US3928139A - Detection of microbial pathogens - Google Patents

Detection of microbial pathogens Download PDF

Info

Publication number
US3928139A
US3928139A US428135*[A US42813574A US3928139A US 3928139 A US3928139 A US 3928139A US 42813574 A US42813574 A US 42813574A US 3928139 A US3928139 A US 3928139A
Authority
US
United States
Prior art keywords
liquid filter
filter medium
microbial pathogens
sample
body fluid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US428135*[A
Other languages
English (en)
Inventor
Gordon L Dorn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
J K & Susie L Wadley Research Institute & Blood Bank
Wadley Res Inst & Blood Bank
Original Assignee
Wadley Res Inst & Blood Bank
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 Wadley Res Inst & Blood Bank filed Critical Wadley Res Inst & Blood Bank
Priority to US428135*[A priority Critical patent/US3928139A/en
Priority to CA190,358A priority patent/CA1021238A/en
Priority to AU65229/74A priority patent/AU487774B2/en
Priority to GB568574A priority patent/GB1458147A/en
Priority to IT48234/74A priority patent/IT1018621B/it
Priority to DE19742406362 priority patent/DE2406362A1/de
Priority to JP1636874A priority patent/JPS5642280B2/ja
Priority to FR7404690A priority patent/FR2217419B1/fr
Priority to BR7500086A priority patent/BR7500086A/pt
Application granted granted Critical
Publication of US3928139A publication Critical patent/US3928139A/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • 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
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/848—Escherichia
    • Y10S435/849—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/852—Klebsiella
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/873—Proteus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/874—Pseudomonas
    • Y10S435/875—Pseudomonas aeruginosa
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/882—Staphylococcus
    • Y10S435/883—Staphylococcus aureus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/885—Streptococcus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/921—Candida
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/921—Candida
    • Y10S435/922—Candida albicans

