WO1998030307A1 - Appareil pour separer des formes de parasite cystique de l'eau - Google Patents

Appareil pour separer des formes de parasite cystique de l'eau Download PDF

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Publication number
WO1998030307A1
WO1998030307A1 PCT/US1998/000212 US9800212W WO9830307A1 WO 1998030307 A1 WO1998030307 A1 WO 1998030307A1 US 9800212 W US9800212 W US 9800212W WO 9830307 A1 WO9830307 A1 WO 9830307A1
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WO
WIPO (PCT)
Prior art keywords
water
centrifuge
filtration
particulate material
sand
Prior art date
Application number
PCT/US1998/000212
Other languages
English (en)
Inventor
Clive J. Shiff
Thaddeus K. Graczyk
Original Assignee
The Johns Hopkins University
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 The Johns Hopkins University filed Critical The Johns Hopkins University
Priority to AU59083/98A priority Critical patent/AU5908398A/en
Publication of WO1998030307A1 publication Critical patent/WO1998030307A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D36/00—Filter circuits or combinations of filters with other separating devices
    • B01D36/04—Combinations of filters with settling tanks
    • B01D36/045—Combination of filters with centrifugal separation devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D24/00—Filters comprising loose filtering material, i.e. filtering material without any binder between the individual particles or fibres thereof
    • B01D24/28—Filters comprising loose filtering material, i.e. filtering material without any binder between the individual particles or fibres thereof with the filter bed moving during the filtration
    • B01D24/32—Rotation
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/38—Treatment of water, waste water, or sewage by centrifugal separation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms

