipe
WO2015154115A3 (en) * 2014-04-09 2016-02-04 Greiner Bio-One Gmbh Receiver container and receiver unit for receiving body fluid
ITUB20156317A1 (en) * 2015-12-04 2017-06-04 Univ Degli Studi Di Catania DEVICE, KIT AND METHOD FOR SEPARATION IN GRADIENT OF DENSITY
CN106000204A (en) * 2016-07-31 2016-10-12 浙江大学 Uniform mixing tube for vortex oscillator
CN106076174A (en) * 2016-07-31 2016-11-09 浙江大学 Coordinate the shearing mixing pipe that vortex oscillator uses
CN106076174B (en) * 2016-07-31 2019-02-01 浙江大学 The shearing that cooperation vortex oscillator uses mixes pipe
WO2019138433A1 (en) * 2018-01-12 2019-07-18 Roberto Guglielmo Method of manufacturing a test tube for use with suction or sedimentation pipettes and test tube so obtained

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