Definitions

  • ABSTRACT A method and apparatus are disclosed which provide for the rapid quantitative detection of microbial pathogens in a sample fluid such as blood.
  • the pathogens are concentrated by depositing the sample fluid on a liquid filter medium such as a concentrated aqueous solution of sucrose or a microporous aqueous solution of a crosslinked polymer having a greater density than the sample, and microporous openings throughout its solubilized network (which'range in size between about 1 micron and about 7 microns) and then the material is subjected to centrifugation to cause the pathogens in the sample to selectively pass into the liquid filter medium.
  • This step not only concentrates the pathogens but also separates them from the other components of the sample such as antimicrobial constituents of blood and medicants present in blood samples, such as antibiotics.
  • the liquid filter medium containing the concentrated pathogens is then added to nutrient media for culturing and quantitative counting.
  • this invention relates to a novel technique and apparatus for diagnosing septicemia.
  • Septicemia which is the presence of .pathogenic microorganisms in the blood, is one of the most serious types of infection encountered.
  • the mortality rate from septicemia isapproximately 25%.
  • shock accompanies septicemia the mortality rate in creases to over 60%.
  • Patients who are suffering from debilitating diseases, undergoing major surgery, receiving immunosuppresive drugs or anticancer. medications are particularly prone to septicemia.
  • pour plate method Another commonly used method is the so-called pour plate method.
  • the pour plate method approximately 0.5 to 1.0 milliliters of blood are suspended in milliliters of nutrient agar media and the contents are poured directly onto a plate. If organisms are present, they should form colonies on the plate in approximately 24 hours.
  • Still anotherv recently developed technique is the filtration method.
  • the red and white blood cells are initially separated from the serum of a blood sample by precipitation or slow speed centrifugation.
  • the remaining serum is then passed through a filter that traps particles the size of bacteria, or larger.
  • the filters are basically solid matrix filters having very small pore sizes extending therethrough.
  • the filter is then placed on a nutrient agar plate. The presence of microorganisms would be detected by-appearance of individual colonies in approximately 24 hours.
  • the liquid broth method is susceptible to overgrowth by faster growing microorganisms.
  • the blood sample may contain two or more types of microorganisms, but if one of these organisms is more prolific and durable than the others, it will have a tendency to reproduce and grow much faster in these media and substantially obscure any other microorganisms which may be present. It is of utmost importance that the phsyician know whether there is more than one type of microorganism present in the blood, not only from the point of view of prescribing the correct antibiotic for all of the microorganisms, but generally the occurrence, of two or more microorganisms simultaneouslyappearing in the bloodstream indicates to the physicianthat the patients defensive systems are rapidly breaking down.
  • the pour plate method is generally considered to be quantitative.
  • a disadvantage of the pour. plate method is that method relies upon upon an open system which is subject to external contamination, e.g., the introduction of pathogens onto the pour plate by the laboratory atmosphere and personnel.
  • the practice of either the liquid broth method or the pour plate method do not result in an efficient separation of the microbial pathogens from antimicrobial factors which may be present in the blood. More specifically, the phagocytic activity of granulocytes and monocytes and the normal antibactericidal activity of serum factors functions to retard the growth of bacteria which have been isolated for cultu,re..Furthermore,in many instances, the patient has received antibiotics prior to the time that the blood culture was drawn and residual amounts of the antibiotics can be present in the samples used in these conventional culture techniques to cause an inhibition of the growth of the bacteria.
  • the filtration method is generally superior to the liquid broth and the pour plate method in determining the presence of more than one type of microorganism in the blood, and in providing a quantitative analysis, it has several drawbacks.
  • the filtration method like the pour plate method, is susceptible to external contamination. It is easy for bacteria to enter into the analysis cycle from .the laboratory environment.
  • the initial precipitation or slow speed centrifugation step utilized with this method to effect a separation of the red and white blood cells from the serum can also prematurely separate microbial pathogens from the serum before it is passed through the filter.
  • a bacteria detection test is needed which is inexpensive, relatively easy to perform using standard laboratory equipment, but yet will: (l) provide a 3 rapid determination of the exact number of microorganisms within a blood sample, (2) identify the exact type of organism present; (3) indicate whether there is more than one type of organism present; (4) provide antibacterial and antifungal sensitivity patterns for each variety of the different organisms isolated; and (5) separate the microorganisms from blood and plasma.
  • one object of this invention is to provide a novel test method and means for rapidly detecting microbial pathogens in a sample fluid.
  • Another object of this invention is to provide a novel procedure for extracting and isolating microbial pathogens from a body fluid.
  • a further object of this invention is to provide a test, the practice of which results in rapid detection of microbial pathogens in a body fluid sample and provides the exact number and the exact type of one or more such pathogens in the body fluid sample.
  • a novel method for the rapid separation of microbial pathogens from other constituents in a sample fluid which includes initially depositing the sample fluid on a liquid filter medium of an aqueous solution which selectively receives microbial pathogens,. and thereafter subjecting the sample and aqueous solution to centrifugation to cause the microbial pathogens to selectively pass into the liquid filter medium.
  • a method of detection of microbial pathogens within a sample of body fluid such as blood which includes initially depositing the blood sample (preferably a lysed blood sample) upon a liquid filter medium in a confined sterile zone, the liquid filter medium having a greater density than the sample fluid and comprising a sterile aqueous solution which will selectively receive the microbial pathogens from the sample fluid; and thereafter subjecting the confined zone to centrifugation, to thereby force the fluid sample against the liquid filter medium and cause the microbial pathogens to selectively pass therein and thereby separate from the mass of the body fluid sample; thereafter separating the remainder of the fluid sample from the liquid filter medium and thoroughly mixing the liquid filter medium containing said pathogens to obtain a substantially uniform distribution of the pathogens therewithin; and then subjecting portions of the liquid filter medium containing the distributed pathogens to culturing conditions.
  • the blood sample preferably a lysed blood sample
  • a novel apparatus used in the detection of microbial pathogens which includes an article comprising an enclosed centrifugation receptacle sealably closed with an injectible closure means and wherein the interior of the receptacle contains the above-described liquid filter medium and the remaining space in the enclosure is maintained at a lower than atmospheric pressure.
  • the aqueous solution further comprises a thermally sensitive gelling agent.
  • the subject invention provides a bacteria detection apparatus and test method which is easy to perform and will provide a rapid determination of the exact number of microorganisms within a blood sample; will indicate whether there is more than one type of organism present; and inherently will separate the microorganisms from the corpuscles and fluid components of the blood and thereby separate the microorganism from antimicrobial properties of the blood and any antibiotics that may have been in the blood at the time that the sample was drawn.