Definitions

  • the invention is related to a continuous centrifugation apparatus and method.
  • a continuous flow centrifuge adapted with an insert of glass beads, sand or the like is used to concentrate cysts of microorganisms such as giardia or cryptosporidium from large volumes of water.
  • the apparatus and methods of the invention facilitate the detection of such contaminating organisms in groundwater and public drinking water.
  • Oocysts of C. parvum are common contaminants of natural waterways and have been found in surveys (Ongerth and Stibbs, Appl . Environ . Microbiol . 53:672-676, 1987); Hansen and Ongerth, Appl . Environ . Microbiol . 57:2790-2795, 1991).
  • Outbreaks of cryptosporidiosis appear to be associated with technical aberrations at water treatment facilities or changes in river flow following drought conditions or high level of snow melt. Such conditions may combine to increase the load of C. parvum oocysts in the natural waterways, as well as allow the organisms to break through the filtration purification barrier and get access to human populations. The extent to which the organism occurs naturally has been explained recently in a review by Lisle and Rose (Agua 44:103-117, 1995) and stresses the gravity of the public health problem.
  • the device requires a pump assisted, pressurized apparatus to extract material from the water sample.
  • the apparatus is recommended for use in clarified water (turbidity ⁇ 1 NTU) .
  • Filtration of 380 L of water constitutes the sample.
  • the fiber filter unit is removed, the apparatus and filter rinsed with elution fluid, and the filter then slit into shreds with a razor knife, washed, kneaded and finally sonicated in 1 L volumes of elution fluid.
  • the cysts which have been recovered from the water are then removed by elution and concentrated by centrifugation and purified on a Percoll-sucrose gradient.
  • the procedure is cumbersome and leaves several procedures vulnerable to loss of material and hence a source of variability in the results.
  • the features of the invention overcome problems associated with other methods which have been used or contemplated for this purpose, for example the problem of clogging which is associated with filtration methods.
  • the invention will be suitable for separating and detecting contaminating microorganisms such as parasite cysts or waterborne bacteria such as fecal coliforms.
  • the apparatus of the invention is centrifuge which is designed to accept a continuous stream of water containing particulate matter through a central hollow cone while the centrifuge is spinning.
  • the water is transferred from the cone to the base of two or more (required for balance) centrifuge tubes via flexible rubber tubing connected to outlets on the lateral margin of the cone.
  • Each tube contains a filtration matrix (glass sand) , which functions as a filtration column. This filtration column is held in place by centrifugal force which also drives the water to the base of the tube, then back up through the filtration column.
  • the water overflows from the tubes and is discarded through a venting port in the centrifuge shroud.
  • the apparatus can process approximately 2 L water per min. which will conveniently enable the examination of samples of 100 L which is normally required for assessing loads of parasite cysts 2-4 micrometers in diameter in natural water of the type processed for drinking. It is expected that an apparatus which can process at least about 1.5 L/min will be useful for the methods of the invention.
  • the apparatus can separate microorganisms of the same size order as Cryptosporidium oocysts (3-7 ⁇ m) from the water.
  • microorganisms and any cystic material are then removed from the filtration matrix and measured or counted.
  • Bacteria may be plated out on isolation media. Cysts may be removed by the use of elution fluid after the method of Aldom and Chagla ⁇ Applied Microbiology 20:186-187, 1995), which has an efficiency of 78%.
  • Centrifuge tube has the standard meaning known to persons of ordinary skill in the art, and includes any container or vessel of a shape and size to be operable in the invention and to hold a filtration column of sand or another matrix suitable for separating cysts according to the method of the invention. Such tubes will generally be cylindrical in shape and enclosed on the bottom.
  • the tubes which are contemplated include, but are not limited to disposable tubes of glass, plastic or other suitable material, which have been specifically designed to fit an apparatus of the invention and are prefilled with matrix material, or reusable tubes which may be prefilled or filled with matrix material "on-site” by the operator.
  • the tubes may attach directly to the rotor, or may be inserted into suitable holders or "buckets" for operation.
  • the invention comprises a continuous flow centrifuge apparatus which is adapted to include a filtration column of particulate material.
  • the invention is an improvement in a continuous flow centrifuge apparatus comprising the addition of a filtration column of particulate material .
  • the particulate material is graded glass beads or sand of a size range which is suitable for trapping the microorganism (s) of particular interest.
  • particulate material with an size range of 120-50 ⁇ m in a column which is at least 7 cm in length will be most useful for these purposes.
  • the invention is considered to be particularly useful for isolating and detecting the cysts of cryptosporidium or giardia.
  • the particulate material is graded glass beads of 120-50 ⁇ m or fine sand of 200-50 ⁇ m in a column of at least about 7 cm in height.
  • the invention also includes a method for concentrating or isolating a microorganism from an aqueous suspension, said method comprising centrifuging said solution using the apparatus of the invention.
  • the method of the invention is an improvement in a method for concentrating, isolating or detecting a microorganism using a continuous flow centrifuge, the improvement comprising using a filtration column of particulate material in the fluid stream of the centrifuge.
  • FIG. 1 Continuous flow centrifuge apparatus.
  • the water sample is introduced into the top of the cone and passes through outlets at the bottom edge of the cone, positioned adjacent to centrifuge tubes through flexible tubing which must fit securely over the outlet ports on the lateral margin of the cone.
  • the flexible tubing extends to the bottom of the centrifuge tube so that as the centrifuge operates the liquid sample passes into the bottom of the centrifuge tube and rises through the filtration matrix to the top.
  • the filtered sample then passes out of the top of the centrifuge tube as overflow, from whence it flows to the bottom of the containment vessel and is removed through a waste outlet.
  • the filtration matrices can be removed and tested for cysts, etc. as described hereinafter.
  • FIG. 1 An exemplary apparatus is shown m Figure 1.
  • This illustrative example is a standard bench top swinging bucket centrifuge (Clinical Model IEC 428) with 6-place horizontal rotor modified to accept a continuous flow of water through an internally machined brass hollow cone. This example operates efficiently at speeds of up to 5,000 rpm. Outlet ports from the cone match the positions of the 6 centrifuge tubes and are connected to the tubes via flexible tubing reaching to the base of each tube.
  • Each centrifuge tube is designed to be filled with glass sand (Microlite) or some similar matrix which will be effectively a filtration column of graded glass beads .
  • Suitable filtration matrices include Superbrite Glass Beads made by 3M Reflective Products Division with particles sized 120 ⁇ m (90%) to 50 ⁇ m (10%) diameter (this material is referred to as glass beads or glass sand) ; filtration sand graded from 200 ⁇ m to 50 ⁇ m diameter in equal amounts (this is referred to as fine sand) .
  • the matrix acts as a particle filter which is coarse enough to allow a flow of water containing clay or suspended colloidal material while still retaining small particles such as C. parvum oocysts.
  • Other suitable materials for the filtration matrix will also be able to be determined by persons of ordinary skill in the art using routine experimentation.
  • the instrument maintains the integrity of the columns while the head is spinning. This can be achieved with the head spinning at 3-4,000 rpm.
  • the apparatus and method thus employ centrifugal force, not only to concentrate particles, but also to maintain the filtration columns (six in this case) composed of glass sand or a similar matrix in a packed form while spinning and which when suspended in liquid phase and spinning at approx 3-4,000 rpm establishes a filter column.
  • Water containing the particulate matter is introduced into the spinning centrifuge through a central hollow cone, and is lead through flexible tubes to the base of each column (tube) , rises through the column while spinning and overflows to waste.
  • water is forced by gravity to the base of each spinning tube, rises through the column and flows to waste through the overflow duct .
  • the sample of water (100 L) is placed in an overhead tank and connected to the centrifuge apparatus by tube via a stopcock.
  • the centrifuge is loaded by adding 15 g of microlite glass sand to each tube ensuring that the central flexible pipe is not obstructed or kinked and that it reaches the base of the tube.
  • the centrifuge is activated and when it reaches constant speed (approx. 4,000 rpm) the stopcock is opened to allow a flow of 1.5-2.0 L/min. directed into the central cone. Overflow is voided to drain. Centrifugation continues until the water is finished and the centrifuge turned off.
  • elution fluid phosphate buffered saline with 0.1 % Tween 80; SDS and 0.001% Sigma anti-foam; pH 7.4 .
  • elution fluid phosphate buffered saline with 0.1 % Tween 80; SDS and 0.001% Sigma anti-foam; pH 7.4 .
  • Each beaker will contain 100 ml of eluent and slurry.
  • the 6 beakers will be gently shaken for 60 min. at 4C, then the glass sand allowed to sediment and the supernatant decanted with washing into a large beaker. All supernatants and washings from the 6 beakers will be combined and this fluid will be passed through a millipore filter (1.2 ⁇ m pore size) under vacuum.
  • the filter membrane will be dissolved in 200 ml acetone, centrifuged (600 x g) 2 min., resuspended and centrifuged sequentially in acetone, 95% ethanol and 70% ethanol and eluting fluid (10 ml) . Aliquot parts of this concentrate which contains all particulate matter from the 100 1 sample are then examined using the Merifluor Cryptosporidium/Giardia test kit.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Mechanical Engineering (AREA)
  • Hydrology & Water Resources (AREA)
  • Environmental & Geological Engineering (AREA)
  • Water Supply & Treatment (AREA)
  • Filtering Materials (AREA)