  • the procedure can be utilized on all types of body fluids, such as blood, bone marrow, spinal and pleural fluids, urine, and the like.
  • the procedure can be utilized on any sample containing microorganisms to concentrate and separate the microorganisms from any antimicrobial factors present in the sample fluid, for example, foodstuff, such as milk, and the like.
  • FIGS. l-l0 schematically illustrate various steps in the preferred analysis procedure of the subject invention utilizing the novel article of the subject invention.
  • FIG. 1 A novel Article 20 utilized in the preferred method of the subject invention is shown in FIG. 1.
  • the Article 20 comprises a glass vial 21 having an opening 22 at its upper end sealed with an injectible stopper 23.
  • the injectible rubber self-sealing stopper 23 is shown in section to illustrate the injectible web 24a which is recessed therewithin.
  • the leading end of stopper 23 preferably carries a frusto-conical recess 24b.
  • the sterile contents of Article 20 comprises an aqueous solution (liquid filter medium) 25 and an evacuated space 26 (complete or partial vacuum).
  • Space 26 is maintained at a lower than atmospheric pressure and at a predetermined value so that space 26 can receive a known amount of liquid (by injection through stopper 23) without excessive pressure being built up within Article 20 which would cause stopper 23 to become dislocated from opening 22.
  • Evacuated containers of this type are well known in the art, and are manufactured to contain various reduced pressures to receive predetermined amounts of liquid through the injectible stopper therefor.
  • a suitable such evacuated container is disclosed in U.S. Pat. No. 2,460,641 which is herein incorporated by reference into this application.
  • the Article can comprise a conventionally produced evacuated tube such as sold under the trademark of Vacutainer by Becton Dickinson Company but further containing the aqueous solution 25.
  • the aqueous solution 25 comprises a liquid filter medium.
  • the liquid filter medium can comprise an aqueous solution of any solute which is nontoxic to the microbial organisms being suspended, and has a density sufficiently high to suspend red and white blood cells, or blood cell debris.
  • the solute is preferably nonionic.
  • the liquid filter medium has a density greater than blood, e.g., greater than about 1.06 gm/cc, and will suspend blood cells or blood cell debris, but yet will receive microbial pathogens.
  • the liquid filter medium preferably contains a minor amount of a thermally sensitive gelling agent.
  • Suitable solutes which can be used in the liquid filter medium include the sugars such as sucrose, glucose, maltose, fructose, mantiol, sorbitol, and the like.
  • the filter solution should be at least about 40 weight percent of the sugar and can contain the sugar up until the saturation limit thereof.
  • the sugars are contained within the liquid filter medium in a range of from about 40 to about 50 wt. thereof.
  • the sugars and especially sucrose is preferred because the solution can be maintained at a physiological pH, i.e., 6.0-7.0.
  • any other solute can be used in the scope of this invention so long as the resulting solution is more dense than blood and will withhold or suspend blood cells, and particularly, red blood cells and red blood cells debris, and is nontoxic 'to the microbial pathogen.
  • suitable such materials include a chemical commonly known as hypaque sodium, C l-l l N NaO (3,5-
  • diacetamido-2,4,6-triiodobenzoic acid sodium salt This material can be utilized in aqueous solution in the same concentrations as sugar as described above.
  • liquid filter medium in the scope of the subject invention includes macromolecular solutes which are capable of producing a liquid gel structure in aqueous media which has a pore size .small enough to preclude red cells or red cell debris but large enough to pass microbial pathogens.
  • a suitable such macromolecular solute is a water soluble crosslinked polymer having microporous openings throughout its solubilized network.
  • a suitable such water soluble polymer includes a copolymer of sucrose and epichlorohydrin which has a weight average molecular weight in the range of from about 300,000 to aboutv 500,000, an intrinsic viscosity of about 0.17 dl/g,'a specific rotation [011 of+ 56.5 and contains dialyzable material in an amount of less than 1 weight percent.
  • a suitable such polymer is sold under the trademark of f FICOLL" by Pharmacia Fine Chemicals Inc., 800 Centennial Avenue, Piscataway, New Jersey.
  • dextran having a weight average molecular weight in the range of from about 10,000 to about 2,000,000 and preferably about 50,000.
  • These polymers when dissolved in water in accordance with the subject invention function as a liquid filter medium for microbial pathogens and apparently ha've microporous openings throughout their solubilized network in the range of between about 1, micron and about 7,microns.
  • the water soluble polymer or macromolecular solute is. preferably present in the aqueous solution in the range of from about 10 to about 40 and more'preferably from about 20 to about 30 weight percent thereof.
  • thermally sensitive gelling agent any agent which will gel the aqueous solution at a temperature generally lower than room temperature butyet will liquefy at higher temperatures which are nondeleterious to the microbial pathogens, e.g., lower than about 50C and preferably no higher than about 42C.
  • Suitable thermosensitive gelling agents include any such gelling agent 60 which is nondeleterious to the solution or to the sample being analyzed. Examples of suitable such materials include the gelatins, i.e., the proteins obtained from collagen by boiling skin, ligaments, tendons, bonds and the like in water.
  • a procedure which is carried out in accordance with one embodiment of this invention for detection of bacteria within a sample of body fluid can be carried out conveniently with the following apparatus:
  • the above-described Article 20 containing the aqueous liquid filter solution the tube can be a 12-14 milliliter volume containing 1 to 2 milliliters of the aqueous liquid filter solution.
  • a material such as sodium polyanethol sulfonate (for example 1.6 ml) or Heparin which acts as an anticoagulant and preferably inhibits phagocytic activity of granulocytes and monocytes and the normal antibacterial activity of serum;
  • EMB enzyme methylene blue dye
  • the blood agar plates suggested are the conventionally utilized blood agar plates which are basically sheep's blood and a base nutritional material such as sugar, which is held together with an agar solidifying agent on a Petri plate.
  • the sabouraud agar plate is specifically designed to grow fungi.
  • the EMB plate is designed to quickly identify certain organisms in that they stain the plate in a unique manner.
  • the liquid thioglycolate medium with added sodium polyanethol sulfonate (SP8) is basically a backup medium which is capable of supporting the growth of both anaerobic and aerobic bacteria and most fungi.
  • Article 20 set forth in FIG. l in the drawing it is initially inverted as shown in FIG. 2 to allow the aqueous solution to pass downwardly against the stopper 23.
  • the article is placed in a suitable cooling unit such as a refrigerator and chilled sufficiently to cause the gelatin to solidify.
  • a suitable cooling unit such as a refrigerator and chilled sufficiently to cause the gelatin to solidify.
  • the tube can be chilled to 4C in the inverted position as illustrated in FIG. 3.
  • a predetermined amount of a blood sample drawn from the patient for example, 8.3 milliliters of the blood is injected into the evacuated test tube containing the sodium polyanethol sulfonate (SPS).
  • SPS sodium polyanethol sulfonate
  • the red cells next are preferably lysed with a suitable agent which is nontoxic to microorganisms e.g., nontoxic saponin. It must be noted that most saponins are toxic to at least some microbial pathogens.
  • a suitable agent which is nontoxic to microorganisms e.g., nontoxic saponin. It must be noted that most saponins are toxic to at least some microbial pathogens.
  • Ser. No. 423,447 filed Dec.
  • the toxic saponin material can be purified in accordance with the invention set forth in my copending patent application and the resulting purified material used in the scope of this invention.
  • saponin preparations are nontoxic, and of course, these materials can also be used in the scope of the subject invention.
  • the 1 ml disposable syringe can be used to inject 0.3 ml of nontoxic saponin (12%) into the blood-SPS mixture within the evacuated test tube. It is noted that the cells can be lysed in any other suitable manner as a 1:1 dilution of distilled water.
  • This prelysis of the blood sample will minimize the possible trapping effect of erythrocytes.
  • This trapping effect would in general comprise the erythrocytes becoming stacked on the top of the liquid filter medium during the centrifugation step and the stacked cells thereby trapping microbial pathogens as they are passed downwardly during centrifugation and thereby prevent them from reaching the liquid filter medium.
  • one of the sterile glass syringe having a 1% inch hypodermic needle is utilized to extract 8 milliliters of the blood-SPS-saponin mixture from the evacuated tube. This mixture is then injected into Article 20 in a manner schematically illustrated in FIG. 4 of the drawing.
  • the needle pierces web 24a of rubber stopper 23, passes through the congealed aqueous filter solution 25 and then the plunger is depressed to deposit the sample 27 on the congealed aqueous filter solution 25 as illustrated in FIG. 4 of the drawing.
  • the turbulence caused by the blood sample passing into the evacuated space 26 will not disturb the aqueous filter solution 25 in the congealed state, and it will remain as a solid bottom layer within Article 20.
  • the hypodermic needle is withdrawn from the rubber stopper 23 and Article 20 containing the congealed aqueous filter layer 25 and the blood sample layer 27 is heated while in the inverted position sufficiently to melt the gelatin and cause the aqueous filter layer 25 is liquefy.
  • the article is heated to a temperature which will not destroy any microbial pathogens which may be present in the blood sample but which will be sufficient to liquefy the gelatin.
  • the tube while inverted can be heated to immersion in a water bath at a temperature set at about 37C 42C as schematically illustrated in FIG. of the drawing.
  • the liquefication of the gelatin in the aqueous solution layer yields liquefied aqueous filter solution which is now ready to function as a liquid filter medium for the microbial pathogens.
  • the separation of the microbial pathogens from the remaining portion of the blood sample is accomplished by placing the Article 20 (while still in the inverted position) into a suitable centrifugation apparatus and subjecting the tube to sufficient centrifugal force and at a temperature below about 42C to separate the microbial pathogens from the remaining constituents in the blood sample.
  • the speed and time of centrifugation can vary widely depending on the construction material of Article 20 and type of centrifugation apparatus.
  • the centrifugation can be conveniently accomplished by imparting from between about 100 and about 6000 gravities and preferably from about 1400-5000 gravities to the container containing the aqueous solution and sample.
  • a more suitable method would include using a swinging bucket centrifuge rotor which imparts between 200 to 4000 gravities for -20 minutes to the 8 particular system described in this preferred embodiment.
  • the second 10 milliliter glass syringe containing sterile air is utilized to withdraw most of the liquid sample 27 from above the aqueous polymer layer 25.
  • 7.5 milliliters of the sample residue can be withdrawn as schematically illustrated in FIG. 7.
  • aqueous filter solution 25 can be conveniently vigorously agitated by touching stopper 23 of Article 20 while inverted, to a vortex mixer for k to 4 minutes. This mixing step is schematically depicted in FIG. 8 in the drawing.
  • the aqueous filter solution 25 containing the uniformly distributed microbial pathogens, if any, is removed from Article 20, using the 3 milliliter disposable syringe as shown in FIG. 9. There should be approximately 1% milliliters of fluid remaining in the tube. This fluid is then distributed on suitable bacterial growth media. This step is schematically illustrated in FIG. 10 in the drawing.
  • the material can be distributed as follows:
  • 1 blood agar plate can receive 0.2 milliliters of the aqueous solution and the plate can be incubated at 37C in an aerobic atmosphere.
  • Another blood agar plate can receive 0.2 milliliters of the aqueous solution and can be incubated at 37C in a candle jar.
  • Another blood agar plate can receive 0.2 milliliters of the aqueous solution and can be incubated at 37C in an anaerobic environment.
  • the Sabouraud agar plate can receive 0.2 milliliters of the medium of the aqueous solution and can be incubated at 25C in an aerobic environment.
  • the EMB plate can receive 0.2 milliliters of the aqueous solution and can be incubated at 37C in a candle jar.
  • the liquid thioglycolate medium can receive 0.5 milliliters of the aqueous solution and can be incubated at 37C.
  • the growth media should be checked daily for the presence of colonies.
  • the number of microbial pathogens in l milliliter of the blood can be determined by multiplying the number of colonies by a correction factor.
  • This correction factor takes into consideration the recovery rate for a given organism, the volumes of blood and liquid filter solutions employed and the amount of the final mixture plated. In the general example set forth above the correction factor is 1.56.
  • any known means can be utilized to admix the blood sample with the anticoagulant and/or lysing agent.
  • the syringe can be utilized to remove only the lower liquid filter layer 25 together with possibly a very small amount of sample from layer 27 from the interior of Article 20 and thereby leave the remaining portion of the sample layer 27 therewithin.
  • Various other modifications can be used in the procedure as desired.
  • Example 2 To illustrate the efficiency of the detection method of the subject invention, 500 blood samples from patients suspected of having bloodstream infections was analyzed by two or all three of the following methods:
  • a. Blood Bottle Method two 5 milliliter samples of the patients blood were sterilly introduced into each of two conventional BBL blood bottles. One bottle was ketp anaerobic and the other bottle was made aerobic. Both bottles were incubated at 37C. The bottles were checked daily for signs of microbial growth. One milliliter samples were taken from each of the bottles if growth was observed or on the second and sixth days after addition of the sample thereto if no growth was observed. The samples were analyzed microscopically andby streaking on conventional bacterial and fungal media (agar plates).
  • I b. Pour Plate Method 0.5 milliliters of a patients blood was aseptically introduced into each of 12 Petri dishes. Next, 20 milliliters per plate of Sabourauds agar medium or blood agar medium was introduced and the contents were vigorously mixed. After this, the plates were incubated under vention:
  • the red cells in this tube were lysed with 0.3 milliliters of 12% with nontoxic saponin aqueous solution and 8 milliliters of this mixture was introduced into a second evacuated tube (Article 20) containing 1.7 milliliters of an aqueous solution comprising 20 weight percent epichlorohydrin-sucrose copolymer, and 1.5 weight percent gelatin, which solution is the same as that described in Example 1 above.,Each Article 20 was then inverted and chilled to 4C until the gelatin solidified the aqueous polymer solution.
  • the blood sample was introduced into each Article 20 in a manner set forth schematically in FIG. 4 of the drawing and described in Example 1.
  • each Article 20 was melted by immersion in a water bath and set at 42C. Each Article 20 while inverted, was then centrifuged 45 minutes in an International U.V. centrifuge at 3000 rpm. After centrifugation 8 milliliters of supernatant above the aqueous polymer layer was removed with a 10 milliliter syringe. The remaining contents of each Article 20 was vigorously admixed with a vortex mixer for l minute and 0.2 milliliter samples thereof were uniformly spread and incubated as indicated with the following media:
  • the centrifugation method appears to be superior to both the blood bottle and pour plate method in terms of Percent Positives de- 65 tected. This includes positives involving more than one organism.
  • the centrifugation method is significantly faster than the blood bottle method.
  • the data in Tables 7 and 8 also illustrate that for a fixed volume of blood, the centrifugation method is more sensitive than the quantitative pour plate technique, i.e., it will recover a higher percentage of the organisms present in the blood sample.