Abstract

L'invention concerne une centrifugeuse à écoulement continu dans laquelle est ménagée un insert de perles de verre, de sable, etc. La centrifugeuse est utilisée pour concentrer des kystes de micro-organismes tels que giardia ou cryptosporidium à partir de grands volumes d'eau. Ledit système et lesdits procédés conviennent pour la détection de micro-organismes contaminants dans les eaux souterraines ou l'eau potable du service public.
PCT/US1998/000212 1997-01-14 1998-01-14 Appareil pour separer des formes de parasite cystique de l'eau WO1998030307A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU59083/98A AU5908398A (en) 1997-01-14 1998-01-14 Apparatus for the separation of cystic parasite forms from water

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3509997P 1997-01-14 1997-01-14
US60/035,099 1997-01-14

Publications (1)

Publication Number Publication Date
WO1998030307A1 true WO1998030307A1 (fr) 1998-07-16

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/000212 WO1998030307A1 (fr) 1997-01-14 1998-01-14 Appareil pour separer des formes de parasite cystique de l'eau

Country Status (2)

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AU (1) AU5908398A (fr)
WO (1) WO1998030307A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10355000B3 (de) * 2003-11-25 2005-09-01 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Filterzentrifuge mit Sensor zur Massenbestimmung von Filtrationsrückständen

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA871263A (en) * 1969-09-05 1971-05-18 H. Wilson Onslow Apparatus for the concentration of macromolecules and subcellular particles from dilute solutions
US4081356A (en) * 1976-09-24 1978-03-28 The United States Of America As Represented By The Department Of Health, Education And Welfare Fecalator, an apparatus and method for concentration of parasite eggs and larvae
US4675110A (en) * 1986-01-31 1987-06-23 Biomedical Polymers, Inc. Filter device and apparatus with multiple gas return passages
US5288415A (en) * 1992-01-28 1994-02-22 Chen Wu Joan L P Apparatus and method for separation of biological agents
RU2011367C1 (ru) * 1991-07-15 1994-04-30 Донской сельскохозяйственный институт Способ диагностики гельминтозов животных
US5556544A (en) * 1995-09-08 1996-09-17 Didier; Emmanuel R. Concentrator & filter

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA871263A (en) * 1969-09-05 1971-05-18 H. Wilson Onslow Apparatus for the concentration of macromolecules and subcellular particles from dilute solutions
US4081356A (en) * 1976-09-24 1978-03-28 The United States Of America As Represented By The Department Of Health, Education And Welfare Fecalator, an apparatus and method for concentration of parasite eggs and larvae
US4675110A (en) * 1986-01-31 1987-06-23 Biomedical Polymers, Inc. Filter device and apparatus with multiple gas return passages
RU2011367C1 (ru) * 1991-07-15 1994-04-30 Донской сельскохозяйственный институт Способ диагностики гельминтозов животных
US5288415A (en) * 1992-01-28 1994-02-22 Chen Wu Joan L P Apparatus and method for separation of biological agents
US5556544A (en) * 1995-09-08 1996-09-17 Didier; Emmanuel R. Concentrator & filter

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BATES T. S., HAMILTON S. E., CLINE J. D.: "COLLECTION OF SUSPENDED PARTICULATE MATTER FOR HYDROCARBON ANALYSES: CONTINUOUS FLOW CENTRIFUGATION VS. FILTRATION.", ESTUARINE, COASTAL AND SHELF SCIENCE, NEW YORK, NY, US, vol. 16., 1 January 1983 (1983-01-01), US, pages 107 - 112., XP002912417, ISSN: 0272-7714, DOI: 10.1016/0272-7714(83)90097-5 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10355000B3 (de) * 2003-11-25 2005-09-01 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Filterzentrifuge mit Sensor zur Massenbestimmung von Filtrationsrückständen

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Publication number Publication date
AU5908398A (en) 1998-08-03

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