  • the method and apparatus of the subject invention can be utilized to separate any microbial pathogen from a sample fluid such as a sample body fluid to thereby detect the presence of microbial pathogens within the body fluid, such as diagnosing of speticemia.
  • the subject invention can be utilized to test blood from supposed healthy blood donors in an effort to screen contaminated blood before it is utilized within a patient.
  • the subject invention can be utilized to test for the presence of microbial pathogens within foodstuffs and the 17 like or any other materialwhichv is destined for mammalian usage.
  • the dense, liquid filter medium which is used in conjunction with centrifugation in the scope of this invention provides a novel method for concentrating microbial pathogens in a sample fluid and for isolating the concentrated microbial pathogens from antimicrobialfactors which are generally present in sample fluids such as blood and other body fluids. Furthermore, the novel method of the subject invention can be used to isolate and concentrate microbial pathogens from a sample of body fluid such as blood for microscopic examination in emergency situations where, a patient has overt acute septicemia.
  • the liquid filter medium in the" scope of. this invention is generally above the density of the sample fluid from which the microbial pathogens are extracted.
  • sucrose-epichlorohydrin copolymer which can be used in the scope of this invention in concentrations ranging from to about 40 weight percent of the, aqueous solution generally has a corresponding density range of from about 1.035. to about 1.16 grams per milliliter.
  • the dense, liquid filter medium not only functions to selectively receive microbial pathogens but also to suspend and protect the pathogens once they have been received and retained therewithin.
  • the high density. polymeric mediums used in the scope of this invention have assisted in preserving the microbialpathogens and particularly those microbial pathogens which have partially damaged cell walls (spheroplasts.).
  • the novel method of the subjectinvention can be utilized to detect any microbial pathogen such as bacteria, fungi, bacterium, Lforms, and mycoplasms.
  • the aqueous sugarsolutions and preferably the aqueous sucrose solutions effectively can be utilized to recover the L-forms and the mycoplasms.
  • Such concentrated sucrose solutions are hypertonic, and prevent the L-forms and mycoplasms from lysing.
  • the concentrated aqueous solutions of sucrose containing a small amount of gelatin, e.g., from l to 2 wt gelatin are the preferred liquid filter solutions which are used in the scope of the subject invention.
  • solutions are hypertonic as described above, but yet have the correct buoyancy to hold up the red and white blood cells and cell debris during centrifugation but not to hold out the pathogens, they provide a cushioning effect for the pathogens andthereby protect the pathogens while suspended therein, and are heat stable.
  • one can autoclave the sucrosegelatin mixture while manufacturing Article without any undesirable breakdown of the gelatin and precipitation thereof.
  • solutionsv can be maintained at near neutral or physiological pl-ls.
  • a major advantage of the subject invention over conventional techniques is that it initially concentrates the microbial pathogens in a closed system which is not subject to the external contamination of pathogens which'may be present inthelaboratory or hospital environment. Furthermore, the concentrated pathogens are separated from the antimicrobial materials that are normally present in sample fluids.
  • the subject invention can be utilized to effectively detect low con centrations in microorganisms such as one microorganism per milliliter of blood, for example.
  • a sample of body fluid is obtained and subjected to analysis for microbial pathogens
  • the improvement comprising: concentrating said microbial pathogens contained within said body fluid and separating said microbial pathogens from the residual of said body fluid after said body fluid is obtained and before said analysis by' the method comprising:
  • liquid filter medium comprises an aqueous solution of a nonionic sugar.
  • liquid filter medium contains at least about 40 wt'% of said sugar therewithin.
  • liquid filter medium comprises a sterile aqueous solution of a macromolecular solute having microporous openings throughout its solubilized network, said openings being of sufficientsize to selectively pass said pathogens from said sample fluid.
  • liquid filter medium comprises an aqueous solution of a copolymer of sucrose and epichlorohydrin which has a molecular weight inthe range of from about 300,000 to 500,000 and a specific rotation [041 of 56.5".
  • microporous body comprises an aqueous solution of dextran which has a molecular weight from about 10,000 to about 2,000,000.
  • said body fluid is selected from blood, bone marrow, spinal fluid, pleu- 19 ral fluid and urine.
  • a method of detecting the presence of microbial pathogens in a sample fluid which is suspected to contain said microbial pathogens and antipathogenic factors comprising:
  • liquid filter medium comprises an aqueous solution of sugar.
  • liquid filter medium is analyzed by initially admixing said liquid filter medium to uniformly distribute said microbial pathogens therein and thereafter quantitatively plating the admixed body on growth media for microbial pathogens.
  • liquid filter body comprises an aqueous solution of'a polymer selected from the group consisting of sucrose-epichlorohydrin copolymer and dextran.
  • sucrose-epichlorohydrin polymer has a molecular weight in the range of from about 300,000 to about 500,000 and a specific rotation [01],, of 56.5.
  • liquid filter medium is an aqueous polymer solution comprising from about 10 to about 40 wt of a polymer selected from a group consisting of epichlorohydrin-sucrose copolymer, and dextran, and from about 1 to about 5 wt of said thermally sensitive gelling agent.
  • liquid filter medium is an aqueous solution comprising at least about 40 wt sugar and from about 1 to about 5 wt of said thermally sensitive gelling agent.
  • An article used for the isolation and concentration of microbial pathogens from a sample of body fluid comprising:
  • an enclosed centrifugation receptacle sealably closed with injectable closure means, the interior of said receptacle comprising an evacuated space maintained at a lower than atmospheric pressure adjacent a sterile aqueous liquid filter solution which is nontoxic to microbial pathogens and has a density sufficient to support all of the sample of body fluid injected therein through said injectable closure means but able to selectively receive substantially all said microbial pathogens upon centrifugation, and said aqueous liquid filter solution further containing a minor effective amount of a thermally sensitive gelling agent therewithin.
  • thermoly sensitive gelling agent is gelatin.
  • aqueous solution is an aqueous solution of a sugar.
  • aqueous solution is an aqueous solution of a macromolecular solute having microporous openings throughout it solubilized network, said openings being of sufficient size to selectively pass said pathogens from said sample fluid.
  • said macromolecular solute is epichlorohydrin-sucrose polymer having a molecular weight in a range of from about 300,000 to about 500,000 and a specific rotation [01],, of 565.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
US428135*[A 1973-02-12 1974-01-09 Detection of microbial pathogens Expired - Lifetime US3928139A (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
US428135*[A US3928139A (en) 1973-02-12 1974-01-09 Detection of microbial pathogens
CA190,358A CA1021238A (en) 1973-02-12 1974-01-17 Detection of microbial pathogens
AU65229/74A AU487774B2 (en) 1974-02-05 Detection of microbial pathogens
GB568574A GB1458147A (en) 1973-02-12 1974-02-07 Detection of microbial pathogens
IT48234/74A IT1018621B (it) 1973-02-12 1974-02-08 Dispositivo per la determinazione quantitativa di patogeni microbici
DE19742406362 DE2406362A1 (de) 1973-02-12 1974-02-11 Verfahren und vorrichtung zur feststellung von pathogenen mikroben
JP1636874A JPS5642280B2 (US07608600-20091027-C00054.png) 1973-02-12 1974-02-12
FR7404690A FR2217419B1 (US07608600-20091027-C00054.png) 1973-02-12 1974-02-12
BR7500086A BR7500086A (pt) 1974-01-09 1975-01-06 Processo de separacao de patogenos microbianos de um fluido de amostra processo de detectar patogenos num fluido do corpo e artigo usado para isolar e concentrar patogenos de um fluido de amostra

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US33169373A 1973-02-12 1973-02-12
US428135*[A US3928139A (en) 1973-02-12 1974-01-09 Detection of microbial pathogens

Publications (1)

Publication Number Publication Date
US3928139A true US3928139A (en) 1975-12-23

Family

ID=26987871

Family Applications (1)

Application Number Title Priority Date Filing Date
US428135*[A Expired - Lifetime US3928139A (en) 1973-02-12 1974-01-09 Detection of microbial pathogens

Country Status (7)

Country Link
US (1) US3928139A (US07608600-20091027-C00054.png)
JP (1) JPS5642280B2 (US07608600-20091027-C00054.png)
CA (1) CA1021238A (US07608600-20091027-C00054.png)
DE (1) DE2406362A1 (US07608600-20091027-C00054.png)
FR (1) FR2217419B1 (US07608600-20091027-C00054.png)
GB (1) GB1458147A (US07608600-20091027-C00054.png)
IT (1) IT1018621B (US07608600-20091027-C00054.png)

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4038150A (en) * 1976-03-24 1977-07-26 J. K. And Susie L. Wadley Research Institute And Blood Bank Sample mixing and centrifugation apparatus
US4131512A (en) * 1976-11-05 1978-12-26 J. K. And Susie L. Wadley Research Institute And Blood Bank Method for detecting microbial pathogens employing a cushioning agent
US4164449A (en) * 1977-11-03 1979-08-14 J. K. And Susie L. Wadley Research Institute And Blood Bank Surface separation technique for the detection of microbial pathogens
US4206282A (en) * 1977-08-29 1980-06-03 Pfizer Inc. Hypertonic culture media
US4212948A (en) * 1978-10-18 1980-07-15 J. K. And Susie L. Wadley Research Institute And Blood Bank Apparatus for detecting microbial pathogens employing a cushioning agent
US4218534A (en) * 1979-02-21 1980-08-19 Dairyland Food Laboratories, Inc. Phage detection
US4503149A (en) * 1983-06-06 1985-03-05 Conoco Inc. Method of microbial assay
US4581331A (en) * 1983-09-29 1986-04-08 E. I. Du Pont De Nemours And Company Method for the rapid detection of virus and viral antigens
EP0113103B1 (en) * 1982-12-27 1986-08-06 EASTMAN KODAK COMPANY (a New Jersey corporation) Inhibition of reduction activities of leukocytes
US4634417A (en) * 1982-12-06 1987-01-06 Georgetown University Process for treatment of tumors and apparatus therefor
WO1989004372A1 (en) * 1987-11-05 1989-05-18 Berg James D Rapid process for detecting pathogenic microorganisms
US4912035A (en) * 1987-06-11 1990-03-27 Eastman Kodak Company Method for minimizing interference by reductants when detecting cells in biological fluids
WO1990010057A1 (en) * 1989-02-22 1990-09-07 A/S N. Foss Electric A method of separating bacteria from a bacteria containing liquid sample and a gradient separation component
US5141855A (en) * 1986-07-28 1992-08-25 Eastman Kodak Company Signal amplifying cobalt (III) redox reagents and methods for the determination of analytes in aqueous fluids
US5171669A (en) * 1987-05-04 1992-12-15 Eastman Kodak Company Cobalt(III) reagents in combination with water soluble polymers
USRE34405E (en) * 1983-08-01 1993-10-12 Abbott Laboratories Determination of analytes in particle-containing medium
US5501960A (en) * 1993-12-03 1996-03-26 Dorn; Gordon L. Method for improving quantitative recovery of microorganisms from specimens containing blood components
US5518894A (en) * 1987-11-05 1996-05-21 Berg; James D. Rapid coliform detection system
US5972641A (en) * 1998-08-28 1999-10-26 Colifast Systems Asa Rapid coliform detection system
US6511819B2 (en) 1998-08-28 2003-01-28 Nye Colifast As Rapid coliform detection system
US20030138906A1 (en) * 2001-11-05 2003-07-24 Ingun Tryland Fluorescence test for measuring heterotrophic bacteria in water
US7107824B1 (en) * 1998-01-14 2006-09-19 Johns Hopkins University Apparatus for the separation of cystic parasite forms from water
US20100120085A1 (en) * 2008-10-31 2010-05-13 Biomerieux, Inc. Method for separation, characterization and/or identification of microorganisms using mass spectrometry
US20100120133A1 (en) * 2008-10-31 2010-05-13 Biomerieux, Inc. Separation device for use in the separation, characterization and/or identification of microorganisms
US20100124763A1 (en) * 2008-10-31 2010-05-20 Biomerieux, Inc. Method for detection, characterization and/or identification of microorganisms in a sealed container
US20100129814A1 (en) * 2008-10-31 2010-05-27 Biomerieux, Inc. Method for separation and characterization of microorganisms using identifier agents
US20100129852A1 (en) * 2008-10-20 2010-05-27 Photonic Biosystems, Inc. Integrated Bioanalyzer
US20100129858A1 (en) * 2008-10-31 2010-05-27 Biomerieux, Inc. Method for separation, characterization and/or identification of microorganisms using spectroscopy
US20100129857A1 (en) * 2008-10-31 2010-05-27 Biomerieux, Inc. Methods for the isolation and identification of microorganisms
US20100133175A1 (en) * 2008-10-20 2010-06-03 Photonic Biosystems, Inc. Filtered Assay Device and Method
US20100136609A1 (en) * 2008-10-31 2010-06-03 Biomerieux, Inc. Method for separation, characterization and/or identification of microorganisms using raman spectroscopy
US20100136608A1 (en) * 2008-10-20 2010-06-03 Photonic Biosystems, Inc. Multiple Filter Array Assay
EP2548591A1 (en) * 2010-03-19 2013-01-23 Asahi Kasei Medical Co., Ltd. Cell removal method, cell removal system, and white blood cell removal method
US8647835B2 (en) 2008-10-31 2014-02-11 BIO MéRIEUX, INC. Methods for separation, characterization and/or identification of microorganisms using spectroscopy
US10870110B2 (en) * 2015-12-11 2020-12-22 Babson Diagnostics, Inc. Specimen container and centrifugation method for separating serum or plasma from whole blood therewith
US11090646B2 (en) 2017-07-27 2021-08-17 Biomerieux, Inc. Isolation tube
US12025629B2 (en) 2023-04-14 2024-07-02 Babson Diagnostics, Inc. Automated centrifuge loader

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3875012A (en) * 1974-01-30 1975-04-01 Wadley Res Inst & Blood Bank Apparatus and method for the detection of microbial pathogens
US3932222A (en) * 1974-12-20 1976-01-13 J. K. & Susie L. Wadley Research Institute And Blood Bank For isolating pathogenic microorganisms
GB1566098A (en) * 1975-11-14 1980-04-30 Nat Res Dev Separation of solid and liquid components of mixtures
SE452336B (sv) * 1978-06-16 1987-11-23 Boehringer Mannheim Gmbh Forfarande for pavisning av sjukdomsalstrande mikroorganismer vilka adsorberats extrakorporalt pa en selektivt bindande adsorbent
DK366778A (da) * 1978-08-18 1980-02-19 Foss Electric As Fremgangsmaade til taelling af bakterier
US9181895B2 (en) 2012-03-01 2015-11-10 Johnson Controls Technology Company Start-stop retrofit systems and methods
WO2020223511A1 (en) * 2019-04-30 2020-11-05 SeLux Diagnostics, Inc. Rapid methods for determining microorganism growth in samples of human origin
US20240133774A1 (en) * 2021-03-31 2024-04-25 Hitachi High-Tech Corporation Method and apparatus for collecting bacteria

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2706702A (en) * 1949-02-23 1955-04-19 Baltimore Biolog Lab Method for culture of specimen
US3072538A (en) * 1960-12-30 1963-01-08 Grace W R & Co Isolation of bacteria

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2706702A (en) * 1949-02-23 1955-04-19 Baltimore Biolog Lab Method for culture of specimen
US3072538A (en) * 1960-12-30 1963-01-08 Grace W R & Co Isolation of bacteria

Cited By (63)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4038150A (en) * 1976-03-24 1977-07-26 J. K. And Susie L. Wadley Research Institute And Blood Bank Sample mixing and centrifugation apparatus
DK153763B (da) * 1976-11-05 1988-08-29 Wadley Res Inst & Blood Bank Fremgangsmaade til paavisning af mikrobielle patogener under anvendelse af et ikke-toxisk, med vand ikke-blandbart, hydrofobt, flydende middel og indretning til brug ved denne fremgangsmaade
US4131512A (en) * 1976-11-05 1978-12-26 J. K. And Susie L. Wadley Research Institute And Blood Bank Method for detecting microbial pathogens employing a cushioning agent
FR2393063A1 (fr) * 1976-11-05 1978-12-29 Wadley Res Inst & Blood Bank Procede de determination de microorganismes pathogenes
US4206282A (en) * 1977-08-29 1980-06-03 Pfizer Inc. Hypertonic culture media
US4164449A (en) * 1977-11-03 1979-08-14 J. K. And Susie L. Wadley Research Institute And Blood Bank Surface separation technique for the detection of microbial pathogens
US4212948A (en) * 1978-10-18 1980-07-15 J. K. And Susie L. Wadley Research Institute And Blood Bank Apparatus for detecting microbial pathogens employing a cushioning agent
US4218534A (en) * 1979-02-21 1980-08-19 Dairyland Food Laboratories, Inc. Phage detection
US4634417A (en) * 1982-12-06 1987-01-06 Georgetown University Process for treatment of tumors and apparatus therefor
EP0113103B1 (en) * 1982-12-27 1986-08-06 EASTMAN KODAK COMPANY (a New Jersey corporation) Inhibition of reduction activities of leukocytes
US4610961A (en) * 1982-12-27 1986-09-09 Eastman Kodak Company Inhibition of reduction activities of leukocytes
US4503149A (en) * 1983-06-06 1985-03-05 Conoco Inc. Method of microbial assay
USRE34405E (en) * 1983-08-01 1993-10-12 Abbott Laboratories Determination of analytes in particle-containing medium
US4581331A (en) * 1983-09-29 1986-04-08 E. I. Du Pont De Nemours And Company Method for the rapid detection of virus and viral antigens
US5141855A (en) * 1986-07-28 1992-08-25 Eastman Kodak Company Signal amplifying cobalt (III) redox reagents and methods for the determination of analytes in aqueous fluids
US5171669A (en) * 1987-05-04 1992-12-15 Eastman Kodak Company Cobalt(III) reagents in combination with water soluble polymers
US4912035A (en) * 1987-06-11 1990-03-27 Eastman Kodak Company Method for minimizing interference by reductants when detecting cells in biological fluids
WO1989004372A1 (en) * 1987-11-05 1989-05-18 Berg James D Rapid process for detecting pathogenic microorganisms
US5292644A (en) * 1987-11-05 1994-03-08 Berg James D Rapid process for detection coliform bacteria
US5518894A (en) * 1987-11-05 1996-05-21 Berg; James D. Rapid coliform detection system
WO1990010057A1 (en) * 1989-02-22 1990-09-07 A/S N. Foss Electric A method of separating bacteria from a bacteria containing liquid sample and a gradient separation component
US5501960A (en) * 1993-12-03 1996-03-26 Dorn; Gordon L. Method for improving quantitative recovery of microorganisms from specimens containing blood components
US7107824B1 (en) * 1998-01-14 2006-09-19 Johns Hopkins University Apparatus for the separation of cystic parasite forms from water
US6165742A (en) * 1998-08-28 2000-12-26 Nye Colifast, As Rapid coliform detection system
US6511819B2 (en) 1998-08-28 2003-01-28 Nye Colifast As Rapid coliform detection system
US5972641A (en) * 1998-08-28 1999-10-26 Colifast Systems Asa Rapid coliform detection system
US20030138906A1 (en) * 2001-11-05 2003-07-24 Ingun Tryland Fluorescence test for measuring heterotrophic bacteria in water
US9068976B2 (en) 2008-10-20 2015-06-30 Photonic Biosystems Inc. Integrated filtration bioanalyzer
US8968681B2 (en) 2008-10-20 2015-03-03 Photonic Biosystems Inc. Filtered assay device and method
US20100133175A1 (en) * 2008-10-20 2010-06-03 Photonic Biosystems, Inc. Filtered Assay Device and Method
US20100136608A1 (en) * 2008-10-20 2010-06-03 Photonic Biosystems, Inc. Multiple Filter Array Assay
US20100129852A1 (en) * 2008-10-20 2010-05-27 Photonic Biosystems, Inc. Integrated Bioanalyzer
US20100124763A1 (en) * 2008-10-31 2010-05-20 Biomerieux, Inc. Method for detection, characterization and/or identification of microorganisms in a sealed container
US9790534B2 (en) 2008-10-31 2017-10-17 Biomerieux, Inc. Methods for separation, characterization and/or identification of microorganisms using spectroscopy
US20100129858A1 (en) * 2008-10-31 2010-05-27 Biomerieux, Inc. Method for separation, characterization and/or identification of microorganisms using spectroscopy
US20100136609A1 (en) * 2008-10-31 2010-06-03 Biomerieux, Inc. Method for separation, characterization and/or identification of microorganisms using raman spectroscopy
US20100129814A1 (en) * 2008-10-31 2010-05-27 Biomerieux, Inc. Method for separation and characterization of microorganisms using identifier agents
US10415075B2 (en) 2008-10-31 2019-09-17 Biomerieux, Inc. Method for separation, characterization and/or identification of microorganisms using mass spectrometry
US8647835B2 (en) 2008-10-31 2014-02-11 BIO MéRIEUX, INC. Methods for separation, characterization and/or identification of microorganisms using spectroscopy
US8652800B2 (en) 2008-10-31 2014-02-18 Biomerieux, Inc. Method for separation, characterization and/or identification of microorganisms using spectroscopy
US20100129857A1 (en) * 2008-10-31 2010-05-27 Biomerieux, Inc. Methods for the isolation and identification of microorganisms
US20100120133A1 (en) * 2008-10-31 2010-05-13 Biomerieux, Inc. Separation device for use in the separation, characterization and/or identification of microorganisms
US20100120085A1 (en) * 2008-10-31 2010-05-13 Biomerieux, Inc. Method for separation, characterization and/or identification of microorganisms using mass spectrometry
US10167494B2 (en) 2008-10-31 2019-01-01 Biomerieux, Inc. Method for detection, characterization and/or identification of microorganisms in a sealed container
US9128058B2 (en) 2008-10-31 2015-09-08 Biomerieux, Inc. Method for separation and characterization of microorganisms using identifier agents
US10059975B2 (en) 2008-10-31 2018-08-28 Biomerieux, Inc. Methods for the isolation and identification of microorganisms
EP2548591A4 (en) * 2010-03-19 2014-09-03 Asahi Kasei Medical Co Ltd CELL DISPOSAL METHOD, CELL DISPOSAL SYSTEM AND METHOD OF REMOVING WHITE BLOOD CELLS
US9474845B2 (en) 2010-03-19 2016-10-25 Asahi Kasei Medical Co. Ltd. Cell removal method, cell removal system, and white blood cell removal method
EP3050583A1 (en) * 2010-03-19 2016-08-03 Asahi Kasei Medical Co., Ltd. Cell removal system
US10117987B2 (en) 2010-03-19 2018-11-06 Asahi Kasei Medical Co., Ltd. Cell removal method, cell removal system, and white blood cell removal method
EP2896416A1 (en) * 2010-03-19 2015-07-22 Asahi Kasei Medical Co., Ltd. Cell removal method, cell removal system, and white blood cell removal method
EP2548591A1 (en) * 2010-03-19 2013-01-23 Asahi Kasei Medical Co., Ltd. Cell removal method, cell removal system, and white blood cell removal method
US10870110B2 (en) * 2015-12-11 2020-12-22 Babson Diagnostics, Inc. Specimen container and centrifugation method for separating serum or plasma from whole blood therewith
US11697114B2 (en) 2015-12-11 2023-07-11 Babson Diagnostics, Inc. Centrifugation method separating serum or plasma from whole blood using a specimen container having a cap to retain blood cells
US11325117B2 (en) 2017-07-27 2022-05-10 Biomerieux, Inc. Centrifugally separating samples in a container having a seal and containing a plunger for opening the seal
US11305273B2 (en) 2017-07-27 2022-04-19 Biomerieux, Inc. Isolation tube with a rheological control member and a plunger
US11383231B2 (en) 2017-07-27 2022-07-12 Biomerieux, Inc. Isolation tube
US11440000B2 (en) 2017-07-27 2022-09-13 Biomerieux, Inc. Isolation tube with an endcap
US11090646B2 (en) 2017-07-27 2021-08-17 Biomerieux, Inc. Isolation tube
US11850584B2 (en) 2017-07-27 2023-12-26 Biomerieux, Inc. Isolation tube
US11883818B2 (en) 2017-07-27 2024-01-30 Biomerieux, Inc. Isolation tube
US11918998B2 (en) 2017-07-27 2024-03-05 BIOMéRIEUX, INC. Assembly comprising a sample collection vessel and a separation container having seal, plunger with seal-piercing point, retainer, and flexible sealing member
US12025629B2 (en) 2023-04-14 2024-07-02 Babson Diagnostics, Inc. Automated centrifuge loader

Also Published As

Publication number Publication date
AU6522974A (en) 1975-08-07
FR2217419A1 (US07608600-20091027-C00054.png) 1974-09-06
IT1018621B (it) 1977-10-20
JPS5642280B2 (US07608600-20091027-C00054.png) 1981-10-03
FR2217419B1 (US07608600-20091027-C00054.png) 1978-01-06
CA1021238A (en) 1977-11-22
GB1458147A (en) 1976-12-08
JPS49110822A (US07608600-20091027-C00054.png) 1974-10-22
DE2406362C2 (US07608600-20091027-C00054.png) 1988-02-18
DE2406362A1 (de) 1974-08-15

Similar Documents

Publication Publication Date Title
US3928139A (en) Detection of microbial pathogens
US3875012A (en) Apparatus and method for the detection of microbial pathogens
US3932222A (en) For isolating pathogenic microorganisms
US4886071A (en) Package including syringe and needle
US4212948A (en) Apparatus for detecting microbial pathogens employing a cushioning agent
CA1098429A (en) Method for detecting microbial pathogens employing a cushioning agent
US4038150A (en) Sample mixing and centrifugation apparatus
Berthrong et al. STUDIES OF THE EFFECT OF BACTERIAL ENDOTOXINS ON RABBIT LEUCOCYTES: I. Effect of Intravenous Injection of the Substances with andwithout Induction of the Local Shwartzman Reaction
Adams et al. Survival and recovery of human platelets stored for five days in a non-plasma medium
US4164449A (en) Surface separation technique for the detection of microbial pathogens
Melsom et al. The cytotoxic effect of mouse macrophages on syngeneic and allogeneic erythrocytes
CA1248435A (en) Process and device for the detection of pathogens
Chandra et al. Extended shelf life of random donor platelets stored for 7 days in platelet additive solution at different temperatures
AU679822B2 (en) Method for improving quantitative recovery of microorganisms from specimens containing blood components
Siegel et al. Action of staphylococcal toxin on human platelets
Dorn et al. Blood culture technique based on centrifugation: developmental phase
EP0107070B1 (en) Detection of microbial pathogens
Goldberg et al. Inhibition of macrophage migration: A test system using human monocytes
Tullis Studies on permeability of the leucocyte
Myhre et al. A rapid method for determining the sterility of frozen‐reconstituted blood
Loos et al. Leukocyte depletion: a biotechnical transfusion story
Conley The LE cell test
Denstedt et al. Problems in the Preservation of Blood
Diepenhorst et al. Removal of Leukocytes from Whole Blood and Erythrocyte Suspensions by Filtration through Cotton Wool: III. Influence of Filtration on the in vivo Viability of Stored Erythrocytes 1
CH615511A5 (en) Device for mixing a dose of sample fluid with a treatment liquid